article
stringlengths 0
682k
| abstract
stringlengths 146
3.69k
|
|---|---|
van der hoeve in 1932 included neurofibromatosis type 1 (nf1), tuberous sclerosis complex (tsc), and von hippel - lindau disease (vhl) in a group named phakomatoses. the conditions were defined as hereditary multisystem disorders, which share the common characteristics of the presence of spots, tumefactions, and cysts which were described as phakomata (from the greek term phakos for mother - spot), which proliferate and carry the risk of malignant transformation. over the years there has been much debate and some authors have suggested the inclusion of other ophthalmic diseases with neurocutaneous manifestations in the group of phakomatoses [26 ], but advances in molecular genetics provide evidence of the role of tumor suppressor genes in the pathophysiogenetic mechanisms of nf1, tsc, and vhl disease, which all have a familial pattern of inheritance, variable expressivity, and multisystem tumors with a risk of malignant transformation and can be considered the recent progress in multimodal imaging techniques, like near infrared reflectance (nir) and optical coherence tomography (oct), has enhanced our knowledge on the retinal manifestations of these diseases, providing unprecedented information on the morphological aspects of hamartomas. these methods have improved early diagnosis and the ongoing surveillance in these conditions. indeed, facilitated and noninvasive imaging with nir has led to propose choroidal nodules as an additional diagnostic criterion in nf1, which aids diagnosis in children and in uncertain cases. peripapillary retinal nerve fiber layer imaging with oct is potentially a rapid tool for monitoring children with nf1 as regarding optic pathway glioma. novel therapeutic strategies involve the tentative use of antivascular endothelial growth factors (anti - vegf) in the management of hamartomas and/or the complications arising from their presence. the present paper is a detailed review of the ophthalmological features in the phakomatoses with particular attention to novel imaging findings and ongoing management methods. nf1 is an autosomal dominant disease due to deletions or mutations of the neurofibromin gene located on chromosome 17p11.2. the prevalence of the disease is about 1 : 3000 and is not based on gender, race, ethnic group, and geographical area. nf1 has complete penetrance and variable expressivity but in about 50% of individuals the condition is due to de novo (spontaneous) mutations [1519 ]. ophthalmological features included in the nih diagnostic criteria of nf1 are lisch nodules and optic pathway gliomas (opg). other ocular structures involved include the orbit, eyelid, conjunctiva, cornea, anterior chamber, ciliary body, and retina. in patients with absence of the greater wing of the sphenoid bone, the most dramatic clinical sign is a pulsating proptosis, which is synchronous with heartbeat. upper eyelid plexiform neurofibromas are unilateral and are observed in the first two years of life., enlargement can lead to elephantiasis and extend to the temporal and frontal areas creating facial asymmetry. although various surgical approaches have been attempted, results are disappointing because of copious intraoperative bleeding and tendency to relapse [6, 2022 ]. sporadic conjunctival neurofibromas and typical asymptomatic hypertrophia of intrastromal corneal nerves (observed in one out of four patients) have been reported. corneal nerve thickening can possibly be due to the intrinsic genetic alteration of the disease with axon and schwann cell abundance. similar to other genetic diseases with corneal nerve thickening, epithelial changes may result from possible corneal nerve dysfunction and should be evaluated when considering refractive corneal surgery in these patients [24, 25 ]. they usually appear from the age of 2 years onwards and approximately 50% of children present nodules ; by adulthood this number increases to over 90% [2629 ]. the number of lisch nodules is correlated with choroidal nodules and we found that lisch nodules are positively correlated with cutaneous neurofibromas with increasing age. lisch nodules are often pigmented and their color may vary from creamy white in dark irides to brown in blue and green irides. we observed that they can vary from dome - shaped nodules to masses with ragged borders and various grades of confluency (figure 1). reported that they were more frequently localized in the inferior hemifield due to the sunlight - shielding effects of the upper eyelid ; however, in our study we did not find a predilection for any specific area. lisch nodules are easily observed with slit lamp examination ; however, they can be difficult to detect due to poor cooperation of young patients, when they are small or flat, or when they are localized in the iris crypts (figure 2). lisch nodules should not be confused with iris mammillations found in ocular melanosis which are smooth and homogenously distributed iris nodules that are commonly unilateral [32, 33 ]. glaucoma onset is rarely seen in nf1, although multiple pathogenic mechanisms related to angle closure have been proposed. the most commonly reported mechanisms are infiltration of the anterior chamber by neurofibromas that obstruct the angle, secondary angle closure by neurofibromatous cysts or increased thickness of the ciliary body and choroid, neovascular glaucoma, and developmental angle abnormalities [3436 ]. optical coherence tomography of the anterior chamber and ultrasound biomicroscopy can show lisch nodules and abnormalities of the ciliary body and chamber angle involved in glaucoma onset [3739 ].. found ipsilateral glaucoma and increased axial length in 23% of 95 patients with orbitofacial involvement and associated findings were pigmentary patches or anterior synechia and thickening of the ciliary body. therefore, tonometry and visual field analysis in patients with nf1 is advisable although this is not always easy to perform in younger patients [4144 ]. choroidal abnormalities of nf1 are often difficult to detect via fundus biomicroscopic examination and fluorescein angiography (figure 3). indocyanine green angiography in nf1 patients shows delayed perfusion of the choriocapillaris in areas corresponding to choroidal nodules [45, 46 ]. yasunari. used infrared monochromatic light to show multiple bright patchy choroidal alterations corresponding to choroidal nodules of the posterior pole in 33 eyes of 17 patients with nf1. evaluated choroidal alterations in nf1 patients using near infrared reflectance (nir) in 190 eyes and found nodules in 82% of patients. they established a cut - off value of 1.5 nodules to make a diagnosis and proposed to include choroidal nodules among the diagnostic criteria (figure 4). nir imaging with oct to detect choroidal alterations has widened the armamentarium of ophthalmologists in the diagnostic process in nf1. oct instrumentation is a valuable adjunct in the examination of children who can not collaborate due to their young age and condition. the enhanced depth imaging function of spectral domain oct allows the visualization of deeper ocular structures than traditional oct as the peak sensitivity is placed behind the retinal pigment epithelium. ayata. performed both longitudinal (b - scan) and transverse (c - scan) oct imaging and observed choroidal nodules at different depths in the choroid and demonstrated that the overlying retina showed changes in thickness. rao and choudhry found that the choroid over nodules had a loss of lucency suggesting vascular compression. we previously reported on choroidal nodules in 38 eyes of 19 patients with nir imaging and cross - sectional edi - oct images and showed that nir detected choroidal alterations corresponded to two types of hyperreflective choroidal nodules on edi - oct. well - delineated, bright, rounded shape alterations on nir corresponded to dome - shaped hyperreflective formations on oct - edi and poorly defined patchy alterations corresponded to rather flat, irregularly shaped, hyperreflective placoid formations (figure 5). moreover, through manual segmentation measurements of retinal layer and choroidal thickness using oct software, we demonstrated generalized choroidal and retinal thinning in patients with nf1. we showed that choroidal vasculature above nodules was altered in nf1 (figure 6) and as the choroidal vasculature is the main vascular support for the outer retina, we hypothesized that compromised choroidal blood flow can lead to an alteration of retinal trophism. retinal microvascular anomalies consisting in small (second or third order venule) tributaries of the inferior or superior - temporal vein denominated corkscrew retinal vessels, hemangioma - like, or ball of thread retinal microvessels have been reported and these seem to be overlying areas corresponding to choroidal nodules as shown by nir imaging (figure 7) [51, 52 ]. other retinal alterations such as multiple retinal capillary hemangiomas, astrocytic hamartomas, and, rarely, combined hamartomas of the retina and the retinal pigment epithelium (chrrpe) were described. oct features of chrrpe have been reported to include preretinal membranes, retinal striae, and a disorganization of the retinal layers including the retinal pigment epithelium and photoreceptors - ellipsoid zone [54, 55 ]. optic pathway gliomas (opg) have the most dramatic impact on visual acuity in nf1 patients. these tumors are mainly grade i glial neoplasms, termed pilocytic astrocytomas, which are histologically similar to gliomas that arise sporadically in individuals without nf1. although many opgs are asymptomatic, one - third to one - half can cause clinical symptoms, usually resulting in reduced vision, proptosis, visual field alterations, color alterations, and precocious puberty. king. studied 51 nf1 children with symptomatic opg where 39% presented decreased visual acuity, 26% had proptosis, 20% had precocious puberty, and 12% presented strabismus. visual field analysis is important in the screening of patients both for glaucoma and for possible opg. however, in young children this test is not always simple to carry out due to the young age of patients, which, however, improves with learning and increasing age [60, 61 ]. several studies demonstrated a correlation between opgs in nf1 affected children and oct evidenced thinning of the retinal nerve fiber layer (rnfl) [57, 62, 63 ]. gu. found a reduction of thickness in the ganglion cell layer - inner plexiform layer (gcl - ipl) in children with nf1 and opgs and suggested this as a more reliable measure of neuronal loss with respect to rnfl thickness. current guidelines for opg management include magnetic resonance imaging (mri) in patients presenting with signs of opg. chang. proposed the use of oct in children with opg, although the opg task force does not make a recommendation for routine oct testing. a variation in opg size is not always well correlated with visual outcomes ; therefore, a reduction in visual acuity or alteration of visual field implicates a need to initiate or alter treatment strategy. results of visual acuity and visual field assessment in children are not always reliable due to their young age. furthermore, children have difficulty in cooperating with standard table mounted oct devices ; thus avery these authors found highly reproducible thickness measurements of the gcl - ipl and suggested that a 10% change in the thickness of this layer should be deemed clinically significant. in a recent investigation at the ophthalmology unit of the university of rome, we used spectral domain oct to evaluate macular neuronal and axonal thickness values in adult subjects with nf1 without opg. we found that the peripapillary rnfl, macular rnfl, and gcl - ipl were reduced in thickness (figure 8). we speculated that this could be due to the age related, chronic nature of nf1 or that choroidal and retinal thinning could also involve the macular and peripapillary rnfl and the gcl - ipl [26, 66 ]. furthermore, we found that the rnfl and gcl - ipl thickness values were correlated in nf1 patients ; thus, we suggested the peripapillary rnfl / gcl - ipl index as a possible parameter in monitoring disease evolution. high levels of vegf have been demonstrated in plexiform neurofibromas and malignant peripheral nerve sheath tumors. interestingly since vegf is produced by schwannoma tumor cells, systemic bevacizumab therapy has been in the management of vestibular schwannomas in neurofibromatosis type 2 [68, 69 ] but further research is warranted to evaluate anti - vegf therapy in nf1. tuberous sclerosis complex is a multisystemic disease outlined by hamartomas involving the central nervous system, eye, skin, kidneys, heart, lung, and liver. this condition was first described by bourneville in 1880 who documented typical tuber - like cerebral lesions in the human brain at postmortem examination. in 1932, van der hoeve described the retinal changes. the mutations for the disease, discovered in 1987, are tsc1 and tsc2 located on chromosomes 9q34 and 16p13, which encode for hamartin and tuberin, respectively. the incidence of the disease is estimated to range from 1/6800 and 1/15000 and the prevalence is 1/10,000 with 50% to 84% of the cases being sporadic. the typical retinal lesions are astrocytic hamartomas, which can be the first manifestation of disease and are among the major criteria for diagnosis. they have been reported in 44% to 48% of patients in two large case series [72, 73 ]. retinal hamartomas characterized by fibrotic astrocytes with small oval nucleus and long cytoplasmic extensions were reported by rowley. in 44 of 100 (41%) patients, with bilateral presentation in 15 cases (34%). these hamartomas are divided into three basic morphological types : smooth, relatively flat, noncalcified, grey, translucent lesions with an oval or circular shape also known as younger tuberous bodies ; elevated, multinodular, calcified, opaque lesions similar to mulberries also named older tuberous bodies ; transitional lesions characterized by both features (figures 9 and 10). the younger tuberous bodies are not easy to identify with ophthalmoscopy due to their translucent or blurry aspect. at times the only sign that can help to identify these lesions is a perilesional circular reflex. the older tuberous bodies are usually peri- or epipapillary and can be confused with optic disc drusen. their structure has a high acoustic density at b - scan ultrasonography due to calcifications in the lesions. the transitional lesions have a smooth margin appearance and can be multinodular in the center. fluorescein angiography can help to recognize the vascularization pattern and to distinguish younger from older lesions. in most cases visual acuity is not affected due to extramacular localization of lesions. visual field defects and a progressive loss of visual acuity in a study on 179 cases reported the coexistence of more than one type of hamartoma in 30% of patients. the relatively flat hamartoma was most frequently observed (70%), followed by the mulberry - like lesion (55%) and the transitional type (9%). other features of the retina found in tsc are retinal pigmentary changes including both hyperpigmented areas (possibly due to congenital retinal pigment epithelium hypertrophy) and areas of hypopigmentation with a punched out appearance that can be found both at the posterior pole and in the midperiphery. rowley. found a statistically significant correlation between punched out areas of chorioretinal hypopigmentation and tsc and showed 39% in the afflicted patients against 6% among controls. shields. studied retinal hamartomas with time domain oct in 15 patients and found typical characteristics such as hyperreflectivity and round, confluent, moth - eaten empty spaces with posterior shadowing on the surface of the tumor and disorganization of the internal retinal layers. moreover, the authors observed a gradual progression from normal to tumorous retina in all cases, inner retinal disorganization in 3 cases (20%), outer retina disorganization in 0 cases (0%), and disorganization of the entire retina in 5 patients (33%). furthermore, oct showed moderate retinal traction over the tumor in 4 cases, optically empty spaces in 10 cases, and shadowing posterior to the tumor in 14 cases (93%). shallow elevation or edema of the retina adjacent to the tumor was found in 13% and 27% of cases, respectively, and macular edema was found in 20% of cases. soliman., using time domain oct, differentiated oct findings in the younger tuberous bodies where alterations were localized at the retinal nerve fiber layer with normal structure of the retinal layers and retinal pigment epithelium below, from oct characteristics of older tuberous bodies where there was hyperreflectivity in the inner retinal layers with total shadowing of the underlying layers. the translucent nature of the younger tuberous bodies could explain the absence of shadowing in these lesions. xu. showed that small retinal astrocytic hamartomas, undetectable with ophthalmoscopy, could be identified with infrared imaging and spectral domain oct. other ocular findings reported by rowley. included palpebral angiofibromas in 39% of patients, nonparalytic strabismus in 5% (4 exotropia and 1 esotropia), iris and choroidal colobomas in 3%, and areas of iris depigmentation in 2%. myopia was present in 27%, hyperopia in 22%, and astigmatism (> 0.75 d) in 27% of patients ; such distribution of refractive errors is fairly similar to the distribution among normal individuals, as assessed by population studies. lopez. reported a case of unilateral eyelid angiofibroma with complete blepharoptosis as the presenting sign of tsc. several case reports document the association between tsc and cataract [78, 79 ], serous retinal detachment [74, 80 ], hamartomas of the iris, and hamartomas of the ciliary epithelium. in some cases ash leaf shaped iris hypopigmentation has been observed, as shown in a patient observed in our institute (figure 11). ocular signs rarely described include corneal leukoma, megalocornea, primary and secondary glaucoma, optic nerve atrophy, papilledema, and vi nerve palsy. table 1 summarizes the clinical findings and diagnostic procedures in tsc. in a review by mennel., flat lesions evolved to more prominent forms without symptoms in about 10% of patients but spontaneous regression was also reported. therefore, observation is only necessary unless complications arise. in cases where exudation occurs, saito. used intravitreal bevacizumab to treat two patients with tumor neovascularization and macular edema and found encouraging results as visual acuity and macular edema rapidly improved, tumor size decreased, and tumor associated neovascularization was rapidly attenuated. retinal toxicity has been associated with the drug vigabatrin, which is often used as first - choice therapy in the treatment of refractory seizures typical of tsc. retinal toxicity is shown with visual field loss that initiates as a bilateral nasal defect and evolves to concentric vision loss. oct of the peripapillary rnfl has shown that the alterations predominantly involve the nasal, superior, and inferior sectors. electroretinogram examination has been held as the optimal method to evaluate vigabatrin toxicity in children under nine years of age where perimetric examination is not reliable, but this frequently requires general anesthesia as some children can not collaborate due to their young age or neurological condition. thus, oct has been proposed as a method to monitor therapy. in young children hand - held oct, especially with the eye tracker system provided by spectral domain technology, is a rapid examination during general anesthesia [11, 90, 91 ]. ophthalmic monitoring is advised upon initiation of vigabatrin therapy and every three and six months during treatment in children and adults, respectively ; treatment should be suspended if there are signs of retinal toxicity. von hippel - lindau (vhl) disease has an autosomal dominant transmission and is caused by a germline variation of the vhl tumor suppressor gene positioned on the short arm of chromosome 3p25 - 26 [92, 93 ]. approximately 1/40,000 cases occur per year, and by age 65 years, there is close to 100% penetrance. the condition is marked by the growth of numerous cysts and benign or malignant tumors in many organs. the most common characteristics of this disease are vascular tumors of the central nervous system hemangioblastoma (hb) and retinal capillary hemangioblastoma (rch) also referred to as hemangioma. lesions are found more commonly in the second or third decade of life although rchs usually occur a decade earlier than cerebellar hemangioblastomas. increase in vascular endothelial growth factors in vhl syndrome is believed to heighten rch formation and growth [94, 95 ]. the most common and, most often, earliest manifestations of vhl disease are rch, which occur in 43 to 85% of patients (figure 12). they develop from the age of 10 until 30 years similarly to hb in the central nervous system. rch may remain asymptomatic for years and can regress spontaneously, but usually their growth leads to visual deterioration. they may be single or multiple tumors and are sometimes the only manifestation of the disease. generally only a single tumor develops in one eye and it is usually asymptomatic in most patients. histopathological procedures show that rchs are made of capillary blood - filled spaces lined by pericytes and endothelial and stromal cells. based on immunohistochemical studies, it has been advanced that this type of cell may represent lipidized fibrous astrocytes and glial cells or more likely vasoformative stem cells that may originate from angioblasts arrested in development. on ophthalmoscopic examination, rchs are confined, globular, red - orange vascular tumors, typically observed in the juxtapapillary or the peripheral retina. peripheral lesions are most commonly found in the superior - temporal quadrant. in 15% of cases rchs the lesions usually have a feeder vessel where exudation and subretinal fluid can be found. ocular complications include macular or extramacular exudation, tractional retinal detachment, new vessels on a peripheral hemangioblastoma, and neovascular glaucoma. small peripheral rchs are sometimes difficult to detect with funduscopy and are better seen with fluorescein angiography where lesions are characterized by hyperfluorescence. the feeder vessel will hyperfluoresce and is well evidenced in the arterial phase whereas the draining vein is clearly seen in the venous phase. in the late phases the tumor shows hyperfluorescence and leakage (figure 13). vessels in hb are fenestrated leading to exudation / leakage in contrast to normal retinal vessels. the differential diagnosis of rch is with wyburn - mason disease (racemose hemangioma), coats ' disease, retinal macroaneurysm, vasoproliferative retinal tumor, and retinal cavernous hemangioma. shields. described oct characteristics of rch, which showed thickening and structural alterations of the retina with mild shadowing of the underlying structures. these authors highlighted the importance of oct in evaluating intra- and subretinal fluid ; longstanding retinal edema in these lesions had features similar to cystoid macular edema whereas the chronic persistence of subretinal edema seemed to have caused a reduction of thickness of the layer corresponding to photoreceptors. rarely exophytic rchs arising from the outer retina, which do not commonly have arteriovenous shunting, have been reported in vhl disease. oct features observed in these forms are location of the lesions in the outer retinal layers, shadowing, and photoreceptor layer rips close to the lesion. the setback for oct is when lesions are large and can not be scanned due to technical limitations of the instrument. treatment of rch usually includes laser photocoagulation, photodynamic therapy, cryotherapy, radiation, and surgical removal. in the literature various authors recommend early treatment of rch when lesions tend to enlarge and become more difficult to treat with time. nevertheless, asymptomatic peripheral rch lesions only require observation and treatment only in case of complications. describe their experience in the management of rch in 77 eyes of patients with vhl. eighty - two percent of rch were initially observed for a period of 84 months and the authors suggested only close observation in lesions smaller than 1500 microns, which are not visually threatening [107, 108 ]. photocoagulation with argon, krypton, and yellow dye and diode laser is effective in extrapapillary or juxtapapillary rch where lesions are 1500 microns or less in size and localized in the posterior retina [107, 108 ]. singh. performed cryotherapy in lesions larger than 3000 microns and treated extrapapillary rch with a mean size of 4500 microns with iodine-125 plaque with good results. thermotherapy, external bean radiotherapy, pars plana vitrectomy, and related procedures are more frequently used to treat the juxtapapillary locations of rch. photodynamic therapy (pdt) is aimed at vascular endothelial cells and has also been employed in the management of rch [109, 110 ]. however, when a hemangioblastoma is associated with the optic nerve, such treatment can result in loss of vision. therefore, vegf - targeted strategies have been tested but therapy outcomes are contrasting [107, 111, 112 ]. su5416 is an inhibitor of vegf receptor-2, which was used through intravenous administration in a case of rch of the optic nerve head and in a single case of a juxtapapillary rch and improved visual acuity and visual field without reducing the size of the tumor [113, 114 ]. found stability or improvement in the ocular lesions in 2 out of 6 patients treated with su5416. intravitreal bevacizumab associated with photodynamic therapy showed tumor regression and reduction of exudation, whereas pegaptanib only reduced exudation with no effect on tumor size. wong. published their results on 5 cases treated with intravitreal ranibizumab and reported that in two patients with large juxtapapillary lesions associated with circinate lipid, intraretinal hemorrhage, and marked retinal edema extending into the macula this therapy did not exert any beneficial effect whereas it was successful in a patient with a juxtapapillary endophytic lesion of one disk diameter with limited retinal edema not involving the fovea. they concluded that intravitreal ranibizumab does not have a beneficial action on the largest and most exudative lesions but is efficacious in small lesions. therefore, effectiveness and appropriateness of treatment are influenced by the site and size of rch ; treatment can not be delivered to juxtapapillary rchs without damage to the optic nerve whereas it can be applied to the peripheral retina. today intravitreal anti - vegf treatment is successfully used in a wider array of retinal pathologies and recently in metastatic tumors of the choroid [118122 ]. a principal motive for unsuccessful outcome of this treatment may be the size of hamartomatous lesions in vhl disease, which can be considerably greater than choroidal neovascular membranes. moreover, the mature vasculature of the hamartomas and genetic alterations underlying the phakomatoses leading to incessant production of vegf can result in poor response [111, 123 ]. the phakomatoses require a multidisciplinary approach and the ophthalmologist plays a fundamental role in diagnosis and management. imaging techniques in ophthalmology have made groundbreaking progress in the past decade and nir may lead to the inclusion of choroidal nodules as the 8th diagnostic criteria in nf1. oct provides novel information on retinal hamartomas and enhanced depth imaging technology enables visualization of the choroid in vivo providing further information on the morphology of the choroid and choroidal nodules in nf1. peripapillary rnfl analysis with oct is a noninvasive tool and is an adjunct in monitoring optic pathway gliomas. furthermore, hand - held oct enables faster examination of the rnfl in children who are unable to collaborate due to their young age and neurological conditions. an enormous bulk of research has led to the establishment of optimal doses and protocols for intravitreal anti - vegf treatment in retinal pathology, but little information is available for this treatment in the phakomatoses and reports on the use of anti - vegf agents are contrasting and incomplete. the future might hold new drug delivery systems for the intravitreal treatment of symptomatic retinal hamartomas. | neurofibromatosis type 1, tuberous sclerosis complex, and von hippel - lindau disease, historically classified as the phakomatoses, are hereditary multisystem disorders characterized by the presence of hamartoma, which carry the risk of malignant transformation. the alteration of tumor suppressor genes seems to be at the basis of their pathophysiogenetic mechanism. lisch and choroidal nodules in neurofibromatosis type 1, retinal astrocytomas in tuberous sclerosis complex, and retinal capillary hemangioma in von hippel - lindau disease are the principal ophthalmic hamartomatous manifestations. the advent of novel imaging techniques such as near infrared reflectance and optical coherence tomography has provided unprecedented insight on the choroidal and retinal features of these diseases. these methods have improved early diagnosis and the ongoing surveillance in these conditions. among an array of treatment modalities, antivascular endothelial growth factor therapy has been used in the management of retinal hamartomas but results have been varied. this review is an update on the pathophysiogenetic mechanisms, ophthalmic manifestations, and novel treatment strategies in the phakomatoses with emphasis on the role of imaging techniques. |
citicoline is chemically identical to cdp - choline, the natural precursor of the major cell membrane phospholipid phosphatidylcholine.given orally or by injection, citicoline is non - toxic and very well tolerated.preclinical experiments with various models of central neurodegenerative diseases have shown that citicoline displays significant neuroprotective properties.however, recent large and well - controlled data have shown no benefit from citicoline in acute ischaemic stroke and traumatic brain injury.the pharmacological actions of citicoline in the central nervous system seem to be pleiotropic and involve, amongst other things, modulation of some kinases and sirtuin-1. however, our understanding of the mechanisms involved is, at most, fragmentary. citicoline is the generic name, or the international nonproprietary name (inn) of cytidine-5-diphosphocholine (cdp - choline, cdpcho), a pharmaceutical substance that is chemically identical to the naturally occurring metabolite, which plays crucial role in the synthesis of phospholipids. the work of kennedy and collaborators in the 1950s showed that this phosphorylated choline nucleotide is a precursor of glycerophospholipid phosphatidylcholine (pc). pc and its twin compound phosphatidylethanolamine (pet) are the two most abundant phospholipids in eukaryotic cells, accounting for more than half of the total phospholipid content in membranes. the pathway of de novo synthesis of pc, the cdp - choline pathway (fig. 1), includes the enzymes cytidine kinase (ck), choline phosphate cytidilyltransferase (cct) and cdp - choline:1,2-diacylglycerol choline phosphotransferase (cpt). choline that is used for synthesis of phosphatidylcholine through the cdp - choline pathway is derived from transport of exogenous choline into the cell or from phospholipase d - mediated turnover of pc.fig. adp adenosine diphosphate, atp adenosine triphosphate, cct choline phosphate cytidilyltransferase, ck cytidine kinase, cmp cytidine monophosphate, cpt cdp - choline:1,2-diacylglycerol choline phosphotransferase, ctp cytidine triphosphate, dag 1,2-dicacylglycerol, pc phosphatidylcholine, pp i pyrophosphate the cytidine-5-diphosphocholine (cdp - choline) pathway of enzymatic synthesis of phosphatidylcholine. adp adenosine diphosphate, atp adenosine triphosphate, cct choline phosphate cytidilyltransferase, ck cytidine kinase, cmp cytidine monophosphate, cpt cdp - choline:1,2-diacylglycerol choline phosphotransferase, ctp cytidine triphosphate, dag 1,2-dicacylglycerol, pc phosphatidylcholine, pp i pyrophosphate the search for relevant literature non - clinical as well as clinical was performed in the pubmed, scopus and web of science databases, using the term cdp - choline, its variants (cdpcholine, cytidine - diphosphocholine, etc.) and citicoline. whereas cdp - choline designates the natural metabolite synthesized inside the cells (a substance of endogenous origin), the term citicoline did not exist until the substance became used as the drug, which occurred in the 1970s. notwithstanding, some papers currently indexed under the term citicoline are dated earlier ; some of them (e.g. the paper by berger and gimenez, describing crystallization of cdp - choline from yeast) could be qualified as concerning citicoline (the compound synthesized exogenously), but others (e.g. the paper by ansell and bayliss, reporting on the concentration of endogenous cdp - choline in the rat brain) undoubtedly concern cdp - choline (the compound synthesized endogenously). the difference between cdp - choline (synthesized endogenously) and citicoline (synthesized exogenously) is not trivial, as will be discussed later. in the anglophone medical literature, the use citicoline as a drug was advocated as early as 1974, by manaka. from japan, for the treatment of parkinson s disease. a few years later, the idea of using it as a neuroprotectant stemmed from the observation, made by horrocks and collaborators, of the reversibility of phosphotransferase enzymes in the brain. the first patent for the use of cdp - ethanolamine, alone or in combination with cdp - choline, to reverse neurodegenerative diseases was issued in 1981. since that time, hundreds of studies concerning various aspects of the preclinical and clinical pharmacology of citicoline have been published. several relevant reviews have also appeared in high - ranking journals, including at least five during the last 4 years [1014 ]. however, the subject of citicoline s mode of action is far from being clarified. the present review is an attempt to classify the major issues concerning the neuroprotective properties of citicoline into three categories : facts, i.e. those that are solved with an acceptable degree of certainty ; doubts, i.e. those that are likely misinterpreted ; and unresolved issues, i.e. those that remain unexplained. needless to say, such classification is subjective : facts, doubts and unresolved issues are, in many cases, intermingled. citicoline displays negligible toxicity. the compound is quickly catabolized (fig. 2), and the products arising are subsequently available for diverse biosynthetic pathways and ultimately excreted as carbon dioxide. the lack of acute and chronic toxicity of citicoline has been repeatedly confirmed in rodents and dogs (see the most recent report by schauss. and the references quoted therein). an impressive example is the median lethal dose (ld50) of an acute single intravenous application of citicoline, which equals 4,600 and 4,150 mg / kg in mice and rats, respectively. the ld50 for ingested citicoline is even higher at approximately 8 g / kg in both mice and rats. for comparison, in mice, the ld50 of an acute single intravenous dose of sodium chloride is 645 mg / kg, and that of vitamin c is 518 mg / kg. in a 90-day rat oral toxicity study of 1001,000 mg / kg daily doses, increases in serum creatinine and in renal tubular mineralization, likely caused by phosphate liberation from citicoline, 2presumed catabolism of citicoline (cyt - p - p - cho) in the rodent intravascular compartment. in the first step, hydrolysis of the pyrophosphate cytidine monophosphate (cyt - p) and phosphocholine (cho - p) are dephosphorylated to cytidine (cyt) and choline (cho), respectively ; supposedly, a large part of the liberated choline is taken up by the liver (which may explain the unexpectedly low cholinergic toxicity of citicoline) presumed catabolism of citicoline (cyt - p - p - cho) in the rodent intravascular compartment. in the first step, hydrolysis of the pyrophosphate bridge takes place. in the second step, cytidine monophosphate (cyt - p) and phosphocholine (cho - p) are dephosphorylated to cytidine (cyt) and choline (cho), respectively ; supposedly, a large part of the liberated choline is taken up by the liver (which may explain the unexpectedly low cholinergic toxicity of citicoline) the usual daily therapeutic dosage of citicoline in humans is 5002,000 mg that is, 728 mg / kg in a person of average bodyweight (70 kg). data from clinical trials have corroborated preclinical toxicological findings, revealing a favourable safety profile, with only a few reports of adverse events, mostly related to digestive disturbances following oral intake. in adult and elderly stroke patients, the drug has lacked significant adverse events (see, for example, the study by cho and kim in 4,191 korean stroke patients), and a meta - analysis of placebo - controlled trials has shown that the overall frequency of adverse effects was comparable between groups comprising 1,652 actively treated and 686 placebo - treated subjects. there are no data concerning the effects of liver or kidney insufficiency on the safety profile and pharmacokinetics of citicoline although, on the basis of the aforementioned toxicology data, an enhanced threat of hyperphosphataemia may be predicted in patients with kidney failure. upon administration, citicoline is relatively quickly catabolized and is the source of choline that appears in the blood. administered parenterally or orally, citicoline is relatively quickly (i.e. within minutes rather than hours) converted to its cholinergic and pyrimidinergic catabolites. in the perfused rat liver, since phosphorylated substrates are considered unable to penetrate cell membranes, it is usually assumed that cytidine monophosphate (cmp) and phosphocholine (pcho) yielded from hydrolysis of citicoline are further dephosphorylated by phosphatases in blood plasma. in agreement with this assumption, citicoline given orally to rats produced pronounced increases in plasma cytidine and choline, although it should be noted that the rise of cytidine was several - fold larger than that of choline. a recently published paper reported on the use of a liquid chromatography electrospray ionization tandem mass spectrometry (lc esi ms / ms) method to evaluate the pharmacokinetics of choline in blood from human volunteers following ingestion of 1,000 mg citicoline tablets. biphasic concentration time curves of choline in plasma have been recorded, with a large peak of 2 g / ml at approximately 2 h and a second, slightly smaller but much broader peak with a maximum at approximately 24 h following ingestion. unfortunately, no data on the initial plasma level of choline were shown, which makes the whole picture a bit unclear. one older source reported an arithmetic mean plasma choline level oscillating around 1.36 g / ml in a healthy human subject, and this was confirmed recently. choline phospholipid metabolism is altered in such a way that cancer cells display elevated levels of phosphocholine, as well as total choline - containing compounds. one may therefore pose a question as to whether increasing choline exposure does nt induce carcinogenesis and/or accelerate cancer growth. however, choline chloride has displayed no mutagenic potential when tested in vitro (using ames testing, yeast gene conversion, clastogenicity and sister chromatid exchange), and similar negative data have been obtained for citicoline. moreover, epidemiological data have shown that the associations between choline intake and cancer if any are weak. for example, johansson. found that elevated plasma concentrations of choline may be associated with a slightly increased risk of prostate cancer, but a similar (even slightly stronger) association has been found for vitamin b2. on the other hand, lee. did not find any association between choline (or betaine) intake and the risk of colorectal cancer, whereas xu. found that dietary choline intake was inversely associated with breast cancer risk. the compound has offered marked neuroprotection in several in vitro and in vivo models of acute and chronic brain ischaemic and neurodegenerative diseases, including brain hypoxia, ischaemia and intracerebral haemorrhage (reviewed by adibhatla and hatcher), brain and spinal cord trauma, in vitro glutamate excitotoxicity [32, 33 ] and in vivo amyloid toxicity. however, the mechanisms of this neuroprotection are far from being understood. one major effect of citicoline is believed to be stimulation of the synthesis and increase in the content of brain phospholipids. increases in brain phospholipids following oral administration of citicoline have also been observed in humans, with use of phosphorous magnetic resonance spectroscopy. the relevant hypothesis assumes that the citicoline breakdown products cytidine and choline enter the brain separately and, inside brain cells, they act as substrates for resynthesis of cdp - choline. this, in turn, is believed to result in slowing down of phospholipid breakdown and acceleration of phospholipid resynthesis necessary for membrane repair. however, since citicoline is devoid of cholinergic toxicity (see below), a significant rise in brain choline following therapeutic doses of citicoline in humans does not seem probable indeed, a decrease in choline in the brains of older subjects and no change in those of younger subjects have been observed following oral citicoline, with use of proton magnetic resonance spectroscopy (mrs). the other mechanisms suggested to be involved in the neuroprotective effects of citicoline in stroke models include prevention of activation of phospholipase a2 (pla2). the related effects comprise attenuation of the increase in hydroxyl radical generation, preventing loss of cardiolipin (an exclusive inner mitochondrial membrane phospholipid essential for mitochondrial electron transport, which is degraded in response to cellular insults and disrupts the mitochondrial respiratory chain). in aged rats, an increase in the brain level of platelet - activating factor (a bioactive phospholipid implicated in neuronal excitotoxic death) has also been noted. in rats, attenuation of mitogen - activated protein kinases (mapks) and caspase activation have been observed following citicoline administration [40, 41 ]. last, but not least, according to the most recent report, treatment with citicoline has been found to increase sirtuin-1 (sirt1) protein levels in cultured neurons, in circulating blood mononuclear cells and in the brain. this effect seems to be of critical importance for neuroprotection in experimental stroke because sirtinol, a specific inhibitor of sirt1 which, by itself, does not influence infarct volume, has been shown to abolish the neuroprotection offered by citicoline. citicoline displayed a potent synergistic effect with resveratrol (which is known to be a sirt1 activator), leading to a 60 % reduction in the experimental infarct volume in rats when both drugs were used in doses that were individually ineffective. moreover, citicoline was ineffective in sirt1 knock - out homozygotic mice subjected to focal brain ischaemia. however, detailed mechanistic explanations for all of these effects are lacking. for example, there is no explanation as to how citicoline administration leads to attenuation of mapk activity and increases sirtuin-1 protein content in brain tissues ; in particular, does the drug act extracellularly, or is resynthesis of cdp - choline inside brain cells a prerequisite ? positive results of preclinical studies with animal models of neurodegenerative diseases have prompted clinical trials with citicoline as a treatment for human brain diseases. whereas several previous small clinical studies had achieved promising results, two recent large randomized multicenter trials the cobrit (citicoline brain injury treatment) trial performed in 1,213 patients with traumatic brain injury, and the international, randomized, multicentre, placebo - controlled sequential ictus (international citicoline trial on acute stroke) trial performed in 2,298 patients with moderate - to - severe acute ischaemic stroke led to the conclusion that citicoline is not efficacious in these clinical settings. the negative outcomes of these studies were deemed surprising and prompted a few comments, which focused mostly, although not exclusively, on methodological aspects of the evaluation of the clinical effects of the drug [4547 ]. what was not commented on was the lack of a mechanistic explanation for the putative neuroprotective properties of citicoline. some recent data are suggestive that prolonged intake of citicoline, given orally or by injection, may be significantly effective in certain slowly developing neurodegenerative diseases. one is glaucoma, currently considered a neurodegenerative disease, which involves the entire central visual pathway. in glaucoma patients with moderate visual defects, citicoline treatment improved retinal function and neural conduction, and continuation of treatment for 28 years significantly slowed, stabilized or even improved glaucomatous visual dysfunction [48, 49 ]. (studio di intervento nel decadimento vascolare lieve) study, oral citicoline taken for up to 9 months significantly improved the mini - mental state examination score and positively influenced mood ; the latter effect could have been related to increases in noradrenaline and dopamine levels, which would be expected on the basis of animal experiments (see the paper by rejdak. and the references cited therein). also, it has recently been shown that in sub - acute ischaemic cerebrovascular disease, administration of citicoline in an intravenous dose of 2,000 mg for 5 or 10 days improves functional independence and reduces the burden of care. the uniqueness of citicoline may lie not only in its negligible toxicity and virtual lack of side effects but also in the fact that it appears to deliver a significant subjective improvement and mood - enhancing effect. where is citicoline catabolized ? while it seems reasonable to assume that, at least in rodents, citicoline given systemically is quickly hydrolysed and further dephosphorylated, the particulars of this decomposition process are uncertain. of note is that, following injection or ingestion of citicoline in the rat, the increase in plasma cytidine was, on a molar basis, several times larger than the concomitant increase in plasma choline. what happened to the domination of the cytidine increase over the choline increase provide an explanation for the lack of cholinergic toxicity of citicoline versus the toxic effects of equimolar choline (see below) ? how is citicoline catabolized ? in humans, an oral challenge with citicoline was accompanied by an increase in plasma uridine instead of cytidine. in this last study, the participants took oral citicoline doses of up to 4 g (still much less per kilogram of bodyweight than the doses used in most rodent experiments), and significant dose - related increases in blood plasma uridine were observed. the data resembled the rat data reported previously by the same laboratory in that the magnitude of the choline increases was several times smaller than the magnitude of the pyrimidine increase. choline increments obtained after 2 or 4 g doses were comparable, on a molar basis, to those seen after similar doses of choline chloride, although the peaks were delayed by one or more hours. the authors failed, however, to detect any significant quantities of cytidine in human blood, either before or after citicoline intake. they interpreted this finding as evidence that the human gastrointestinal tract and liver quantitatively transform cytidine liberated from citicoline to circulating uridine. would this indicate that hydrolysis of citicoline to cmp and pcho and consecutive dephosphorylations of these products to cytidine and choline occur almost immediately, followed by conversion of cytidine to uridine ? but why is the final effect namely, an increase in circulating choline delayed by one or more hours ? resynthesis of cdp - choline in the brain following citicoline intake and the pharmacodynamics of citicoline. it is usually assumed that following citicoline intake, cytidine and choline enter brain cells separately and are used for intracellular synthesis of cdp - choline.. stated it is believed that when citicoline is exogenously administered as sodium salt, it is hydrolysed into choline and cytidine to be re - synthesized later in the brain. these statements seem to imply that resynthesis of citicoline from cytidine and choline in the brain is the only event or at least the most important event of systemic citicoline application. at this point, the difference between citicoline (synthesized exogenously) and cdp - choline (synthesized endogenously) acquires its key importance, for at least three reasons. first, it must be appreciated that only a minor fraction of the choline dose administered as citicoline enters the brain. investigated the biodistribution and biokinetics of [c]choline (a radio - pharmaceutical used for oncological positron emission tomography [pet ] studies) in rats and humans following intravenous injection. they found that the highest uptake of the tracer was by the kidney, lung and adrenal glands, whereas the brain cortex and cerebellum were the organs taking up less than 0.1 % of the tracer dose. second, stimulation following citicoline intake of at least two other major synthetic pathways of choline in the brain, one leading to betaine (an important donor of methyl groups) and the other leading to acetylcholine (an important cns neurotransmitter), should also be taken into account. third, the ischaemic heart is the source of increased choline in the blood of patients with an acute coronary syndrome related to coronary plaque instability. moreover, the authors mention small unpublished pilot studies in which elevated levels of whole blood choline were also found in patients with stroke or cerebral ischaemia in combination with advanced plaques in the carotid artery. it is reasonable to assume that degradation of membrane phospholipids and elevation of blood choline levels occur in all cases of brain ischaemia, i.e. ischaemic stroke. is there any difference between the metabolic effects of choline increases in plasma consequential to citicoline intake and those resulting from a heart attack or stroke ? the systematic review and meta - analysis of data obtained with preclinical models of embolic stoke provided evidence that citicoline does indeed deliver some neuroprotection ; however, the effect is stronger for infarct volume reduction and more limited for neurological outcome. the authors concluded that factors shown to be important for translation into human studies are multiple - dose administration, large infarct size and/or neurological deficit. however, the most important cause of the irreproducibility of preclinical results in a clinical setting may be the use of excessively large doses of citicoline in most animal experiments. for example, in a series of papers authored by savci and collaborators (see the paper by eyigor. and the references quoted therein), various cardiovascular and endocrine effects of large, although subtoxic, doses of citicoline (0.52 were an increase in blood pressure and large rises in plasma levels of catecholamines and several pituitary hormones, including vasopressin and oxytocin. can these effects be of potential benefit, e.g. in hypovolaemic shock (as suggested by savci.), or are they instead an early sign of toxicity ? (on the other hand, it would be of importance to find an explanation for the observation that in rats both normotensive and haemorrhagic these massive doses of citicoline increased blood pressure, whereas no such effect occurred following equivalent doses of either cytidine or choline.) similar doubts may be raised against the recent report by gutirrez - fernndez., who compared the effects of citicoline (1,000 mg / kg intraperitoneally) and recombinant tissue plasminogen activator [rt - pa ] (5 mg / kg intravenously) in a rat model of embolic stroke. the problem with this paper was that whereas the rt - pa dose used per kg of bodyweight was 5 to 8 times the dose used in the treatment of human embolic stroke (0.61.1 mg / kg intravenously ; see wardlaw.), the citicoline dose was 15 to 30 times the dose used in clinical trials of citicoline in stroke. the rationale for investigating the effects of injecting citicoline in doses corresponding to 30150 g per person (assuming an average human bodyweight of 70 kg) is doubtful. why is citicoline so much less toxic than choline ? the acute toxicity of citicoline and choline after oral and intravenous application was compared by agut.. these authors concluded that cdp - choline given either orally or intravenously did not cause any cholinergic intoxication in the treated groups, whereas such toxic effects were observed after administration of an equimolar dose of choline. apparently, cdp - choline given by the oral or intravenous route yields toxicological consequences that are different from those yielded by choline. a mechanistic interpretation of the substantially lower toxicity of citicoline compared with that of choline is lacking to date. used citicoline doubly - labelled with carbon-14 at the methyl groups of choline and tritium at c5 of pyrimidine, and obtained evidence of cdp - choline being broken down in the intestine, resulting in release of choline and cytidine, likely due to the actions of intestinal esterases and pyrophosphatases. these split compounds as well as the original cdp - choline may be absorbed from the intestinal mucosa as such. apparently, they could not rule out the possibility that following oral intake, some fraction of intact citicoline is absorbed. the consequences of the assumed fast hydrolysis and subsequent dephosphorylation of citicoline after injection or oral intake are usually interpreted in terms of a prodrug, which is administered in an inactive or less than fully active form and is subsequently metabolically converted (bioactivated) to the active pharmacological agents, cytidine and choline. however, one may assume that the reverse is true, i.e. the most active form is unhydrolysed (intact) citicoline, whereas cytidine and choline are its pharmacologically less active metabolites. although the prodrug concept clearly prevails in the scientific literature, several observations may indicate that intact citicoline molecules, or perhaps cmp and/or pcho intermediates, display their own activities, which are different from and/or more potent than the actions of cytidine and choline. one reason for not discarding the idea of intact citicoline being significantly neuroprotective is the magnitude of the protective effect of citicoline in vitro versus in vivo. neuroprotective effects in vitro occurred upon exposure of retinal cells or brain neurons to citicoline concentrations as low as submicromolar to micromolar [66, 67 ], whereas in the in vivo (animal) experiments, the minimal doses necessary to produce appreciable neuroprotection were within the range of 0.31 g / kg, an amount that could be equivalent to 0.62 mmol / kg, assuming even distribution throughout the body. thus, citicoline seems to be a neuroprotectant that acts weakly in vivo but is much more potent in vitro. a few studies have indicated that in experimental ischaemic stroke, liposomal formulations of citicoline are significantly more neuroprotective (i.e. by means of decreasing the ischaemic infarct volume) than equivalent doses of free citicoline (see, for example fresta and puglisi [69, 70 ] and adibhatla. [69, 70 ]). the most recent study of this kind indicated that liposomal citicoline is more neuroprotective than the equivalent intravenous dose of the free drug which, in turn, is more neuroprotective than the equivalent intraperitoneal dose. although other explanations may be speculated upon, the aforementioned result is compatible with the idea that intact citicoline is pharmacologically more active than its metabolites. interesting observations have been reported on the modulation by citicoline of the activity / expression of some protein kinases involved in neuronal death namely, mapks in the postischaemic brain and extracellular signal - regulated kinase 1/2 (erk1/2) in the rat retina after kainic acid (ka) treatment. modulation of activity of cellular kinases and, in particular, members of the erk / mapk family, occurs through transduction of extracellular signals [72, 73 ]. perhaps the neuroprotective actions of citicoline are exerted not by its hydrolysis products but by the unhydrolysed molecules acting extracellularly as signalling molecules. in this context, one should consider the possibility of binding of citicoline to plasma proteins such as albumin. albumin binding has been reported for cytidine and also for sphingosylphosphorylcholine, a compound remotely similar to citicoline. if intact citicoline binds to albumin, its hydrolysis could be retarded and its action as a signalling molecule could be prolonged. in spite of the negative results of recent pivotal studies in acute ischaemic stroke and traumatic brain injury, there is continuing interest in the neuroprotective properties of citicoline. the drug is non - toxic, and numerous preclinical data support the view that it displays neuroprotective properties. the most frequently presented explanation for the neuroprotective effects of citicoline on the brain is based on the assumption that it is a prodrug which, following injection or ingestion, is sequentially hydrolysed and dephosphorylated, finally, to cytidine (or uridine in humans) and choline. then these two metabolites separately enter the brain tissues and are used to resynthesize cdp - choline, which exerts neuroprotection intracellularly by supporting biosynthesis of cellular phospholipids. an alternative explanation that unhydrolysed citicoline, or perhaps phosphocholine and/or cytidine monophosphate, are pharmacologically active metabolites of citicoline has never been considered. | citicoline is the generic name of the pharmaceutical substance that chemically is cytidine-5-diphosphocholine (cdp - choline), which is identical to the natural intracellular precursor of phospholipid phosphatidylcholine. following injection or ingestion, citicoline is believed to undergo quick hydrolysis and dephosphorylation to yield cytidine and choline, which then enter the brain separately and are used to resynthesize cdp - choline inside brain cells. neuroprotective activity of citicoline has been repeatedly shown in preclinical models of brain ischaemia and trauma, but two recent, large, pivotal clinical trials have revealed no benefits in ischaemic stroke and traumatic brain injury. however, the substance seems to be beneficial in some slowly advancing neurodegenerative disorders such as glaucoma and mild vascular cognitive impairment. this paper critically discusses issues related to the clinical pharmacology of citicoline, including its pharmacokinetics / biotransformation and pharmacodynamics / mode of action. it is concluded that at present, there is no adequate description of the mechanism(s) of the pharmacological actions of this substance. the possibility should be considered and tested that, in spite of apparently fast catabolism, the intact citicoline molecule or the phosphorylated intermediate products of its hydrolysis, cytidine monophosphate and phosphocholine, are pharmacologically active. |
mast cells are normally present in small numbers in the connective tissue of all organs and more particularly (around blood vessels and nerves) in the dermal layer of skin, ranging from 5 to 15 m in diameter, and in histologic sections often appear ovoid, tadpole, or spindle shaped cells with cytoplasmic granules of 0.2 to 0.5 cm in size. they exert their influence locally and systemically by releasing a variety of potent mediators like histamine, leukotrienes, and cytokines through degranulation and cause neovascularization by producing angiogenic mediators such as fibroblast growth factor (fgf), transforming growth factor- (tgf), tumor necrosis factor- (tnf), and vascular endothelial growth factor (vegf). an attempt has been made to quantitatively estimate the number of mast cells in oscc and to signify their role in tumor growth and progression [13 ]. 20 paraffin embedded specimens of oscc of age groups from 3080 years with no systemic illness but having the habit of smoking and alcohol consumption for a period of 510 years were retrieved from the archives of the department of oral pathology, among which seven cases were of well differentiated squamous cell carcinoma (n = 7 ; females = 4, males = 3) and 13 were moderately differentiated squamous cell carcinoma (n = 13 ; 4 = females, 9 = males). 20 normal oral mucosal biopsies from age groups of 1520 years with no systemic illness were obtained from 20 adult patients undergoing extraction for orthodontic treatment. serial sections of 5 m thickness were made from paraffin embedded tissue blocks using semiautomatic microtome (thermo scientific microm hm340e). all the sections were stained with 1% toluidine blue as per churukian and schenk method. mast cells were counted under light microscope at a magnification of 400x in a z - pattern from left to right and the data obtained was statistically analyzed using anova. mast cell granules are purplish red and the nuclei of mast cells appear sky blue in color. toluidine blue stains the mast cell granules metachromatically due to its reaction with sulphated mucopolysaccharides. a highly significant (p = 0.00) threefold increase in the average number of mast cells / slide was observed in oscc when compared to controls (table 1, figure 3). no statistical difference (p = 0.058) was found in the average number of mast cells / slide when moderately and well - differentiated oscc were compared. average count of mast cells / microscopic field under 400x magnifications was found to be 3 in oscc and 2 in controls. paul ehrlich in 1877 discovered a granular cell of loose connective tissue and named it as mastzellena well fed cell. studies on mast cells in normal and various pathologic conditions have shown them to be complex, well - engineered, and multifunctional cells playing an essential role in acquired and innate immunity. they take origin from multipotent cd 34 + precursor in the bone marrow, later circulate in the peripheral blood as agranular monocytic cell, and then migrate into tissues, assuming their typical granular morphology from their immature state. they are normally distributed throughout the connective tissue, adjacent to blood or lymphatic vessels, and near or within peripheral nerves. they are numerous especially beneath the epithelial surfaces of the skin, in the respiratory system, gastrointestinal and genitourinary tracts. many of the mediators are stored within cytoplasmic granules and these include preformed mediators like histamine, heparin, and tryptase ; lipid derived mediators like leukotriene 's b4 (ltb4), ltc4, ltd4, and lte4 ; proinflammatory cytokines like tnf-, il-1 ; mitogenic cytokines like il-3, il-5 ; and immunomodulatory cytokines like il-4, il-10, and serotonin and other mediators are produced at the time of mast cell stimulation such as il-1 [5, 6 ]. mast cells have been considered the tissue equivalent of the circulating basophils though they arise from a common precursor cell in the bone marrow. the two cell types are readily distinguished by their morphology on light microscopy and the presence of chloroacetate esterase activity in mast cells. they have been studied in normal gingiva, chronic inflammatory gingivitis, desquamative gingivitis, lichen planus, oral submucous fibrosis (osmf), and oscc. they exhibit phenotypic plasticity and variation in the mast cell mediators with the change in the microenvironment. volumes of literature speak about their role in the inflammatory reactions and neovascularization. in some malignancies, large numbers of mast cells were detected before the occurrence of neovascularization [2, 7 ]. in carcinogenesis, angiogenic regulation is biphasic. in the premalignant early phase of hyperplasia and dysplasia, infiltrating mast cells degranulate and activate dermal fibroblasts which intensify angiogenesis. they even activate progelatinase b (a member of the matrix metalloproteinase (mmp) family) which is involved in both extracellular remodeling and regulation of angiogenesis. the mast cells activate and progressively intensify angiogenesis by releasing sequestered angiogenic activators. as neoplastic progression proceeds, angiogenic growth factor gene expression is upregulated in the cancer cells, marking progression to the second cancer phase, wherein the tumor cells control their angiogenic phenotype directly instead of depending on the manipulation of inflammatory cells to indirectly affect neovascularization [1014 ]. this implies that mast cells have significant role in the early stages of cancer progression and increase in mast cells is observed in the initial stages whereas, in second cancer phase, the mast cells decrease because the tumor cells are not dependent on them anymore for the neovascularization effect and this could be the reason for the increase in the mast cells in moderate when compared to well - differentiated oscc in our study and also might be due to interobserver mystification in classifying the well- and moderate differentiated forms of oscc when compared to poorly differentiated. though many studies have proven the presence and significance of mast cells, their mediated angiogenesis is complex and not completely understood. in harmony with the literature, our study also showed a high mast cell count in oscc than in control tissue indicating their supportive role in tumor progression and metastasis. this study reveals that there is a definitive increase in mast cells count when compared to normal mucosa substantiating their contributing role in tumor progression. | mast cells are regarded as complex and multifunctional cells, playing a significant role in immunopathology and a substantial role in tumor angiogenesis. angiogenesis is a complex process that is tightly regulated by various growth factors in which mast cells act directly by releasing angiogenic factors and henceforth promoting tumor growth and metastasis. the aim of this study is to evaluate the number of mast cells in tissue sections of oral squamous cell carcinoma (oscc) in comparison with normal mucosa. a total of 40 cases (20 oscc and 20 normal mucosa) were stained with 1% toluidine blue and the quantitative analysis was done by using light microscope under 400x magnification. a significant increase in the mast cell count was observed in the sections of oscc when compared to normal mucosa suggesting their contributing role in tumor growth and progression. |
due to the progressive increase of life span and the improvement of the quality of life (qol) of the elderly, the surgical indications for degenerative and trauma lumbar spine in the aging population is increasing. the current elderly population desires to remain active and resists the acceptance of disability and low back pain. it becomes unavoidable for a spine surgeon to encounter patients with osteoporosis or other decreased bone quality who require spinal decompression and stabilization for degenerative spinal diseases, spinal trauma, infection, tumor, or inflammatory spinal diseases [13 ]. in the young population, the conventional posterior pedicle screw arthrodesis associated with lumbar interbody fusion (lif) is widely used in spinal surgery to attain rigid stabilization after surgical intervention in situations leading to a progressive mechanical instability [4, 5 ]. despite the demonstrated efficacy, some drawbacks are currently reported associated to the extensive soft - tissue dissection that is necessary to facilitate the insertion of the screws and prepare the fusion bed. the muscular incision increases perioperative blood loss, the postoperative pain, and the hospitalization time increases the risk of failed back surgery syndrome [69 ]. as a result, interest has increased for less traumatic surgical approaches that are associated with minimally invasive techniques for pedicle screw placement and lif, with less postoperative pain and blood loss than conventional open procedures. in the aging population, this interest for minimal invasive techniques is not as evident, probably because the conventional spinal arthrodesis is already considered as challenging [11, 12 ]. it has been well documented that bone mineral density (bmd) is one of the main factors related to spinal instrumentation failure. the ability of screws to resist pullout from bone is directly related to the bmd. many potential complications, such as screw loosening, migration, or pullout, compromising the surgical outcome have been described. several authors reported the efficiency of the augmentation techniques by injecting pmma into the vertebral body through the pedicle before inserting the screw. also, introduction of pmma through a tapped hole can increase the risk of pmma leakage through potential breaches that could occur in the pedicular wall during the tapping before screw insertion. to avoid this, a novel - concept cannulated screw with fenestrations in the distal portion of the screw has been designed. after insertion of the screw into the pedicle, cement can be injected and will distribute evenly around the thread of the screw to improve fixation performance [15, 16 ]. the purpose of this paper is to describe a novel technique using cannulated and fenestrated pmma augmentable screw in percutaneous and minimally invasive spinal posterior arthrodesis and to report the safety and efficiency of this technique in a prospective patient series. a consecutive prospective series of 15 osteoporotic patients operated on between march 2010 and july 2011 (12 female, 3 male, mean age 71.2 years (6088)) with osteoporotic compression / burst fracture (4 patients), degenerative spondylolisthesis (5 patients), and spinal and/or foraminal stenosis (6 patients) underwent mis posterior pedicle arthrodesis with or without interbody fusion with pmma cement augmentation of pedicle screws. all patients were included in this study based on the results of a dexa bone mineral density examination showing osteopenia to severe osteoporosis according to the who criteria. figure 1 shows the new model of cannulated and fenestrated pedicle screw featuring fenestrations that allows cement injection through the implant. expedium fenestrated screws (depuy spine, johnson & johnson) was used in all cases. inclusion criteria were as follows : (1) patient over 60 years of age ; (2) demonstration by dexa bone mineral density examination of osteopenia to severe osteoporosis according to the who criteria ; (3) evidence of spinal trauma, degenerative or deformative spinal disorders with an indication of stabilization and realignment of the thoracolumbar or lumbar spine. patients were excluded from the study in case of (1) previous history of spinal infection ; (2) spondylolisthesis > grade iii ; (3) severely increased risk for surgery under general anaesthesia due to cardiovascular, pulmonary, or other concomitant diseases. the mean follow - up period was 13,3 months (6 to 24 months). all patients were evaluated using ct scan or mri to define the surgical indication and to measure the pedicle diameter and length prior to surgery. in all cases, preoperative clinical data were collected : pain intensity was evaluated by the vas and the function was assessed by the odi. dose of antibiotic (cephalosporin) was injected intravenously 1/2 hour before the incision and renewed once the surgery lasted longer than 3 hours. patients were placed in a prone position on a radiolucent standard operating table with chest and pelvis supported to gain correction of kyphotic deformity when needed. conventional c - arm fluoroscopy was used for the entire procedure (arcadis ; siemens ; munich, germany). the novel pedicle screw used in this series was the titanium expedium fenestrated screw (viper mis spine system, depuy spine, johnson & johnson) which is a polyaxial and fully cannulated screw with six fenestrations in the grooves of the distal portion of the thread and an opening at the distal tip (figure 1). a specific delivery system, including alignment guides, cement delivery cannula for use with the v - max mixing, and delivery system, was used to inject the cement under controlled pressure through the cement cannula. pmma bone cement (vertebroplastic, depuy spine, johnson & johnson) (figure 2) was extruded through the fenestrations to fill the spaces inside the osteoporotic cancellous bone. under exact fluoroscopic antero - posterior view of the vertebral body, depending on the surgical plan, a pure bilateral percutaneous pedicle screw arthrodesis or a combination of unilateral percutaneous associated with a contralateral mini - open (modified wiltse) can be realised. for the pure percutaneous fenestrated screw placement, a skin incision is made 10 to 20 mm lateral to the pedicle 's upper quadrant projection. the thoracolumbar fascia is split and a targeting needle is used to introduce a k - wire guide inside the pedicle. successive ap and lateral fluoroscopic images are taken to accurately identify the pedicle entry point, the optimal position of the needle at the posterior wall of the vertebral body, and the good alignment of the needle with the desired screw trajectory. a k - wire guide is then placed in the needle and advanced in the two - thirds of the vertebral body. we placed pedicle k - wire guides in all target pedicles as during the first step of the procedure. dilators of progressively larger sizes are used to create the working channel by dilating the muscle tissue. a tap (undersized to the screw) the fenestrated screw is inserted into the pedicle guide over the k - wire with a selected length of screw and the position of the holes, located as far as possible from the posterior wall to prevent possible pmma leakage into the spinal canal (figure 3). when all the fenestrated screws are optimally placed, we suggest to make a trial of the unconstraint placement of the rod to avoid positioning issues during the definitive rod placement after cement injection. after pmma augmentation, alteration of the screw position is no longer possible (figures 4(a) and 4(b)). the rod insertion is done through one of the percutaneous skin incisions under the muscular fascia. when a central canal decompression or a transforaminal interbody fusion (tlif) is planned, the described percutaneous procedure is done unilaterally along with a mini - open approach as illustrated by holly. using a multiple blade retractor before the placement of the pedicle screws. the bone graft used for the tlif or for the posterolateral fusion is a mixture of (1) autologous local bone shavings, (2) allograft from cadaver bone bank, and (3) bone marrow aspirated from the posterior iliac crest. when the canal recalibration or the placement of interbody cage filled with bone graft is done, the fenestrated screws are placed over the k - wire using the same steps as described before. the screw and the cement delivery system the pmma bone cement is delivered through the cement cannula placed within the cannulation of the fenestrated screws under continuous image intensifier visualization (figure 5). the amount of cement injected into each screw varies from 1.5 to 3 ml. we experienced that the ideal amount of cement to inject was 2 ml. to prevent cement leakage, the injection was done in a higher viscosity state (started 5 minutes after mixing). the cement injection was stopped in case of any leakage of cement (anterior, posterior, or into an adjacent disc) (figure 6). a total of 78 fenestrated screws were implanted (min 4 ; max 10 per patient), in combination with standard cannulated viper screws (when sacral screws were placed bicortically). depending on patient 's clinical situation, patients were allowed to ambulate with protected thoracolumbar - sacral orthosis or lumbar - sacral orthosis 48 hours after surgery. the orthosis was maintained until the confirmation of the optimal screw placement and the absence of radiological complications on a postoperative thoracolumbar ct scan. all patients were followed up at the outpatient department at 3, 6, and 12 months, and then regularly every year. in addition, the patients had to assess their radicular and low back pain on a 10 cm vas between 0 (no pain) and 10 (maximal pain). a radiographic evaluation was also performed at each followup based on standard radiographs for signs of screw loosening, loss of sagittal alignment (kyphosis), and screw migration. optimal intervertebral or posterolateral fusion was considered on radiographs if (1) presence of bone bringing inside and/or around the cage and (2) absence of radiolucency lines around screws or cages were noted at 12-month follow - up radiographic control. all 15 patients had osteoporosis with a dexa bone mineral density examination showing moderate to severe osteoporosis. seventy - eight cement - augmented fenestrated screws were placed on a total of 82 screws (4 bicortical standard screws were placed in s1 without injection of pmma). the surgical indication was degenerative in 73.3% (11/15 patients) and osteoporotic burst fracture in 26.6% (4/15 patients). short segment fusions were performed in 3 patients to reduce operative times and minimize potential morbidity. medical history of previous spinal surgery was noted in 6/15 patients (2 disc herniation surgeries, 2 decompression laminectomies, 2 arthrodesis). the surgical procedure consisted of percutaneous stabilisation using the augmented fenestrated screws in 6 cases and an unilateral percutaneous stabilisation associated with a contralateral tlif or bone graft placement through a miniaccess approach in 9 patients. the mean operative time was 165 min 54.4 (range, 80275 min), and the mean perioperative blood loss was 261.4 ml 195 (range, 30600 ml). the mean cement injection per pedicle was 2.02 ml 0.56 (range, 1.53.0 ml). the injection of pmma was done in a minimum of 5 minutes after mixing to obtain a high viscosity consistency of the cement. despite this waiting time pmma extravasations were posterior towards the spinal canal (n = 2), in the intervertebral disc (n = 1), and into the external venous plexus (n = 2). pmma extravasations were noted in 4 of the 78 fenestrated screws placed (5% of screws). there were no cases of severe morbidity post - operatively (no death, no myocardial infarction, no pulmonary emboli, or intraoperative hypotension). two postoperative complications related to the procedure were noted : and one s1 screw misplacement associated with a nerve radiculitis (no cement injected through this screw), one subcutaneous infection with multisensible staphylococcus epidermidis treated with 2 weeks of antibiotherapy. the mean follow - up period in this study was 13.3 months (range, 624 months). at the end of follow - up period we noted no construct failure, no screw fractures, no loss of correction, or screw pullout. based on the vas for back pain and leg pain, pain intensity was significantly improved at discharge, 6 months and 1-year followup (table 2), the back function evaluated by odi score showed significantly improvement when compared between preoperative and discharge period including 6-month and 1-year followup (figure 7). based on the 1-year follow - up rx control, the fusion was considered as completed in all cases where tlif or posterolateral bone graft were placed (7 patients). in fracture cases, nevertheless, the burst fracture was consolidated in all patients. in patients 7 and 10, despite the absence of interbody bone grafting, a spontaneous progressive interbody fusion was noted. this 83-year - old woman presented with more than 5-year history of low back pain, more significant left buttock, lateral calf, and foot pain, as well as intermittent claudication. the pain increased while walking, but the pain was reduced when sitting or bending forward. on physical examination, hypoesthesia was noted in the l5 dermatome bilaterally. the pinprick sensation was decreased in the l-5 dermatome and no motor weakness was detected. the deep tendon reflexes were reduced in the left leg and the straight leg - raising sign was negative. sagittal mr imaging revealed l4-l5 and l5-s1 discopathy and disc herniation, spinal stenosis, and bilateral foraminal stenosis more marked at the level (figures 8(a) and 8(b)). a right percutaneous arthrodesis with augmented fenestrated pedicle screws in l4-l5 and s1 combined with a contralateral minimal access total l4 - 5 and l5-s1 facetectomy and tlif (with interbody cages filled with a mixed allograft and autologous bone marrow) was performed. control lumbar spine radiography confirmed the stability of the fusion, as well as the absence of hardware failure (figures 8(c) and 8(d)). clinically, the patient noted a significant reduction of the preoperative pain and a walking perimeter objectively increased. in recent years, minimally invasive surgical techniques to perform spinal stabilization have gained in popularity due to the demonstration of reduced perioperative muscular damage, blood loss, postoperative pain, and rehabilitation time [1924 ]. reported as safe and effective in the normal population, those techniques have been referred to the aging population with poor bone quality as a contraindication. indeed, in elderly patients, the conventional open procedure of arthrodesis using posterior pedicle screws are considered as a challenge. many complications have been reported and correlated with decreasing bone mineral density [1113 ]. carreon. reported after lumbar arthrodesis that at least 1 major complication occurred in 21% and at least 1 minor complication in 70% of elderly patients. dong. was the first to analyse the potential interest of a mini - open tlif approach for single - level instrumentation degenerative spondylolisthesis and stenosis with instability in elderly adults and reported a good clinical and radiological outcome associated with a low rate (7.4%) of minor complications. nevertheless, more recently, in a larger retrospective series, lee and fessler reported an overall rate of perioperative and postoperative complications of 20% without significant difference comparing with a young population. karikari. retrospectively reviewed their series of elderly patients who underwent minimally invasive lumbar interbody fusion and found an overall rate of major complications of 7.4% and a total complication rate of 32.4%. unfortunately, they failed to distinguish posterior and lateral based approaches in their analysis of minimally invasive lumbar interbody fusion, limiting the applicability of their results. none of the above - mentioned studies reported their fusion rate at the end of followup. in our study, we firstly describe the different surgical steps of the percutaneous (or through an miniopen access) placement of a novel cannulated and fenestrated screw designed to allow the injection of a pmma bone cement through the implant following the optimal positioning of the screw inside the pedicle and the vertebral body. this augmentation technique was already reported in conventional open approach to reduce the complications related to the bone - implant interface (pullout of screw, implant fracture) [15, 30, 31 ] but never through a percutaneous or minimally invasive approach. various studies demonstrate that pmma bone cement used to augment screws in osteoporotic bone enhance the screw - bone fixation by 49 to 162% [32, 33 ]. fransen suggests that the direct injection of cement through the screw can provide to the implant an immediate improved anchoring and that the filling of the vertebral body (vb) can decrease the risk of compression fractures at the treated levels. this technique can also be used in association with kyphoplasty of the fractured vb, allowing correction of the kyphosis with short - length constructs. cement extravasation was observed when a screw was inserted inside a screw hole prefilled with cement. in 2005, yazu. published an experimental study conducted on osteoporotic cadaveric vertebrae and compared the performance of fenestrated screws with traditional screws without cement augmentation. concluded that cement injection could be controlled more accurately using fenestrated screws, reducing the risk of leakage into the canal and/or foramina. recently, amendola. confirmed in a prospective cohort series of 21 patients that fenestrated screws for cement augmentation provided effective and long lasting fixation in patients with poor bone quality due to osteoporosis or tumors. no cases of loosening were recorded after a mean followup of 36 months. in our series, no major complication was reported. to the best of our knowledge, this paper is the first report of a cement augmentation technique of pedicle screws through a percutaneous or minimally invasive approach. in this technique, first, the positioning of the screw must be perfectly aligned with the pedicle with a good convergent trajectory. no fractures of the anterior and lateral cortex of vertebral body can be tolerated to avoid cement extrusion in the retroperitoneal space. secondly, to avoid breakage of the cement bridges between the screw and the bone, a definitive positioning of the screw must be controlled and the fixation system should be locked and the rods tested in position before injecting. thirdly, the cement injection started only when the cement reached a high viscosity state to avoid extravasation. finally, cement injection must be performed under continuous fluoroscopic imaging to provide immediate visual feedback and control to stop the injection in case of any sign of extravasation. despite this caution technique, as it has been demonstrated that the pullout strength did not significantly increase with the volume of cement injected over a range of 1.5 ml [37, 38 ], we suggest to inject maximum 1.5 to 3.0 ml of cement per screw. in this serie, the mean volume of injection was 2.02 ml 0.56 per screw. in table 3 similarly, as described for the young population, in our elderly population the mis procedures were associated with a low rate of peri- and postoperative blood loss, postoperative pain, hospital stay, and recovery time. the clinical state of the patients was significantly improved and this improvement was maintained during the short followup of this clinical series. tested the biomechanical removal of cement augmented pedicle screws in cadaver spines. in the majority of screws, the removal was easy ; in two removals, some bone cement remained attached to the screws and created secondary fractures to the pedicle. they suggested to control this potential removal in a real clinical situation under fluoroscopic control to prevent inadvertent damage on pedicle. in this primary experience, nevertheless, we highly suggest to monitor the annual radiation exposure of surgeons and to apply all recommendations to reduce this exposure. the need for lead shielding can not be overstated. the use of thyroid shielding, leaded glasses, and radiation attenuation gloves is absolute. despite the interest of this study, a longer followup would be important in order to consider this novel technique as an effective one. a controlled randomized study could be suggested. the pmma augmentation technique of fenestrated pedicle screws is a safe technique to increase the pullout strength of screws placed in osteoporotic spines. this is the first clinical report of this augmentation technique through a percutaneous and/or a minimally invasive approach. we can confirm the safety and efficacy of this technique to prevent the short - time complications as described in performing arthrodesis in aging populations. the ultimate safety of using this technique in this vulnerable population needs of course to be confirmed in a larger series with a longer followup. the risk associated to pmma extravasation remains the critical part of this technique. at the start of injecting the high viscosity consistency of the cement, the strict usage of fluoroscopic control should be used to immediately detect any radiological sign of extravasation to prevent severe complications. | we describe a percutaneous or minimally invasive approach to apply an augmentation of pedicle fenestrated screws by injection of the pmma bone cement through the implant and determine the safety and efficiency of this technique in a clinical series of 15 elderly osteoporotic patients. clinical outcome and the function were assessed using respectively the visual analogue scale (vas) score and the oswestry disability index (odi). peri- and post - operative complications were monitored during a minimum of 2 years of follow - up. radiographic follow - up was based on plain fluoroscopic control at 3, 6 and 12 months and every year. in this approach, four steps were considered with care : optimal positioning of the screws, correct alignment of the screw heads, waiting time before the injection of cement, fluoroscopic control of the cement injection. using these precautions, only 2 minor complications occurred. vas scores and odi questionnaires showed a statistically significant improvement up to 13.3 months postoperatively. no radiological complications were observed. based on this experience, pmma augmentation technique through the novel fenestrated screws provided an effective and long lasting fixation in osteoporotic patients. applying this procedure through percutaneous or minimally invasive approach under fluoroscopic control seems to be safe. |
the online version of this article (doi:10.1007/s00125 - 009 - 1592 - 4) contains supplementary material, which is available to authorised users. type 2 diabetes mellitus and its common complication, peripheral neuropathy, affect a large population [1, 2 ]. peripheral neuropathy leads to sensory and motor deficits, which often result in mobility - related dysfunction, alterations in gait characteristics [3, 4 ] and balance impairments [5, 6 ]. diabetic patients with peripheral neuropathy have lower gait velocity, decreased cadence, shorter stride length, increased stance time and higher step to step variability compared with healthy controls. moreover, these patients have less ankle moment and ankle power [7, 8 ], as well as a different onset and cessation time of muscle activity compared with healthy controls. patients present more co - contractions of agonist and antagonist muscles at the ankle and knee joints during stance phase. speculate that the co - contraction mechanism may enable these individuals to adopt a safer, more stable gait pattern to compensate for diminished sensory information. the same authors have reported reduced ankle strength and mobility, which they considered to be the primary factors contributing to gait alterations. allet. have also found lower limb strength, fear of falling and sensory problems to be related to spatiotemporal gait alterations. additionally, individuals with peripheral neuropathy show postural instability with a larger centre of pressure displacement, higher sway area and greater instability when standing still with eyes closed. in addition to these gait and balance impairments, diabetic patients are known to suffer from increased risk of injurious falls. fall - related injuries are often assumed to trigger a vicious circle because of their potentially detrimental influence on the physical activity levels of affected patients. public health guidelines for diabetes management recommend that patients perform at least 30 min of physical activity a day six times a week, requiring adequate gait security and balance. however, little is known about treatment strategies that could improve patients gait and balance, thereby also reducing the risk of falls. although there is evidence that an exercise regimen improves clinical measures of balance in patients with peripheral neuropathy, clinical trials investigating the gait of diabetic individuals generally focus on increased foot pressure, another major problem in this population and related to the high risk of ulcers in such patients. only few studies have evaluated treatments that aim to improve gait and balance and decrease fall risk [16, 17 ]. tested an insulin sensitiser, rosiglitazone, which promises to reverse some of the circulatory impairments seen in diabetes, thereby improving patients gait. they reported encouraging results after administering rosiglitazone (decreased step width, reduced reaction time and less acceleration at the joints). however, rosiglitazone was recently associated with increased risk of myocardial infarction and death from cardiovascular incidents. evaluating patients with various form of peripheral neuropathy (30 of 42 of whom had diabetic peripheral neuropathy), found that the use of a cane, ankle orthoses or touching a wall improved step - width range, step - time variability and speed while walking under challenging conditions. and orr. have investigated the effect of a specific physical training programme not only on the activity level and quality of life of diabetic patients, but also on their habitual and maximal walking speed. however, both studies seem to have evaluated the same group of participants. in these studies, the effect of a tai chi for diabetes programme (twice a week for 16 weeks) on gait, balance, musculoskeletal and cardiovascular fitness, self - reported activity and quality of life was compared with that of sham exercises. nevertheless, several studies [2123 ] have shown positive training effects on gait speed, postural stability and mobility of elderly individuals. additionally, a meta - analysis evaluating fall prevention studies for the elderly showed a 4% decrease in the fall - rate of individuals who were in a treatment group. since elderly people often show symptoms similar to those in diabetic patients (i.e. de - conditioning, muscle weakness, decreased joint mobility and decreased foot sensibility), we assumed that programmes developed for the elderly might also improve gait and balance of diabetic patients. thus, this study aimed to evaluate the effect of a specific training programme, which was based on previously elaborated core components of successful fall prevention programmes for the elderly, on the gait and balance of diabetic patients. gait speed and coefficient of variation of gait cycle time (cvgct) were selected as primary outcomes because they have been shown to be related to increased risk of falls [22, 26 ] (slow gait speed and high cvgct indicate increased fall risk). balance control, muscle strength and joint mobility are also important fall risk factors that may be influenced by exercise. for these reasons, they were chosen as secondary outcomes. fear of falls is a cognitive behavioural component, which was recently shown to be related to the gait velocity of diabetic patients. we hypothesised that diabetic patients participating in a training programme would significantly improve their walking speed and gait variability (cvgct). we further assumed that such patients would significantly : (1) improve their balance ; (2) increase their lower limb strength (in particular hip extensors, knee extensors and ankle flexors) ; (3) increase their ankle joint mobility ; and (4) decrease their fear of falling. study design this randomised controlled prospective trial with two arms (intervention group and control group) was conducted at the university hospital of geneva, switzerland. the study was approved by the local ethics committee.a sample of 71 diabetic patients was recruited from patients consulting either the service of therapeutic education for chronic diseases or the service of endocrinology at the university hospital in geneva. of these, 35 patients were allocated to the intervention group and 36 to the control group. patients were included if they were medically diagnosed with type 2 diabetes (fasting blood sugar 7 mmol / l). only patients without medical contraindications for engaging in physical activity and with a clinically diagnosed neuropathy clinically diagnosed neuropathy was evaluated on the basis of a vibration perception threshold of 4 with a rydel patients were requested to indicate when they could no longer feel the vibration. at this point the investigator rated the vibration on a 9-point scale (0 = severe neuropathy ; 8 = no neuropathy). exclusion criteria were : patients with concomitant foot ulcers, orthopaedic or surgical problems affecting gait variables, non - diabetic neuropathy (due to charcot marie tooth disease or alcohol), other neurological pathologies (other than peripheral neuropathy) that could influence gait variables or inability to walk a minimum of 500 m without a walking aid.training programmes started as soon as enough patients (20 to 28 per group) had been recruited, i.e. in september 2007, january 2008 and april 2008. for each of these starting points a new randomisation list was electronically generated and was used by a person not involved in the recruitment, evaluation or treatment processes. each list ensured equal distribution over groups.all eligible patients identified by the medical staff were asked to participate. they were informed that if willing to participate, they would be randomly assigned to an intervention group or control group. patients who agreed to join the study were contacted by the study coordinator for an individual appointment, at which the informed consent document was signed and baseline evaluation performed. after this initial appointment, during which patients underwent a clinical examination, a gait analysis and both static and dynamic balance tests, and also filled in a fear of falls questionnaire, patients were randomly allocated to the intervention group or to the control group. all outcome measures were assessed at baseline, after 12 weeks and after 6 months by the same experienced physiotherapist each time.patients allocated to the intervention group received a timetable containing all planned sessions over 12 weeks. however, the nature of the study made it impossible to blind patients and therapists. the assessor was kept as much as possible unaware of the group assignment. however, as she worked in this hospital during the experiment, she saw some patients arriving for training sessions.after the 12 week programme patients were encouraged to continue with the learned exercises during the next 6 months. test description and measures prior to the clinical examination, the physiotherapist checked the vibration perception threshold with the tuning fork. maximum isometric strength of the hip, knee, ankle flexors and extensors was then measured with a hand - held dynamometer (microfet ; hoggan health industries, inc., west joint mobility of the hip and both knee and ankle flexion and extension were measured with a manual goniometer. patients worry of falling when performing different activities was assessed with the falls efficacy scale international (fes - i). afterwards patients underwent the following functional tests : performance - oriented mobility assessment (poma), which scores 16 items (nine for evaluation of balance, seven for assessment of gait).outdoor gait assessment recorded with a device (physilog ; bioagm, lausanne, switzerland). participants were asked to walk, wearing four miniature gyroscopes (adxrs 250 analogue device) attached to both shanks and thighs with velcro straps, on a specific walkway at their preferred walking speed. the walkway consisted of two 50 m tarred pathways, two 50 m grass pathways and two 20 m cobblestone pathways in the hospital s backyard. for this study a detailed description of this gait assessment has been published elsewhere.dynamic balance test in which participants had to walk as fast and as precisely as possible on a 5 m beam (15 cm high, 15 cm wide). patients had one practice trial before the test started. if a patient had to step off the beam he or she was allowed to do the test again. in the event that this patient stepped off the beam again, he or she was asked to resume the exercise at the stepping off point, continuing from there to finish the test.static balance test evaluating postural control by means of a device (biodex balance system ; biodex medical systems, new york, usa). the level of difficulty while standing on this platform can be manipulated by altering the resistance of the platform to deviations. the balance test is most difficult when the platform provides the least resistance to tilting and is therefore the least stable. each participant stands barefoot on the platform and performs two different tests (level 8 = easiest level, level 6 = a more difficult level). the foot position was standardised using a pre - formed triangle (heels together with feet forming an angle of 20 degrees). they were permitted to touch handrails, but only to re - establish balance during extreme postural deviations. the participants were then instructed to find a position at which they could maintain platform stability. each recording lasted for 60 s with a 60 s rest between each trial. we used a single recording per test condition and only one attempt per condition to reduce fatigue. all tests were done at baseline, after the 12 weeks intervention and at 6 months after the intervention. a balance index was calculated using the time and deviation (in degrees) of the platform away from a level position. performance - oriented mobility assessment (poma), which scores 16 items (nine for evaluation of balance, seven for assessment of gait). outdoor gait assessment recorded with a device (physilog ; bioagm, lausanne, switzerland). participants were asked to walk, wearing four miniature gyroscopes (adxrs 250 analogue device) attached to both shanks and thighs with velcro straps, on a specific walkway at their preferred walking speed. the walkway consisted of two 50 m tarred pathways, two 50 m grass pathways and two 20 m cobblestone pathways in the hospital s backyard. for this study only the tarred terrain was evaluated. dynamic balance test in which participants had to walk as fast and as precisely as possible on a 5 m beam (15 cm high, 15 cm wide). if a patient had to step off the beam he or she was allowed to do the test again. in the event that this patient stepped off the beam again, he or she was asked to resume the exercise at the stepping off point, continuing from there to finish the test. static balance test evaluating postural control by means of a device (biodex balance system ; biodex medical systems, new york, usa). the level of difficulty while standing on this platform can be manipulated by altering the resistance of the platform to deviations. the balance test is most difficult when the platform provides the least resistance to tilting and is therefore the least stable. each participant stands barefoot on the platform and performs two different tests (level 8 = easiest level, level 6 = a more difficult level). the foot position was standardised using a pre - formed triangle (heels together with feet forming an angle of 20 degrees). they were permitted to touch handrails, but only to re - establish balance during extreme postural deviations. once in this position, the participants were then instructed to find a position at which they could maintain platform stability. each recording lasted for 60 s with a 60 s rest between each trial. we used a single recording per test condition and only one attempt per condition to reduce fatigue. all tests were done at baseline, after the 12 weeks intervention and at 6 months after the intervention. a balance index was calculated using the time and deviation (in degrees) of the platform away from a level position. treatment description the training took place twice a week for 60 min over 12 weeks. this intensity was chosen on the basis of previously developed successful interventions in pre - frail elderly persons [25, 3638 ]. each session was conducted in groups (five to eight participants) in order to promote long - term participation. four different physiotherapists and four assistants were trained to direct the sessions in order to guarantee continuity. a session consisted of a warm up (5 min), followed by a circuit training (40 min) including gait and balance exercises. balance and walking tasks (stance on heel / toes, tandem stance, one leg stance, different kinds of walking) alternated with functional strength and endurance exercises (sitting to standing, walking up and down a slope, stair climbing and mini hops). each task was carried out twice for 1 min each time, and the complexity of the task could be progressively increased, e.g. changing from stable to unstable surfaces (wobble board), increasing step height. sessions were completed with interactive games (e.g. badminton, obstacle race in teams) (10 min) and a short feedback session with suggestions for individual home exercises (5 min). as currently no specific gait and balance programme is offered to diabetic patients, the control group received neither treatment nor specific advice. both the intervention group and control group were allowed to continue their usual leisure activities. sample size calculation in previous studies, gait velocity of diabetic patients was 1.25 0.19 m / s compared with 1.45 0.14 m / s in a healthy control group. allowing that diabetic patients might improve their mean speed from 1.25 m / s to 1.35 m / s, 64 patients were needed to have an 80% probability that the study would detect a treatment difference at a two sided 5% level of significance. a 10% drop - out rate was hypothesised and therefore we aimed to include 71 patients in total. data analysis statistical analyses were performed using spss version 15 for windows (spss, chicago, il, usa). the outcomes at 12 weeks (i.e. post - treatment) and 6 months were dependent variables. the intervention allocation was considered to be a dichotomous independent variable in the analysis. the baseline values of the outcome measures were incorporated in the linear regression model as covariates.to reduce the risk of type i error, a bonferroni corrected alpha level of p = 0.0026 (alpha divided by number of tests per follow - up) was used to determine a significant difference between groups. however, to allow identification of areas of interest for future investigations and to reduce the risk of type ii errors, the results are presented with the corrected (p = 0.0026) and uncorrected (p = 0.05) significance levels. an intention to treat analysis was performed and in the event of missing values for any variables, these values were imputed by means of the last observation carried forward method. this clinical trial is closed to recruitment and closed to follow - up. the flow chart (fig. 1) provides a detailed description of drop - outs and loss to follow - up, as well as the number of participants analysed in each group. descriptive statistics show patients characteristics (table 1) and illustrate the improvement of the intervention group in all variables post - intervention (electronic supplementary material [esm ] table 1 ; fig. 2). two patients developed pain in their achilles tendon, obliging us to slow down the progression for intervention group participants partially lost their treatment benefit in the 6 months of follow - up, but their performance level remained superior to that at baseline. 1flow chart with detailed description of recruitment, number of drop - outs and reasons for not continuing. the flow chart is based on the consort statement recommendations in a previous publication table 1description of patients baseline characteristicsbaseline evaluationintervention groupcontrol groupage (years)63 (7.99)64 (8.89)bmi (kg / m)30.46 (6.03)31.46 (5.25)falls in 12 months (n)0.71 (1.07)0.45 (0.89)test with tuning fork3.23 (1.26)3.32 (1.32)height (cm)166.14 (8.5)168.56 (8.64)weight (kg)83.62 (16.56)89.17 (12.33)values are mean (sd)a fall was defined as an unexpected event in which the participant comes to rest on the ground, floor or lower levelscale 08fig. 2plots illustrating the baseline difference of (a) gait speed, (b) cvgct, (c) poma score, (d) time to walk over a beam, (e) biodex sway index (level 6) and (f) fear of falls for each group at baseline (ba), after the intervention (pi) and at the 6-month follow - up (fu). values are adjusted mean and 95% white squares and dotted line, control group ; black circles and continuous line, intervention group flow chart with detailed description of recruitment, number of drop - outs and reasons for not continuing. the flow chart is based on the consort statement recommendations in a previous publication description of patients baseline characteristics a fall was defined as an unexpected event in which the participant comes to rest on the ground, floor or lower level plots illustrating the baseline difference of (a) gait speed, (b) cvgct, (c) poma score, (d) time to walk over a beam, (e) biodex sway index (level 6) and (f) fear of falls for each group at baseline (ba), after the intervention (pi) and at the 6-month follow - up (fu). white squares and dotted line, control group ; black circles and continuous line, intervention group control group patient variables all progressively deteriorated compared with their baseline performance (esm table 1). after intervention, the intervention group increased their habitual walking speed by 0.149 m / s (0.54 km / h ; p < 0.001) compared with the control group. in addition, a majority of secondary outcome variables showed significant between - group differences (at the bonferroni corrected significance level of p < these outcome variables were : the dynamic balance test (time to walk over a beam), the poma test (total score and sub - scores), the biodex sway index recorded at level 6, the fes - i score, the hip and ankle plantar flexor strength, and hip flexions mobility. after 6 months, the differences in all these variables remained significant (p < 0.0026) except for the biodex sway index (p = 0.005) and ankle plantar flexor strength (p = 0.217) (esm table 1). this study aimed to evaluate the effect of a training programme, which was based on core components for successful fall prevention in the elderly, on gait and balance of diabetic patients. our results confirm that gait and balance in these patients can be concurrently improved by a targeted intervention. patients in the intervention group increased gait velocity, balance, muscle strength and joint mobility, and decreased their fear of falling. however, post intervention evaluation showed no difference between groups for the primary outcome cvgct (esm table 1). possibly the tarred surface was not challenging enough to fully reveal the gait problems of our diabetic patients. the cvgct was relatively low at baseline (2.75% for control group, 2.6% for intervention group) and consequently did not improve (decrease) after treatment, probably due to a floor effect. step time variability on smooth and irregular surfaces was recently shown to be most strongly associated with reduction in step length on an irregular surface as compared with a smooth surface. richardson. have shown that the greater the decrease in step length on an irregular surface, the greater the step time variability on both surfaces, and the greater the increase in step time variability on the irregular surface. thus, analysis on a more challenging surface, e.g. cobblestone pathway, which was beyond this paper s aim, might have revealed differences between groups. despite the above, the degree of improvement achieved by the intervention group in gait velocity (0.54 km / h or 11.6%) and dynamic balance (time to walk over a beam) (3.39 s or 34%) was not only significant but also clinically relevant. a decrease in gait speed of 0.1 m / s in the elderly has been associated with a 10% decrease in the ability to perform daily living activities. these values are just within the critical range (19 to 24 points) that implies a moderate risk of falling. intervention group patients passed this moderate risk of falling cut - off point after the training. in addition, their change of 2.0 points signifies a real improvement considering that effects greater than 0.8 (with a group size of n = 30) reputedly represent a significant improvement unrelated to chance fluctuations. the more difficult biodex balance test (level 6) also highlighted patients progress in postural control. importantly, all gait and balance variables (except the biodex balance test) remained significant at 6-month follow - up, although some treatment benefit was lost. increased hip and ankle strength as well as ankle mobility may explain the progress in gait velocity and both static and dynamic balance, although the improvement of ankle dorsal flexor strength and ankle dorsiflexion mobility just failed to be significant at the bonferroni corrected significance level. these two values further decreased at 6 months, possibly explaining the regression of gait and balance measures. knee flexion mobility showed normal baseline values, thereby explaining the absence of improvement after training. we decided not to calculate the post - treatment group effect on knee and hip extension mobility as none of the patients had a flexor contracture. regarding the fes - i, patients showed a relatively low level of concern about falling. perhaps this result reflects our study population s relatively good gait function and functional capacity. this could have been influenced by the inclusion and exclusion criteria used and must be considered in the clinical decision - making process. it may be hypothesised that patients with more severe diabetic peripheral neuropathy or with more impaired functional capacity would benefit less from training, due to the fact that, from a functional point of view, the detrimental effects of the disease are impossible to reverse or compensate. patients suffering from more severe peripheral neuropathy could benefit even more from a structured exercise regimen, as their condition provides more scope for improvement. moreover, even though balance and gait changes seen in diabetic patients are probably primarily due to neuropathy, there are other potential contributors. gait impairments are also observed in diabetic patients without clinically detectable neuropathy, raising the question of whether it would be worthwhile to test a similar, but more intensive programme on diabetic patients without neuropathy in order to prevent further aggravation of gait and balance. all exercises were taught by qualified physiotherapists and were function - orientated. to boost patient motivation and enable them to interact with other patients, for further discussion of the results, the effect of our primary outcome was compared with the results of a meta - analysis evaluating the effect of exercise on gait speed in the elderly. this meta - analysis reported a success rate of 57% for exercise training on habitual gait speed. the overall gait speed change reported was 0.01 m / s compared with our 0.15 m / s. however, the range of velocity change was large (0.2 to 0.34 m / s), which could be due to the various studies included and the evaluation of populations with diverse co - morbidities under varied conditions (e.g. 6 m distance compared with a 6 min walk). the values of our population (control group 2.6%, intervention group 2.75%) are between those reported for elderly non - fallers (2.1% ; mean age 76 years) and those for elderly fallers (3.8% ; mean age 82 years), which was somewhat better than expected. however, this may be explained by the fact that our population was about 15 years younger than participants in hausdorff s trial. despite the beneficial effect of our programme on gait speed and balance, its influence on fall frequency in diabetic patients although the cvgct did not improve, our results provide encouraging data, which justify further studies with larger sample sizes focusing on fall frequency itself, physical activity level and quality of life. our baseline evaluation contains no information about patients cardiac status, severity of retinopathy, visual acuity, disease duration, orthostatic hypotension, medications or other possible factors related to gait and balance impairments, and to treatment responsiveness. the fact that the control group was given no attention at all may represent a confounder for this study s results. the degree to which an improvement depends on personal attention from the therapists, vs the effect of the actual exercise regimen, needs to be considered. however, the positive result on all measured outcomes clearly points to a beneficial effect due to therapeutic exercise. muscle strength was measured with a hand - held dynamometer, which gives a more precise measurement than manual muscle testing. however, the reliability and accuracy of the measurement may be limited by the investigator s ability to hold the dynamometer stationary and by the fact that participants may overpower the testers. we tried to minimise this problem by ensuring that the same person always carried out the tests. nevertheless, the relatively low ratio between ankle plantar flexor and ankle dorsal flexor strength could indicate an underestimation of plantar flexor strength. in addition, not only sagittal but also frontal plane muscle strength (i.e. hip abductors and adductors), which is involved in frontal plane stability during gait, should be evaluated. although our study showed positive results, clinicians should be aware of possible adverse events. two patients developed pain in their achilles tendon, obliging us to slow down the progression for to the best of our knowledge, this is one of the first randomised controlled trials to describe an effective physiotherapy training programme geared to concurrently improving the balance and gait of diabetic patients. future studies should examine the effect of exercise regimens on patient groups differentiated by neuropathy status (patients without, with mild or with severe peripheral neuropathy, identified by a more complex instrument for peripheral neuropathy screening). in addition, outcomes such as functional capacity, the number of falls or physical activity level should be considered in order to draw meaningful conclusions about exercise efficacy among patients with diabetes, thereby facilitating medical and clinical decision - making. the appraisal of (1) advantages, difficulties and feasibility of treatment, and/or (2) prevention of gait and balance problems, as well as (3) the related fall risk in diabetic patients may be another interesting issue for further quantitative and qualitative studies. a specific gait and balance training programme based on a circuit approach and including gait and balance exercises combined with function - orientated strengthening can improve gait speed and balance, and increase both muscle strength and joint mobility of diabetic patients with a vibration perception threshold 4. further studies with a larger sample size are needed to explore the influence of these improvements on the number of reported falls, patients physical activity levels and quality of life. | aims / hypothesisgait characteristics and balance are altered in diabetic patients. little is known about possible treatment strategies. this study evaluates the effect of a specific training programme on gait and balance of diabetic patients.methodsthis was a randomised controlled trial (n = 71) with an intervention (n = 35) and control group (n = 36). the intervention consisted of physiotherapeutic group training including gait and balance exercises with function - orientated strengthening (twice weekly over 12 weeks). controls received no treatment. individuals were allocated to the groups in a central office. gait, balance, fear of falls, muscle strength and joint mobility were measured at baseline, after intervention and at 6-month follow-up.resultsthe trial is closed to recruitment and follow - up. after training, the intervention group increased habitual walking speed by 0.149 m / s (p < 0.001) compared with the control group. patients in the intervention group also significantly improved their balance (time to walk over a beam, balance index recorded on biodex balance system), their performance - oriented mobility, their degree of concern about falling, their hip and ankle plantar flexor strength, and their hip flexion mobility compared with the control group. after 6 months, all these variables remained significant except for the biodex sway index and ankle plantar flexor strength. two patients developed pain in their achilles tendon : the progression for two related exercises was slowed down.conclusions/interpretationspecific training can improve gait speed, balance, muscle strength and joint mobility in diabetic patients. further studies are needed to explore the influence of these improvements on the number of reported falls, patients physical activity levels and quality of life.trial registration : clinicaltrials.gov nct00637546funding : this work was supported by the swiss national foundation (snf) : pbskp-123446/1/electronic supplementary materialthe online version of this article (doi:10.1007/s00125 - 009 - 1592 - 4) contains supplementary material, which is available to authorised users. |
a number of surgical techniques for treating disc herniation recently have been developed and applied to patients who do not respond to conservative treatment. although various surgical options exist for lumbar disc herniation patients, open discectomy has been proven to have lasting benefits in numerous cases, but with diverse outcomes1,8,28). some patients suffer from persistent or recurrent radiating pain after open discectomy ; this pain can be accompanied by a recurrence of the disc herniation. recurrent herniation is one of the most common causes for an unsatisfactory outcome following lumbar discectomy and occurs in 5 - 15% of patients2,5,17,24,26). recurrent lumbar disc herniation has been defined as disc herniation at the same level after previous discectomy, regardless of ipsilateral or contralateral herniation, with a pain - free interval greater than 6 months26). there have been many studies on recurrent disc herniation reporting diverse risk factors including age, gender, body mass index (bmi), smoking, traumatic events, operation time, level of herniation, type of disc herniation, degree of disc degeneration, and the duration of symptoms5,6,9,17,26). in this study, the authors analyzed surgical outcomes after open discectomy and the risk factors for recurrent disc herniation. in this study, we included 178 patients who had undergone open discectomies for lumbar disc herniation between may 2006 and december 2009. hospital records and imaging studies, including magnetic resonance imaging (mri) of all patients, were retrospectively reviewed. in all patients with new symptoms, mri images (t1- and t2-weighted sagittal and axial view) were obtained and assessed for recurrent disc herniation. the mri scans revealed 18 patients who appeared to have recurrent disc herniation at the same level and who were unresponsive to conservative treatment. eighteen patients underwent reoperation and were reviewed retrospectively during the mean follow - up period of 16.5 months. the non - recurrent group included 98 patients who had experienced no recurrence of leg pain until the most recent follow - up ; the mean follow - up period was 27.9 months. visual analogue scales (vas) and modified macnab criteria were used to compare the clinical outcomes between the recurrent and the non - recurrent group. sex, age, discectomy level, degree of disc degeneration, type of disc herniation, smoking status, and traumatic events were considered risk factors for recurrent lumbar disc herniation. the pain - free interval after the first operation was considered a clinical parameter for the prognosis of recurrent lumbar disc herniation. degeneration of the nucleus pulposus was classified according to the method of horton and daftari9), in which a point value is given to the signal intensity of the nucleus (white, 0 points ; speckled, 1 point ; dark, 2 points)4). in addition, a score was given for the presence of a high - signal intensity line within a disc with reduced signal intensity (absent, 0 points ; present, 1 point) ; this finding is related to the presence of fluid within cystic spaces or fissures in markedly degenerated discs. annular degeneration was graded on the basis of the height of the disc (normal or slightly decreased, 0 points ; moderately or markedly decreased, 1 point). the overall disc degeneration score was then calculated on a scale of 0 - 4 (0, no degeneration ; 4, marked degeneration) using the sum of the nuclear and the annular scores. the extent of disc herniation was rated on t1- and t2-weighted images according to the methods of masaryk.14) and modic.16), differentiating among protrusion, extrusion, and sequestration. on axial and sagittal mri sequences, the type of disc herniation was defined as " protruded " if there was a focal extension of the disc beyond the posterior margin of the vertebral body, " extruded " if disc tissue had migrated through a defect in the outer annular fibrosus but was connected to the disc, and " free fragment of sequestrated " if the herniated tissue was no longer connected to the disc. the pain - free interval after first operation in the recurrent group was divided into four groups according to recurrence within the first year, at 1 - 3 years, at 3 - 5 years, or more than 5 years post - surgery. the mann - whitney u - test and the chi - square test were used for statistical analyses ; results were considered significant when the probability value (p - value) was less than 0.05. the patients in the non - recurrent group were older than the patients in the recurrent group at the time of diagnosis (56.6 years versus 46.4 years), but this was not considered statistically significant (p>0.05). the recurrent group was composed of 5 men and 13 women ; the gender distribution difference was statistically significant (p0.05). most patients in the two groups had a score of 3 points on the mri disc - degeneration scale. the average score was 2.8 points in the non - recurrent group, and 2.7 in the recurrent group. no statistically significant difference be- tween the two groups was noted in the disc - degeneration score (p>0.05). there were three protruded (17%), seven extruded (39%), and eight sequestrated discs (44%) at primary discectomy in the recurrent group. in the non - recurrent group, preoperative mris showed that protruded discs were most common in the recurrent group, sequestrated discs were most common. the increased incidence of recurrent disc herniation among patients with previous extruded and sequestrated disc compared to protruded discs was statistically significant (p0.05). radicular pain associated with traumatic events was reported by 26 patients (27%) in the control group and 11 patients (61%) in the recurrent group. traumatic events occurred at a significantly higher rate in the recurrent group compared to the control group (p<0.05). thirteen patients underwent reoperation within 1 year after the first operation, three patients from 1 to 3 years, one patient from 3 to 5 years, and one patient more than 5years later (table 2). the mean vas score was 2.3 in the non - recurrent group and, 4.0 in the recurrent group. in addition, according to the modified macnab criteria, 71% of patients in recurrent group and 81% of patients in the control group showed a good or excellent result. the clinical outcomes in the recurrent group were worse than those in the control group (table 3). the recurrence of lumbar disc herniation in patients after surgery ranges from 5 to 15% in various studies, although - these studies varied in terms of their patients populations, follow - up periods, the definitions of recurrence, and types of procedures13). in this study, the recurrence rate was 10.1% at a mean follow - up period of 27.9 months. cinotti.6) reported that in males, the presence of markedly degenerated discs may be a risk factor for recurrent herniation after microdiscectomy. suk.26) reported that young age, the male gender, smoking, and traumatic events are risk factors. according to kim.12) old age and higher bmi are risk factors for recurrence after percutaneous endoscopic lumbar discectomy. however, swartz.28) reported that age, gender, smoking status, level of herniation, and duration of symptoms were not associated with a higher rate of recurrence after partial laminectomy and discectomy. kim.13) also reported that the recurrence rate in young active men was similar to that of the general population. cinotti.6) and kim.12) reported that older patients generally have more degenerated disc - s, which may explain their higher incidence of recurrent herniation. however the degree of disc degeneration was not associated with a higher rate of recurrence in this study, nor was age associated with a higher rate of recurrence after open discectomy. unlike suk.26) and cinotti.6), we found that female gender was a statistically significant risk factor for recurrence. to explain the higher incidence of recurrent herniation in young men, some authors have suggested that the annular incision made at primary surgery makes the operated disc more susceptible to sudden prolapse, particularly under conditions of mechanical overload during sports activity or weight - lifting6,13). because of their body mass, mechanical overload could be relatively higher in women than in men. without taking into consideration individual occupations or physical activity levels, it may be difficult to conclude that the male gender is a significant risk factor for recurrence. in our opinion, physical factors such as bmi, occupation, and the patient 's general medical condition may be important factors influencing the recurrence of disc herniation, although we did not analyze these characteristics because of the lack of relevant date in our clinical records. although we found female gender to be a significant risk factor, there is limited value in this result because the number of patients with recurrent disc herniation was too small compared to other studies to evaluate the female preponderance. suk.24) and terhaag.27) reported that isolated trauma or injury does not seem to play a prominent role in the etiology of disc herniation ; this has been reported in only 0.2 - 10.7% of adults with documented herniation. conversely, cinotti.6) found that 42% of patients with recurrent herniation had associated the onset of radicular pain with an isolated injury or a precipitating event ; this is higher than in previously reported papers (15 - 32%). we found that a traumatic event was associated with recurrent herniation in 61% of patients, which was much higher than measured by cinotti.6) and enough sufficient to consider a traumatic event as a risk factor of recurrence. cinotti.6) found that disc degeneration as seen on preoperative mri was more severe on average, in patients who developed a recurrent herniation than in the control group. to explain the higher incidence of recurrent herniation in the discs of patients with degenerative changes, cinotti.6) suggested that the healing processes that occur in the outer lamellae after annular injury may lead to a less effective reconstitution of the annulus in markedly degenerated discs, so that a recurrent herniation may be more likely to occur. dora.7) reported that a minor degeneration of the disc represents a risk factor for recurrent disc herniation after discectomy. dora.7) suggested that cinotti.6) had included patients with abnormalities such as lateral stenosis, narrowed intervertebral foramina, and epidural scarring, and excluded additional confounding factors, such as the morphology of disc herniation, which might have influenced the risk of recurrence. although we found that the degree of disc degeneration was not associated with a higher rate of recurrence, it is difficult to compare our results with those of dora.7) because they used a five - point grading system based on the method of pfirrmann.22) the results of dora.7) combined with our findings suggest that recurrent disc herniation may be more likely to occur in the discs of patients with little or no degenerative changes. a prospective study design based on single shared parameter one controversial risk factor for recurrence is the shape of the disc itself28). in a study of 531 patients, morgan - hough.18) reported a revision rate of 7.9% over a period of 16 years and concluded that a contained disc protrusion was almost three times more likely to require revision surgery compared to extruded or sequestered discs. kim.13) reported that the recurrence rate after open discectomy was significantly higher in the protruded type than other types. they suggested this may be due to the incomplete removal of the herniated disc in protrudedtype injuries13). however, cinotti.6) found no difference in the rates of recurrence associated with partial or complete discectomy. although suk.26) asserted that disc shape plays no part in recurrence, carragee.5) prospectively evaluated disc herniation types, the rate of reherniation, and the rate of reoperation, and assessed the type of disc herniation using a penfield dissector, unlike kim.13) and this study, which used mri images. in the study of carragee.5), patients in a fragment - defect group, who had extruded fragments and massive posterior annular loss, had higher rates of reherniation and reoperation than other groups (a fragment - fissure group, a fragment - contained group, and a group without contained fragments). carragee.5) found that an extruded fragment with massive posterior annular loss had the highest prevalence of recurrent herniation. in this study, recurrent disc herniation developed at a higher incidence with extruded and sequestrated discs than with protruded discs. similar to carragee.5), we conclude that the annular defects in extruded disc and sequestrated discs are the risk factors for recurrent disc herniation because the intervertebral disc is the most avascular tissue in human body, with low regenerative ability19). when nucleotomy is performed, there is no regeneration of the nucleus pulposus including the annulus fibrosus3,25,27). the absence of healing in defective annulus tissue may increase the recurrence rate with extruded and sequestrated types of herniation. suk.26) used the percentage difference between the preoperative and the postoperative pain scores to assess clinical improvement. they reported no significant differences in clinical improvement after revision and the previous surgery26) cinotti.6) used a 100-point grading system to assess the overall clinical outcome, with 20 points assigned to the severity of pain, 30 to functional status, 20 to patient satisfaction, and 30 to the results of physical examinations. the study found no significant difference in the rate of satisfactory results in recurrent and control groups after primary discectomy and reoperation. in this study, we assessed clinical outcomes using the mean vas score and modified macnab criteria and found poorer clinical outcomes in the recurrent group than in the control group. many institutions recently have reported favorable outcomes after revision surgery for the treatment of relapsed disc herniation21). earlier reports suggesting less favorable outcomes from revision surgery may have been flawed because they included broader post- operative diagnoses such as foraminal stenosis, epidural fibrosis, herniations at other levels, and segmental inst- ability11,21). in this study, no patients had foraminal stenosis or segmental instability at the time of their revision surgery, but the degree of epidural fibrosis was unclear. repeated surgery in the lumbar spine for recurrent sciatica after a previous discectomy has been associated with poor results when fibrosis is present and the pain - free interval after primary discectomy is shorter than 6 - 12 months21). quimjian and matrka20) also reported that a longer pain free interval after the first operation is associated with a better prognosis after recurrent lumbar disc herniation. in this study, 72% of the patients experienced the recurrence of symptoms within 1 year. the clinical outcomes in this study might have been affected by the short pain- free interval before revision surgery, which may have produced the unsatisfactory results of the patients of the recurrent group. one of the most important factors influencing the recurrence rate of disc herniation is the thoroughness of follow - up and the number of cases17), and our study was limited by a high rate of follow - up loss, an insufficient follow - up period, and a small number of patients. ; these factors may have prevented us from detecting significant differences among the groups and from evaluating the risk factors for recurrent herniation and clinical outcomes. in addition, our study was limited by a lack of accurate hospital records to review retrospectively. finally, we did not include other factors such as diabetes, obesity, and hospitalizations, which might be more applicable to clinical parameters17,26,28). fewer patients had satisfactory results after reoperation for recurrent radicular pain than after the primary operation. the risk factors for recurrent lumbar disc herniation in this study were female gender, the type of disc herniation (extruded, sequestrated), and traumatic events. | objectiverecurrent lumbar disc herniation has been defined as disc herniation at the same level, regardless of ipsilateral or contralateral herniation, with a pain - free interval greater than 6 months. the aim of this study is to analyze outcomes and identify the potential risk factors for recurrent lumbar disc herniation.methodsthe authors retrospectively reviewed the cases of 178 patients who underwent open discectomy for single - level lumbar disc herniation. visual analogue scales and modified macnab criteria were used to compare the clinical outcomes between the recurrent group and the non - recurrent group.sex, age, discectomy level, degree of disc degeneration, type of disc herniation, pain - free interval after first - operation, smoking status, and trauma were investigated as potential recurrence risk factors.resultsof the 178 patients for whom the authors were able to definitely assess symptomatic recurrence status, 18 patients (10.1%) underwent revision surgery for recurrent disc herniation. the most common level involved was l4-l5 (61%) and the mean period of time to recurrence was 18.7 months (6 - 61 months). there were 17 cases of ipsilateral herniation and 1 case of contralateral herniation. the types of herniation for which revision surgery was done were protrusion (3 cases), and transligamentous extrusion (14 cases). there were five excellent, eight good, and two fair results.conclusionrepeated discectomy for recurrent disc herniation produced unsatisfactory outcomes. factors such as sex, type of disc herniation and traumatic events were found to be significant risk factors. |
a female neonate, one hour after delivery, was admitted at pathology ward of ghaem hospital for autopsy. she was born by a preterm delivery at 32 weeks of conception, weighing 1300 g, from a 39-year - old g1p0a0 woman with no significant finding in her medical history. immediately after delivery, the neonate transmitted to nicu, but she died one hour after delivery. further 3d sonogram showed a fetus with a defect in the body wall and sticking out liver (figure 1 and 2). color doppler showing ectopia cordis 3 dimention sonogram showing sticking out liver in physical examination, there were a defect in thoracoabdominal wall with ectopia cordis, an absence of pericardium, an evisceration of the intestines and liver, and a bilateral cleft lip and palate. radiographic finding showed an absence of mid portion and the distal segment of the sternum. in autopsy, a bilateral cleft palate and lip, an evisceration of the liver and intestines, and a thoracoabdominal schisis with ectopia cordis were seen (figure 3 and 4). the extension of the thoracoabdominal wall defect was from the midsternum to the supraumbilical area. additionally, left sided clubfoot was seen, and there was an anterior diaphragmatic defect. ectopia cordis, midline supraumblical defect of thoracoabdominal wall and evisceration of liver and intestines bilateral cleft lip and palate there was a thoracic ectopia cordis of the heart with a rotation of 100 degrees infer superiorly, and its apex toward the midline of the neck. there were defects in the atrial and ventricular septum, and a patent ductus arteriosus. microscopic examination revealed chronic inflammation and congestion of the liver, epicardium, and bowel serosa. cantrell pentalogy is a rare congenital thoraco - abdominal disruption, first described by cantrell with five characteristic : 1) ectopia cordis and intracardiac anomalies ; 2) lower sternal defect ; 3) midline supraumbilical thoraco - abdominal wall defect ; 4) anterior diaphragmatic defect ; and 5) defect of diaphragmatic part of pericardium that results in relation between pericardial cavity and peritoneum.1 prevalence of pentalogy of cantrell is about 1 per 65000 live births and classified as a developmental defect of midline anterior body wall. full pentalogy of cantrell is a severe and rare syndrome, but incomplete forms with combination of two or three defects were reported frequently.2 intracardiac anomalies that are constant portion of pentalogy of cantrell are vsd (in 100% of cases), asd (52%), pulmonary stenosis (33%) and tetralogy of fallot (20%).1 also sternal fusion defect is a rare malformation and an inferior type is seen in pentalogy of cantrell.3 cantrell offered a developmental failure in lateral mesoderm during day 14 - 18 as a reason for indecision of transverse septum of diaphragm, therefore migration of paired mesodermal fold does not occur.1 failure of the transverse septum to develop, as well as abnormal development of the myocardium, cause diaphragmatic and cardiac defect, respectively.4 because of various phenotypes of abdominal wall defect in cantrell 's pentalogy, multiple factors is said to be responsible, including mechanical teratogens, major gene mutation, chromosomal abnormalities such as trisomy 13 and 18 and disrupted vessels defects.5 mutation of tas gene which mapped at xq25-q26.1 area, is mentioned to has a roll in fusion of sternum, multiple cardiac, diaphragmatic and anterior abdominal wall defects, and also additional abnormalities reported in some cases of cantrell 's pentalogy.67 carmi reported some cases with encephalocele and cleft lip with or without cleft palate, in association with abdominal wall defects such as pentalogy of cantrell.7 in some cases of cantrell of pentalogy, aggregation of fluid in the chest and neck cavity was reported as a result of venous congestion because of cardiac failure, increased mediastinal pressure due to diaphragmatic herniation or omphalocele.5 abnormalities of the extremities are also reported in associated with few cases of pentalogy of cantrell.810 one study reported arthrogryposis, left thumb defect and shortening of left upper limb together with exencephaly.11 peixoto - filho mentioned that clubfoot was seen in few cases.12 also in the presented case clubfoot was seen. intrauterine diagnosis of this pentalogy is impossible before 12 week of gestation, because of herniation of bowel out of abdomen is a normal event in fetal development at this time, but after that ultrasonography is a useful method even in the first trimester.1213 differential diagnosis of fetal abdominal wall defect after 12 week is omphalocele, pentalogy of cantrell and gastroschisis. if midline abdominal wall defect is present together with other abnormalities specially ectopia cordis one should consider pentalogy of cantrell.14 both 2d and 3d obstetric ultrasonography are recommended, but 3d ultrasonography is not necessary in first trimester.12 other diagnostic methods including ct - scan and mri can be used for confirmation.41516 prognosis of pentalogy of cantrell depends on severity of intra and extra cardiac defects, pulmonary hypoplasia, extent of abdominal wall defect, cerebral anomalies and diaphragmatic herniation. the mean survival rate without any interventional surgery is about 36 hours.3 studies showed that even with care monitoring in professional centers and multiple corrective surgeries, they had high morbidity and mortality rate and long time prognosis is poor.17 the presented case had all portions of pentalogy of cantrell as well as midline supraumbilical wall defect and ectopia cordis, lower sternal, pericardial and diaphragmatic defects together with bilateral deep cleft lip / palate and left side clubfoot which were reported in other case reports. prenatal routine obstetric ultrasonography of the case revealed abdominal wall defect with evisceration of intestines and liver which confirmed by 3d ultrasonography and gastro - schisis was offered. but diagnosis of pentalogy of cantrell was made after autopsy. | cantrell 's pentalogy (cp), a rare congenital malformation, consists of the supraumbilical abdominal wall defect, the sterna lower part defect and agenesis of the anterior portion of the diaphragm, an absence of the diaphragmatic part of the pericardium, and a malformation of cardia. this case report presents a female neonate, who was born at 32 weeks of conception, weighing 1300 g and was admitted one hour after delivery. she had the five anatomical defects known for cantrell 's pentalogy. moreover, autopsy revealed a bilateral cleft lip and palate, a patent ductus arteriosus, and an atrial and ventricular septal defect. |
a 39-year - old woman presented to our hospital with a history of photorefractive keratectomy (prk), performed two weeks prior. the best corrected visual acuity (bcva) of the affected eye was limited to hand motion. slit lamp examinations revealed diffuse conjunctival congestion and a corneal ulcer with stromal infiltration [fig. (a) diffuse conjunctival congestion and corneal ulcer with stromal infiltration, (b) large full thickness infiltrate with 1 mm hypopyon, (c) endothelial dusting, aqueous flare and cells the ulcer bed and edges were scraped and were sent for bacteriological and mycological examination analysis. treatment with corticosteroid eye drops was suspended and the patient was started on the following drugs : topical fortified 5% vancomicin, tobramicin 0.3%, moxifloxacin 0.5%, amphotericin b 0.15% at 4 h intervals ; systemic antifungal and antibacteric drugs consisted of oral itraconazole at a dose of 200 mg daily with oral ciprofloxacin 1 g daily, and intramuscolar ceftriaxone 1 g daily respectively. after two days the cornea revealed a full thickness infiltrate with 1 mm hypopyon [fig. after 5 days, the patient underwent a therapeutic penetrating keratoplasty with an 8-mm graft on a 7.75-mm bed using barron corneal trephine. post - surgery treatment consisted in topical norfloxacin 0.3% (6 times per day) and amphotericin b (4 times per day) ; systemic ciprofloxacin 1 g daily, ceftriaxone 1 g daily, itraconazole 200 mg daily and intramuscolar metilprednisolone 20 mg daily [fig. 1 ]. after 8 days, the culture of corneal scraping revealed the presence of fungal filaments. we decided in agreement with the infectious disease specialist, to add therapy with oral voriconazole, (800 mg per day on the first day, followed by 400 mg per day on successive days) and topical amphothericin b eye drops 6 times per day. the donor cornea and anterior chamber remained free from infiltrates until day 12, after keratoplasty, when the patient showed eye redness, pain, 1/50 of visual acuity, endothelial dusting and aqueous flare and cells [figs. oral voriconazole was changed to intravenous formulation, 8 mg / kg twice per day. (a) eye redness, endothelial dusting, aqueous flare and cells, (b) fungal infiltrate in with wound dehiscence, (c) eradication of infection twenty days after surgery the patient had recurrent fungal infiltrate in the donor cornea with wound dehiscence [fig. because of the high degree of homology in culture between fusarium solani or oxysporum, it has been difficult to rule out the two similar opportunistic pathogens from the biopsies performed for microbiological analysis. in order to have a proven and rapid differential diagnosis we sent biopsy material to the institute of microbiology of bellinzona (switzerland), where a matrix - assisted laser desorption ionization - time - of - flight analysis (maldi - tof) has been performed. the fungal isolate was identified as fusarium solani, occurring in both the first and second explanted corneas. after specific antifungal sensitivity testing, we decided to start the patient on the following agents : 3 mg / kg twice a day of intravenous amphothericin pb ; netilmycin 0.3%, moxifloxacin 0.5%, voriconazole 1% (drops every 2 h) and diclofenac 0.1% (drops every 8 h). during the following days, we performed a combination of intracameral / intrastromal voriconazole (50 g/0.1 ml) in addition to intracameral amphothericine b (10 microg/0.1 ml), in three different administrations. after 6 m from the last surgery the infection was eradicated, the donor cornea was clear, and the bcva was 7/10 [fig. 2c ]. the incidence of infectious complications post prk is relatively low and the pathogens primarily involved are gram - positive organisms. to our knowledge, only few cases of fungal keratitis are reported. the use of bandage contact lens (bcl) and topical steroid therapy, provided in the postoperative course of the prk together with the breakdown of the barrier function of the corneal epithelium, may cause a predisposition to fungal corneal infection. a variety of factors can determine the course of a fungal keratitis such as early diagnosis, sensitivity to drug therapy and virulence of the fungus. in our case, the natural history of the infection was influenced by appropriate timing of intervention ; in fact, the choice to perform a penetrating keratoplasty proved to be effective in containing the infection and preventing corneal perforation. our patient had a recurrence of the infection that can be explained by the delay in starting the specific therapy with voriconazole, [due to the time required for microbial culture (8 days) ] and the need to perform the maldi - tof in order to identify the species of fusarium. the long use of topical steroid after prk might be another cause of fungal infection recurrence after the first prk, as reported in the literature. the presence of hypopyon before surgery has also been reported as a risk factor for recurrent fungal infection after pk. the management of fungal keratitis after prk depends on many factors : in our experience, the decision of performing a prompt keratoplasty was aimed at reducing the corneal infection in the first occurrence, and to solve it in the second manifestation. finally the use of intracameral antifungal medication proved to be very effective in avoiding the recurrence of infection. | a 39-year - old woman presented to our hospital with a history of photorefractive keratectomy (prk), performed two weeks prior ; slit - lamp examination revealed diffuse conjunctival congestion, corneal ulcer and stromal infiltration. after 5 days of antifungal and antibacteric treatment, the infiltrate progressively increased so that a therapeutic penetrating keratoplasty was necessary. the microbiological analyses revealed the presence of fungal filaments. twenty days after surgery the patient had recurrent fungal infiltrate in the donor cornea with wound dehiscence. we performed a second penetrating keratoplasty. with the matrix - assisted - laser - desorption - ionization - time - of - flight analysis (maldi - tof) we identified a fusarium solani. intravenous amphothericine b, a combination of intracameral and intrastromal voriconazole and intracameral amphotericine b were administered. after 6 months from the last surgery the infection was eradicated. the management of fungal keratitis after prk depends on many factors : in our experience, a prompt keratoplasty and the use of intracameral antifungal medication proved to be very effective. |
desmoplastic fibroma of bone is a well differentiated, locally aggressive fibrous tumor that histologically resembles the extra abdominal desmoid tumor of soft tissues. it is extremely rare, and less than 200 cases have been reported so far.12 the most common site of affection is the jaw, followed by pelvis and long bones.34 forearm bones are rarely involved.58 the mainstay of treatment is surgical excision. wide excision followed by reconstruction of the bony defect using fibular graft has been widely described in tumor surgery. fibular graft functions well as an intercalary graft ; even, there are reports of reconstruction of the distal radius (after excision of giant cell tumor) using fibular graft where the fibular head functions as an articular part of the wrist joint. however, there are no reports of the use of fibular graft in reconstruction of the elbow joint. we report a case of giant desmoplastic fibroma of ulna that has involved the proximal two thirds. the unique technique of reconstruction of the olecranon process with the articular surface of the fibular head has been described. a 15-year - old child presented with a 3-year history of progressively increasing swelling in the right forearm. there was no significant pain or constitutional symptoms. local examination revealed a diffuse swelling over dorsomedial and medial aspects of the forearm, extending from just below the elbow joint to about 8 cm above the wrist. swelling was firm in consistency, with a bosselated surface and was arising from ulna. plain radiograph showed an expansile osteolytic growth involving the proximal ulnar metaphysis and two - third of the diaphysis [figure 1a ]. there was honeycomb pattern with coarse ridge like trabeculae, and matrix was purely lytic. magnetic resonance imaging showed a large circumferential growth arising from the central medullary canal, expanding the bone [figure 1b ]. surrounding cortex was intact and there was no soft tissue component. elbow and wrist joints were free of tumor. the tumor appeared iso- to hypointense to muscle on t1-weighted images and the majority of the tumor areas were isointense to surrounding muscle on t2-weighted images. (a) preoperative anteroposterior and lateral radiographs showing a well demarcated expansile osteolytic growth involving about two - thirds of ulna. (b) magnetic resonance imaging showing a large circumferential growth from the central medullary canal, expanding the bone. surrounding cortex is intact and there is no soft tissue component the tumor was excised including half of the olecranon process and distally up to about 9 cm from the wrist joint. cut surface showed a homogenous grayish white appearance destroying the entire bone with intermittent ballooning of the cortex. the head of fibula was cut transversely to match the remaining half of the olecranon process to recreate the elbow joint. this was fixed with the remainder of the olecranon process with tension band wires and a square nail. the head of the fibula was cut transversely, and the distal part of the head and shaft was used for reconstruction. the transverse cut in the head of fibula was aligned at the articular surface with the remaining proximal olecranon. distally fibular diaphysis was fixed with ulnar shaft with a dynamic compression plate [figures 2, 3 ]. the functional attachment of the triceps was that which remained attached to the proximal olecranon. some part of the triceps attachment was also freed from the resected part of the ulna. postoperative radiograph anteroposterior and lateral views of the same patient showing restoration of the proximal ulnar anatomy with fixation hardware in situ 20 microscopic field of the tumor section showing monomorphic cells without any anaplastic changes, interspersed between the strands of collagen, arranged in a parallel fashion histopathology of the tumor revealed a moderately cellular lesion on low - power microscopy, with spindle - shaped cells interspersed between the strands of collagen, arranged in a parallel fashion. on high - power microscopic field, there was clustering of monomorphic cells without any anaplastic changes or mitosis [figure 3 ]. patient was followed up for 2 years with no evidence of recurrence. the fibular graft united with the olecranon proximally and distally with remaining part of ulna. followup radiographs at 2 years showing union of the fibular graft with proximal ulna and some restoration of the elbow joint (a, b). desmoplastic fibroma of bones is considered to be locally aggressive with a high recurrence rate. radiological differential diagnosis include other lytic lesions such as aneurysmal bone cyst, giant cell tumour, chondromyxoid fibroma, and low - grade fibrosarcoma. treatment of choice is wide local excision through the normal soft tissue.78 recurrence rate is 17% following resection and as high as 55% following curettage.2 various options have been used in literature [table 1 ] for the reconstruction of bone defects in forearm following tumor resections, such as creation of single bone forearm from radius and ulna and centralizing the carpus over the single bone, free fibular graft, fibular head graft for distal radius, and microvascular fibular graft and allografts. free fibular graft has proved to a good option for reconstruction of bony defects and has been extensively used in tumor surgery. the technique we have used to create the articular surface of the olecranon using the fibular head articular surface has not been previously described. though a normal joint surface can not be expected, this reconstruction is an acceptable option considering the morbidity of the tumor resection for elbow function. elbow is not a weight - bearing joint and some degree of degenerative changes can be tolerated. this will provide a more stable and mobile elbow joint compared to the single bone forearm. review of literature on desmoplastic fibroma arising from bone in forearm proximal half of the olecranon has been preserved during excision based on intraoperative frozen section. thus, the fibular graft functioned as an intercalary graft between the olecranon proximally and distal third ulna distally. firstly, it helped to maintain some integrity of the extensor apparatus of the elbow to have some useful elbow function also to and counterbalance the elbow flexors. secondly, it provided a surface to articulate with the trochlea to provide some stability and mobility of the elbow joint. most of the elbow articulation was made from this segment of the olecranon that was preserved. following principles of olecranon reconstruction from trauma surgery, elbow joint can be reasonably restored even if a large part of olecranon is sacrificed, provided the triceps mechanism is restored.910 this explains the reasonable range of motion achieved in elbow in this case. thus, desmoplastic fibroma is a rare tumor of bone with a benign but locally aggressive behavior. | desmoplastic fibroma is a rare, well differentiated, locally aggressive fibrous tumor usually arising from soft tissues, and rarely from bones. involvement of forearm bones is extremely unusual. we present a large desmoplastic fibroma of right ulna in a 15-year - old male. the tumor was excised with a wide margin, and the bony defect was reconstructed with nonvascular autologous fibular graft. reconstruction of the olecranon process was attempted using the fibular head and the remaining olecranon. at 2-years followup, there was no recurrence, flexion extension arc of the elbow joint was 40130 and there was no restriction of activities of daily living. |
obsessive compulsive disorder (ocd) is an illness that is characterized by recurrent obsessions and compulsions that cause marked distress and impairment. it has a lifetime prevalence of 2 - 3% and is the fourth most prevalent psychiatric disorder. estimates suggest that it is the tenth leading cause of disability in the world. up to 3040% of patients fail to respond significantly to current treatments and those who do respond often experience only partial remissions [4, 5 ]. patients who fail to respond to two serotonin reuptake inhibitors (ssris) are considered refractory. refractory ocd is regarded as an especially refractory disorder [7, 8 ] with severe cases being among the few psychiatric disorders that continue to be treated by irreversible procedures such surgical interventions, gamma knife ablation, and deep - brain stimulation with permanently implanted electrodes [911 ]. given the nature of these treatments, there is an urgent need for effective pharmacotherapy. however, studies of clomipramine and desipramine revealed efficacy only for clomipramine [12, 13 ]. since clomipramine has ssri activity, it has been suggested that abnormalities of the serotonin system play a causal role in ocd. although ssris remain first - line treatment for ocd and attempts have been made to associate polymorphisms in serotonin - related genes with ocd, no specific abnormality of the serotonin system has been definitively identified as a cause of ocd. the role of the dopamine system has also been considered and ssris have been augmented with neuroleptics, including atypical neuroleptics. however, no specific abnormality of the dopamine system in ocd has been firmly identified. recently, abnormal glutamatergic neurotransmission has also been considered as a basis for ocd. in a theoretical analysis, carlsson concluded that ocd and attention deficit hyperactivity disorder (adhd) are antithetical with regard to phenomenology and with regard to prefrontal glutamatergic neurotransmission, with ocd being hyperglutamatergic and adhd being hypoglutamatergic. an experimental study using single - voxel proton magnetic resonance spectroscopy found that the combined glutamate / glutamine peak (glx) in the anterior cingulate cortex is reduced in ocd. another study suggested that sequence variations in the 3-untranslated region of the gene for the nmdar-2b subunit (grin2b) are associated with ocd. more recently, polymorphisms in the gene for the glutamate transporter (slc1a1) have been associated with ocd in two studies [17, 18 ]. taken together, these findings raise interest in abnormal glutamate neurotransmission as an aspect of ocd, but much work needs to be done to clarify the reproducibility of the associations and the functional significance of the polymorphisms. therapeutic approaches to ocd have also been attempted with reagents that modify glutamate neurotransmission. for example, riluzole was used to augment pre - existing medication in an open 12-week trial with 12 treatment - resistant subjects. n - acetylcysteine has been used with reported success to augment an ssri in a case of refractory ocd. topiramate augmentation of paroxetine has been reported to be beneficial in one case of refractory ocd ; whereas it appeared to cause ocd in two other cases [22, 23 ]. all of the foregoing trials are confounded by the use of more than one psychotropic reagent. to our knowledge, there are no reports of modulators of glutamate neurotransmission being used alone in a sustained fashion to treat ocd. in contrast to approaches based on neurotransmitters, swedo and coworkers have proposed that a subtype of ocd is the result of an antigen - specific autoimmune response that is stimulated by gabhs infection [2426 ]. this diagnostic subtype is referred to as (pediatric autoimmune disorders associated with strep) pandas and may include ocd or tourette syndrome as well as other comorbidities. predictions of the pandas hypothesis for therapy have been tested in trials with ivig and plasma exchange. positive findings were initially reported [25, 26 ]. however, in the nine years following the placebo - controlled trial, these treatments do not appear to have become widely used, raising questions about the generality of the initial findings. body dysmorphic disorder (bdd) is an illness of unknown etiology that is characterized by excessive preoccupation with an imagined or minor defect in body appearance. bdd is often a severe disorder and has been associated with an elevated suicide rate. in a nationwide study in germany, bdd is a poorly studied disorder for which available treatments are frequently inadequate, with 2747% failing to respond to adequate trials of ssris. it is of interest that antipsychotics, either as monotherapy or as ssri augmenters, are reported to be ineffective for bdd. bdd is not unusual as a comorbidity of ocd, occurring in 12% of cases. the combination of ocd and bdd is usually found to exhibit more impairment and a poorer prognosis than ocd alone [3032 ]. in this paper, we describe a case of refractory ocd and bdd in which infection - triggered exacerbations appear to have played a prominent role. a serendipitous observation of an exacerbation that followed treatment with a commonly used antibiotic has led to an hypothesis that postulates a deficiency in the nmdar signal transduction cascade. this hypothesis points to a new direction of research on the molecular basis of ocd and bdd and makes the clear prediction that enhancement of nmdar neurotransmission should provide amelioration of symptoms. in this paper, we present the observations from a five - year period in which glycine, an nmdar coagonist, was used as the sole treatment of previously refractory ocd and bdd. this study has been conducted under a protocol approved by the institutional review board of the institute for health sciences of st. consent under the aegis of this protocol has been obtained for the publication of the historical information in this paper. in addition, the subject (referred to as o in what follows) has read this paper and has given additional written informed consent for the publication of his case details. to protect subject identity, precise values of clinical tests with numerical results, which could be used as identifiers, differences between modified and actual values are sufficiently small to be of no scientific significance. a key resource for this naturalistic study has been a family - compiled archive that includes behavioral and medical information from birth to the present time. this information includes extensive written and recorded oral diary entries by o 's father as well as written entries by o. in addition to blood test results, there are either reports or copies of charts from specialists and copies of charts of primary care physicians (from birth to the present). photocopies of some prescriptions and pharmacy records covering the period when psychotropic medications were used are also available. in addition, tablet containers for psychotropic medications and some other medications have been retained as a primary record of medication prescribed. academic reports from kindergarten to the present, many examples of school work done by o, records of formal cognitive testing at ages 7 and 30, and results on standardized, us academic tests are also available. a valuable feature of o 's archive is the existence of redundant information on many issues of critical importance. an important category of information in the archive is objective information from individuals or organizations that are unconnected with this study (referred to in what follows as third - party objective information). this category is free of concerns about bias arising from the fact that the subject, the subject 's parents, and the investigators were not blinded in relation to glycine treatment. this category of information has been essential to establish the claim that there was major and persistent impairment before glycine treatment and substantial reduction of impairment following treatment. it has also been essential for establishing the occurrence of a substantial relapse in a period of nontreatment. the archive also contains recorded observations of objective phenomena made by the subject or his parents. o 's father reports that he has given emphasis to objective and qualitative indications of changes in behavior that were sustained over time. this information has been evaluated for its internal consistency and its consistency with third - party objective information. subjective information (e.g., o 's symptom reports and parental judgments) has also been evaluated for internal consistency and consistency with third - party objective information. in cases where observations were recorded or recalled from memory at substantial time intervals after events occurred, this is explicitly indicated in the text. moreover, a major effort has also been made to determine when observations could represent concept - driven perceptions or represent concept - driven selection of data. examples of relevance to the conclusions of this paper are discussed in the text. general medical history, radiological findings, and descriptions of ocd- and bdd - related behavior are included in an appendix, since this information may be useful for future definitions of glycine - responsive subtypes of ocd and bdd and as constraints on molecular models. o 's parents recall their child as happy and contented for the first two years of his life. however, at age 2, they recall the sudden eruption of separation anxiety that was much more intense than what they saw in other children in their social group. the prodrome of o 's illness became more clearly apparent at age 7 when he received psychotherapy from a psychiatrist whose chart noted anxiety enormous. at this time, learning difficulties in school prompted a request for cognitive testing, which revealed evidence for mild cognitive deficits. with improved school performance and social adjustment, psychotherapy was discontinued after one year. as an adult, after developing an understanding of his illness, o has recalled wall - touching rituals as early as age 9, but these were not noticed by parents. o 's parents believe they can recall several very mild examples of obsessive mentation that involved reassurance rituals as early as age 9. at age 14, they clearly recall a wall - touching ritual. although they report that they were unaware of the concept of obsessive - compulsive disorder at that time, they regarded this behavior as abnormal and initiated a psychiatric consult. however, parental records and the psychiatrist 's chart indicate that a diagnosis of ocd was not obtained at this time. the mild prodrome in the prepubertal years was followed by frank illness that emerged rather abruptly at age 15 when school attendance was disrupted. after attending ~1/2 of ninth grade, o and his parents report that he refused to attend school any longer after developing the fear that students in the school had weapons. this fear of crime was later found to be associated with a powerful line - crossing obsession that involved the fear that individuals likely to commit crimes were entering into his neighborhood. this obsession was associated with reassurance rituals referred to as talk - throughs (appendix a.1.1). at this time, cognitive behavior therapy was attempted by a psychologist who is reported by o 's parents to have found a mirror phobia and mild anxiety. attempts by the psychologist to treat the putative mirror phobia with a desensitization protocol are reported to have failed, and both o and his parents currently consider that working on the mirror intensified rather than reduced the mentation and behavior that was later diagnosed as bdd. with the endorsement of the psychologist, o was sent to boarding school where fears of ugliness and the mirror phobia are reported by both o and his parents to have intensified. when o returned home on vacation, he refused to return to school because of the mirror problem. additional efforts by the psychologist to achieve mirror desensitization were again unsuccessful and led to psychiatric consultations that resulted in diagnoses of ocd and bdd at age 17. o currently recalls being mildly disturbed by his seventh grade class picture (age 13). in any case, available evidence suggests that bdd was preceded by obsessive - compulsive behaviors, which were preceded by cognition deficits. in response to the diagnoses of ocd and bdd, a trial of fluoxetine (4080 mg / day, 134 days) was ineffective and was followed by a long trial of fluvoxamine (200300 mg / day, 251 days). a second opinion from a research psychiatrist specializing in anxiety disorders confirmed the diagnoses of ocd and bdd and recommended risperidone as an adjunct to fluvoxamine. at age 19, during the trial of fluvoxamine (300 mg / day) + risperidone (2 mg / day), a severe exacerbation occurred that involved intensification of pre - existing symptoms and the emergence of terrifying, violent imagery, a symptom which had not been reported before. after full completion of the recommended 30-day risperidone trial, diary records indicate a decision by o 's parents to suspend pharmacotherapy on the basis of their concern that this exacerbation was a reaction to the medication. psychotherapy was subsequently initiated with a different psychiatrist and was continued for about nine months. following the age-19 exacerbation, o is reported to have become largely housebound as a result of the line - crossing obsession associated with a fear of crime (appendix a.1.1). the addition of bdd by proxy to pre - existing self - related bdd also occurred at this time. bdd by proxy involved o 's fear / belief that his parents were ugly and that he looked like them (appendix a.1.2). also present were somatic / cognitive preoccupations that were very mild in relation to other symptoms (appendices a.1.3 and a.1.4). one year after the age-19 exacerbation, there was another exacerbation similar to the previous one in its severity and in the emergence of terrifying violent imagery. however, in this exacerbation, no psychotropic medication had been taken in the past nine months. paroxetine (5070 mg / day) and olanzapine (5 mg / day) were prescribed and o was referred to another psychiatrist for behavior therapy and weekly monitoring. the latter soon added clonazepam (0.54.0 mg / day). in spite of an initial, apparent improvement that permitted o to go for short, unescorted walks in his neighborhood during a three - week period, a clear deterioration was apparent after 3.5 months, leading the psychiatrist to terminate olanzapine and to continue paroxetine at 6070 mg / day for another 9 months. again no improvement was perceived. in response to the failure to make progress with established pharmacotherapy, o and his family began to search for newly emerging treatments. when o was 21, they became aware of the pandas concept of swedo and coworkers [2426 ]. consultation with o 's pediatrician revealed that two severe cases of bilateral ear infections with -hemolytic - positive throat cultures had occurred approximately 1 - 2 months before the peaks of the age-19 and age-20 exacerbations (-hemolytic - positive throat cultures are ~50% probable gabhs infection in temperate climates, j.b. o 's father 's diary contains a detailed description of the age-19 exacerbation, which began as soon as the infection cleared. seeing their son 's further deterioration o 's father emphasizes that at that time he had no knowledge of the pandas concept and therefore did not associate the deterioration with the -hemolytic - positive infection that was discovered retrospectively two years later. the retrospective discovery at age 21 that probable gabhs infections preceded the age-19 and age-20 exacerbations led to consultations with a rheumatologist. in response to a report that ivig treatment was effective with pandas, paroxetine was tapered from 60 mg / day to zero and overlapped ivig for only two months. ivig therapy was initiated at 160 grams for the first month and 80 grams / month for an additional 17 months. although there was an apparent behavioral improvement after the first infusion, this improvement was not sustained and ivig was eventually terminated for lack of efficacy. about 3 months after the ivig trial began, 500 mg / day of amoxicillin was added as a prophylaxis against gabhs. o 's parents report no obvious benefit, and also no obvious deterioration, from amoxicillin prophylaxis, which was terminated after about 6 months to permit initiation of a different antibiotic regimen to remove h. pylori. nine months into the ivig treatment, removal of h. pylori using a combination of clarithromycin (1000 mg / day), amoxicillin (1500 mg / day), and lansoprazole (30 mg / day) for 12 days was associated with a severe exacerbation of existing obsessions and compulsions and emergence of highly distressful violent imagery. episodes of highly disturbing violent imagery are reported to be extremely distinctive, occurring only three times in o 's life, namely in this exacerbation at age 22 and in the two major exacerbations at ages 19 and 20 that followed severe bilateral ear infections. another distinctive feature of this exacerbation was a much stronger presentation of ritualistic behaviors than ever seen before, either during baseline illness or during previous exacerbations. o 's father 's diary indicates that the main ritual was a prayer ritual that was performed for many hours a day, leading initially to knee erosions that were followed by calluses. another distinctive feature of this exacerbation is its similarity to the previous ones in which there was a gradual rise in symptoms over a period of approximately 1 - 2 months. before, during, and after the antibiotic treatment, measurements of streptolysin o titers were negative. after the peak of the exacerbation, records indicate that the prayer ritual gradually declined over a period of approximately 12 months to generate a new baseline in which ritualistic behaviors are reported to have been increased relative to pre - exacerbation levels. after the termination of ivig treatment, o 's parents report that they decided to support their son at home on their own until a new treatment was developed. while waiting for the development of new treatments (from age 23 to 25), o and his parents tried on their own initiative and in consultation with physicians various nutraceutical treatments, (e.g., [33, 34 ]) that had been developed for other illnesses that are sometimes associated with ocd, such as huntington 's disease. the glycine trial described below represented another of their efforts involving the use of a nutraceutical. the diagnoses of ocd and bdd that were given at age 17 by a psychiatrist were subsequently confirmed by several other psychiatrists, including two ocd specialists. no signs or symptoms of other major psychiatric illnesses have been noted by the multiple psychiatrists who have examined o. there is no evidence of substance misuse, as noted in general medical history (appendix a.2). in o 's continuous and generally increasing illness, individual signs and symptoms. however, the basic spectrum of signs and symptoms appears to have been unchanged since the emergence of frank illness at age 15, suggesting stable diagnoses from age 17 to age 30. at the time of the exacerbation that followed h. pylori eradication, it was considered that the exacerbation could have been due to one or more of the three agents used to achieve eradication (amoxicillin, lanzoprazole, clarithromycin). clear precedents for psychiatric sequelae of lanzoprazole and amoxicillin were not found in the literature at that time. more recent literature suggests that beta - lactams can modulate glutamate neurotransmission but that doses substantially above the therapeutic range appear to be required. consistent with this finding is the observation that o has taken amoxicillin on multiple occasions before and after the exacerbation without adverse effects. in contrast to amoxicillin and lanzoprazole, there were reports describing substantial cns and psychiatric sequelae from macrolide antibiotics, including clarithromycin. however, a majority of reports on psychiatric sequelae from clarithromycin claimed remission immediately after cessation of treatment. in o 's case, the exacerbation continued to increase over a period of approximately 2 months after the 12-day treatment period. consequently, the role of clarithromycin was discounted until a reanalysis three years later in 2002. in 2002, one of us (w. l. cleveland) became aware of studies of phencyclidine 's effects on patients with medication - controlled schizophrenia. a remarkable finding is that a subpopulation of these patients can exhibit a prolonged exacerbation following a single, brief exposure to the drug. drugs interacting with other receptors, such as lsd, failed to exhibit protracted exacerbations (see [37, 38 ] and references therein). the similarity of the protracted kinetics of the phencyclidine response to the kinetics of the antibiotic - induced exacerbation seen in o led to a literature search for evidence that clarithromycin could influence nmdar neurotransmission. a paper by manev. provided evidence that clarithromycin could inhibit cell death from glutamate - induced excitotoxic injury in cultures of human cerebellar neurons. activation of the nmdar as indicated by calcium influx was not inhibited, suggesting that clarithromycin (and the other macrolides studied) inhibited a downstream aspect of the nmdar signal transduction (nmdar - st) cascade. the concentrations used in their in vitro studies were in the range of 100 m. the literature indicates that clarithromycin can reach comparably high concentrations in tissues and cells, with concentration increasing in proportion to the duration of treatment. moreover, access of clarithromycin and its main, active metabolite, 14-oh - clarithromycin, to the brain is suggested by the observation that clarithromycin is useful in the treatment of central nervous system toxoplasmosis infections [41, 42 ]. it should be noted that low concentrations of macrolides in cerebrospinal fluid (csf) are an indication of a marked tendency for macrolides to accumulate in cells rather than poor access to the brain. the macrolide affinity for cells is also seen in a comparison of clarithromycin and azithromycin, which reached levels of 940 m and 520 m, respectively, in alveolar macrophages. the above considerations led to the hypothesis that clarithromycin induced an exacerbation by inhibition of a downstream aspect of the nmdar - st cascade. moreover, it seemed plausible that this inhibition modulated, either directly or indirectly, the intrinsic defect responsible for baseline symptoms. an immediate prediction of this hypothesis is that an nmdar agonist (or coagonist) would lead to symptom improvement. this prediction motivated consideration of the high - dose glycine treatment developed for schizophrenia, which has been used with apparent safety as an adjunct to other medication in trials beginning in 1988 [44, 45 ]. after consultation with physicians, o decided to initiate a trial of glycine at 0.8 grams / kilogram of body weight / day, the dose used with apparent safety in schizophrenia trials. the initial response to glycine was monitored in relation to major life disruptions (see below) and in relation to the specific signs and symptoms of preglycine illness (appendix a.1). schoola sign of major impairment is no attendance of high school after failing to complete tenth grade. an attempt to continue o 's education with one - on - one tutoring was discontinued six months after stopping regular school. this led to an unsuccessful attempt to take ged examination (for high school equivalency diploma) at age 25, several months before initiation of glycine treatment. records indicate that when o arrived at the test site, he refused to enter, giving an explanation related to his line - crossing obsession. at this time, he also refused to handle photo - identification for entry into the test site. thus, in the 8-year period preceding glycine treatment (age 17 to age 25), there was no regular school or formal tutoring. a sign of major impairment is no attendance of high school after failing to complete tenth grade. an attempt to continue o 's education with one - on - one tutoring was discontinued six months after stopping regular school. this led to an unsuccessful attempt to take ged examination (for high school equivalency diploma) at age 25, several months before initiation of glycine treatment. records indicate that when o arrived at the test site, he refused to enter, giving an explanation related to his line - crossing obsession. at this time, he also refused to handle photo - identification for entry into the test site. thus, in the 8-year period preceding glycine treatment (age 17 to age 25), there was no regular school or formal tutoring. social lifethe diary maintained by o 's father indicates that by age 19, o had become housebound except for doctor visits, visits to his father 's place of work, and rare visits to an art museum, all of which required his father 's escort and door - to - door taxi travel along routes that were perseveratively selected by o because of the fear that his line - crossing obsession would be triggered by people he might see on alternate routes. at age 21, there was a brief period of about 3 weeks duration, during treatment with paroxetine + olanzapine, in which o went for short, unaccompanied walks in his neighborhood. otherwise, records indicate that o was housebound in the six - year preglycine period that followed the age-19 exacerbation. there were no other social contacts except for telephone contact with a grandmother in another city. the diary maintained by o 's father indicates that by age 19, o had become housebound except for doctor visits, visits to his father 's place of work, and rare visits to an art museum, all of which required his father 's escort and door - to - door taxi travel along routes that were perseveratively selected by o because of the fear that his line - crossing obsession would be triggered by people he might see on alternate routes. at age 21, there was a brief period of about 3 weeks duration, during treatment with paroxetine + olanzapine, in which o went for short, unaccompanied walks in his neighborhood. otherwise, records indicate that o was housebound in the six - year preglycine period that followed the age-19 exacerbation. there were no other social contacts except for telephone contact with a grandmother in another city. behavior at homeo 's disorganized lifestyle is reported to have created disarray in his home that prevented o 's parents from bringing guests to their home for at least seven years preceding glycine treatment. o 's made some effort to do school work, but he was generally resistive to parental tutoring. any attempt to discuss academics was thwarted by the tendency to discuss an obsession. o 's disorganized lifestyle is reported to have created disarray in his home that prevented o 's parents from bringing guests to their home for at least seven years preceding glycine treatment. o 's made some effort to do school work, but he was generally resistive to parental tutoring. any attempt to discuss academics was thwarted by the tendency to discuss an obsession. the last exacerbation in the preglycine period occurred 3 years before initiation of glycine treatment. as noted previously, the decline of this exacerbation over about one year led to a baseline in which ritualistic behaviors were increased relative to the pre - exacerbation baseline. this new baseline remained stable in the two years preceding the initiation of glycine treatment. it was against this stable baseline of clear and substantial impairment involving the above life disruptions that the response to glycine treatment was initially detected. in this 5-year study of glycine treatment, decisions to start and stop glycine treatment were made by o in consultation with physicians. target doses varied from 50 to 66 g / day (0.60.8 grams of glycine per kilogram of body weight). as an indication of compliance, glycine treatment was typically initiated by increasing the dose over 12 days from 10 g per day to the target dose. in observation period 1 (op1), the full dose was initially taken t.i.d., but, for reasons of convenience, was subsequently taken b.i.d. the daily target dose was varied from 50 to 66 g / day in this period. glycine was abruptly terminated on day 204 due to an upper respiratory infection with high fever. o 's father 's diary indicates that the first improvement was seen 23 days after reaching target dose on day 34-op1(34) (day 34 of observation period 1(day 34 of study)). for the first time in 5 years, o left his apartment building without parental escort and went for a short walk in front of his building. soon after this, he began daily attendance a nearby library where he prepared for the high school equivalency exam (general education development (ged) test of the american council of education). this is reported to have been done on a 9 am to 5 pm weekday schedule without parental escort. official scores on the five ged subtests ranged from the 67th to the 99th percentile on scales normalized to scores of graduating high school seniors in the us. reduced intolerance to mirrors is supported by a sustained resumption of barbershop haircuts after approximately five months of glycine treatment. o was escorted to barbershops by his father, who reports that he observed no obvious distress over mirrors. six months after initiation of glycine treatment, o was encouraged to study in a different library on the campus of a university near his home. reaching the campus required paternal escort and crossing a busy street that had previously been avoided due to the line - crossing obsession. this was an improvement over the preglycine period in which o insisted that his father handle his photo - identification. o 's parents consider that the partial tolerance of the street and campus indicated a significant and further reduction of the line - crossing obsession. at this time, o 's father 's diary indicates that talk - throughs, which had been daily before glycine treatment, had become infrequent. the willingness to handle a photo - identification card although substantial illness remained, the data for op1 suggest steady and significant progress during the first 200 days of glycine treatment. on day 1-op2(204), o developed an upper respiratory infection with fever as high as 104.2f (oral, hg thermometer). a commercial lateral flow immunoassay for gabhs antigens (one - step clearvue, quidel, san diego, ca) was negative. was thought to be unusually high in relation to o 's prior history, o and his parents became concerned that it could be due to glycine. detailed notes in o 's father 's diary suggest a gradually rising exacerbation following the infection. on day 21-op2(224), the line - crossing obsession was activated on the way home from the library, leading to the first talk - through in quite sometime. the next day, it took an hour of parental urging to get o to leave his home. o reported the emergence of a hand - washing ritual driven by fear of harm. although unequivocal, they described this exacerbation as distinctly less intense than the major exacerbations at ages 19, 20, and 22. most notably, it did not involve terrifying violent imagery. by the time of this exacerbation, both o and his parents were aware of the concept that exacerbations could result from infections. therefore, there is the possibility that the exacerbation described for this period reflects concept - driven perceptions or selection of data. however, o 's father notes that, at that time, they expected exacerbations only from gabhs infections, since the two previous infection - associated exacerbations were associated with positive throat cultures. therefore, as a result of the negative test for gabhs done at the beginning of the infection in the office of o 's primary care physician, an exacerbation is described as unexpected. in response to the deterioration seen in op2, o and his family decided to resume glycine treatment on day 1-op3(245). after 5 days at 25 g / day, glycine was again terminated because of another infection with chills and vomiting and a fever of 102.4f. glycine was again resumed at full dose (50 g / day) on day 13-op3(257). on day 21-op3(265), o reported a reduction in rituals. fewer rituals and fewer complaints related to the line - crossing obsession were noted on day 29-op3(273). other observations suggest a rapid recovery of lost gains. on day 31-op3(275), o was escorted only to the edge of the campus, walking to the library by himself. the trend to improvement is further suggested by the fact that by day 85-op3(329), o began freely exploring the entire campus. on day 91-op3(335), he began working with a tutor (other than his parents) for the first time in 9 years. increased tolerance of his mother 's face is suggested by attendance with her at movies and restaurants and acceptance of her help with sat preparation. third - party records indicate that a major activity during this period was a preparatory course for the sat, a reasoning test of the college board required for admission by many us colleges. o 's tolerance of a classroom with many students suggests a reduction of bdd - related social discomfort. travel to the course site was by taxi with parental escort, often o 's mother. moreover, the course site was on a street that was avoided in the preglycine period due to the line - crossing obsession. third - party course records indicate that o 's math scores in the practice tests increased by 100 points and his verbal scores by 60 points. according to the college board, a difference of 60 points is evidence of a true difference in ability (standard error of the difference). in this period, landmark events in which o resumed an activity that had been prevented in the preglycine period. even though they acknowledge that substantial illness remained, o 's parents report that they considered the progress achieved by the end of this observation period to be a dramatic improvement over preglycine illness. given that the exacerbation of op2 was associated with both an infection with high fever and a cessation of glycine treatment, it was unclear if glycine cessation contributed to the exacerbation. therefore, o and his family decided to stop glycine treatment during a period without infections. o made his final efforts to prepare for the sat that was taken on day 68-op5(479). scores on the actual tests, which were taken in the standard time intervals, surpassed all practice test scores. his verbal score was in the 90th percentile and was 60 points higher than the best practice test and 120 points higher than the first practice test. his math score was at the 50th percentile and was 30 points better than the best practice score and 130 points higher than the lowest practice test. o 's improved life style at home led his parents to renovate their home and resume normal family social life for the first time in 8 years. diary records indicate that he was present for the first visit of a family friend to his home in five years. in this observation period, this step forward is among the 10 landmark occasions in the archive where o resumed an activity prevented in preglycine period. op5 is important because it reveals the robustness and stress - resistance of o 's improvements. on day 68-op5(479) there was the sat. this was followed by application to college which required an interview with an admissions official on day 112-op5(523). although the interview is reported to have gone well, o 's application was rejected on day 123-op5(534). on day 137-op5(548) o attended an alternative college that was located at a site that would have been a major line - crossing trigger in the preglycine period. in response to this sequence of major stressors, taken together, the data for op5 suggest further substantial improvements that were a continuation of gains seen in op3. as described in op2, an upper respiratory infection with high fever was followed by a behavioral exacerbation. a concern that glycine might have caused the unusually high fever led to the decision to stop glycine during the flu season. the extension beyond the flu season was due to o 's reluctance, arising from the unknown safety of long - term, high - dose glycine therapy. it was at the beginning of this observation period that o resumed his formal education after not attending school for an entire decade. as noted above, o was not accepted in the college of his choice. however, the letter from the admissions office encouraged reapplication after demonstrating competence at another institution. the archive indicates that o attended all classes, completed all assignments on time, and received no special accommodations. he is reported to have tolerated the stress associated with academic deadlines, including all - night sessions to complete papers. academic records document very high grades that permitted a successful reapplication to the college of his first choice. in this 4.5-month period of glycine cessation, there were two indications of a loss of prior gains : a decline in cognition (see section 3.5.11) and a mild increase in the line - crossing obsession. there was also an intensification of somatic / cognitive preoccupations to the point that they generated (for the first time) mild impairment. a notable feature of op6, the first lengthy (220-day) off - glycine period is that the observed, mild relapse in ocd was circumscribed and occurred slowly. in addition to a general perception of relapse by o 's parents, there was also a major concern about the impact of the apparent cognitive decline seen in op6 (see section 3.5.11) on academic performance in a new college with a more demanding curriculum. while o 's parents report that they strongly encouraged resumption of glycine therapy, the final decision was left to o as in the past. attendance at the new college began on day 13-op7(781) with o taking two courses. one course proved to be quite difficult for o. a low grade on the first exam of this course was obtained on day 40-op7(808). this is reported to have generated considerable stress, since o as a new student with an unusual academic and personal history was under pressure to demonstrate his ability to handle the curriculum. o 's father 's diary indicates that there were no signs of a behavioral relapse from this stressful experience. he is reported to have handed in all assignments on time and to have attended all classes except for one. stresses from academic deadlines, for example, all - night sessions to complete papers, are reported to have been handled without any sign of a behavioral deterioration. in the second semester, academic records show that the same consistently adequate performance continued with respectable grades. an important bdd - related gain in this period is o 's reported resumption of hair combing in front of a mirror. although o covered his nose and mouth with one hand, this maneuver suggests an increased tolerance of mirrors, since, prior to this period, hair combing was reportedly done with parental assistance or by manual feeling without visual feedback. o 's father reports that this maneuver did not involve any repetitive working on the mirror, as hair combing was done quickly. another bdd - related landmark event in this observation period is seen in an entry in o 's father 's diary on day 34-op7(802), which indicates that o began to tolerate direct observation of his father 's face in a sustained manner. prior to this, tolerance of direct observation is reported by o to have occurred but only sporadically. however, he continued to maintain that he kept her face in peripheral vision. o 's father 's diary notes only a single talk - through dealing with this obsession. the only obvious remnant of the line - crossing obsession was a partial restriction of the use of public transportation. in summary, there were notable gains that involved coping effectively with a new and challenging academic curriculum, a further reduction in the line - crossing obsession and reductions in self - related bdd and bdd by proxy. importantly, multiple stressful events did not lead to any obvious loss of behavioral improvement. the decision to stop glycine was as always made by o. the reasons were not recorded. o 's father reports that he was not able to detect any consistent trend toward deterioration or toward improvement. eighty three days after glycine resumption (day 83-op9(1151), o began the third semester at his new college, registering for two courses. academic records indicate that o obtained the highest possible grade in one course and next to the highest possible grade in the other course. as before, two courses were taken and the same excellent grades were obtained. diary entries and email correspondence indicate that social activities occurred in classes, in student centers, at social events for students, and at athletic events. all of these activities, except for in - class socialization, are reported to have occurred for the first time in this semester. on day 202-op9(1270), o went on his first date with a girlfriend in 13 years. also of interest in op9 are records indicating a substantial reduction in the signs of bdd by proxy involving o 's mother 's face and in the signs of bdd involving his own face. on day 41-op9(1109), o announced that he was able to look directly at a mirror image of his face with only his mouth and the bottom surface of his nose covered. previously, he had found it necessary to cover his nose and lower part of his face. another indication of reduced bdd intensity is the occurrence of very few bdd - related talk - throughs in op9. o 's father 's diary lists 13 talk - throughs for the entire 47-week period. only several of these were bdd - related. taken together, as noted above, the line - crossing obsession was considered by o and his parents to be substantially reduced by the end of op7. in op9, there are indications for the further reduction of this obsession. most notably, o is reported to have begun freely traveling about his city using bus and subway transportation. this was done either alone or with peers without restrictions on time or areas traversed. o 's father 's diary records no talk - throughs regarding the line - crossing obsession in op9. as in op7, there were no signs of any sensitivity to crime news. both o and his parents currently report the complete disappearance of the line - crossing obsession in op9. somatic / cognitive preoccupations are reported in o 's father 's diary to have been the subject of most of the thirteen talk - throughs listed in this 331 day period. o 's parents consider that somatic / cognitive preoccupations became milder in the final parts of op9. this is supported by the absence of medical consults for somatic preoccupations in these periods. on the basis of the excellent progress in this period, o and his parents report that glycine treatment would have been continued had it not been for the occurrence of unexplained weight loss. op9 was terminated on day 332-op9(1400) to observe weight fluctuations in the absence of glycine therapy. taken together, the above observations for op9 suggest the addition of major gains to those seen in op7 and prior treatment periods. by the end of op9, these gains represented a largely complete remission of ocd and a large fractional reduction in bdd - related impairment relative to preglycine illness. the reduced impairment reflected in the high level of o 's academic and social activities represented a major reversal of the life disruptions of preglycine illness. although, unexplained weight loss initiated this period, o reports that concerns about the unknown safety of long - term treatment led him to extend it. available evidence for this long off - glycine period suggests the occurrence of a clear but mild relapse in bdd, a milder relapse in ocd, and a major relapse in cognition (see section 3.5.11). specifically, o refused the opportunity to take driving lessons after the end of the academic semester (day 372-op10(1772)) on the basis that he might see his face in the mirrors or the windshield of the automobile. the opportunity to travel to a foreign country was refused on the grounds that he would not be able to handle his passport without seeing his picture, although he continued to handle old photo - identification. there was also a return of a hair - loss preoccupation seen in the preglycine period. o 's parents consider the above signs to be a clear increase in bdd intensity and impairment that was modest compared to pretreatment illness. additional diary entries suggest an increase in mother - related bdd by proxy that is reflected in a loss of o 's ability to tolerate his mother 's presence and the risk of seeing her face. in op10, there were no signs of a major return of the line - crossing obsession. however, two episodes suggest the beginning of a restriction in mobility. on day 380-op10(1780) o refused to meet his father in an area of a public park which he had previously visited without complaint. in a recent interview, o explained that he was afraid that he would see someone in that location who would trigger the return of the line - crossing obsession. on day 362-op10(1762) o refused to accompany his father to a restaurant in an area that had been highly sensitive in relation to the line - crossing obsession in the preglycine period. diary records indicate that in op9, o had gone to restaurants in the same area with a girlfriend. some weeks later on day 411-op10(1811), a similar episode occurred. the above observations suggest a small restriction in mobility resulting from a mild return of the line - crossing obsession. on days 279-op10(1679), 380-op10(1780), 388-op10(1788), o 's father 's diary contains descriptions of episodes which suggest a return of concerns about crime - related news. in a recent interview somatic / cognitive preoccupations in the first part of op10 were not greatly changed from the previous observation period. however, complaints about speech difficulties increased in the last three months of this period, leading to a consult with a clinical neuropsychologist. the consult failed to identify speech problems but did identify memory deficits, which are discussed below in the section on cognition. this observation period again revealed the slow nature of the off - glycine relapse that was seen in op6, the previous lengthy off - glycine period. bdd exhibited a more unequivocal relapse that clearly represented increased impairment, but the relapse was far from the preglycine baseline. formal cognitive testing toward the end of this period revealed a much more dramatic relapse of the cognitive dimension that is likely to be close to preglycine baseline (see below). thirteen days after ending a glycine treatment period of 138 days (op5), o demonstrated rapid learning and retention of new math concepts in a tutoring session for a math placement exam given at the time of college enrollment. in this session with his father, his work was largely free of the types of errors seen by his father at 14 and also in formal cognitive testing at age 7. this parental impression was supported by higher than expected performance on the placement exam. during the ensuing algebra course, after an additional 2126 weeks of glycine cessation, retention difficulties and the characteristic pattern of errors reappeared. these informal observations suggested the possibility of a cognitive benefit from glycine treatment that slowly declined after treatment was stopped. the possibility of a cognitive benefit from glycine treatment was much more strongly suggested by third - party objective data derived from formal cognitive testing done at the end of op10. toward the end of op10, o was evaluated by a neuropsychologist who was blinded to psychiatric and treatment histories during testing and the preparation of a preliminary report. the evaluation indicated that fine motor skills and speed were within normal limits as were simple attention, mental manipulation, and sequencing. however, with more complex tasks requiring manipulation of nonsequential items, self - monitoring, response inhibition, and set shifting, performance declined considerably. visual memory was also severely defective, especially when learning material that lacked structure or associative quality. verbal memory was described as impaired when dealing with material that was unstructured or unrelated. oral expression received the highest score (94th percentile). in this analysis of the clinical neuropsychological data, we focus on test results that are relevant to memory function. of special interest are results from the wechsler memory scale - iii (wms - iii) and the california verbal learning test - ii (cvlt - ii). the wms - iii auditory immediate and auditory delayed subtest scores were in the ~1st percentile. memory deficits were also revealed by the cvlt - ii, where all but two z - scores ranged from ~1 to ~4. in our view, these substantial off - glycine deficits are highly unlikely to have been present in the on - glycine periods when o obtained a verbal sat score in the 90th percentile and a 99th percentile score in the reading and social studies subtests of the ged. here we emphasize that both the ged and the sat scores were obtained in the standard time periods. these test scores also seem to be in marked contrast with the psychologist 's impression that attention and executive deficits at the time of testing (off - glycine for 17 months) exerted considerable impact on tasks requiring complex mental manipulation, self - monitoring, multitasking, and working memory. further support for a cognitive improvement from glycine treatment can be found in evidence that the off - glycine deficits seen in op10 represent a decline from a prior time. such evidence derives from the wechsler test of adult reading (wtar) in which word reading scores (thought to be resistant to brain injury or other causes of cognitive decline) are used to predict premorbid iq scores. clear evidence for achievement of adult reading levels can be seen in o 's 99th percentile score on the reading and social studies subtests of the ged (age 26) and his 90th percentile score on the verbal part of the sat (age 27). wtar results (at age 30) obtained 17 months after glycine cessation indicate that o 's actual verbal standard score, full - scale standard score, perceptual organization index score for the wechsler adult intelligence scale - iii were 1417 points below wtar predictions (cumulative percentage = 59%). a more dramatic difference of ~30 points between predicted and actual was seen for the working memory index scores (cumulative percentage = 1%). here it should be noted that working memory is central to cognitive functioning and is strongly correlated with reasoning ability. moreover, working memory is currently considered to be a key determiner of fluid intelligence [51, 52 ]. given the central importance of working memory and o 's high verbal score on the sat (reasoning test of the college board) at age 27 in op5 (137-days on glycine) and the absence of any infection - triggered or other obvious exacerbations since that time, we suggest that the wtar data are most consistent with a cognitive decline as a result of glycine cessation in the 17-month period that preceded the test. his recorded notes indicate that o reported no obsessions or mental rituals or any other discomforts or distractions in either testing session. the clinical psychologist 's report noted that o was euthymic and appeared to make a full effort and that test results for depression, anxiety, and adhd indicated that these factors did not impact test results. ingested glycine appears to enter circulation rapidly, generating a sharp rise in plasma glycine concentration. in o 's case, multiple measurements at approximately two hours after completion of consumption have yielded values ranging from ~2600 to ~3300 m, with an average of ~2800. the time at which peak concentration is reached is unknown, but may be less than 2 hours after ingestion and may depend on the rate at which glycine is consumed and on the presence of other food in the stomach. on one occasion, a plasma value obtained at 3 hours after glycine ingestion (~1350 m) was substantially less than values obtained at 2 hours (~20003000 m) (normal reference 151490 m). on another occasion, 24 hours after terminating an extended period of glycine consumption, plasma glycine was ~280 m, a base - line value in the reference range (151490 m). this observation is consistent with studies of healthy volunteers by hahn and sandfeldt, who found the half - life of excess plasma glycine to range from 40 to 100 minutes in many individuals to 6 hours in one individual. hahn and sandfeldt also found that glycine infusion in amounts comparable to those used in this study induced little or no increase of plasma ammonia in most individuals. however, about 1015% of healthy volunteers developed plasma ammonia levels in the 100 m range after i.v. for example, in a typical measurement, plasma ammonia about 2 hours after glycine ingestion was ~120 m (reference : 47 m). orotic acid in urine at this time was ~15 mmol / mol / creatinine (reference : 0.41.2). several physicians, including specialists on hepatic encephalopathy and urea cycle disorders, have suggested that the mild hyperammonemia seen in o is unlikely to cause harm. nonetheless, hyperammonemia represents an issue that, to our knowledge, has not been studied in prior trials. as noted previously, op1 was terminated because of a fever of 104.2f during an upper respiratory infection. at that time, o and his parents could not recall a fever this high in previous infections and worried that glycine might cause elevated temperatures. however, no high fevers have been experienced in the subsequent four years, during which glycine was taken for 850 days. in this period, the only precaution has been to terminate glycine at the first sign of an infection. although there is no clear evidence for glycine inducing higher - than - normal fevers, we include these findings for future reference, since there is literature suggesting a role of nmdar - st in the fever response to bacterial toxins. mild weight loss with no obvious cause led to the termination of op9. on the basis of sporadic measurements, weight declined from 184 pounds to 165 pounds over a period of 22 weeks. weight then increased to 172 pounds over a period of 8 weeks. over the next 6 months, as noted in the section for op10, there were no signs of anorexia nervosa or other eating disorders. at this point there is no clear evidence for glycine as the cause of weight loss. in summary, available evidence suggests that high - dose glycine treatment used for a total of 947 days in a five - year observation period has not produced any detectable, serious adverse affects in an individual closely monitored by his physicians. this observation is in agreement with the apparent absence of serious adverse effects in the 20 years of glycine trials with schizophrenia. there is substantial third - party objective evidence that o experienced unrelenting illness that generally increased in the 10 years that preceded glycine treatment and that o 's education and social life were profoundly disrupted. evidence for disruption of education resides in a comprehensive set of third - party records on school attendance and nonattendance, academic performance during attendance, results from standardized academic tests, financial records from academic institutions, and written correspondence from academic administrators and city truancy officials. for 8 years before glycine treatment, there was no attendance of school or commercial tutoring. resumption of education following the initiation of glycine treatment is likewise supported by comprehensive third - party objective evidence. over a period of approximately 2 years, evidence for sustained and increasing improvement resides in the fact that o passed the high school equivalency exam, prepared for the sat test in a commercial course, preformed well on sat test and began college. evidence for a resumption of social life following glycine treatment is likewise supported by substantial third - party objective evidence. for example, evidence for a resumption of social life resides in email correspondence with classmates that relates to social activities at specific events and locations in his city and another city. credit card records indicate the purchase of clothes, attendance at restaurants and concerts in his city and another city. thus, there is clear, third - party objective evidence for resumption of an active social life and normal mobility in his city that is not subject to concerns about subject, parental, or researcher bias relating to outcome assessment. this evidence for a major reduction in preglycine life disruptions suggests a major improvement in the ocd and bdd dimensions of o 's illness. however, before this major improvement can be considered a genuine effect of glycine, it is necessary to rule out placebo effects and a spontaneous remission. given that placebo effects derive from patient anticipations for treatment, there is the a priori expectation that placebo effects will not be durable in individuals experiencing powerful, life - disruptive psychiatric illness. this expectation has been confirmed by quitkin and coworkers in systematic studies of placebo effects in depression [5761 ]. specifically, there is a marked tendency for placebo responses to occur abruptly within the first two weeks and to disappear within the first 12 weeks of treatment. this placebo response pattern is in contrast to what the authors regard as the response pattern for a true pharmacologic response, where the initial effects appear after a period of two weeks and tend to last 12 weeks or longer. the placebo response pattern can, of course, be seen either with a true placebo or with an active drug. these authors conclude that a significant portion of relapses within the first 6 weeks of treatment with an active drug are not related to loss of a true drug effect. rather some are related to loss of nonspecific placebo effects, and abrupt nonpersistent responses during drug treatment are most likely the result of placebo effects. they note that the early occurrence of placebo effects is consistent with the notion that they derive from patient expectations. others have made similar observations. in relation to studies of ocd patients, ackerman and greenland have noted that the observation of less improvement in longer trials of active drugs may be due to placebo effects from expectations generated by side effects : it is possible that in the early 10-week clomipramine and 8-week fluvoxamine trials, some of the subjects in the active treatment arms responded to nonspecific study effects (such as side effects). in longer studies,. suggest that precise assessment of drug effects probably requires several months of observation. in view of the above considerations, we suggest that it is highly unlikely that the objective, major, and long - lasting changes in life activities that followed glycine treatment represent a placebo effect. unlike placebo effects, the change that followed initiation of glycine treatment was not abrupt. the subject and his parents report that the first sign of improvement (leaving home without parental escort for the first time in 5 years) occurred 5 weeks after initiation of glycine consumption. further improvement was also gradual, with third - party objective evidence documenting a gradual resumption of education and social life over treatment periods totaling 150 weeks (in an observation period of 5.3 years). described by quitkin. as sufficient to detect true drug effects. although the above studies clearly suggest that effects generated by patient expectations tend to be transient, it is nonetheless possible to observe some cases in which improvement from a known placebo appears to be enduring. for example, quitkin observed sustained improvement in placebo - treated individuals with depression. in these cases, the authors make the reasonable suggestion that the improvement was more likely to have been a spontaneous remission than a placebo effect. therefore, it is necessary to consider the possibility that o 's enduring improvement following initiation of glycine treatment was due to spontaneous evolution of illness. we first note that a spontaneous remission appears unlikely on the basis of prior longitudinal studies, which suggest that both ocd and bdd generally have a continuous course, with spontaneous remissions being rare, especially in ssri - refractory cases [6469 ]. moreover, the occurrence of both disorders together tends to be associated with a worse prognosis. although suggestive, these considerations do not constitute definitive evidence against a spontaneous remission in a specific case. one approach to distinguish true efficacy from spontaneous remission in a specific case is to determine if deteriorations occur during periods of nontreatment. to pursue this approach, this study was continued until we obtained objective, third - party evidence for an unequivocal deterioration during nontreatment that avoided concerns about subject expectations and outcome assessment bias in the observations made by the subject, his parents and researchers. as noted for the long off - glycine periods, op6 and op10, modest relapses were seen for ocd and bdd. however, the clearest evidence of deterioration was for the cognitive dimension of o 's illness. most notably, third - party objective evidence derived from clinical neuropsychological testing in op10 suggests substantial deficits in selective aspects of cognition by the end of this period. this testing with formal methods was done by a neuropsychologist who was blinded to psychiatric and treatment histories. a comparison of this off - glycine data with the excellent sat scores obtained in the on - glycine period, op5, suggests a substantial decline in selective aspects of cognition in op10 relative to op5 levels, as discussed in section 3.5.11. all of this evidence is objective except for some neuropsychological tests that required subjective evaluation by the clinical neuropsychologist, such as the rey - osterrieth test. in these cases, any subjective judgments were made under blinded conditions that precluded expectations of the neuropsychologist that were based on treatment history or psychiatric diagnoses. the possibility that treatment cessation could have generated expectations in our subject that somehow affected his performance on the neuropsychological testing can also be considered. we first note that the subject reports that he initiated testing because of speech difficulties. the point here is that the deficits found were different from the subject 's initially expressed complaints. further evidence against expectations for a decline in memory after glycine cessation reside in the fact that in serial interviews, the subject has expressed doubts about the validity of the tests for memory deficits and has noted that friends have commented on his excellent memory of the distant past. moreover, he has not resumed high - dose glycine consumption in response to the test results. it can also be noted that the neuropsychologist reported that the subject was euthymic and appeared to make a full effort. on the basis of the above considerations, we suggest that the third - party evidence that we present for a cognitive deterioration is either fully objective evidence or is evidence that is not compromised by an absence of blinds or by subject expectations. an important point is that the selective cognitive deterioration was substantial, since some wms - iii scores were in the first percentile and since the rey - osterrieth copy score was more than 8 standard deviations below the mean. we suggest that it is unlikely that scores on these tests would have been much lower had they been measured before the initiation of glycine treatment. in summary, we suggest that the bias - free evidence that we present for a major improvement of ocd and bdd during treatment and for a major deterioration of selective aspects of cognition during nontreatment is consistent with the possible efficacy of glycine. in response to the success achieved in the first year of this study, the first aspect of this initiative has been a placebo - controlled study in which glycine was used as an adjunct to pre - existing pharmacotherapy. the 5 individuals who were treated with glycine had a mean decrease in y - bocs score of 6.04 points where as the 9 subjects treated with placebo experienced a 1.00 point decrease (p =.053) (greenberg, benedict, doerfer, perrin, panek, cleveland, and javitt, adjunctive glycine in the treatment of obsessive - compulsive disorder in adults [71, 72 ]). although much larger studies are needed to determine the frequency of responders, the results of greenberg. are in agreement with this case study and raise the expectation that additional ocd patients will be glycine responders. to facilitate large trials the comprehensive case description in this paper has been motivated, in part, by this goal. to our knowledge, this case study represents the first use of high - dose glycine with ocd or bdd and the first use of glycine alone with any psychiatric disorder. in addition, there was no psychotherapy or behavior therapy during the 5-year period of monitoring. thus, this study is free of factors that confound the interpretations of many studies. given the magnitude of the improvement seen in this study, we suggest that trials of glycine alone be considered for future studies. in agreement with prior studies of glycine as an adjunct to conventional medication in schizophrenia, an initial response to glycine treatment was detected in this study only 23 days after reaching the target dose. however, the improvement reported for the first 12 weeks of treatment was only a small fraction of the improvement reported for the five on - glycine treatment periods that extended over a period of almost four years. this suggests that the short clinical trials used in the past, for example, 12 weeks, may have failed to reveal the full therapeutic potential of high - dose glycine treatment in the disorders studied. the results of this high - dose glycine trial with refractory ocd, bdd, and cognition deficits are in unequivocal agreement with the main prediction of the hypo - nmdar - st hypothesis that inspired its initiation. however, it is also necessary that this hypothesis be compatible with prior, well - established findings for nmdar neurotransmission. since our hypothesis was constructed to explain the apparent behavioral effects of clarithromycin, which has been reported to inhibit nmdar - st at a downstream point, it is reasonable to consider if other downstream nmdar - st inhibitors have similar effects. there are many studies of nmdar inhibition on human behavior, but, to our knowledge, all employ global inhibitors, such as phencyclidine and ketamine, that act on the nmdar itself. it is nonetheless of interest to consider known effects of ketamine and phencyclidine in relation to this study. to our knowledge, controlled studies with phencyclidine and ketamine have considered either healthy subjects or subjects with schizophrenia [38, 74 ]. in healthy subjects, the basic findings are deficits of cognition and other signs and symptoms that are schizophrenia - like [38, 74 ]. in subjects with schizophrenia, a worsening of existing or reactivation of previously controlled symptoms is the main presentation [38, 74 ]. in these very short - term experiments, signs and symptoms of ocd, to our knowledge, have not been reported. although visual distortions of body parts are a prominent consequence of ketamine and phencyclidine [38, 74 ] and somatosensory abnormalities have been suggested to be an aspect of bdd deriving from parietal - occipital abnormalities [75, 76 ], full - fledged dsm - iv bdd has not, to our knowledge, been reported in these experiments. at first sight however, general considerations suggest that local defects in nmdar - st could be specific for ocd / bdd. the absence of such defects in normal individuals and in individuals with schizophrenia would explain the absence of ocd / bdd in the reported ketamine and phencyclidine experiments. here it can be noted that disorder - specific defects appear to have influenced the exacerbations induced by clarithromycin in other cases. for example, in one case, previously controlled bipolar disorder was reactivated. in another, previously controlled posttraumatic stress disorder was reactivated. it can also be noted that disorder - specific defects for ocd and bdd are plausible in relation to known neurobiology. given that ssris are the first line drugs for both ocd and bdd, it is possible that abnormal serotonin neurotransmission plays a causal role in these disorders in some individuals. this raises the possibility of a local, ocd / bdd - specific defect that is restricted to serotonergic neurons of the raphe nuclei. recent evidence indicates that nmdar - st regulates the firing rate of these neurons, suggesting that deficient nmdar - st could lead to an abnormal firing rate. the more downstream the defect the more likely it will involve the molecular systems that produce, release, and transport serotonin. recent clarifications of mechanisms regulating serotonergic neurotransmission [8084 ] reveal a complex picture in which multiple processes couple nmdar input with serotonin release / reuptake. defects occurring in these processes could represent ocd / bdd - specific defects in nmdar - st. another possibility is a deficiency of nmdar - st in gaba - ergic inhibitory interneurons (abundant in both the dorsal and median raphe nuclei) that regulate serotonergic neurons. this scenario can be seen as a variant of the elegant and influential theory of olney. models, is that glutamate functions not only as a straightforward excitatory agent in the brain, but as a major regulator of inhibitory tone. local nmdar - st deficits restricted to interneurons that regulate serotonergic neurons could reasonably be expected to lead to abnormal serotonergic tone and the emergence of ocd / bdd. in both of the above scenarios, there is an absence of the more widespread perturbations expected for global nmdar - st inhibition by ketamine and phencyclidine. the absence of these perturbations would be compatible with the emergence of ocd / bdd without the schizophrenia - like symptoms seen with ketamine and phencyclidine. also, in both scenarios, it would be reasonable to expect worsening of signs and symptoms by an inhibitor and improvement from an enhancer of nmdar - st. however, it should be emphasized that the above analysis is intended only to suggest plausibility of disorder - specific defects, not to propose a specific mechanism for the case under study. although ocd and bdd have not been seen in ketamine experiments with humans, some of the cognitive deficits seen in o show substantial overlap with the cognitive effects of ketamine. a consistent finding in multiple ketamine studies is a robust, dose - dependent decrease in verbal declarative memory that occurs at doses below those that induce schizophrenia - like symptoms [38, 86 ]. deficits in verbal memory and learning in ketamine experiments have been assessed with story recall and list learning tests. for example, newcomer. found a dose - dependent decrease in verbal memory under ketamine using a paragraph recall test from the wechsler memory scale - revised. immediate and delayed recall scores from this study can be compared with o 's corresponding scores on a revised version of this test, the wms - iii. in other ketamine studies [8890 ], a robust decline in verbal memory has been found with list learning tests such as the hopkins verbal learning test (hvlt). these results can be compared with the significant deficits seen in o 's scores on the cvlt - ii, where the t score for trials 15 was ~36 (z = 1.4) and z - scores for delayed recalls (free and cued) ranged from ~2 to ~2.5, except for the short delay free recall, which was ~1. thus, o 's off - glycine deficits in verbal memory are strongly reminiscent of those induced by ketamine. are o 's very high on - glycine scores on the ged and verbal sat tests (99th and 90th percentiles, resp.), which suggest much improved verbal learning and memory in an on - glycine state, as discussed above. the finding that ketamine - induced cognition deficits appear at doses lower than those needed for other types of symptoms [38, 86, 9294 ] is also of relevance to our hypothesis. assuming that an endogenous nmdar - st defect would mimic ketamine, this finding leads us to expect that the return to a deficient nmdar - st state after cessation of glycine would manifest itself with a cognitive relapse whose emergence is earlier and more pronounced than the relapses for other categories of signs and symptoms. our data from ops 6 and 10 appear to be consistent with this prediction. also of relevance is the fact that in the initial emergence of illness in early childhood, difficulties in cognition appeared before obsessive - compulsive behaviors and bdd - related signs and symptoms. finally, we note that additional support for our hypo - nmdar - st hypothesis can be found in the course of o 's illness. specifically, it is known that nmdar inhibition has mild behavioral effects in childhood and much larger effects after puberty [95100 ]. assuming that an endogenous nmdar - st defect would imitate an inhibitor, it follows that o 's very mild illness in childhood and the emergence of much more incapacitating illness after age 15 are consistent with an nmdar - st deficit. although the findings of this study support the hypo - nmdar - st hypothesis on which it is based, it is appropriate to consider our findings in the light of alternative hypotheses proposed by others. one of the defining features of the pandas subtype is the occurrence of exacerbations with sudden, abrupt onset. the exacerbations are described as typically quite dramatic, with patients reporting that their symptoms appeared all of a sudden a few days after i had a sore throat. the fine details of o 's exacerbations are not congruent with this picture. for o 's age-19 exacerbation that followed a positive throat culture, detailed notes of o 's father indicate an improved relationship with his son during the acute phase of the infection. the behavioral deterioration is described as beginning as soon as the infection cleared. worsening of existing symptoms (e.g., line - crossing obsession) occurred 24 days after the positive throat culture. episodes of violent imagery appeared for the first time approximately 40 days after the positive throat culture. in addition to the slower kinetics, o 's nonresponse to ivig therapy is unlike some pandas patients. although, o 's case can not be classified as a clear case of pandas, it is still possible that it involves an antigen - specific autoimmune response induced by gabhs. there are, of course, multiple precedents, both experimental and theoretical, for the induction of antigen - specific anti - self - immune responses by foreign antigens that cross - react with self - epitopes or interact with self - receptors [101103 ]. thus, the occurrence in o of two exacerbations following probable gabhs infections is immediately suggestive of an antigen - specific autoimmune mechanism. this follows from the major precedent residing in rheumatic fever (rf) as an antigen - specific autoimmune disease primed by gabhs. as expected for a pathogen - induced, antigen - specific autoimmune disease, recurrences of rf in adulthood are associated with probable reinfection with gabhs, often as a result of known, close contact with children having gabhs infections. the time interval between an initial occurrence of acute rf in childhood and a subsequent occurrence in adulthood can be multiple decades. during these long periods, there is an annual occurrence of upper respiratory viral infections, some of which are flu - like. as expected for infections which are unlikely to be antigenically related to gabhs, there is no retriggering of rf. in contrast to rf, exacerbations in o 's case and in some pandas cases appear to follow both gabhs and non - gabhs infections. it appears that sydenham 's chorea, a sequela of rf, can reoccur without gabh infection. one might propose that, in these cases, antigen - specific clones primed by an initial gabhs infection are somehow retriggered by other pathogens, such as viruses. however, the failure to see this phenomenon in rf represents a major challenge to this proposal. one possibility is that the putative brain injury responsible for o 's case arises from nonantigen - specific consequences of gabhs and other infections. such a mechanism would be compatible with recurrence from both gabhs and non - gabhs infections and would not be incompatible with the peripheral antibrain antibodies reported in the recent literature [107111 ], since the latter could be a result (rather than a primary cause) of brain injury due to the release of antigens from an immunologically protected site. here it is of interest that husby. who in 1976 found antistriatal antibodies in patients with sydenham 's chorea, also found similar titers of antistriatal antibodies (using identical techniques) in patients with huntington 's chorea. this led in 1977 to the suggestion that huntington 's chorea could be an infection - triggered autoimmune disease. the latter is now known to be a polyglutamine disease that involves preferential death of striatal neurons. in brain - injury models of this type, it is formally possible that antibrain antibodies arising from brain injury could play a secondary role in pathogenesis. however, the demonstration that such antibodies play a causal role is a difficult task that is technically inaccessible in the absence of validated animal models [116, 117 ], which are unavailable for psychiatric disorders that involve high - level human thinking. the four spect scans done over five years provide evidence for stable and sustained hypoperfusion (figure 1, appendix a.3). one possibility is that it reflects a primary vascular defect of long standing that creates a susceptibility to psychiatric illness. in this context, one is reminded of the interesting proposal by hanson and gottesman that vasculature - inflammatory defects are a root cause of psychiatric illness in subsets of patients with schizophrenia and other psychiatric illnesses, including those associated with gabhs infections. they postulate a chronic smoldering inflammation of the vasculature that is episodically triggered to cause brain injuries leading to psychiatric illness. o 's abnormal spect scans (with a differential diagnosis that includes vasculitis) and his exacerbations following infections suggest consideration of the theory of hanson and gottesman in future studies. in the literature on ocd, spect scans most commonly show hyperperfusion in frontostriatal areas, but hypoperfusion is prominent in a significant fraction of patients [119, 120 ]. to our knowledge, in cases where hypoperfusion was seen, heterogeneity was not noted. in a rare spect study of bdd, carey. reported that both regional hypoperfusion and hyperperfusion could be seen in six subjects with bdd. of special interest are 2 young adult male bdd patients with no other current comorbidity who showed heterogeneous hypoperfusion in the occipital and parietal lobes. as seen in figure 1 (appendix a.3), heterogeneous hypoperfusion is a distinctive feature of o 's spect scans, suggesting that his scans most closely resemble the bdd scans. the detection of brain glycine by single voxel mrs was initially accomplished only in individuals with genetic or other defects that lead to unusually high levels of glycine [121, 122 ]. our preliminary results in 2004 with a 3 tesla machine (figure 2, appendix a.4) raise the possibility that high - dose glycine treatment causes sufficient elevation of forebrain glycine to permit detection using standard procedures with optimal echo times. future attempts to confirm this finding will benefit from recent improvements in techniques and the emergence of 7 tesla scanners [123125 ]. the opportunity to monitor glycine concentrations in specific brain regions will permit the exploratory studies needed to identify the oral dose required to get the desired concentrations in relevant brain regions. studies in rodents suggest that in much of the forebrain, endogenous glycine concentration is substantially lower than that in the hindbrain and spinal cord, making the ability of mrs to measure glycine concentrations in small voxels an important advantage. as noted previously, high - dose glycine treatment may lead in some individuals to mildly elevated plasma ammonia. this raises the possibility that optimal glycine therapy in individuals with elevated ammonia would require a coadministered agent to control ammonia. there is also the possibility that excess levels of glycine in the brain may lead to increased in situ production of ammonia. these issues can likely be addressed with the new high - field scanners [128130 ]. trials of glycine with schizophrenia have prompted a search by pharmaceutical companies for drugs that enhance nmdar neurotransmission. while this study suggests that these drugs may also be useful with ocd and bdd, it is likely that a significant period of time may elapse before they become available. we therefore emphasize that glycine is available here and now and at very low cost. this point is of great significance to patients, who need immediate relief from what is often severe and unrelenting illness. with these considerations in mind, we suggest that the findings of this study motivate further exploration of the efficacy and safety of glycine as a treatment of ocd and bdd as well as further study of the hypo - nmdar - st hypothesis that motivated this treatment. obsessionsin the preglycine period, o and his parents report that obsessive mentation was continuously present from the time of eruption of frank illness at age 15. the most impairing obsession was a line - crossing obsession, which would be triggered (or intensified) when o would see a person in his neighborhood whom he considered to be likely to commit a violent, personal crime. reports of crime in the news media also activate the obsession, provided that they occur in a neighborhood are of no concern, since, in the latter case, there is evidently no line - crossing. it is also a reactive obsession in the classification framework of lee and kwon, in which obsessions responsive to perceived environmental stimuli are called reactive. by not leaving his home o was able to reduce distress by avoiding exposure to environmental intensifiers, but this generated major impairment by disrupting education and social life. in the preglycine period, o and his parents report that obsessive mentation was continuously present from the time of eruption of frank illness at age 15. the most impairing obsession was a line - crossing obsession, which would be triggered (or intensified) when o would see a person in his neighborhood whom he considered to be likely to commit a violent, personal crime. reports of crime in the news media also activate the obsession, provided that they occur in a neighborhood are of no concern, since, in the latter case, there is evidently no line - crossing. it is also a reactive obsession in the classification framework of lee and kwon, in which obsessions responsive to perceived environmental stimuli are called reactive. by not leaving his home o was able to reduce distress by avoiding exposure to environmental intensifiers, but this generated major impairment by disrupting education and social life. violent imagery involved images of a knife cutting his father causing extreme fear and distress. as o 's father noted in a letter to a psychiatrist : the terrifying thoughts are truly agonizing and he begs for relief while holding my hand and literally trembling. it should be noted that two psychiatrists, who were consulted at this time, concluded that the violent imagery did not involve hallucinations. violent imagery with intense distress has occurred only during the three major exacerbations and thus represents a distinctive signature of such exacerbations. since these episodes are not triggered by perceived environmental stimuli. o 's episodes of violent imagery can be classified as autogenous obsessions in the classification framework of lee and kwon. it is of interest that o reports that episodes of violent imagery are not associated with anger, which is in contrast to reactive obsessions. ritualistic behaviorsaccording to o 's father, activation of the line - crossing obsession would invariably lead to a stereotypic conversation that was highly repetitive due to frequent but incomplete attempts to make a particular point. o 's father would provide repeated reassurances that o 's obsession - related fears were unfounded. talk - throughs were not restricted to the line - crossing obsession, but could concern any ocd - related obsession or the bdd - related excessive preoccupations (see below). o 's father emphasizes the distinct, qualitative difference between a talk - through and a normal conversation that progresses in a linear fashion from one point to another. according to o 's father, activation of the line - crossing obsession would invariably lead to a stereotypic conversation that was highly repetitive due to frequent but incomplete attempts to make a particular point. o 's father would provide repeated reassurances that o 's obsession - related fears were unfounded. o 's father refers to this type of conversation as a talk - through. talk - throughs were not restricted to the line - crossing obsession, but could concern any ocd - related obsession or the bdd - related excessive preoccupations (see below). o 's father emphasizes the distinct, qualitative difference between a talk - through and a normal conversation that progresses in a linear fashion from one point to another. although talk - throughs have features of reassurance rituals, they are more complex as they frequently involved an outburst of verbal anger. o 's father notes that when talk - throughs were avoided for several days there would be a stronger outburst of anger when they were resumed. after about 1 hour of the repetitive conversation and after an outburst of anger, the psychic distress, including anger, would usually be dissipated until they returned the next day. talk - throughs were a daily feature in much of the six year period that preceded high - dose glycine treatment. at times of peak illness, they consumed multiple hours per day. other ritualistic behaviors are reported to have included prayer, wall - touching, jumping into bed, and turning lights on and off and were done in response to fear of harm. o indicates that fear of harm could either be for himself or for his parents, especially his father. just right. in some cases, rituals had to be done a large number of times or according to a numerical pattern (in groups of 30 in one case). prior to the age-22 exacerbation that followed antibiotic treatment for h. pylori, there were a number of just right rituals but they were minor in relation to the reassurance rituals associated with the line - crossing obsession. following the antibiotic treatment for h. pylori, o 's father 's diary indicates a very large increase, over a period of about two months, in a prayer ritual in which o would suddenly drop to his knees. this ritual was frequently performed throughout an entire day, leading to knee calluses. in periods when o 's bdd signs and symptoms were at their peak, o 's father reports that o would spend most of a day working on the mirror, an activity that would involve repeatedly checking his appearance in a mirror in order to get a good impression of his face. rare good impressions were always followed by numerous disturbing impressions that were often associated with verbal anger. o 's parents report that this activity clearly appeared to intensify distress and to increase o 's preoccupation with his presumed facial defects. specifically, there was the fear / belief that they were ugly and that he looked like them. bdd by proxy first emerged at age 19, several months after the first infection - associated exacerbation and led to a general avoidance of his mother and restricted viewing of his father. in periods when o was not spending multiple hours per day likewise there was avoidance of social events as a result of a fear that someone would say that he was ugly or looked like his mother or father. all of this reinforced o 's tendency to be housebound, adding to the similar effects of the line - crossing obsession. being housebound reduced distress but led to major impairment by disrupting education and social life. somatic complaints associated with dysphagia, muscular discomforts and discomforts at joints and the vasculature (cold hands, hot hands, palpitations) are another distinctive aspect of o 's behavior. the sites of discomfort are reported to have been stable over time, although complaints at a particular time usually involve only a subset of sites. the possibility that o 's somatic complaints represent hypochondriasis was reviewed by the ocd specialist consulted at age 19. o 's father 's notes and the chart for these consults indicate that hypochondriasis was not found. thus, o 's somatic concerns are not presented as improperly held beliefs or fears about specific illnesses ; rather, they are presented as complaints about discomforts at body sites with claims that they impede his functioning. thus, o 's somatic complaints are different from the somatic obsessions often reported for ocd, which are typically concerned with fears of illness rather than discomforts at body sites. in the recent interviews, o has reported that discomfort in a particular joint increases gradually until it reaches the point where he feels a need to pop the joint to eliminate the discomfort. it appears that the action of joint popping does not in itself generate impairment, since it is done only a few times per day. moreover, it has not been possible to identify any tic - like behaviors for o 's other somatic preoccupations. one possible explanation of o 's somatic preoccupations is that they reflect, at least in part, an intrinsic hypersensitivity to body sensations. this has been described for individuals with tourette syndrome, which, historically, has been known to occur often with ocd and which may therefore share causal factors. the joint discomfort and its relief by patients with ts are remarkably sensitive to and are easily captured by changes in the sensory world although, o has not received a diagnosis of ts and does not appear to cross the threshold for a tic disorder, it is possible that ts and ocd as it occurs in o share causal factors, one or more of which generate the sensory hypersensitivity. this possibility is supported by a more recent characterization of sensory phenomena in ocd, ocd+ts, and ts. it should be emphasized that in the preglycine period, somatic preoccupations were seen by o 's parents to be almost negligible in relation to major ocd and bdd presentations. it was during op6 (the first lengthy off - glycine period) that these preoccupations were first seen to cause a mild but clear impairment. in addition to somatic complaints, o has frequently complained since age 20 about difficulties in speech production, specifically an inability to find words and physical difficulties in speech production. at the end of observation period 10 (see section 3.5.11), o initiated an examination by a speech pathologist, which revealed no speech deficits but did reveal a memory deficit. in a subsequent neuropsychological evaluation, superior range and represented the highest of all scores. on this basis, we tentatively refer to o 's speech complaints as cognitive preoccupations. however, it is possible that the speech - related complaints are an inaccurate description of genuine cognitive deficits that were revealed in the neuropsychological evaluation (see section 3.5.11). in our view additional documentary material on methods and behavioral phenomena is at www.informaticpsychiatry.org/glycine. except for his psychiatric disorder, o has been in good general health all of his life. during pregnancy, ultrasound - guided amniocentesis apgar scores were 9, 9, and 9 at 1, 5, and 10 minutes, respectively. raynaud 's disease was first noticed at age 21 just after the end of a taper of paroxetine from 70 mg / day to 0 mg / day. it has been witnessed on several occasions in clinical exams and continues to the present day. at age 26, orthopedic and genetic consultations both revealed a mild connective tissue disorder (megacephaly, arched palate, striae distensae, lax joints, and pectus excavatum) that could not be identified with known disorders. three echocardiograms, done at ages 21, 25, and 29, were described as normal except for a trivial aortal insufficiency that was noted for the first two but not the last, which was done four years after initiation of glycine treatment. other mild conditions are recurrent otitis externa and recurrent oral stomatitis that was noted as early as age 7, when parents recall a prescription for lidocaine. in general, neurological exams have been unremarkable except for the confirmation of psychiatric diagnoses. however, at age 15, an initial diagnosis of temporal lobe epilepsy was discounted after discovering an electrode artifact in the eeg. trigeminal neuralgia, a diagnosis that was described as unusual for a 15-year old, was suggested as a possible alternative. subsequent recurrences of these symptoms, if any, appear to have been very minimal. also, at this time, a neuroopthalmologist found a convergence insufficiency that gradually normalized without treatment, as found in a later examination at age 18 by the same neuroopthalmologist. objective evidence for a cns abnormality resides in four spect scans (technetium-99m - hexamethyl propylamine oxime (tc-99m - hmpao)) done at ages 22, 23, 25, and 27. approximately 20 mci of tc-99m - hmpao were injected into the patient while eyes were open in a quiet, dimly lighted room. approximately one hour after tracer injection, scans were done with a three - headed camera. images were reconstructed in the transaxial, coronal, and sagital planes. according to the report for the age-22 scan, marked heterogeneous and decreased cortical uptake the report also indicates that the observed pattern is often secondary to vasculitis or encephalopathy of an infectious or toxic nature, for example, shearing injuries, cocaine abuse or lyme disease. o was tested three times for lyme disease with negative results and is reported to have never experienced a shearing injury. systemic lupus erythematosus (sle) is another illness that can be associated with heterogeneous brain spect scans. a representative scan is shown in figure 1. a semiquantitative analysis of the age-22 spect scan using the procedure of mountz. the right medial frontal region showed the lowest level of perfusion, which was 26.5% below the average of the cerebellar values. as indicated, the frontal, parietal, right posterior temporal and occipital areas are down more than 12%, whereas the basal ganglia, thalamus, anterior temporal, and left posterior temporal are not. a second scan was done at age 23 and a third at age 25 just before the initiation of high - dose glycine treatment. the fourth scan was done three days after stopping a 138-day glycine treatment period. at the level of clinical evaluation, these three scans are equivalent to each other and to the prior scan. in summary, the spect scans show substantial abnormality of unknown cause that is stable over a five year period. however, it should be noted that 3 days after stopping glycine, the brain glycine level may have returned to normal. further study is required to determine if elevated brain glycine affects regional cerebral blood flow. magnetic resonance imaging (mri) studies were obtained at ages 15, 20, 25, and 27 and proton spectroscopy (mrs) studies were obtained at ages 25 and 27. the mri studies at ages 15, 20, 25, and 27 showed an ovoid focus of increased proton density and t2 signal in the right periatrial white matter. the long axis of this lesion is perpendicular to the ventricle and measures less than 1 centimeter. in the age-25 scan mr evaluation was performed with and without contrast using the following sequences : t1 sagittal, t1, t2, proton density, flair and diffusion weighted axial images. following contrast administration, sagittal, coronal, and postcontrast magnetization transfer axial sequences were also obtained. precontrast magnetization transfer axial sequence was also performed as a control for contrast enhancement on the postcontrast magnetization transfer axial sequences. the focus of high t2 signal was found to suppress on flair sequence and showed no signs of marginal gliosis to suggest prior ischemia, inflammation, demyelination or infection. single voxel magnetic resonance spectroscopy (mrs) was done on the ovoid lesion and on a corresponding region on the left side. values of n - acetylaspartic acid, choline, and creatine were within normal limits on both sides. taking all of the evidence together, it is most likely that the t2 hyperintensities are prominent virchow - robin spaces. significant changes are not seen in the 12 year period bracketed by the scans. the age-25 mri / mrs study demonstrated normal blood volume throughout both cerebral hemispheres and the posterior fossa. on the diffusion - weighted sequence, other brain scans include a transcranial doppler sonogram at age 25 (day 85-op1(85)), which was unremarkable. a magnetic resonance angiography scan with and without contrast was done at age 26 (day 19-op2(222). an mri / mrs study was done during a period of high - dose glycine consumption (day 50-op7(818)). on this occasion, 25 grams of glycine supplemented with 3 grams of arginine were taken over a period of one hour. three hours after consumption of glycine + arginine, blood was drawn for measurement of plasma glycine (~1350 m) and plasma ammonia (~30 m). approximately 1.25 hours later, the proton magnetic resonance spectrum (3 tesla, press (point - resolved echo single - shot techninque), te (echo time) = 35 msec) of figure 2 was obtained. the voxel was located in the right medial frontal region, the region of greatest hypoperfusion on the spect scan of figure 1. of special interest is a shoulder of signal at 3.55 ppm that is adjacent to the major myo - inositol peak at 3.61 ppm. this shoulder is expected to reflect contributions from the glycine resonance at 3.55 ppm and the minor myo - inositol resonance at 3.52 ppm. in prior studies with 1.52.0 tesla scanners and short echo times, a clearly detectable 3.55 ppm shoulder has been seen only in conditions where there is a known excess of glycine (e.g., nonketotic hyperglycinemia) [121, 122 ]. more recent studies suggest that this is also the case with 3 tesla scanners. for example, in an mrs study of grey matter in a healthy individual (3 tesla, te = 40 msec), no shoulder at 3.55 ppm was seen. similarly, in a study of the medial frontal cortex in an individual with ocd (3 tesla, te = 30 msec), no shoulder at 3.55 ppm was seen. the absence of 3.55 ppm shoulders in these spectra of individuals not taking glycine and the presence of such a shoulder in o 's spectrum (3 tesla, te = 35 msec) are consistent with an elevated glycine level arising from the elevated plasma glycine that followed glycine consumption 4.25 hours before the scan. however, definitive detection of elevated glycine will require on - glycine and off - glycine measurements, and monitoring of the expected metabolism of elevated levels to baseline levels. o 's 3 tesla spectrum is of also of interest in relation to the prominent glx peak at 2.022.5 ppm. an enlarged glx peak is consistent with an increased contribution from the glutamine resonance at 2.45 ppm as a result of ammonia production from glycine metabolism in the brain. note that plasma ammonia is unlikely to be an issue in this instance, since its measured value ~1.25 hours before mrs was well within the normal reference range. a peak at 0.91.2 ppm is also seen and is likely to be of lipid origin. cleveland, delapaz, and fawwaz are contributors to a patent application filed by columbia university that is based on this study. | this paper describes an individual who was diagnosed with obsessive - compulsive disorder (ocd) and body dysmorphic disorder (bdd) at age 17 when education was discontinued. by age 19, he was housebound without social contacts except for parents. adequate trials of three selective serotonin reuptake inhibitors, two with atypical neuroleptics, were ineffective. major exacerbations following ear infections involving group a -hemolytic streptococcus at ages 19 and 20 led to intravenous immune globulin therapy, which was also ineffective. at age 22, another severe exacerbation followed antibiotic treatment for h. pylori. this led to a hypothesis that postulates deficient signal transduction by the n - methyl - d - aspartate receptor (nmdar). treatment with glycine, an nmdar coagonist, over 5 years led to robust reduction of ocd / bdd signs and symptoms except for partial relapses during treatment cessation. education and social life were resumed and evidence suggests improved cognition. our findings motivate further study of glycine treatment of ocd and bdd. |
the purpose of this study was to define the frequency of fasting and incidence of complications among libyan diabetic patients during ramadan. benghazi is the second largest city in libya with population of about 670,797 inhabitants and 14.1% prevalence rate of diabetes mellitus (dm) (6), with type-1 constituting about 6% of all cases. we interviewed and reviewed the records of 493 consecutive diabetic patients who attended the benghazi diabetes and endocrine center (bdec) during the first week after eid elfitr 1429 hijri (2008), and a standard form was filled by the authors. the following parameters were assessed : age, sex, type of diabetes, duration of diabetes, type and doses of treatment before and during ramadan, level of hba1c within three months before ramadan, number of non - fasted days, causes of breaking the fast, number and time of self - reported hypo- and hyperglycemic episodes, and any history of hospital admission during the preceding ramadan and the reason for it. hypoglycemia was defined as symptoms that were perceived by the patient as hypoglycemia and urged him / her to break fast with or without documented low blood glucose, and it was categorized as severe or mild. a severe hypoglycemic episode was defined as the episode that could not be treated by the patient alone, but required assistance from another person, whether a physician or not. mild hypoglycemia was defined as a hypoglycemic episode that was treated by the person with diabetes alone without need for assistance. severe hyperglycemia was defined as a self - reported blood glucose level 300 mg / dl. data were expressed as meanstandard deviation (sd) and differences between groups were tested using the chi - squared test and independent - samples t - test. data were expressed as meanstandard deviation (sd) and differences between groups were tested using the chi - squared test and independent - samples t - test. of the 493 patients, 95% were type-2 diabetics, 50.3% were males and 39% had their hba1c checked within three months before ramadan. mean age was 5911.7 years, mean duration of diabetes was 11.310 years, and mean hba1c (for those who had hba1c results) was 7.81.6. about 70.4% of patients completed 30 days of fasting and 97.5% fasted at least 15 days (table 1). the main reason of breaking the fast was hypoglycemia (43.4% of causes) followed by severe hyperglycemia (27%), while other causes constituted about 30% (fig. about 14.6% experienced mild hypoglycemia, 3.2% had severe hypoglycemia and 11.2% had severe hyperglycemia. there was no significant difference between type-1 and type-2 diabetics regarding number of days fasted, frequency of hypoglycemia or severe hyperglycemia, or admission rate during ramadan (table 2). among type-2 diabetic patients during ramadan fasting, 77.3% were on insulin therapy, 20.5% were on oral anti - diabetics, and 2.2% were on diet control alone. there was no significant difference between those who were treated with insulin or with oral anti - diabetic agents in the mean hba1c, mean number of fasted days, admission rate or incidence of hypoglycemia, or severe hyperglycemia (table 3). hba1c checked 3 m : percent of patients who had their hba1c checked within three months before ramadan. comparison between type-1 and type-2 diabetics comparison between type-2 patients according to the type of treatment the incidence of hypoglycemia during ramadan was 31 episodes/100 patients, while the incidence of severe hyperglycemia was 17 episodes/100 patients (table 4). about 74% of hypoglycemic episodes and 79% of hyperglycemic episodes occurred during the first two weeks of ramadan (fig. 2), and about 90% of all hypo- and hyperglycemic episodes occurred during the daytime. females had a significantly higher frequency of severe hypoglycemic episodes (4.9% vs. 1.6%, p=0.04) and a lower mean number of fasting days than males (27.85.9 vs. 29.242.6, p=0.001). patients who experienced severe hyperglycemia had a significantly higher baseline hba1c than other patients (8.41.1% vs. 7.61.6%, p=0.02). they also had a significantly lower mean number of fasting days (26.95.7% vs. 294, p=0.001). both type-1 and type-2 diabetic patients in benghazi managed to fast more frequently than diabetics in other muslim countries, with a remarkably lesser frequency of hypo- and hyperglycemia (table 5). it is possible that libyan diabetics are more motivated to fast than those in other muslim countries. more likely, however, is that about three - quarters of the libyan patients had some treatment adjustment during ramadan as compared to less than one - third of those in the epidiar study (1), which probably contributed to the lower rate of hypo- and hyperglycemia, and hence to a lower frequency of breaking the fasting. comparison between bdec study and epidiar study the most common complications that occurred during ramadan were hypoglycemia followed by hyperglycemia, and these were the main two reasons for breaking the fast, as reported elsewhere (1). however, both types of episodes were remarkably less frequent during the last two weeks as compared to the first two weeks, perhaps because patients adjust their dietary habits and schedules to suit their condition as the days go by. the definition of hypoglycemia used in this study depended mainly on patients ' perception of symptoms, which means that in principle episodes of hypoglycemia might go unnoticed. however, patients are probably unlikely to miss feeling a hypoglycemic episode because it would become exacerbated as the fasting continued during the day. on the other hand, we might have included some non - hypoglycemic episodes that were interpreted as hypoglycemia by the patients. therefore, we believe that the reported figure of the frequency of hypoglycemic episodes might be a slight overestimate rather than an underestimate of the frequency of these episodes. there was no significant difference between type-1 and type-2 diabetics regarding fasting rate, admission rate and frequency of hypo- and hyperglycemic episodes during ramadan. these results are contrary to the reported higher risk of complications in type-1 patients during ramadan fasting (7). the mean hba1c in type-1 patients was 8.21.7%, which is above the currently recommended goal of < 7%. if type-1 patients had a strict glycemic control with mean hba1c level lower than 7%, it would be more likely that they will get a more frequent hypoglycemic episodes. nevertheless the number of type-1 diabetics in this study was too few to draw conclusions. patient education and adjustment of doses throughout ramadan is needed to decrease the risk of both hypo- and hyperglycemia. further large studies are needed to study the effects of ramadan fasting on type-1 diabetic patients in libya. the authors have not received any funding or benefits from industry to conduct this study. | backgroundthe epidemiology of diabetes and ramadan fasting was reported from many muslim countries, but not from libya.methodologywe interviewed 493 consecutive diabetic patients at benghazi diabetes and endocrine center for the potential complications of fasting during ramadan.resultswe found 70% of diabetic patients completed the 30 days of ramadan fasting. hypo- and hyperglycemia was the most commonly reported complications especially during the first two weeks of ramadan month.conclusionit seems majority of diabetic patients in libya manage to fast during ramadan month. patient education and early planned adjustment of diabetic medication is needed to decrease the frequency of diabetic complication during ramadan month. |
type 1 diabetes mellitus (t1 dm) is an autoimmune disorder and individuals with this disorder are prone to develop high anion gap metabolic acidosis with ketonuria, known as diabetic ketoacidosis (dka). we report a case of normal anion gap metabolic acidosis without ketonuria during the management of hyperglycemia in a patient of t1 dm. although t1 dm and drta are both associated with autoimmune disorders, their coexistence has been very rarely reported. our case represents the second of its kind to the best of our knowledge and emphasizes the need to screen for drta in patients of t1 dm, justified by the fact that different treatment strategy will be required for the management of diabetic ketoacidosis in t1 dm associated with drta. our patient was a 26-year - old male, a known case of t1 dm for 6 years on insulin, who presented to our outpatient department (opd) with complaints of epigastric pain, postprandial fullness, and nausea. the patient was not compliant with the treatment and was coming erratically for follow - up to the opd. on examination, the patient was conscious, oriented, dehydrated, and had mild pallor. examination revealed a pulse rate of 60/minute, blood pressure (bp) of 100/60 mm hg, respiratory rate (rr) of 17 breaths / minute, and temperature of 98.6f. investigations revealed the following : hemoglobin (hb) 10.2 g / dl ; total leukocyte count (tlc) 5.3 10/l ; differential leukocyte count (dlc) : n 71%, l 20%, m 7.5% ; platelet 273 10/l ; erythrocyte sedimentation rate (esr) 24/1h ; urea 37 mg / dl ; creatinine 1.09 mg / dl ; bilirubin 1.34 mg / dl ; aspartate transaminase (ast) 34 u / l ; alanine transaminase (alt) 50 u / l ; alkaline phosphatase (alp) 450 u / l ; total protein 5.9 g / dl ; and albumin 3.7 g / dl. arterial blood gas analysis revealed the following : ph 7.20, po2 72 mm hg, so2 95 mm hg, pco2 36 mm hg, na 137 meq / l, k 2.5 meq / l, hco3 12.1 meq / l, cl 116 meq / l (100112 after initial resuscitation with iv fluids, and potassium replacement, the patient 's blood sugar stabilized to 172 mg / dl random ; however, metabolic acidosis and hypokalemia persisted. [hco3 + cl ], which was normal : 137 [12.1 + 116 ] = 8.9 meq / l. the patient was diagnosed as drta in view of normal anion gap metabolic acidosis with hypokalemia with inability to acidify urine in the presence of systemic metabolic acidosis. meanwhile, the patient started tolerating oral intake and was started on oral feeds with pre - meal subcutaneous regular insulin. subsequently, the patient was put on oral sodium bicarbonate tablets at a dose of 2 meq / kg and oral potassium replacement. it is thought to be caused by interaction of genetic, environmental, and immunological factors, which leads to the pancreatic beta cell destruction. t1 dm is associated with many autoimmune diseases as almost 1530% patients have autoimmune thyroid disease, 49% have celiac disease, and 0.5% have addison 's disease. t1 other uncommon associated autoimmune diseases include pernicious anemia, juvenile rheumatoid arthritis, psoriasis, vitiligo, etc. type 1 rta or drta is a rare disease which can be either inherited, sporadic, endemic, or acquired secondary to a variety of conditions. the most common causes of secondary drta are autoimmune disorders like sjgren syndrome, autoimmune thyroiditis, chronic active hepatitis, primary biliary cirrhosis, systemic lupus erythematosus, hypothyroidism, and vasculitis.[811 ] it has been seen that in some patients drta may be possibly caused by autoantibody against renal collecting duct. the clinical spectrum of drta in t1 dm is similar to that of diabetic ketoacidosis complicating t1 dm, except for urine ph > 5.5 in the former and presence of ketonuria in the latter. however, the management of acidosis in drta is contrary to that in diabetic ketoacidosis due to the controversial role of alkali therapy in the latter situation. although t1 dm and drta are both associated with autoimmune disorders, their coexistence has rarely been reported. whether this coexistence is purely coincidental or is because of common autoimmune pathogenesis is unclear. interestingly, till date, diabetic ketoacidosis complicating t1 dm associated with drta has not been reported in literature as the diagnosis and management of such a case will be challenging because the results of most clinical trials do not support the routine use of bicarbonate replacement, as in one study in children which showed that its use was associated with increased risk of cerebral edema. on the other hand, soda bicarbonate is the cornerstone therapy for management of hypokalemia due to drta, and without correction of hypokalemia, the use of insulin is contraindicated in diabetic ketoacidosis. therefore, it is necessary to first explore the possible mechanism involved in drta in t1 dm in order to draw a definite association of the two autoimmune disorders and secondly plan a treatment strategy during diabetic ketoacidosis in a patient of t1 dm associated with drta. | a 26-year - old male patient suffering from type 1 diabetes mellitus got admitted with abdominal pain and high blood sugars. on further evaluation, he was found to have normal anion gap metabolic acidosis without ketonuria and urinary ph was alkaline. the patient was diagnosed as type 1 renal tubular acidosis (rta) (distal rta) and was managed by alkali replacement in addition to control of blood sugars. the association of type 1 rta with type 1 diabetes mellitus has been rarely reported in the literature. the association needs a different attention as diagnosis and management of diabetic ketoacidosis in such cases will be tricky. the case presented here is the first of its kind from our part of the world and second as far as english literature is concerned. |
thyroid cancer is more common in females, and is currently the seventh leading cause of new cancer diagnosis in women. these cancers can range from papillary or follicular thyroid carcinoma, both well - differentiated tumors with excellent prognosis and indolent course, to poorly differentiated, and often lethal, anaplastic form. currently, known risk factors for thyroid cancer include previous high - dose environmental radioactive iodine exposure, previous childhood radiation to the head and neck, iodine deficiency, female gender, and family history of thyroid cancer [3, 4 ]. in 2007, there were estimated 33 550 new cases of thyroid cancer diagnosed, with estimated 1530 total deaths. the incidence of new thyroid cancer diagnosis has increased by 6.2% from 1997 to 2004, with a concomitant increase in mortality by 0.3% from 1985 to 2004. it is unknown whether this rise in thyroid cancer is due to increased imaging of the neck resulting in increased diagnosis of smaller, nonpalpable cancers (mainly papillary thyroid carcinoma) that would have previously gone undiagnosed or whether this is due to a true rise in thyroid cancer from an undetermined etiology. recently, studies have shown associations between vitamin d deficiency and breast, prostate, and colon cancers. there are animal models and in vitro studies that have shown vitamin d to have an antiproliferative effect on thyroid cancer [1012 ]. this study evaluates vitamin d deficiency as not only a potential novel risk factor for thyroid cancer but also a potential therapeutic target. if thyroid cancer is similar to other solid tumors regarding its relationship to vitamin d, we hypothesize that vitamin d levels will be lower in patients with active thyroid cancer than patients with thyroid cancer in remission, and that both thyroid cancer groups will be lower than thyroid nodules. this study was approved by the university of nebraska medical center irb to use stored blood specimens from the thyroid cancer collaborative registry (tccr). the tccr was established at unmc / the nebraska medical center in march 2008 and has accrued over 300 patients to date. all patients who give informed consent to be a part of the registry complete a standardized questionnaire that contains demographic information, medical history, family history, diet and lifestyle habits, past or current environmental exposures, and occupation. blood and tissue samples are collected and stored for all patients with thyroid cancer and thyroid nodules. this study is a pilot study designed to provide preliminary data, which will allow us to design a larger study. to design a larger trial, using the outcome the proportion of persons with vitamin d deficiency (defined as a baseline vitamin d level of 6 months since initial treatment or > 12 months since repeat treatment ; (2) total thyroidectomy and no radioactive iodine remnant ablation and negative neck ultrasound ; (3) less than total thyroidectomy and no radioactive iodine remnant ablation and negative neck ultrasound ; (4) detectable antithyroglobulin antibodies and negative imaging (radioactive iodine whole body scan or ultrasound). patients with a history of thyroid cancer were considered to have active disease if they met one of the following criteria : (1) total thyroidectomy and radioactive iodine remnant ablation and unstimulated thyroglobulin > 1 ng / ml and/or stimulated thyroglobulin > 5 ng / ml ; (2) less than 6 months from initial treatment or 800 iu vitamin d per day. stored serum from the tccr was assessed for serum calcium, albumin, creatinine, and 25-hydoxy - vitamin d (25-oh - d). serum utilized to run laboratories was obtained from participants over a period starting 4/1/2008 and ending on 2/6/2009. the serum calcium, albumin, and creatinine were evaluated at the unmc / nmc laboratories utilizing ortho clinical diagnostics vitros 950 system. for those individuals taking supplemental vitamin d exceeding 800 iu per day, we utilized baseline 25-oh - d levels prior to vitamin d supplementation, if available. baseline 25-oh - d levels were obtained from the electronic medical record, and extend from 12/15/2001 through 2/2/2009. however, 25-oh - d levels prior to 07/22/2008 were sent to arup for analysis that used either diasorin or nichols assays during that period. all 25-oh - d levels starting 07/23/2008 were done through the unmc / nmc laboratories, utilizing tandem mass spectrometry on the applied biosystems api3000 ms / ms equipped with agilent 1100 lc. baseline 25-oh - d levels prior to july 2008 (arup assays) were obtained from 11 thyroid nodules, 10 thyroid cancer in remission, and 2 active thyroid cancer patients. there were 2 thyroid nodules, 1 thyroid cancer in remission, and 4 active thyroid cancer patients who did not have baseline 25-oh - d levels available, or were on an unknown dose of vitamin d, and stored serum was used to evaluate 25-oh - d levels. patient characteristics for each group were assessed using descriptive statistics, including medians, range, frequencies, and proportions. continuous outcomes were compared between the groups using the kruskal wallis test with subsequent pair - wise comparisons performed using a wilcoxon rank sum test and a bonferroni - adjusted alpha level of 0.017 when the overall test was significant. a logistic regression modeling procedure was used to compare the odds of low 25-oh - d levels (vitamin d 45 years), bmi (30 kg / m), season of vitamin d measurement (winter : october march ; summer : april september), and tnm staging. only risk factors significant at the 0.10 level in univariate analysis were included in the multivariate model. tandem mass spec for vitamin d assay was used for all patients that were not on supplemental vitamin d > 800 iu per day. if they were on vitamin d > 800 iu per day, baseline vitamin d levels, utilizing either diaosrin or nichols assays, because of the potential for differences in vitamin d levels obtained with different assays, we also analyzed our data using only the patients with vitamin d levels assessed by the tandem mass spec method, which included the majority of patients in the study (81%). we did have a small number of patients (13%) with samples run on both the diasorin and mass spec assays available, so we also compared the mean sd of vitamin d of the 2 assays. this research was funded by a grant from the university of nebraska medical center center for clinical and translational research. a total of 111 patients were evaluated, 42 (38%) nodules, 45 (40%) thyroid cancer in remission, and 24 (22%) active thyroid cancer. gender, age, season, and bmi were not significantly different between groups, however, there was a trend toward more males in the active thyroid cancer group. ninety percent of the nodule group was female compared with 87% in remission and 71% percent in the active thyroid cancer group (p =.11). fifty - seven percent of the nodule group was > 45 years, compared with 44% of the remission group and 37% of the active group (p =.28). summer season of vitamin d measurement was 62% in the nodule group, 64% in the remission group, and 67% in the active group (p =.94). there was no significant difference between the remission and active thyroid cancer groups when evaluating for tnm stage (p =.20) or histologic type of thyroid cancer (p =.35). calcium, albumin, and creatinine were not statistically different between groups (data not shown). after removal of the diasorin and nichols assays, a total of 90 patients were evaluated. this included 33 (37%) nodules, 35 (39%) thyroid cancer in remission, and 22 (24%) active thyroid cancer patients. using only the mass spec vitamin d samples, gender, age, season, and bmi were not significantly different. however, tnm stage was significantly different between the remission and active cancer groups showing a higher percentage of the remission group was stage i. in the remission group, 82% were stage i, 9% were stage ii, 3% were stage iii, and 6% were stage iva, while in the active cancer group, 55% were stage i, 15% were stage ii, 22% were stage iii, and 5% were stage iva (p =.05). vitamin d deficiency was defined as 25-oh - d 75 nmol / l), there again was no significant difference (p =.74) (table 2). in the nodule versus remission versus active groups, 2%, 4%, and nmol / l, respectively. however, 52%, 40%, and 46% had 25-oh - d levels between 2575 nmol / l, respectively, and 45%, 56%, and 54% had 25-oh - d levels of > 75, there was still no significant difference when evaluating each group for percentage of patients with vitamin d deficiency or when stratifying each group by tertiles (table 2). when evaluating median (range) 25-oh - d for the entire group (using all vitamin d assays), the summer group (n = 71) had a median 25-oh - d of 73 (30 kg / m was the only significant risk factor for vitamin d deficiency in univariate analysis (or 6.98, 95% ci:2.9516.52, p 30 kg / m, consistent with previous studies [13, 14 ]. these results are different from other studies that have shown a relationship between vitamin d deficiency and other solid tumors. one recent study evaluated the incident risk of developing nonskin cancers of all types when vitamin d supplementation was administered over four years. this study compared individuals receiving calcium plus 1000 iu of vitamin d supplementation to calcium supplementation only and placebo controls. all individuals had similar baseline mean 25-oh - d levels around 70 nmol / l, with a mean increase in 25-oh - d levels of 24 nmol / l at 12 months in the group receiving vitamin d only. in comparison to the placebo group, the calcium plus vitamin d group had a lower relative risk of developing cancer over the course of the study. epidemiological studies have also shown an increased incidence of colon cancer with lower levels of vitamin d. vitamin d levels in the lowest quartile of vitamin d, or below 75 nmol / l, have been associated with approximately twice the risk of colon cancer than those with higher levels [9, 16 ]. levels of 25-oh - d in postmenopausal women with breast cancer have been shown to be lower than women without breast cancer and there is a significant inverse association between 25-oh - d levels and postmenopausal breast cancer. another study evaluating prostate cancer and vitamin d also demonstrated risk was inversely related to 25-oh - d, but the risk in this study was only seen at levels 75 nmol / l range had a higher percentage of individuals from the summer group. although it is possible that having more summer specimens could affect these results, post - hoc analysis did not show a statistically significant effect of season on vitamin d levels. previous reports of vitamin d deficiency in healthy populations have been roughly 1434% [14, 18, 19 ]. we overall found higher rates of vitamin d deficiency in all three of our groups. the prevalence of vitamin d deficiency in nodules was 48%, thyroid cancer in remission was 56%, and active thyroid cancer was 58%. even though the rates were not statistically different between groups, all three groups were higher than what has been previously reported in healthy subjects. ethnicity has been associated with vitamin d levels [20, 21 ], so it is important to consider when comparing data. our study participants were predominantly caucasians, so should be compared to normal controls with similar ethnicity. recently, nhanes data showed there has been an overall decline in 25-oh - d levels from the 19881994 census compared with the 20012004 census. this could explain the increased rates of overall vitamin d deficiency in all three of our groups, however, there are many potential reasons for this change, one of which includes changes and overall improvement in the vitamin d assay. other potential causes could be difference in ethnicity, season of measurement of vitamin d, and bmi of the population as a whole. one of the main limitations of this study is the lack of healthy (no thyroid disease) controls. however, in a separate, published study, healthy controls were recruited and vitamin d levels obtained at our institution, and we use them as a basis for comparison since they are demographically similar to our current study group. in that study, there were 41 healthy control, nonobese participants with a mean age was 46.3 years. however, seventy - eight percent were female, 98% were caucasian, and they had a mean bmi of 24.7 kg / m. that group is also representative of our study group because it represents patients from the same latitude, and included vitamin d levels from both summer (54%) and winter (46%) months. the prevalence of vitamin d deficiency in that healthy control population was 32% as compared with our data showing 48% of thyroid nodules with vitamin d deficiency and 5658% of thyroid cancer. another limitation is the small sample size. as this is a pilot study, our numbers are small, which may have prevented us from detecting a statistical difference. our results, therefore, will need to be confirmed with a larger, potentially multicenter study. it is important to consider that some autoimmune conditions have been reported to be associated with vitamin d deficiency, which may play a role in our thyroid nodule group. thyroid antibodies are not routinely obtained for all thyroid nodule patients, so were not available from all patients. however, when evaluating those patients that did have antibodies available, there was only 14% of the entire group that had positive antibodies. we recognize that using different vitamin d assays may result in different vitamin d levels reported. to account for this potential difference, we have analyzed our data using results combined from the three different assays, as well as tandem mass spec alone, which make up the majority of specimens (81%). we did not find any significant differences in our results when analyzing data from the whole group and mass spec assay only, and there was no significant difference between vitamin d levels when comparing simultaneous diaosrin and mass spec results. in conclusion, we did not find a significant association between vitamin d levels and thyroid nodules, thyroid cancer in remission, and active thyroid cancer. we also found the overall prevalence of vitamin d deficiency to be higher than expected for the general healthy population, which also needs to be studied further in a larger population that controls for issues known to affect vitamin d such as ethnicity, bmi, season of measurement, autoimmunity, and vitamin d supplementation. | introduction. there are reported associations between vitamin d deficiency and breast, prostate, and colon cancer, but the relationship in thyroid cancer has not been evaluated. methods. we evaluated serum calcium, creatinine, albumin, and 25-hydroxy vitamin d (25-oh - d) in 42 thyroid nodule, 45 thyroid cancer in remission, and 24 active thyroid cancer patients. results. 25-oh - d was not different between groups. the percent with 25-oh - d levels < 75 nmol / l was not significantly different between groups and was not affected by season of measurement, age, or cancer stage. multivariate regression showed a bmi of 30 kg / m2 to be the only significant predictor of vitamin d deficiency. conclusions. rates of vitamin d deficiency are similar in thyroid nodules and thyroid cancer, although higher than the general population. this is different than previous studies for other cancers, which show higher rates of vitamin d deficiency. bmi was the only predictor of vitamin d deficiency. |
nave cd8 + t cells become activated when their receptors recognize antigens presented by professional antigen - presenting cells (papcs) in the context of mhc - i molecules. upon recognition of target cells, such primed cytotoxic t - lymphocytes (ctls) are able to limit the spread of virus infection through the lysis of host - infected cells. moreover, along with helper t cells they orchestrate the induction of key cytokines such as interferon- (ifn-) and tumor necrosis factor- (tnf-) needed for an optimal immune response. in addition to their importance in halting virus replication, ctls play a central role in the specific immune response and are essential in the elimination of intracellular pathogens and limiting the potential escape of tumor cells [2, 3 ]. the cross - presentation pathway allows for exogenously - derived antigens to be presented on mhc - i molecules to ctls [47 ]. to induce antitumor immune responses, or to prime ctls for viruses that inhibit direct presentation thus, cross - presentation represents a promising mechanism for strategies that target the induction of ctl responses for vaccine development to induce both effector and protective memory t - cell responses. in order for us to discuss the role of cross - presentation in future vaccine developments all nucleated cells express mhc - i molecules and are capable of presenting antigens to ctls. however, the priming stage or activation of nave cd8 + t cells requires peptide - mhc class i complexes presented by the papc in addition to co - stimulatory signals such as interaction with b7 molecules, cd40, cd70, and the 4 - 1bbl family members, as well as the secretion of key cytokines such as il-12 and ifn- [1113 ]. the cd8 + t cells priming step can occur via two different mechanisms of antigen presentation : the direct- and cross - presentation pathways. in the direct or endogenous presentation pathway, antigens are derived from endogenously synthesized proteins, improperly translated proteins, and, or unstable defective ribosomal proteins [15, 16 ]. these cytosolic proteins are targeted for proteasomal degradation after their polyubiquination. proteasomal degradation products are then transported into the endoplasmic reticulum (er) via the transporter associated with antigen processing (tap), moving through the golgi complex to the cell surface (figure 1). alternatively, the cross- or the exogenous- presentation pathway occurs when uninfected papcs present exogenously - derived antigens [4, 5, 7 ] after uptake of soluble or cell - associated antigens. the latter antigenic form can be derived from different sources such as cellular fragments [1719 ], intracellular bacteria, virus - infected or tumor cells [2126 ], and as reported recently from parasitic infections. it appears that protein stability in these models is critical for efficient cross - presentation to occur [17, 2831 ]. on the other hand, soluble proteins tend to be cross - presented, but with much lower efficiency than cell - associated proteins. although efficient presentation of exogenous antigens was originally attributed to macrophages, it is now clear that such function can be achieved by different bone marrow - derived apcs, including dcs and macrophages [3436 ], as well as spleen - derived macrophages. interestingly, it appears both spleen and bone marrow - derived macrophages down regulate their ability to cross - present cell - associated antigens during differentiation. in cross - presentation, several processing mechanisms have been proposed, including the canonical model where antigens inside the endosomal / phagosomal vesicles are translocated into the cytosol before they follow the regular proteasome / er / tap route (figure 1). in addition, it was proposed that papcs possess organelles, such as the phagosome, optimized for the cross - presentation of exogenous antigens, by functioning autonomously to generate mhc class i - peptide complexes from proteins internalized into the same phagosome. moreover, soluble antigen cross - presentation was recently found to involve early endocytic compartment trafficking that is aided by tap recruitment and signaling through tlr4 and myd88. however, whether these antigen processing models are applicable to exogenous antigens other than ova proteins has yet to be investigated. as this paper focuses on exogenous antigen delivery in vaccine development, we will concentrate on cross - presentation. regardless of the exact processing mechanism in play within the cell, the cross - presentation pathway can result in one of two outcomes : cross - tolerance or cross - priming. inadequate activation of ctls may result in tolerance, which is an ideal situation for self - antigens. thus, under normal circumstances, cross - presentation of peripheral self - antigens from normal healthy tissue will induce cross - tolerance [40, 41 ]. this outcome could depend on many factors such as which apc is presenting the antigen [4143 ], and how the antigen is being cross - presented [44, 45 ]. on the other hand, if immunity is to be induced, signals necessary for t - cell activation leading to cross - priming will be provided by papcs, leading to clonal expansion, differentiation, and establishment of robust memory cells. memory t cells are long - lived cells that allow for an efficient adaptive immune response upon re - exposure to a pathogen. such memory cells can be divided into two categories depending on their trafficking and effector functions. the effector memory t cells (tem) are found in peripheral tissues, can respond rapidly to infections, and are characterized by the low expression of certain markers such as cd62l, ccr7, and cd27. upon antigen recognition, they immediately produce effector cytokines, such as ifn- and tnf-, conferring a state of protection. in contrast, central memory t cells (tcm), which are found mainly in secondary lymphatic organs such as lymph nodes, express high levels of the cd26l, ccr7, and cd27 markers, and are slow to exhibit their effector functions. an important goal of any vaccination protocol would be the induction of both sets of cd8 + t - cell memory. another critical factor to consider in t - cell activation focuses on the profile of t - cell immunodominance hierarchy that a vaccine is likely to induce. with regard to immunodominance, epitope - specific cd8 + t cells can be organized into a hierarchy, in which certain immunodominant epitopes will cause a set of t cells to expand extensively compared to subdominant epitope - specific t cells. a ctl response could be more effective when generated against a greater number of epitopes, as it is more diverse and should lead to the formation of a wider number of specific memory cd8 + t - cell populations that can confer protective immunity. in evaluating the physiological relevance of cross - priming in vivo to vaccine development, it has been demonstrated that cross - priming is a robust process that induces significant ctl responses to multiple epitopes, both in viral and tumor models. in a related study, cross - priming of inactivated flu virus resulted in a broad and balanced ctl responses compared to live virus. interestingly, it appears that the ability of an epitope to access cross - priming may support its immunodominance position when considering the overall hierarchy [5052 ]. during virus challenge experiments, it was found that an initial cross - priming with a lymphocytic choriomeningitis virus (lcmv)-nucleoprotein (np), that normally cross - primes ctls for the np396 epitope, resulted in increasing the magnitude of np396 epitope - specific t cells. thus, the immunodominance hierarchy was modified so that the np396 epitope, an immunodominant epitope found within the lcmv - np, was favored in a subsequent virus challenge. these effects were maintained over time and may have important implications for vaccination protocols such as the currently administrated inactivated flu vaccines. in this paper, it was speculated that the initial cross - priming with lcmv - np resulted in the enhanced ability of np396-specific clones, but not the np205-specifc clones, to expand and out compete other t - cell clones specific for epitopes generated from other proteins. importantly, these changes only occurred when cross - priming preceded viral infection and thus when t - cell frequencies competing for resources were lower. such findings were not observed in the vaccinia virus model when peptides were used prior to infections to prime ctl responses. these diverse findings may be related to the different replication profiles of both viruses considering that vaccinia does not replicate as efficiently as lcmv in mice. it would also be interesting to compare immunodominance data in a virus challenge experiment obtained after priming with peptide versus proteins, where in the latter condition cross - priming is expected to take place [50, 53 ]. finally, with regard to cross - priming and vaccination, the discovery and development of adjuvants that are able to regulate cross - presentation is of utmost importance. when co - administered with an antigen, adjuvants assist in the generation of an immune response. adjuvants can elicit their immune enhancing effects in a variety of ways, including targeting pattern recognition receptors such as toll - like receptors (tlrs). tlr signaling can result in dc maturation, leading to upregulated mhc and co - stimulatory molecule expression, and increased priming of t cells without the need for cd4 t - cell help [26, 5457 ]. for example, it has been shown that the synthetic tlr9 ligand, cpg - odn (cytosine - phosphorothioate - guanine oligodeoxynucleotides), is readily taken up by receptor - mediated endocytosis and was found to enhance ctl responses. furthermore, the antigen - specific primary and secondary expansion of t cells was better established when tlr9 ligand was cross - linked to the ova antigen [55, 5860 ]. thus, based on many models of cross - priming, researchers are logically aiming to develop novel vaccine vectors that are capable of inducing ctl responses in vivo by utilizing exogenous antigen delivery. in the latter part of this paper, we will discuss some examples of such vectors and contemplate on other future developments in the vaccine field. in vaccination, delivering exogenous antigens via cross - presentation should ideally induce both humoral and ctl responses to improve effective immunity. as the major papc in vivo, dcs are able to stimulate nave cd8 + t cells and are important when considering precise targeting of delivery vectors. a key area of research involves the modification of vector surfaces with ligands for dc receptors, such as tlr ligands, as well the application of natural substances that can allow for effective immune modulation. for example, the active metabolites of vitamin d3 can influence adaptive immune responses to peripherally administered antigens during vaccination / infection. in these experiments, the ability of murine dcs to migrate from skin sites of vaccination to mucosal lymphoid organs seem to be dependent on the production of active vitamin d3 metabolites produced locally at the vaccination site, which was associated with the application of specific tlr (tlr3/tlr4) ligands [61, 62 ]. these findings support previous studies, which demonstrated that both systemic and common mucosal immune responses developed in adult mice upon administration of a subcutaneous or intradermal vaccine containing active vitamin d3. generally, vaccines should induce effective protective ctl immunity, and the production of immunological memory. potent ctl responses have been reported to be limited to live attenuated viral or bacterial vaccines. however, the use of such vaccines is offset by the risk of reinitiating virulence. thus, a key target of novel vectors, such as virus - like particles, microparticles, and archaeosomes, is to efficiently access the cross - presentation pathway. as a result, these vectors will induce ctl effector and memory t - cell responses without risking the health of the host. such vectors have shown interesting immunological properties, discussed below, that enable them to be potent in inducing cross - priming. virus - like particles (vlps) are formed from viral structural proteins that lack a viral genome and thus assemble into nonreplicative particles [64, 65 ]. vlps are safe, stable, and extremely immunogenic due to their highly repetitive molecular structures [64, 65 ]. in addition to their highly immunogenic nature, vlps can be easily modified to increase their immunogenicity by packaging cpgs into the core antigen particle, which results in higher frequencies of peptide - specific cd8 + t cells being induced. since vlps are exogenously acquired by papcs, they are able to use the cross - presentation pathway [6769 ] and can induce both ctl and memory responses. for example, a recombinant parvovirus- (ppv-) vlp, encoding a known lcmv cd8 + t - cell epitope, was able to stimulate the immune system up to 8 months after the last immunization. this long - lasting effect was characterized by protection against a lethal viral (lcmv) infection, in addition to responses against peptide - coated or virus - infected target cells. the high particulate nature of ppv - vlps was hypothesized to be associated with its optimal delivery to the mhc - class i antigen presentation pathway, resulting in high immunogenicity. this ability to induce ctls via cross - priming was demonstrated in another study with hiv p55gag - vlps immunizations. in this study, immunized animals were efficiently primed (> 8.5 months) for p55gag - specific ctl responses that recognized multiple hiv p55gag epitopes. altogether, these studies demonstrate that vlps are able to prime strong mhc - i restricted cd8 + t - cell responses through cross - presentation. the exact mechanism of cross - presentation is not fully defined ; however, recent data suggests that the processing pathway involving vlps may vary according to the type of vector employed. for example, cross - presentation of papaya mosaic vlps, which induces robust ctl responses, was proteasome independent. in the case of parvovirus - like particles (ppv - vlps) without adjuvant it is important to note that presence of potential adjuvants, such as tlr ligands, enhances the efficiency of cross - presentation as it allows for the relocation of the tap molecules to the early endosomes. as one would expect, the processing of the ppv - vlps required vacuolar acidification, proteasome activity, and tap translocation, but not mhc class i molecule recycling. altogether, the data shows that the cross - presentation of ppv - vlps occurs via an endosome - to - cytosol processing pathway. interestingly, the uptake and capture of ppv - vlps involved macropinocytosis and lipid rafts participation. in contrast when employing hepatitis b virus (hbv) vlps, it was found that efficient ctl cross - priming by dcs occurred either in a tap - dependent or tap - independent manner. the hbv (vlps) are also taken up by macropinocytosis, but rely on endosomal processing and recycling mhc i molecules in dcs. thus it may be necessary to evaluate each vlp type independently, as each vector will have a unique nature that may be closely associated with the protection it can elicit against its targeted pathogen or disease. it is also important to note that not all vlps are able to activate papcs by themselves. this was highlighted by data showing that a ctl epitope from lcmv (p33-vlps) was efficiently processed for mhc class i presentation but induced weak ctl responses. the ctl response failed to mediate effective protection from viral challenge in the absence of external substances that activate apcs, such as anti - cd40 antibodies or cpg oligonucleotides, which engage tlr9. clearly, further research is required to fully optimize this technology, however, vlps represent a promising avenue for vector development. recently, a novel approach utilized hiv-1 vlps of a mutant hiv-1 nef, which acts as an anchoring element for foreign proteins. immunization with such vlps incorporating a hpv-16 e7 protein, fused to the mutant hiv-1 nef, resulted in an robust anti - e7 cd8 + t - cell response, and protection against an hpv - e7 expressing tumour. furthermore, it was suggested that co - inoculation of vlps with diverse epitopes of the same pathogen could be used to generate ctls with a wider range of specificities to attain enhanced protection. microspheres (mss) are composed of biodegradable polymers, such as poly (lactic acid) (pla), poly(glycolic acid) (pga), and polylactic - co - glycolic acid (plga), and can be formed through processes such as solvent evaporation or spray drying [54, 75 ]. depending on their diameter, loaded mss can be taken up by apcs through phagocytosis. this uptake could be also enhanced due to the depot formation at the site of injection. mss can be administered orally because they can protect their cargo including adjuvants (e.g., cpg - odns) from enzyme - mediated degradation. upon hydrolysis of their polymeric bonds by phagosomal enzymes after phagocytosis, the ms polymers are disrupted, causing the release of the encapsulated antigen. biodegradable microspheres (mss) are useful to microencapsulate antigens alone or combined with adjuvants. their ability to allow for the controlled release of the antigen or to allow for the formation of a depot at the site of injection is particularly useful. poly (d, l - lactide - co - glycolide) (plga) has been used to prepare microspheres that possess tremendous potential to release encapsulated antigens in a controlled manner, thus facilitating the protection of the antigen cargo from immediate degradation in vivo. a key advantage of mss is the flexibility in their design, which allows for a variety of possibilities in combining different antigens and adjuvants. moreover, specific cell targeting is feasible through the addition of ligands to the vehicle surface that are able to specifically bind receptors on the surface of the targeted cell type [78, 79 ]. mss can deliver exogenous antigens to the cross - presentation pathway, but appear to require additional signals, such as adjuvants, to trigger a ctl response. this process may involve t - cell help which can be provided via cd40l interactions on helper t cells, and is needed to promote the costimulatory activation state of dcs required for optimal cross - priming [81, 82 ]. as mss are unable to trigger dc maturation by themselves, cross - presentation of antigens by immature dcs can result in t - cell ignorance. data examining this phenomenon concur that additional help signals are required to enhance the cross - priming of mss. for example, mss encapsulating an antigen with an immunodominant cd8 + t - cell epitope (b - ovap) failed to induce antigen - specific cross - priming of cd8 + t cells, unless recombinant ppv - vlp were co - injected, which served as an adjuvant to induce a potent ctl response. interestingly, the ppv - vlps enhanced this ctl response in mhc class ii and cd40 mice indicating that the cd4 + t - cell help was bypassed under these conditions. furthermore, such adjuvant activity did not require tlr2, tlr4, or tlr9 stimulation. in accordance, recent research has focused on examining the influence of adjuvants on cross - priming within the antigen - containing mss. these studies used ova incorporated into mss and examined cross - primed antigen - specific t cells by the secretion of ifn- in peptide specific assays [60, 84 ]. the ctl response elicited when tlr ligands were coencapsulated with ova was stronger compared with ova - ms alone or if antigen and adjuvants were separately encapsulated. in a study examining the coencapsulation of ova and a tlr9 ligand (cpg - odn), the authors found that both ova and cpg - odn were translocated into lysosomal - associated membrane protein-1 (lamp1)-positive phago - endosomal compartments of dc. the processed ova peptides on mhc class i and ii molecules on dcs allowed for robust antigen - specific cd4 + and cd8 + t - cell proliferation that were tlr9-dependent. the processing route for mhc - i antigen presentation in this study required endosomal acidification, tap translocation, and proteasomal processing. in a separate study, it was found that cpg - odn in the mss induced dc maturation, characterized by increased mhc and co - stimulatory molecule expression, which may have aided the increased cross - priming. in both reports [60, 84 ], induction of ova specific tcells, especially after boosting, was comparable to what is observed when live vectors encoding ova are employed [85, 86 ]. in addition, the induced immunity was protective against a challenge of l. monocytogenes, vaccinia virus and protected against ova expressing tumors [60, 84 ]. for example, liposomes are vesicles composed of lipid bilayers that are separated by aqueous regions, and are comparable to mss as they are also biodegradable and safe, making them attractive vectors for vaccine delivery [75, 87 ]. liposomes were one of the early vectors developed in order to co - deliver adjuvants, such as cpg odn, and antigens, and have been used in therapy of diseases such as leishmaniasis. in addition, the adjuvant effects of liposomes complexed to tlr agonists were found to induce effective cd4 + and cd8 + t - cell responses against peptide and protein antigens. in particular tlr3 or tlr9 agonists effectively cross - primed cd8 + t - cell responses independently of cd4 + t - cell help. nonetheless, despite the advantages of using mss in vaccine delivery, additional data explaining the exact processing mechanisms is needed. in addition, further research is required to reveal how this generates protective potential when applying different antigenic determinants in different infections models. regardless, the simultaneous inclusion of antigen and adjuvant appears to hold promise to optimize future vaccination strategies that use mss. archaeosomes are a novel generation of liposomes that are composed of the polar lipids of archaea, which leads to immune - stimulating interactions with apcs. the archeal lipid cores offer archaeosomes long - lasting stability at a variety of environmental extremes, including temperature, ph, and oxidative conditions. these properties allow them to induce strong memory responses and avoid fusion or aggregation, thus reducing antigen leakage. the entrapment of antigen in archaeosomes has been documented to facilitate mhc - i cross - presentation, resulting in the activation of long - lived cd8 + t - cell immunity [85, 8991 ]. for example, a single immunization with a methanobrevibacter smithii - ova (m. smithii) archaeosome was found to prime 25% of antigen - specific t cells by day 7, while boosting on day 21 resulted in an expansion to about 20%. furthermore, after two immunizations of low doses m. smithii - ova archaeosomes, re - stimulated spleen cell effectors were able to exhibit strong cytolytic activity of target cells labeled with the specific peptides. this ability to activate ctls with archaeosomes was demonstrated in cd4 and il-12 mice. when examining the quality of archaeosome - primed cd8 + t - cell responses, it was reported that the responses were superior in magnitude to other particulate vesicular systems such as liposomes. in addition, a single injection of the vector induced a profound primary response, leading to the formation of around 1% of cd8 + tcm which exhibited a phenotype (cd44cd62l) typical of the central memory cells. the responses were comparable to vaccination with live vectors encoding ova such as l. monocytogenes vector and induced protective cd8 + t - cells responses that were tlr2-independent. unlike the mss delivery system that we discussed above, archaeosomes can target antigen for cross - presentation while simultaneously activating papc, thus allowing for proper induction of co - stimulatory molecules and cytokine production. ova entrapped in m. smithii is the best - characterized model with respect to studying the mechanisms of archaeosome cross - presentation. due to the presence of exposed archaetidylserine head groups on their surfaces, thus, they are able to utilize the same pathway that relies on the phosphatidylserine (ps) receptor - mediated clearance of apoptotic cells by papcs. however, after phagocytosis, it was seen that the presentation of archaeosomal - antigens on mhc - i relied on cytosolic proteasomal processing and tap - activity. interestingly, it was demonstrated that the blocking of endosomal acidification resulted in the block of mhc i processing, which can be due to the fact that acidification in the endosomes or phagolysosomes helps antigen escape from the archaeosomes. it is important to note that other archaeosome types lacking ps are also phagocytosed efficiently and can induce strong cd8 + t - cell immunity. thus, different archaeosome vectors can be expected to rely on different receptors for cellular entry. overall, archaeosomes provide promise to the field of vaccine development, and appear to represent versatile, potentially universal vectors. this research field can only benefit from the involvement and collaboration of more research groups. the majority of vaccines utilized in the past were attenuated vaccine strains and, although critical in the prevention of several infectious diseases, the inherent risk that they carry necessitates the development of alternative vaccines. the advent of nonreplicating vaccine vectors (key findings summarized in table 1) has revealed the importance of efficiently targeting exogenously derived antigens to immune cells, which would allow for the cross - priming of cd8 + t cells rather than cross - tolerance. the attractiveness of this mechanism is offset by the need to optimize such vectors, in order to maximize the immune response. in order to achieve this goal, more research is needed to dissect the cellular and molecular factors that regulate cross - presentation events during the initiation of ctl immune responses. we have come far since the observations made by edward jenner and the field is now ripe for developing novel vaccine - adjuvant complexes to make the next step forward that improves on the initial discovery of the small pox vaccine. | vaccines intended to induce a cytotoxic cd8 + t - cell response are highly sought after. however, some of these vaccines can be problematic if they replicate in the host. an alternative strategy is to exploit cross - presentation of exogenous antigens to express peptides on major histocompatibility complex (mhc) class i molecules. during cross - presentation, the delivered exogenous antigen can be taken up and processed through diverse mechanisms. here, we will discuss the recent advances regarding the complex nature of the cross - priming process and the models that reflect its relevance in vivo. moreover, we summarize current data that explore potential adjuvants and vaccine vectors that deliver antigens to activate cd8 + t cells relying on cross - presentation. |
there is now a wide choice of methods and software available for mapping genes by linkage. although the method of analysis is often determined by the experimental design, there is less guidance regarding the most appropriate software. here, the most well - known packages for linkage analysis will be briefly reviewed and some directions and standards for future work will be suggested. at one extreme, linkage analysis is applied to a small number of large pedigrees in which the trait exhibits a strongly mendelian mode of inheritance. methods for this type of data are usually termed ' parametric ' because an explicit penetrance model defining the relationship between genotype and disease must be specified. the most flexible package for these analytical methods remains fastlink, which is functionally equivalent to the original linkage package. for most pedigree structures, whether one applies single- or multi - point analysis of a disease or quantitative trait, vitesse is a faster package ; however, fastlink continues to be more efficient for pedigrees containing inbreeding loops. at the other extreme, linkage analysis is also applied to a large number of small pedigrees with unknown mode of inheritance. ' non - parametric ' allele - sharing methods are usually preferred here, for which the most well - known program is genehunter. genehunter contains an extensive set of linkage and association tests and, as such, is a de facto standard for statistical genetics analysis. a disadvantage of this position is that any new program will aspire to improve on genehunter, so that for many of its functions there are now other programs with better performance. an important example is allegro, which is faster for most pedigree structures, includes a wider range of scoring functions and computes more accurate significance levels for non - parametric statistics. the latter feature is also available in genehunter - plus, but this is only available for version 1.3 of genehunter and so does not access the speed - ups available in later versions. another recent competitor is merlin, which employs a still faster algorithm that is particularly useful in dense marker maps, for which the number of recombinations allowed between markers can be constrained. the range of analyses is similar to genehunter, merlin also provides the linear - model lod score available in allegro but not the exponential model. merlin does not calculate parametric lod scores -- which are available in genehunter and allegro -- but for non - parametric analysis, error checking and haplotyping, it will often be the fastest program. all three of these programs handle x - linked data, although this also is only available in version 1.3 of genehunter. an alternative approach for an unknown mode of inheritance is to perform parametric analysis over a range of models and then adjust the best lod score for this optimisation. this approach is implemented in mflink. in small pedigrees, there seems to be little to choose between this approach and the allele - sharing methods discussed above ; however, currently mflink can only perform two - point analysis. a promising new model is implemented in superlink. fishelson and geiger show that the algorithms used by fastlink and genehunter are instances of a more general model, under which a more efficient order of computation is determined at run - time according to the input pedigree. for parametric linkage analysis, some impressive speed - ups over vitesse future versions will include allele sharing and other statistics (m. fishelson, personal communication). haseman - elston regression is a sib - pair method available in genehunter with heuristic adjustments for general pedigrees. recently, the regression framework has been extended to more general pedigrees, and this is implemented in merlin. this approach now has comparable power to variance - components methods, with less dependence on trait normality and some computational advantages. merlin and genehunter also provide rank - based tests (confusingly also termed ' non - parametric '), which are appropriate for non - normally distributed traits. again, note that for genehunter the test is a sib - pair method, with heuristic adjustments for general pedigrees, whereas for merlin the test is immediately applicable to general pedigrees. variance - components methods are more powerful than regression, provide parameter estimates and easily accommodate a wide range of null hypotheses ; the cost is stronger dependence on trait normality and higher computational burden. implementations are available in merlin, provided that no dominance variance is assumed, and in genehunter. merlin is currently the only program that can perform multipoint variance components analysis on the x chromosome. allegro also contains undocumented implementations of various quantitative trait methods. exact multipoint analysis is limited either by the number of markers that can be included (fastlink, vitesse) or the pedigree size (genehunter, allegro, merlin). with current microsatellite markers, large pedigrees usually contain enough information from a small number of markers for current software to be adequate. this will change with the move to automated single nucleotide polymorphism typing for linkage studies, so it is becoming more important to have software that can handle large numbers of markers in large pedigrees. currently, this is only generally possible through the approximation methods of simwalk2, which nevertheless has good reported accuracy. although the program has a lot of tuning parameters, the mega2 utility program provides a reasonably easy route to a default analysis which is suitable in most cases. more efficient approximation methods are an area of current research, for example morgan, which currently only allows fully penetrant recessive traits but shows promise for more general models. modern computing favours graphical user interfaces (guis), which allow mouse - driven input ; but these are conspicuously absent from linkage software. descendents of linkage have essentially no user interface, although the terminal - based tool lcp is available to set up analysis scripts ; genehunter and solar run their own interactive command shells, whereas allegro and merlin use a single command with optional arguments and auxiliary input files. on the plus side, all of these interfaces are amenable to scripting -- for example to allow one to repeat the same analysis on multiple input files -- but the single - command interface of allegro and merlin is easily the most convenient to use in scripts. with the availability of java, html and tcl as cross - platform languages for gui development, it is hoped that future versions of these packages will incorporate simpler user interfaces, as well as scriptable back ends. the linkage input file format is recognised by many programs but is by no means universal. mega2 is a useful utility for converting between formats, but even this requires an additional map file which duplicates information contained in the locus file. it is hoped that the linkage format, however imperfect, will eventually be recognised by all programs that perform linkage analysis, without the need for supplementary conversion scripts. genehunter, allegro, merlin and solar can all output multipoint identical - by - descent (ibd) distributions, which are valuable for gaining insights into the segregation patterns in pedigrees. none can input this information, however : it is not possible, say, to calculate the ibd distribution under the recombination restrictions of merlin and then use this to obtain an exponential - model lod score from allegro. furthermore, sometimes different analyses result in the same distribution, and it is inefficient to recompute it each time. with some caveats, it is possible to avoid this recomputation in solar, but simple input of ibd, haplotype and recombination information would still generally be a useful feature for future versions. this survey has necessarily been cursory, and there is a wealth of other good linkage software available. a comprehensive list of statistical genetics software can be found at http://www.nslij-genetics.org/soft/, with links to their sources. this list continues to be mirrored at its previous site, http://linkage.rockefeller.org / soft/. it is perhaps over - inclusive, containing a number of obsolete programs, and it makes no recommendations. by contrast, the collection at http://www.hgmp.mrc.ac.uk/registered/menu/linkage.html contains only the most popular programs, but provides executable files, browsable documentation and a web - based graphical interface for the most common applications. | there is now a wide choice of software available for linkage analysis. the most well known packages are briefly reviewed here. the package with the most extensive range of analyses is genehunter, but for many of its functions there are other programs with better performance. these include fastlink and vitesse for parametric analysis allegro and merlin for non - parametric analysis and solar for variance components analysis. the computational limits of current approaches can be improved with simwalk2 and the promising new superlink program. directions for future work include improved user interfaces and consensus formats for data input and exchange. |
during the last several years numerous transgenic animal models of alzheimer 's disease (ad) have been engineered to examine the effects of the two major ad neuropathological hallmarks, amyloid plaques, and neurofibrillary tangles (nft) on neurodegeneration. the vast majority of these ad transgenic mice overexpress a mutant human amyloid - beta (a) precursor protein (app) gene alone or in combination with a mutated presenilin (ps) gene resulting in the presence of brain extracellular amyloid plaques, which are mainly formed by the accumulation of insoluble a species [16 ]. since overexpression of amyloid - beta peptide did not recapitulate all of the neuropathological features of ad, additional models were created adding mutant tau transgenes. for example, to further evaluate the pathogenic mechanisms underlying nft formation, transgenic mouse models have been generated to harbor a mutant human tau gene found in frontotemporal dementia or pick 's disease (p301l or p301s). these mutant mouse models display nft - like structures consisting of abnormal cytoskeletal tau protein aggregates in the central and peripheral nervous systems [4, 710 ]. recently, a triple transgenic mouse (3xtg - ad) harboring the human appswe, ps1m146v, and taup301l gene mutations was developed, displaying accumulation of both intracellular a and tau in an age - dependent manner within the cortex, hippocampus, and amygdala [1114 ], and to a lesser degree, in the brainstem. immunohistochemical studies using immersion - fixed 3xtg - ad mouse tissue have shown that intracellular a precedes the appearance of tau pathology, developing a deposits at 6 months and intraneuronal tau pathology at 9 months of age [11, 12, 14 ]. recent reports have demonstrated that the development of a plaques differed between 3xtg - ad mouse colonies as well as between male and female mice [16, 17 ]. furthermore, rostral - caudal differences in the onset of tau pathology have been reported, but only in male 3xtg - ad mice. therefore, in the present study we performed a systematic detailed evaluation of the evolution of tau conformation and phosphorylation events beginning at 3 weeks of age using perfusion - fixed tissue at the light and electron microscopic level to more completely define the cascade of amyloid and tau pathology in male and female 3xtg - ad mice. the data derived from this study provide novel information underlying the temporal progression of amyloid and tau pathology within the cortex, hippocampal / subicular complex, and the amygdala that is pivotal in determining the selective vulnerability of neurons during the life span of male and female 3xtg - ad mice. this data is critical for the design of future experiments to address pharmacological, mechanistic, behavioral, and gender questions in studies using this widely used mouse model of ad. a colony of homozygous 3xtg - ad and nontransgenic (ntg) mice were generated from breeding pairs provided by dr. these transgenic mice harboring the human appswe, ps1m146v, and taup301l mutations exhibit intraneuronal and extracellular amyloid pathology as well as tau pathology. at least 4 male and 4 female juvenile (3 weeks), young (2 - 3 months), adult (46 months), middle - aged (8 - 9 months), and old (1820 months) 3xtg - ad and non - transgenic (ntg) mice were examined. in addition young, middle - aged and old female 3xtg - ad mice were used for electron microscopic examination. animal care and procedures were conducted according to the national institutes of health guide for care and use of laboratory animals. mice were anesthetized with an injection of ketamine / xylazine (100 mg / kg/5.0 mg / kg) and transcardially perfused for 2 minutes with 0.9% physiological saline followed by a solution containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 m phosphate buffer (pb) for 5 minutes (~50 ml) and then post - fixed in the same solution for 24 hours at 4c. since many transgenic mice studies use immersion - fixed brain tissue and considering that tau antigenicity is time and fixation sensitive [1826 ], another group of mice was transcardially perfused with physiological saline and their brains hemisected and immersion - fixed for 24 hours in the same fixation solution. all brains were cryoprotected in 30% sucrose, sectioned on a sliding microtome at 40 micron thickness, and stored in a solution consisting of 30% glycerin, 30% ethylene glycol, in 0.1 m phosphate buffer at 20c until processed for immunohistochemistry. tissues from immersion or perfusion fixed brains were processed as free - floating sections and immunostained for anti - happ / a reactive amino acid residue 117 of beta - amyloid (6e10 ; 1 : 2,000 dilution, covance, n.j.), the tau conformational antibodies alz50 (66kd), mc1 (66kd) (1 : 10,000, 1 : 250 dilution, resp., both gifts from dr. peter davies, albert einstein school of medicine, n.y.), and the phosphoepitope tau antibodies at180 (~66 kd), at8 (~66 kd) (both at 1 : 1000 dilution, thermofisher, waltham, ma) and phf-1(5767 kd) (1:10000 dilution ; gift from dr. peter davies, the specificity of each of the antibodies used in this study was previously characterized by western blot by others (6e10 (covance), at180 [27, 28 ], at8, phf-1 [3032 ], alz50 [3336 ], and mc1 [34, 35 ]). prior to staining, sections were washed 3 10 min in phosphate buffer and 3 10 min in tris - buffered saline (tbs) to remove cryoprotectant before a 20-minute incubation in 0.1 m sodium metaperiodate (sigma, st. tissue was then permeablized 3 10 minutes in tbs containing 0.25% triton - x (thermofisher, waltham, ma) and blocked in the same solution containing 3% goat serum for 1 hour. sections were incubated with appropriate antibody dilutions overnight on an orbital shaker at 45 rpm at room temperature in 0.25% triton x-100, 1% goat serum solution. the next day, tissue was washed 3 10 min in tbs containing 1% goat serum prior to incubation with appropriate secondary antibody (see table 1) at a 1 : 200 dilution for 1 hr. following 3 10 minutes washes in tbs, sections were incubated in vectastain abc kit (vector labs, burlingame, ca) in tbs for 1 hour. tissue was then rinsed 3 10 minutes in 0.2 m sodium acetate, 1.0 m imidazol buffer, ph 7.4, and developed in acetate - imidazol buffer containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride (dab, sigma, st louis, mo). for comparison across ages sections from animals of different ages were immunostained at the same time and with the same duration of dab reaction. the reaction was terminated in acetate - imidazol buffer, tissue mounted on glass slides, dehydrated through graded alcohols (7095100%, 3 5 min), cleared in xylenes (3 5 min), and coverslipped with dpx (biochemica fluka, switzerland). cytochemical control sections consisted of (1) tissue from ntg mice which were processed in a manner identical to the immunohistochemical procedures described above for 3xtg - ad animals, (2) tissue from 3xtg - ad as previously described with the exception of the primary antibody, and (3) a preadsorption control consisting of a 100 fold amount of a 1 - 42 (us peptides, rancho cucamonga, ca) incubated with 6e10 in tbs containing 0.25% triton x-100, 1% goat serum overnight at room temperature. the preadsorbed serum was used in place of the 6e10 antibody in the immunohistochemistry protocol. since the antigen used to create the tau monoclonal antibodies obtained from commercial sources was unavailable, only the 6e10 preadsorbtion control was performed. selected sections were fluorescently double - labeled for 6e10 and alz50 using the above protocol with the following modifications. all steps prior to incubation of primary antibody were as described above with the exclusion of the quenching of endogenous peroxidase activity with 0.1 m sodium metaperiodate. tissue was incubated with primary antibody overnight (6e10 ; 1 : 200 dilution). after rinsing 3 10 minutes in tbs, tissue was incubated with cy3-conjugated goat antimouse igg fc subclass 1 specific secondary antibody (jackson immunoresearch ; west grove, pa) for 2 hours in the dark. sections were washed 3 10 minutes in tbs and then incubated with alz50 (1 : 1000) overnight in the dark. tissue was again rinsed 3 10 minutes and then incubated in cy2-conjugated goat antimouse igm -chain specific secondary antibody (1 : 200 dilution) for 2 hours in the dark. immunofluorescence was visualized using a zeiss axioplan 2 microscope using excitation filters at wavelengths 489 and 555 nm and emission filters at 505 and 570 nm for cy2 and cy3, respectively. florescent images were stored on a computer and brightness and contrast was enhanced using gimp version 2.6.7. double immunofluorescence staining for 6e10 and phosphotau markers were not successfully achieved using methods to block the cross reactivity between monoclonal igg1 subclass primary antibodies (see table 1). to determine ultrastructural localization of intraneuronal a and tau immunoreactivity, we performed an immunoelectron microscopic analysis of hippocampal / subicular neurons obtained from female homozygous at 2- (n = 2), 9- (n = 2), and 23- (n = 2) month - old 3xtg - ad mice. each animal was transcardially perfused with 4% paraformaldehyde/0.1% glutaraldehyde in phosphate buffer, brains were removed from the skull and immersion post - fixed in the same fixative overnight at 4c and cut into 80 micron thick horizontal sections using a vibratome-1500 (vibratome, saint - louis, mo). tissue containing the hippocampal / subicular complex was immunostained using the antibodies directed against a/app (6e10, 1 : 2000 dilution), the conformation specific tau antibody alz50 (1 : 10,000 dilution), and the phosphospecific (ser202/thr205) antibody at8 (1 : 1000 dilution). the chromogen dab was used to visualize each antibody. immunostained sections were post - fixed in 1% oso4 for 4560 minutes, dehydrated and embedded in epoxy resin. immunoreactive profiles within the hippocampal / subicular complex were selected using light microscopy, ultrathin sections (5070 nm) were cut using a leica ultracut uct (leica microsystems inc, bannockburn, il) microtome, and the majority of sections counter - stained with 2% uranyl acetate and lead citrate. immunostaining appeared as an electron - dense precipitate (dark - black) in neurons and other profiles within hippocampal - subicular complex of mutant mice. cortical brain tissue obtained at autopsy from an 82-year - old male ad patient was immersion fixed in 4% paraformaldehyde and processed for tem without antibody staining to compare the ultrastructure of human ad pathology to that found in 3xtg - ad mice. in the present study forebrain sections from male and female 3-week, 24, 5 - 6, 8 - 9, and 1820-month - old 3xtg - ad and age - matched ntg mice were immunostained using well - characterized antibodies directed against 6e10, tau conformational epitopes (alz50 and mc1), and phosphotau epitopes at180 (phosphothreonine 231), at8 (phosphoserine 202/205), and phf-1 (phosphoserine 396/404). in general, an age - related increase in intraneuronal and neuropil immunoreactivity in the hippocampal - subicular complex and amygdala were seen for all antibodies examined (figure 1). the exception was mc1, where immunoreactivity decreased after 24 months of age (see figure 6). our findings revealed that transcardial but not immersion fixation dramatically enhanced the immunovisualization of at8 (figures 1(e)1(h)) and phf-1 (figures 1(f)1(l)) as early as 3 weeks of age. by contrast, at180 and alz50 immunoreactivity was not affected by the fixation procedure. therefore all morphological analyses and photomicrographs subsequent to figure 1 were derived from transcardially fixed tissue. the colocalization of 6e10 and tau antibodies using fluorescence was achieved successfully for 6e10 (igg1) and alz50 (igm), but not for 6e10 and at180 (igg1) or at8 (igg1) due to the cross reactivity of their primary antibodies. therefore, the comparative analyses of the distribution of 6e10 and phosphotau markers were performed using dab - reacted tissue sections at the light microscopic level. all immunohistochemical controls failed to display cellular or plaque reactivity beyond background levels for any tau antigen or 6e10 independent of fixation, age, and gender. the 6e10 antibody, which recognizes amino acids 38 of the a sequence, revealed the greatest number and topographic distribution of immunoreactive (-ir) neurons in 3xtg - ad mice compared to the other antibodies we examined. there was an age - related increase in the extent of 6e10-ir neurons in all regions examined (figure 2). beginning at 3 weeks of age, intraneuronal 6e10-ir neurons were found in lamina 3 and 5 of the fronto - parietal cortex, deep layers of the cingulate cortex, ca fields of the hippocampus, subiculum, and the basolateral amygdala (bla) in 3xtg - ad mice at each age independent of gender (figure 2). 6e10-ir neurons, particularly in the hippocampal ca1 subfield, appeared round or oval in shape with many cells showing a thin ring of immunoreactivity located within the somatodendritic compartment (figures 3(a), 3(d), and 3(g)). however, a number of cells in the cortex and amygdala had thickened perinuclear staining (figures 3(d) and 3(a)), which became more prominent by 9 months of age (figure 3(b) and 3(h)). beginning at 9 months of age there was neuronal shrinkage of 6e10-ir neurons in 3xtg - ad mice (figures 3(b), 3(e), and 3(h)), which continued at least until 1820 months of age where many neurons displayed blunted dendrites, as well. although no dramatic gender differences in intraneuronal 6e10 staining were seen prior to 8 - 9 months, a plaque deposits were visualized for the first time in the subiculum of 8 - 9-month - old female, but not male mutant mice (data not shown). by 1820 months both genders displayed a plaque deposits in the subiculum (figures 3(j), 3(k), 3(m), and 3(n)) and hippocampus (data not shown). a plaques appeared smaller and more diffuse in male than seen in the female subiculum of 3xtg - ad mice (figures 3(j) and 3(k)). in fact, female ca1 neurons lost their dendritic 6e10-ir extensions into the substantia radiatum (figure 3(o)) while similar processes remained present in male mutant mice (figures 3(l) and 3(o)). the alz50 antibody recognizes an early stage conformational tau epitope that labels neurons undergoing early degenerative events associated with nft formation but has also been shown to label neurons in normal brains [3337 ]. similar to 6e10 staining, the number of alz50-ir neurons increased with age in all regions examined (figure 4). as early as 3 weeks of age alz50-ir staining was seen predominantly within the more caudal and ventral aspects of the hippocampal ca1 pyramidal and subicular neurons, whereas a few scattered alz50-ir neurons were found in the neocortex and bla (figures 4(a)4(d)). in addition, alz50-ir neurons were also found in the septal but not temporal portions of the hippocampal formation (data not shown). beginning at 24 months of age a few scattered alz50-ir neurons were found in layers 3 and 5 of the fronto - parietal and the deep layers of the cingulate and retrosplenial cortex in 3xtg - ad mice (figures 4(e)4(h)). by 5 - 6 months of age there was an apparent increase in the number of alz50-ir neurons in the ventral hippocampus and amygdala (figures 4(i)-4(l)), as well as more intense neuropil staining (figures 1(q) and 1(r)). however, unlike the continuous distribution of 6e10-ir pyramidal neurons in the hippocampal complex, the topographic distribution of labeled alz50-ir ca1 pyramidal neurons spread in dorsal and anterior directions from the ventral and caudal regions of the brain (figure 4). at 3 weeks of age alz50-ir neurons in the cortex, hippocampus, and amygdala appeared round or oval and displayed a dense rim of perinuclear staining (figures 5(a), 5(d), and 5(g)) with long dendrites extending into the neuropil. at 9 months of age many alz50-ir neurons appeared shrunken with a distorted cellular morphology in the hippocampal complex and amygdala (figures 5(e) and 5(h)). interestingly, some cells no longer displayed perinuclear staining but became completely filled with alz50 reaction product (figure 5(h)) or were dystrophic (data not shown). while the presence of alz50-ir dystrophic neurites appeared concomitantly with the onset of a plaque deposition between 8 and 9 months of age in female mutant mice, dramatic differences between male and female alz50 staining were only apparent by 1820 months of age. the dorsal and anterior subicular region adjacent to the ca1 neurons was most affected with alz50-ir dystrophic neurites in male and female 1820-month - old 3xtg - ad mice (figures 5(j) and 5(m)). in general, numerous large and globular dystrophic alz50-ir neurites were seen surrounded by plaques in both male and female mice (figures 5(m) and 5(n)). in female transgenic mice many plaques displayed a rim of dark alz50-ir dystrophic neurites with a clear center (figure 5(k)), while in males this neuritic organization was less obvious (figure 5(n)). in addition, in 1820-month - old female 3xtg - ad mice dark, swollen, alz50-ir dystrophic neurons were found in abundance among more lightly stained ca1 pyramidal cells (figures 5(m) and 5(o)). furthermore, the amygdala displayed similar neuronal perinuclear staining in male and female transgenic mice with an occasional rim of alz50-ir dystrophic neurites surrounding plaques only in female mutant mice (data not shown). the mc1 antibody recognizes early stage conformational epitopes similar to alz50 but does not react with normal neuronal tau [34, 35, 37 ]. in contrast to alz50 staining, lightly stained mc1-ir neurons were found only in the ca1 pyramidal cells of the ventral hippocampus in 3-week - old 3xtg - ad mice (figure 6(a)). by 24 months, lightly stained mc1-ir neurons were seen throughout the ca1 subfield of the ventral hippocampus (figure 6(b)). a few darkly stained mc1 neurons were seen, some of which appeared shrunken with blunted dendrites within the hippocampus. on the other hand, no mc1-ir neurons were seen in the bla at 3 weeks of age (figure 6(d)). in 24-month - old mutant mice, scattered round - or - oval shaped lightly stained mc1-ir neurons were found embedded within the mc1 immuopositive neuropil of the bla. interestingly, mc1-ir neuropil and neuronal labeling decreased in the hippocampus and bla in 8 - 9-month - old mutant mice (figures 6(c) and 6(f)). mc1-ir profiles were not detected in the neo- and limbic cortex up to 9 months of age. by 1820 months, mc1 labeling matched alz50 immunoreactivity in male and female 3xtg - ad mice. although plaque - like structures were hard to detect, globular mc1-ir dystrophic neurites and neurons were found mainly throughout the female subiculum (figures 6(j), 6(k), and 6(l)). some dystrophic neurons appeared to have eccentrically located dark cytoplasmic neuronal inclusions (figures 6(i), 6(k), and 6(l)). hyperphosphorylated microtubule - associated protein tau is the major component of the paired helical filament seen in the nft. at8-ir neurons were found in the neocortex, amygdala, and hippocampal formation in both male and female 3xtg - ad mice at all ages examined (figure 7). like the distribution of alz50-ir neurons, hippocampal and subicular regions contained the highest number of at8-ir neurons compared to the cortex and amygdala in 3-week - old 3xtg - ad mice (figures 7(a)7(d)). a few at8-ir neurons were observed in layer 5 of the fronto - parietal cortex and bla (figures 7(a)7(c)). as mutant mice age, the extent of neuronal at8 labeling in the hippocampal complex increased in a ventral to dorsal and posterior to anterior gradient (figures 7(e)7(l)), but unlike the alz50-ir profiles at 5 - 6 months of age, anterior portions of the hippocampal complex did not appear to be affected (figures 7(c), 7(g), and 7(k)). at 3 weeks of age, at8-ir neurons displayed more diffuse cytoplasmic staining in the cortex, hippocampus, and bla (figures 8(a), 8(d), and 8(g)), rather than the dense perinuclear staining observed in alz50-ir neurons in these regions. however by 9 months of age at8-ir neurons appeared shrunken, distorted, and showed thicker perinuclear staining, especially in the amygdala (figures 8(b), 8(e), and 8(h)). some neurons in the hippocampus (figure 8(e)), bla (figure 8(h)), and subiculum displayed blunted at8-ir dendrites. dystrophic neurons were more abundant in the female subiculum and constituted the greatest difference between male and female pathology at 1820 months of age (figures 8(j)8(o)). while the entire extent of the female subiculum showed numerous at8-ir dystrophic neurites, the male subiculum contained less neuritic dystrophy (figure 8(j) versus figure 8(m)). at8-ir pyramidal cells displayed an asymmetric accumulation of dense perinuclear staining (figures 8(k) and 8(o)), and at8-ir dystrophic neurites were associated with plaques (figures 8(l), 8(n)). at180 detects tau phosphorylation at the thr231 site, an early event in the assembly of tau into filaments [27, 28 ]. at180-ir neurons displayed the greatest number and spatial distribution of all tau antibodies examined at 3 weeks of age in 3xtg - ad mice. at180-ir neurons were found in layer 5 of the fronto - parietal cortex, bla, ca1 ventral hippocampus, and subiculum (figures 9(a), 9(d), and 9(g)). qualitatively there was an age - related increase in the number of at180-ir neurons within each brain region examined as well as in the intensity of neuropil staining in the ventral hippocampus and bla (figures 1(s)1(x)). at 3 weeks of age at180 immunostaining appeared as diffuse cytoplasmic reactivity in round or oval pyramidal cells of the neocortex, hippocampus (figure 9(a) and 9(d)) and bla (figure 9(g)) in 3xtg - ad mice. some neurons embedded in the ca1 pyramidal cell layer, bla, and subiculum displayed perinuclear staining (figures 9(d) and 9(g)). by 9 months, at180-ir hippocampal and bla neurons revealed blunted staining in dendrites (figure 9(e)). in the bla, differences between male and female at180 staining occurred in the hippocampal ca1 neurons and the subiculum at older ages. dystrophic neurites were more widespread within the subiculum in female compared to male 3xtg - ad mice (figures 9(j) and 9(m)). however, in male mice, at180-ir dystrophic pathology was mainly found in the subiculum closest to the border with the ca1 pyramidal cell layer. in addition, ca1 pyramidal neurons displayed a dense perinuclear staining and blunted dendrites (figures 9(l) and 9(o)). phf-1 recognizes tau phosphorylated at serine residues 396 and 404 and is generally considered a late marker in the evolution of phf positive nfts [3032 ]. at 3 weeks of age only a few lightly stained phf-1-ir neurons were observed in the ca1 region of the ventral hippocampus and the bla in perfusion fixed 3xtg - ad mice (figures 10(d) and 10(g)). at 2 - 3 months, a few phf - ir neurons were seen at the ca1/subicular interface of the dorsal hippocampus (data not shown). by 9 months, the number of phf-1-ir ventral hippocampal ca1 pyramidal and bla neurons increased (figures 10(e) and 10(h)). the distribution of phf - ir neurons in the hippocampal complex followed a ventral to dorsal gradient. up to 9 months of age, an occasional phf-1-ir neuron was visible in the neocortex (figure 10(b)). phf-1 dystrophic neurons and neurites were more prevalent in the subiculum of aged females (figures 10(m) and 10(n)) compared to aged males (figures 10(j) and 10(k)). phf-1-ir ca1 neurons displayed distorted cellular morphology, with broken, asymmetric, but strongly stained perinuclear staining in the female subiculum (figure 10(o)). in contrast, phf-1-ir neurons in the male subiculum showed similarly stained dark neurons but retained somatodendritic staining (figure 10(l)). to evaluate whether neurons expressing 6e10 also contained tau epitopes we performed dual immunofluorescence and comparative light microscopic experiments on adjacent hippocampal subicular sections at 3-week, 9-, and 1820-month old male and female 3xtg - ad mice. although it would have been interesting to double stain for 6e10 and other tau makers (at180 and at8), all three antibodies are murine igg1s. therefore, dual immunofluorescence experiments were performed only for 6e10 and alz50, an igm. in these experiments we observed two bands of 6e10 immnoreactivity located within ventrally located within ca1 hippocampal and subiculum neurons of female mutant mice at all ages examined (figures 11(a), 11(d), and 11(g)). by contrast, alz50-ir neurons were only observed in the ca1 pyramidal cell layer, and as neuropil staining in the subiculum (figures 11(b), 11(e), and 11(h)). in general, alz50-ir neurons were found to be colocalized with 6e10-ir neurons at each age examined in the ventral hippocampus (figures 11(c), 11(f), and 11(i)). because 6e10, at180, or at8 are all of the murine igg1 subclass, adjacent sections through the ca1 and subicular fields were reacted with individual antibodies and visualized using peroxidase / dab. in contrast to the single layer of alz50-ir neurons in the ca1 pyramidal cell layer (figures 12(b), 12(e), and 12(h)), numerous at180, at8, and 6e10 immunopositive neurons were seen in both ca1 and subicular fields in the ventral hippocampus at each age examined (figure 12). at the ultrastructural level, unstained tissue sections revealed plaques within the hippocampal / subicular complex that displayed numerous fibrillar branches, which were surrounded by dystrophic neurites in 9-month - old 3xtg - ad mice (figure 13(a)). in 23-month - old female 3xtg - ad mice, the majority of neuritic plaques displayed a central core surrounded by many more fibrillar branches and dystrophic neurites, similar to that seen in human ad plaques (figure 13(c)). the neuropil contained dystrophic neurites of different sizes displaying various accumulations of electron - dense cytoplasmic material, multilaminar, multivesiclular, and dense - core bodies in both 9-and 23-month - old 3xtg - ad mice (figures 13(a) and 13(b)), similar to that seen in an ad plaque (figure 13(d)). tem analysis of intraneuronal 6e10 immunoreactivity revealed staining mainly confined to the periphery of the nucleus within ca1 hippocampal / subicular neurons in 2-month - old 3xtg - ad mice (figure 14(a)). at 9 months of age this perinuclear 6e10 reaction product appeared more intense and extended into the cytoplasm (figure 14(b)). at the ultrastructural level, alz50 and at8-ir were seen in the soma and dendrites of ca1 neurons in female 2-month - old mutant mice (figures 14(c) and 14(d)). intraneuronal electron dense alz50 and at8-ir cytoplasmic staining was more widespread and increased in intensity in 9 month - old - mutant mice (figures 14(e) and 14(f)). interestingly, cytoplasmic alz50-ir appeared more extensive than at8 immunoreactivity in these neurons at both 2 and 9 months of age (figures 14(c), 14(d), 14(e), and 14(f)). despite the presence of at8 and alz50-ir cytoplasmic labeling within hippocampal / subicular ca1 neurons, tem failed to reveal any type of filamentous aggregates in 2- or 9-month - old 3xtg - ad mice (figures 14(c), 14(d), 14(e), 14(f), 14(l), and 14(m)). in contrast, at 23 months of age both male and female 3xtg - ad mice displayed alz50 and at8-ir straight filaments with a 1920 nm diameter within the hippocampal - subicular complex (figures 14(i)14(m)). previous studies have shown widespread amyloid but not tau pathology at 6 months of age in the hippocampus [3, 12, 13, 17 ], and behavioral studies using the morris water maze have demonstrated memory retention deficits dependent upon the appearance of amyloid pathology at 6- but not 2-month - old 3xtg - ad mice. our study found that (1) the appearance of 6e10 immunoreactivity was concomitant with conformation (alz50 and mc1) and phosphorylation (at8, at180, and phf-1) events in the neurons of the hippocampal - subicular complex and amygdala as early as 3 weeks of age ; (2) the detection of the two phospho - epitopes at8 and phf-1 was fixation dependent at all ages ; (3) the number of a plaques, as well as neuritic and neuronal dystrophy, increased with age in the cortex, hippocampus, and subicular area in 3xtg - ad mice ; (4) aged female mice displayed more plaque and tau pathology than aged male 3xtg - ad mice ; a finally (5) straight tau filaments were found only in 23-month - old female 3xtg - ad mice. in the present study, 6e10-ir neurons were seen as early as 3 weeks of age in lamina 3 and 5 of the fronto - parietal cortex, deep layers of the cingulate cortex, ca1 pyramidal neurons of the hippocampus, subiculum, and bla in 3xtg - ad mice independent of gender. these data indicate that intraneuronal 6e10 immunoreactivity occurs extremely early in the development of these mice (3 weeks) and are in contrast to previous investigations showing that 6e10 intraneuronal staining occurred between 3 and 6 months of age in the hippocampus of these mice [12, 17 ]. because the 6e10 epitope resides in amino acids 38 of the n - terminal portion of the a sequence, it does not preclude binding to full length app or its amyloidgenic derivatives. consequently, the exact amyloid species revealed within neurons at 3 weeks of age in 3xtg - ad mice and at other ages examined is unclear. oligomeric a species may, in part, underlie cellular degeneration [3, 11, 3848 ]. we also observed many shrunken 6e10-ir neurons beginning at 8 - 9 month of age in each brain area examined in both male and female 3xtg - ad mice. this phenotype was most pronounced in ca1 hippocampal neurons of older female 3xtg - ad mice. these neurons exhibited loss of dendritic 6e10 immunoreactivity when compared to ca1 neurons in male mutant mice. these gender differences in a intraneuronal pathology lend support to prior investigations showing that female 3xtg - ad mice display earlier and more severe plaque pathology, which may be related to progesterone and estrogen - mediated signaling. however, other factors may contribute to the discrepancies in ad - like pathology reported in studies using different colonies of 3xtg - ad mice, including a loss of phenotype related to successive breeding, founder effects between colonies, or differential expression of transgenes. together or separately, variations in transgenic animal strain or amyloid species may affect both pathological and behavioral observations reported in studies using 3xtg - ad mice. of interest was the observation that the immunohistochemical localization of phosphospecific tau proteins at8 and phf-1 was fixation dependent, whereas alz50, mc1, at180, and 6e10 immunoreactivity was fixation independent in young, juvenile, and middle age 3xtg - ad mice. notably, neurons containing the phospho - epitope at8 were seen in layer 5 of frontoparietal cortex, ventral hippocampal pyramidal, and amygdala neurons, whereas phf-1-ir intraneuronal staining was seen only in the ventral hippocampus in perfusion - fixed 3-week - old 3xtg - ad mice. previous biochemical and immunohistochemical studies indicate that fixative composition affects the ability to detect various cytoskeletal proteins [18, 22, 23 ]. the process of aldehyde fixation relies upon a slow cross - linking of carbonyl aldehyde to functional protein groups. while perfusion fixation rapidly exposes proteins to fixative through the vasculature, immersion fixation relies upon a slow 1 mm per hour diffusion rate to penetrate tissue. it is possible during immersion fixation that proteolysis or dephosphorylation of the tau phospho - epitopes recognized by at8 and phf-1 occurs prior to the full penetration of the fixative. since immersion - fixed tissue reacts with c - terminal, n - terminal, and internal tau antibodies, proteoloysis of tau is unlikely [25, 50 ]. a more plausible possibility is the destruction of at8 and phf-1 epitopes by the action of endogenous phosphatases during slow immersion fixation [5153 ]. likely, phosphatases act differentially depending on the individual tau phosphoepitopes ; hence, it is possible that the at180 phospho - epitope is less sensitive to phosphatase activity than either at8 or phf-1 phosphorylation sites, resulting in a more robust staining in immersion - fixed tissue. although long postmortem intervals reduce phospho - tau antigenicity due to active phosphatases, [19, 50, 55 ], this was not a confounding variable in the present study since all animals were sacrificed and perfused with fixative within 5 minutes of anesthetization. the potential effect of fixation on the immunolocalization of tau and other proteins represents an important caveat that may provide an explanation for the underlying differences in tau antigenicity reported in previous studies using the 3xtg - ad mouse model [3, 1214, 16, 57, 58 ]. although a previous report described intraneuronal mc1-ir ca1 dorsal hippocampal neurons only in 1215-month - old 3xtg - ad mice, the present study displayed weak mc1 immunoreactive neurons in the ventral hippocampus even as early as 3 weeks of age. we also observed alz50 immunoreactive ca1 pyramidal and subicular neurons in the ventral aspect of the hippocampal / subicular complex at 3 weeks of age. overall, alz50 immunoreactivity was much more intense and labeled a greater distribution of immunoreactive neurons than with the mc1 antibody. while alz50 immunoreactivity increased in an age dependent manner in the neocortex, hippocampus, and amygdala, mc1 immunoreactivity exhibited a biphasic response, peaking initially between 2 and 4 months, declining at 9 months and then peaking again at 1820 months in numerous swollen, tangle - like appearing ca1 hippocampal and subicular neurons. interestingly, both mc1 and alz50-ir neurons were found in the ca1 pyramidal but not the subicular cell layer in the ventral hippocampal formation at the ages examined suggesting that alz50 is labeling similar populations of neurons. unlike previous studies where various phosphotau epitopes were immunohistochemically detectable by 15 months of age in 3xtg - ad mice [12, 17, 49 ], we observed tau - ir neurons primarily in the ventral ca1 hippocampal pyramidal and subicular neurons at 3-weeks of age. this occurred well before the onset of plaque deposition in 8 - 9-month - old 3xtg - ad mice. the current findings indicate that intraneuronal amyloid and tau co - occur or appear concurrent in the same neurons at least as early as 3 weeks of age suggesting that these mutant mice are born with the dual expression of these proteins within select populations of brain cells. the early appearance of tau - ir neurons in the forebrain of 3xtg - ad mice is not surprising since overexpression of mutant p301l four repeat tau and the appswe amyloid mutation together or tau p301l alone induces an age dependent onset of mc1, at8, and phf - ir neurofibrillary tangles (nfts) in the telencephalon of mutant mice as young as 2.5 months of age. however, it remains to be determined whether amyloid interacts with tau over time to induce the apparent age - related perikaryal degeneration seen in these mice. studies are underway to test the hypothesis that depletion of intraneuronal amyloid at an early age would prevent or slow the degeneration of neurons in these mice. it is interesting to note that tau containing straight filaments were seen in 1820-month - old mutant mice suggesting that late - stage tau neuronal aggregation also plays a key role in the ultimate demise of neurons in 3xtg - ad mice. although no studies to date have shown frank neuronal cell loss, a recent report described a reduction in the number of neurons containing 6e10 and tau markers in the brainstem of these mutant mice. to more fully understand the vulnerability of hippocampal complex neurons a previous study colocalized ht7, a human specific pan tau antibody and m71/3 (oligomeric a) in the dorsal hippocampus in 3xtg - ad mice. our immunofluorescence colocalization experiments demonstrated that virtually all ventral ca1 pyramidal but not all subicular neurons contained both alz50 and 6e10 from 3 weeks to at least 1820 months of age, suggesting that ca1 neurons are more vulnerable to alz50 pathology. although we were unable to perform colocalization experiments between 6e10 and our tau antibodies, we performed a comparative light microscopic analysis of adjacent hippocampal sections. this study revealed at180 and at8-ir neurons in the caudal aspects of the subicular region where we failed to visualize alz50-ir neurons across all ages examined. the reason for the selective appearance of at180 and at8 in the ventral subiculum remains unknown. one possibility is that neurofibrillary pathology proceeds according to a sequence of tau conformational and phosphorylation events [6164 ], which may vary between neuronal populations. in ad, as tau transitions to a more hyperphosphorylated state, it undergoes a self - assembly process forming phf structures reducing the microtubule stability of the neuronal cytoskeleton [65, 66 ]. more specifically, the evolution of nfts in ad follows a progression of phosphorylated tau modifications from tg3 (phosphospecific at thr231), found in preneurofibrillary tangles (prenfts), to at8 and phf-1 in intraneuronal and extraneuronal nfts. this progression may be dynamic since intraneuronal at180 and at8 precedes alz50 tau formation before the appearance of fibrillar tau [63, 64 ]. we suggest that the sequence of cellular tau conformation and phosphorylation events may also be dynamic in 3xtg - ad mice since alz50-ir neurons were found only in ca1 neurons in the hippocampus but not in the underlying subiculum. despite differences in tau intraneuronal epitope expression, we found age - related changes in neuronal morphology in the 3xtg - ad mouse to be similar to the nft neuronal phenotypes described in the human ad brain [67, 68 ]. some at8-ir neurons at 9 months of age displayed blunted dendrites (see figure 8(e)) or tortuous proximal dendrites and thickened perinuclear staining reminiscent of braak type 2 neurons. in our aged mutant mice, proximal and distal dendrites lost at8 immunoreactivity and neurons appeared more globose and dystrophic similar to braak type 3 neurons, which accumulate fibrillar material in the human ad brain. furthermore, the present ultrastructural analysis revealed, for the first time, the presence of at8 and alz50 positive straight tau filamentous aggregates in female aged 23-month - old, but not in young or middle - age 3xtg - ad mice of the same gender, which are similar to those seen in tangle bearing neurons in the human ad brain. similar tau filaments have been described in single transgenic mice overexpressing the mutant p301l human tau gene [10, 59 ] as well as in the double mutant (tapp) mouse expressing appswe and p301l mutant forms of tau. nft formation was enhanced in young and middle age tapp mutant mice with respect to single tau transgenic mice. although young and middle - aged 3xtg - ad mice carry the same appswe, and taup301l mutations as the tapp mouse, 3xtg - ad mice did not accumulate filamentous aggregates at younger ages. in this regard, mice overexpressing human genomic and cdna tau genes also do not display intraneuronal filamentous structures at young ages. these findings suggest that factors other than the accumulation of tau underlie the filamentous tangle bearing phenotype seen in human ad. factors that may influence the formation of ad filaments include differences in the abundance or ratio of specific tau isotypes in different cohorts of neurons and/or minor alterations in proteases, kinases, phosphatases that alter the structure of these proteins during the life span of the neuron, and/or the promoter system designed for specific transgene expression in particular brain regions. since age is a major risk factor for ad, aging itself may be a crucial variable in the formation of intraneuronal filaments in 3xtg - ad mice. despite differences in the evolution of tau biochemistry, there are morphological similarities between neurons containing tau in 3xtg - ad mice and human ad indicating the usefulness of these mice for studies of the mechanism(s) underlying select aspects of ad pathology. | the age - related pathological cascade underlying intraneuronal tau formation in 3xtg - ad mice, which harbor the human appswe, ps1m126v, and taup301l gene mutations, remains unclear. at 3 weeks of age, at180, alz50, mc1, at8, and phf-1 intraneuronal immunoreactivity appeared in the amygdala and hippocampus and at later ages in the cortex of 3xtg - ad mice. at8 and phf-1 staining was fixation dependent in young mutant mice. 6e10 staining was seen at all ages. fluorescent immunomicroscopy revealed ca1 neurons dual stained for 6e10 and alz50 and single alz50 immunoreactive neurons in the subiculum at 3 weeks and continuing to 20 months. although electron microscopy confirmed intraneuronal cytoplasmic alz50, at8, and 6e10 reaction product in younger 3xtg - ad mice, straight filaments appeared at 23 months of age in female mice. the present data suggest that other age - related biochemical mechanisms in addition to early intraneuronal accumulation of 6e10 and tau underlie the formation of tau filaments in 3xtg - ad mice. |
bipolar disorder (bipolar affective disorder [bpad ]) is one of the most complex psychiatric conditions characterized by recurrent mood episodes and longitudinally varied course. it affects at least 1% of the population and according to the world health organization (who), bpad is the sixth leading cause of disability among illnesses worldwide. the traditionally accepted clinical conception of the course of bpad is that it is marked by time - limited acute episodes of mania and major depression, with occasional hypomanic and mixed episodes, with recovery back to euthymia. the classical concept also suggests favorable functional adaptation between episodes, with a marked decrease in acute morbidity with effective mood - stabilizing treatments. however, a number of recent studies have indicated that several patients with bpad, who no longer met the syndromal or symptomatic criteria following recovery from an acute affective episode, nevertheless continue to display functional impairment. quality of life (qol) and mental health have a mutual correlation in the sense that qol is a direct consequence of mental health. a study done by xiang. in 2010 concluded that social functioning is the main predictor of qol in psychiatric patients. several qol studies on bipolar patients revealed that they experience lower functioning and well - being even in the euthymic phase of the disorder. functional impairment in vocational and social adjustment is commonly encountered among patients diagnosed with bpad. the link between bpad and the important outcome measure of social disability is under - researched in india. this study attempted to assess the level of disability and qol in outpatients with bpad in remission and to identify the sociodemographic, clinical, illness, and treatment - related characteristics associated with poor outcome. consecutive patients, who satisfied the inclusion criteria, and attending the psychiatry outpatient department at mosc medical college, kolenchery, between august 2014 and august 2015 were contacted for participation. patients between 20 and 60 years belonging to either sex who agreed to give an informed consent, with a diagnosis of bpad currently in remission as per the international classification of diseases diagnostic criteria for research (icd-10 dcr) were recruited. to ensure remission, they required scores of < 8 and < 12 on the hamilton depression rating scale (ham - d) and the young 's mania rating scale (ymrs), respectively. also, episodes of mood disturbance should not be present over the past 2 months. organic mood (affective) disorders, seizure disorder, mental retardation, persistent neurological deficits, and other chronic debilitating medical illnesses such as chronic obstructive pulmonary disease, coronary artery disease, valvular heart disease, chronic liver disease, chronic kidney disease, and arthritis were exclusion criteria. the presence of any other psychiatric comorbidity or psychoactive substance use other than nicotine, amounting to harmful use / dependence was also considered exclusion criteria. the international classification of diseases-10 diagnostic criteria for research, derived from chapter v (f) of icd-10 was used to diagnose bpad. the criteria being deliberately restrictive were intended to maximize homogeneity of study groups and comparability of findings in various studies. the hamilton rating scale for depression-21 item developed by max hamilton in 1960, total scores range from 0 to 53 (the sum of the first 17 items). young 's mania rating scale (ymrs) is a clinician - rated scale to assess the severity of manic symptoms. information for assigning scores is gained from subjectively reported symptoms over the past 48 hours and observation during the interview. world health organization quality of life scale bref malayalam version (who qol - bref) is based on four domains physical, psychological, social relationship, and environmental. there are also two items about individual 's overall perception of qol and his / her health. world health organization disability assessment schedule 2.0 (who das) is constructed on the conceptual framework of the international classification of functioning, disability, and health. it assesses the level of functioning in the six major life domains : (i) cognition, (ii) mobility, (iii) self - care, (iv) getting along, (v) life activities, and (vi) participation in society. for ease of administration in the outpatient setting, the 12-item interviewer version was used. kuppuswamy 's socio - economic status scale is the most widely accepted scale in india to assess the socio - economic status of the study group. individuals were classified as belonging to lower, upper lower, lower middle, upper middle, or upper socio - economic status. a specially designed semi - structured proforma was used to collect sociodemographic, illness related, and treatment - related variables. consecutive patients who met the inclusion and exclusion criteria were recruited. the socio - demographic, illness related, and treatment - related variables were collected from the patients and/or the family members. mean and standard deviation were used to describe continuous variables, whereas frequencies and percentages were obtained for categorical data. pearson 's correlation coefficient and student 's t - test were employed to assess the statistical significance. statistical analysis was performed using the statistical package for social sciences (spss for windows, spss inc., patients between 20 and 60 years belonging to either sex who agreed to give an informed consent, with a diagnosis of bpad currently in remission as per the international classification of diseases diagnostic criteria for research (icd-10 dcr) were recruited. to ensure remission, they required scores of < 8 and < 12 on the hamilton depression rating scale (ham - d) and the young 's mania rating scale (ymrs), respectively. also, episodes of mood disturbance should not be present over the past 2 months. organic mood (affective) disorders, seizure disorder, mental retardation, persistent neurological deficits, and other chronic debilitating medical illnesses such as chronic obstructive pulmonary disease, coronary artery disease, valvular heart disease, chronic liver disease, chronic kidney disease, and arthritis were exclusion criteria. the presence of any other psychiatric comorbidity or psychoactive substance use other than nicotine, amounting to harmful use / dependence was also considered exclusion criteria. the international classification of diseases-10 diagnostic criteria for research, derived from chapter v (f) of icd-10 was used to diagnose bpad. the criteria being deliberately restrictive were intended to maximize homogeneity of study groups and comparability of findings in various studies. the hamilton rating scale for depression-21 item developed by max hamilton in 1960, total scores range from 0 to 53 (the sum of the first 17 items). young 's mania rating scale (ymrs) is a clinician - rated scale to assess the severity of manic symptoms. information for assigning scores is gained from subjectively reported symptoms over the past 48 hours and observation during the interview. world health organization quality of life scale bref malayalam version (who qol - bref) is based on four domains physical, psychological, social relationship, and environmental. there are also two items about individual 's overall perception of qol and his / her health. world health organization disability assessment schedule 2.0 (who das) is constructed on the conceptual framework of the international classification of functioning, disability, and health. it assesses the level of functioning in the six major life domains : (i) cognition, (ii) mobility, (iii) self - care, (iv) getting along, (v) life activities, and (vi) participation in society. for ease of administration in the outpatient setting, the 12-item interviewer version was used. kuppuswamy 's socio - economic status scale is the most widely accepted scale in india to assess the socio - economic status of the study group. individuals were classified as belonging to lower, upper lower, lower middle, upper middle, or upper socio - economic status. a specially designed semi - structured proforma was used to collect sociodemographic, illness related, and treatment - related variables. consecutive patients who met the inclusion and exclusion criteria were recruited. the socio - demographic, illness related, and treatment - related variables were collected from the patients and/or the family members. mean and standard deviation were used to describe continuous variables, whereas frequencies and percentages were obtained for categorical data. pearson 's correlation coefficient and student 's t - test were employed to assess the statistical significance. statistical analysis was performed using the statistical package for social sciences (spss for windows, spss inc., majority of patients were males, and were middle - aged, married, educated, employed, hailing from nuclear families in rural habitat, and belonging to lower socio - economic class. they had early age of onset of illness, with multiple affective episodes, with psychotic symptoms during these episodes. socio - demographic and clinical characteristics the patients had a mean total who - disability assessment scale score of 19.2 2.09. the maximum subscore was in domain 4 (4.04 0.57), i.e. domain of getting along (ability to interact with other people) followed by domain 2 (mobility, i.e., ability to move and get around). disability was least in the domain 5, i.e., life activities (mean total score of 2). the subjects had a poor qol with a mean total who - qol bref score of 54.26 2.85. the qol was worst in the domain 3, i.e., social interactions (11.99 1.79) and was better was in the domain 1 (physical, 14.83 1.12). the following variables were statistically significantly related to disability on bivariate analysis : gender, age, marital status, not an earning member, age of onset of illness, duration of illness, number of depressive and hypomanic episodes, history of deliberate self harm, and qol score. negative earning member status, and qol scores were the predictive variables as per multiple linear regression done after adjusting for age and gender. disability scores increased with increasing age, among females and in those who were not the bread - winners of their family, while it was inversely related to qol scores. factors associated with world health organization disability assessment schedule ii score table 3 includes the factors associated with qol score. the following variables were significantly related to qol on bivariate analysis : age, duration of illness, total duration of ill period, history of deliberate self - harm, and who das score. however, only age and who das scores remained statistically significant after adjustment using multiple linear regression. factors associated with world health organization quality of life - bref score therefore, disability worsened with increasing age, among females and in those who were not bread - winners of their family, while it was inversely related to qol scores. a significant proportion of patients with bipolar disorder in remission had disability and poor qol. this has been demonstrated in previous studies which suggested the need to identify disability and qol issues among bipolar patients who are in remission and are asymptomatic. contrary to a previous study, increasing age was associated with increased disability and low qol. disability was also associated with gender and not being an earning member of the family. social pressures based on gender and livelihood issues form important stress for people with mental illness. women living within patriarchal societies are prone to extreme stress and need help and support in addition to attempts at changing social norms. livelihood issues, particularly in countries without welfare measures, are sources of severe stress. examining and evaluating interventions to reduce disability and to improve qol are crucial in the successful management of people with bipolar illness. while much has been achieved in reducing symptoms using psychotropic medications, a lot more is needed to be done to reduce disability and to improve qol in people with mental illness. the strengths of the study are the inclusion of consecutive cases and a relatively larger number of people with bipolar disorder in remission. however, being a male who could earn for himself and family was found to be associated with lower disability level and better qol. planning and implementing measures to empower bipolar patients to meet the demands of day to day living and identifying the reversible physical and psychological causes of poor outcome in females may help to improve the outcome. health insurances and policies should be adopted to meet the treatment expenses, and opportunities for better education and employment of the bpad population as well as the ' at - risk ' group are areas that need to be worked upon. to conclude, the authors identify the need for more longitudinal studies to generalize these findings. | background : despite significant advances in pharmacological and psychological therapies for bipolar disorder, many people continue to have less than optimal outcomes, which are associated with significant disability and poor quality of life (qol). this study aimed to assess the disability and qol and factors associated with such suboptimal outcomes in subjects with bipolar disorder in remission.methods:consecutive patients diagnosed to have bipolar disorder in remission attending the department of psychiatry, mosc medical college, kerala, india were recruited for the study. they were assessed using the international classification of diseases diagnostic criteria for research-10, hamilton scale for depression, young 's mania rating scale, world health organization - qol (who qol - bref), who - disability assessment scale (who - das), and kuppuswamy 's scale for socioeconomic status assessment.results:eighty-four patients were evaluated. the mean total who - das score was 19.2 2.09, the maximum disability in domain 4 (getting along) followed by domain 2 (mobility). the mean total who - qol bref score was 54.26 2.85, the lowest subscore in domain 3 (social interactions). disability scores were significantly associated with increasing age, female gender, not being an earning member of the family, and lower qol scores. poorer qol scores were significantly associated with increasing age and higher disability score.conclusions:many bipolar patients in remission have significant disability and poorer qol. there is a need for longitudinal studies to explore such associations and develop interventions to reduce the disability thereby enhancing the qol. |
the boston type 1 keratoprosthesis (kpro) has undergone significant advances in prosthetic design and surgical technique in the last decade. these changes have been accompanied by rising numbers of kpro implantation for expanding etiologies, and even the adoption of the kpro as a primary procedure for selected indications. internationally, single and multicenter groups have published short- and intermediate - term kpro outcomes with attention to specific conditions. we will review special considerations regarding patient selection, preoperative evaluation, surgical planning, and postoperative follow - up for boston type 1 kpro. the success of the device begins with appropriate patient selection for kpro implantation. for a potential candidate, the surgeon should consider the etiology of vision loss, visual acuity of both the operative and fellow eye, lid position and ocular surface status, and concomitant ocular and systemic diseases. patient reliability is an important factor, as compliance with medications and follow - up is critical for device retention. outcomes of the boston type 1 kpro have been associated with the preoperative diagnosis (table 1). studies suggest patients with stevens johnson syndrome and ocular cicatricial pemphigoid fare worse than those with non - cicatrizing etiologies, such as repeated graft failure for infections or dystrophies, with chemical burns and aniridia falling somewhere in the middle of the spectrum.1,2 the boston type 1 keratoprosthesis study group identified three risk factors for kpro device loss, including autoimmune etiology (eg, ocular cicatricial pemphigoid, stevens johnson syndrome), ocular surface exposure requiring tarsorrhaphy, and increased number of prior failed penetrating keratoplasties.3 given the poor prognosis of a traditional penetrating keratoplasty in some conditions, such as those with limbal stem cell deficiency, kpro as a primary procedure may be considered in certain patients.4 device survival is shortened in patients with poor ocular surface stability, whether from cicatrizing disease or limbal stem cell dysfunction (lscd), with increased rates of complications. johnson syndrome and ocular cicatricial pemphigoid, is an identified risk factor for endophthalmitis following kpro placement.5 similarly, increased rates of sterile keratolysis are reported in patients with underlying autoimmune disease and retroprosthetic membranes (rpms).6 rpm development, in turn, is associated with aniridia and infectious keratitis as the indication for kpro placement.7 although autoimmune diseases were initially considered a relative contraindication due to the heightened risk of complications, recent improvements in postoperative management have allowed increased confidence in the placement of kpro in very select patients with reasonable tear production and controlled ocular surface inflammation.8,9 however, the surgeon should proceed with caution, armed with a tailored management plan, after discussing the associated risks with the patient.8,9 regardless of the specific indication, a detailed discussion of the procedure should be carried out with all patients prior to surgery, including the risks of device extrusion, endophthalmitis, and glaucoma progression, as well as realistic expectations of visual rehabilitation. of particular importance is that the patient understands the commitment to frequent postoperative visits, as well as the necessity for indefinite postoperative prophylactic topical medications. a thorough office examination is a key step in successful kpro placement, including an assessment of visual potential. the world health organization criteria stipulates that a patient be monocular or functionally blind in both eyes to be considered eligible for a boston kpro ; however, many surgeons now offer kpro surgery despite relatively good vision in the contralateral eye. this evolution in practice pattern is largely due to improving rates of postoperative visual function, restoration of binocularity, and enhanced cosmesis.10,11 a recent study evaluated patient responses to the national eye institute visual function questionnaire 25 (nei vfq-25) following kpro implantation, and found a significant improvement in quality - of - life measures even in those patients with vision better than 20/200 in their nonsurgical eye.12 to optimize outcomes, patients should possess certain essential characteristics, including adequate lid anatomy, good blink function, controlled ocular surface inflammation, and a sufficiently moist ocular surface. fornices should be evaluated and able to accommodate a large bandage contact lens after surgery. long - term use of a bandage lens postoperatively can prevent drying of the ocular surface, dellen formation, and corneal melts, and absence of a lens is an independent risk factor for postoperative complications of infections and corneal melts.13 thus, some patients may benefit from fornix reconstruction as a first stage procedure in preparation for implantation of the boston kpro (figure 1). oculoplastic consultation may also be indicated in patients with poor lid closure or lagophthalmos to minimize exposure. ocular surface stability is integral to device survival.5 tear production can be measured with schirmer testing, and goblet cell and meibomian gland function evaluated to assess all three layers of the tear film. limbal stem cell function is also critical, and often compromised in patients with stevens johnson syndrome, aniridia, chemical burns, and ocular cicatricial pemphigoid. clinical findings of lscd include peripheral corneal vascularization, advancement of conjunctival epithelium onto the cornea, and the presence of goblet cells on the corneal surface.14 if lscd is suspected, strategies for epithelial healing must be part of the surgical plan. conjunctival scarring should be noted, as it may affect contact lens fit or placement of a planned tube shunt. a thorough history can help the surgeon determine the possibility of significant optic nerve dysfunction, retinal pathology, or dense amblyopia. b - scan ultrasonography should be performed to evaluate retinal anatomy if the posterior segment can not be visualized. it is critical to assess the adequacy of intraocular pressure (iop) control preoperatively to determine the need for additional glaucoma procedures. a more complete picture can be established by collecting multiple iop measurements, the number and duration of topical antihypertensives, and history of any prior filtering procedures or glaucoma lasers. physicians may attempt to quantify preexisting optic nerve damage, but optic nerve head optical coherence tomography (oct) and automated visual field testing are often precluded by poor preoperative vision and a limited view to the posterior segment. a confrontation visual field should be done to ensure a patient has light perception with projection. peripheral anterior synechiae and angle anatomy can be evaluated by anterior segment oct (as - oct), if available. lens status must be determined to order the boston type 1 kpro with the appropriate refractive power. the boston kpro is available in either an aphakic or pseudophakic version, and surgeons should consider preoperatively which of these will be implanted. if a patient is phakic, a lensectomy must be performed, and an aphakic kpro is implanted or an intraocular lens with a pseudophakic model according to the surgeon s preference. as in standard cataract surgery, the goal should be total preservation of the posterior capsule. if the patient is pseudophakic, implant stability and chamber depth should be assessed and can be evaluated with as - oct or ultrasound biomicroscopy. stable posterior chamber lenses may be left in place ; however, anterior chamber or unstable lenses should be explanted. if an aphakic kpro is planned, the axial length of the eye should be measured by a scan. the boston type 1 kpro is available in 0.5 mm increments of axial length (figure 2).15 the kpro surgeon and patient benefit from access to a multispecialist team familiar with kpro implantation and follow - up, as these patients commonly need care for glaucoma, oculoplastic, and retinal issues. often, several procedures can be combined to allow the best chance for visual recovery, device retention, and iop control. procedures can be carried out during or prior to kpro implantation, in efforts to minimize postoperative inflammation. several strategies are available to promote epithelial healing in patients with lscd or a dry, inflamed ocular surface. persistent epithelial defects, a frequent complication in lscd patients, must be taken seriously as they are associated with sterile keratolysis and device extrusion.16 temporary or permanent partial tarsorrhaphies may be considered at the time of kpro implantation. ocular surface toxicity from the postoperative drop regimen can be minimized by using preservative - free formulations and limiting the number of topical medications. an adequate contact lens fit is necessary, and a contact lens specialist familiar with keratoprostheses is invaluable, as often multiple fittings or a custom lens may be required.17 scleral or hybrid lenses may be useful in challenging patients in whom other measures fail. uncontrolled iop or the use of maximum medical antihypertensive therapy preoperatively should alert the clinician to consider a simultaneous glaucoma procedure, such as a tube shunt, at the time of kpro implantation. in addition, glaucoma drainage device implantation can be also considered for aniridic patients without a history of glaucoma given that their high risk of glaucoma development is further increased by kpro implantation. tubes may be implanted either in the anterior chamber or ciliary sulcus without vitrectomy, or as a pars plana tube with vitrectomy (figure 3).18,19 conjunctival scarring, crowded anterior chambers with iridocorneal adhesions, and the need for a good bandage contact lens fit postoperatively are considerations in deciding the position of tube placement. reported outcomes from these combined procedures have been successful in controlling iop with low risk of complications.20 similarly, patients who have significant retinal history or an unclear retinal status given poor view to the posterior segment preoperatively may benefit from concurrent vitrectomy.21 the back plate is available in either polymethyl methacrylate (pmma) or titanium in the snap - on version. originally the back plate was made of pmma, but titanium, a newer option, can be machined to be thinner than its pmma counterparts, potentially reducing the risk of rpm formation and anterior chamber congestion.22 titanium is nonferromagnetic and compatible with magnetic resonance imaging, and studies demonstrate that the back plate can be colored to achieve better cosmesis via electrochemical anodization.23 potential disadvantages of titanium are the metallic artifact created on as - oct that makes visualization of the angle anatomy more difficult, unlike pmma, and a worse cosmetic appearance. two size options exist for the diameter of the back plate : 7 or 8.5 mm. in patients with small corneas, short axial lengths or a crowded anterior chamber, the surgeon may request the 7 mm back plate for better sizing. the newer click - on boston type 1 kpro is only available with an 8.5 mm titanium back plate, and does not require a separate locking ring. implantation is only one aspect of kpro management, and close, lifetime follow - up is critical for device survival. patients must understand preoperatively that a commitment to frequent monitoring and drop maintenance is linked to implant success. more than a decade of international experience with postoperative complications offers insight into their prevention and management. in addition, improved imaging techniques provide a better understanding of the interactions between the device and host anatomy. behlau demonstrated that daily, topical antibacterial prophylaxis in kpro patients significantly reduces the risk of endophthalmitis. in the usa, the rate declined by 75% following the widespread adoption of long - term daily antibiotic use.24 regimens differ among kpro surgeons, but agents should cover for gram - positive organisms, the most common culprit, and gram - negative microbes. massachusetts eye and ear infirmary (meei) has recommended daily dosing of compounded vancomycin (concentration of 14 mg / ml) plus a fourth - generation fluoroquinolone, to be increased to twice daily in patients with autoimmune disease. the fluoroquinolone may be substituted for another antibiotic, such as trimethoprim / polymyxin b, and long - term vancomycin use may be limited to cases with autoimmune disease, chemical burns, and only eyes.25 cost, frequency of dosing, and patient compliance may influence drop selection.24, 26 fungal keratitis and endophthalmitis remain rare, and no consensus exists regarding routine fungal prophylaxis. in climates in which fungal keratitis is endemic, natamycin 5% or compounded amphotericin 0.15% may be given twice daily for 1 week every 23 months.27 an additional option to augment antimicrobial preventive care is to instill one drop of 5% povidone iodine at each visit.28 the value of a long - term bandage contact lens in kpro patients is manifold. as discussed earlier, a contact lens protects the corneal surface surrounding the anterior implant from desiccation, epithelial breakdown, and melt.29 these complications, in turn, place the patient at risk of infectious keratitis, sterile keratolysis with implant extrusion, and endophthalmitis. in addition, a lens often improves patient comfort, cosmesis, and glare, as well as addresses any refractive errors. tinted contact lenses may be effectively used to reduce light scatter and glare (figure 4).30 bandage lenses, however, are not without complications. protein deposits and inflammatory biofilms may develop, and lenses must be routinely changed. no consensus exists as to the ideal wear schedule of bandage contact lenses in kpro. typically, contact lenses are exchanged every 34 months, but may be discarded at different intervals depending on individual patient characteristics.28 advances in the imaging techniques of as - oct and ultrasound biomicroscopy allow in vivo imaging of the boston type 1 kpro. spectral domain, high - resolution as - oct can demonstrate the extent and thickness of rpms, epithelium over the anterior kpro plate, periprosthetic gaps, thinning in the corneal graft, angle closure, peripheral anterior synechiae, and associated anterior chamber shallowing.31,32 often, these entities may not be easily appreciated on clinical exam. an imaging protocol consisting of cross - sectional scans of the device and anterior chamber over 360 allows serial comparison over time.33 in patients with persistent epithelial defects, as - oct can assess for corneal graft thinning and imminent back plate exposure. in some patients, pooling of fluorescein may be seen beneath the anterior optic, suggestive of a gap between the corneal carrier and posterior surface of the device front plate.34 as - oct may be helpful to distinguish corneal thinning as the cause versus asymmetrical seating of the device. development or progression of glaucoma is one of the leading causes of vision loss following kpro implantation, and its detection may be difficult.35 progressive closing of the angle on as - oct has been demonstrated in the majority of kpro patients in several studies (figure 5).33,36 the correlation between angle closure and increased iop is still unclear and the subject of further study, but it may represent one mechanism of progression in these patients. serial optic nerve oct and optic disc photos as well as functional testing with visual fields are recommended at intervals shorter than those utilized for the follow - up of other forms of glaucoma. posterior segment oct can monitor for cystoid macular edema, an often under - recognized but treatable complication in this group of patients. as the as - oct can not visualize posterior to the pigmented iris tissue, ultrasound biomicroscopy may be used to image glaucoma tube shunts, posterior chamber intraocular lens implants, and other structures behind the iris plane.37 in conclusion, careful preoperative evaluation, surgical planning, and postoperative management improve outcomes of the boston type 1 kpro implantation. treatment plans should be tailored for each patient based on their presenting diagnosis and clinical features. multidisciplinary care, involving glaucoma, retinal, oculoplastic, and contact lens specialists, is often required. finally, the patient is a key member of the team, and must be committed to long - term care of their prosthetic. | for the anterior segment surgeon, the implantation of boston type 1 keratoprosthesis is a multistep process that begins with careful patient selection. success depends on thorough preoperative evaluation, detailed surgical planning, and frequent postoperative follow - up. new practice patterns have emerged for each of these phases as the international experience with keratoprosthesis grows. this review details special considerations that can improve outcomes and also allow surgeons to consider its use in challenging patient populations at each step. |
high mechanical properties, chemical stability, and biocompatibility make zirconia an attractive core material for fabrication of all - ceramic restorations. bonding techniques of all - ceramic restorations are dependent on chemical compositions of each ceramic system. hydrofluoric acid etching and silanization are mandatory steps to achieve a durable resin bonding to silica ceramics. on the other hand zirconia ceramic requires alternative techniques for long - term durable resin bonding. therefore several surface treatments are used to improve bonding to zirconia ceramic, such as selective infiltration etching (sie) technique, laser etching, alumina coating, silica ceramic coating, tribochemical silica coating or airborne - particle abrasion. several ceramic primers have been introduced into the dental market recently to enhance chemical bonding to zirconia ceramic, such as primers containing a phosphonic acid monomer, 6-mhpa (6-methacryloxyhexylphosphonoacetate), (az, primer) or 3-trimethoxysilylpropyl methacrylate, mdp, ethanol (clearfil ceramic primer) and organophosphate monomer, carboxylic acid monomer and other monomers (z - prime plus). however, there are obvious problems in obtaining a durable bonding to zirconia ceramics. conventional luting agents, such as glass ionomer cement (gic), could be used for cementation of zirconia ceramic full - coverage restorations. however adhesive cementation is preferred in case of compromised retention and resin - bonded fixed dental prosthesis. self - adhesive resin cements have been introduced into the dental market to simplify bonding procedures. however bond strength results to zirconia ceramics in the literatures using different categories of luting agents are very controversial. moreover, chemical composition of zirconia ceramic and intaglio surface morphology are unique for each commercial system. therefore, conclusions drawn considering bonding to one zirconia ceramic system may not be applicable to other systems. recently, a new zirconia ceramic (ice zirconia, zirkonzahn) has been introduced in the dental market. according to its manufacturer, this zirconia ceramic has bending strength over 1,400 mpa and could be used for fabrication of 16-unit fixed dental prosthesis. however, no independent data considering bonding to this zirconia ceramic has been published yet. several studies have evaluated bond strength of adhesives in vitro in terms of shear, tensile and microtensile bond strength (tbs). however, tbs test is considered as the most accurate one. moreover, aging and thermocycling are two important factors that have been shown to decrease the bond strength in in vitro studies. the purpose of this study was to investigate in vitro the influence of different surface treatments, storage in water and thermocycling, on the tbs of 3 luting agents to this zirconia ceramic. the null hypotheses of the study were (1) a durable bonding to the zirconia ceramic would be achieved regardless of the surface treatments, (2) self - adhesive resin cement would provide a durable bonding to this zirconia ceramic similar to multistep adhesive resin cement, and (3) resin - modified gic (rmgic) would provide a durable bonding to this zirconia ceramic compared to both adhesive resin cements. a total of 18 fully - sintered zirconia blocks (5x5x4 mm) in dimension were used for this study. each ceramic block was duplicated in light - polymerized hybrid, type 2 restorative composite resin (alphadent composite, shade a2) using vinyl polysiloxane material (president, coltne whaledent, altsttten, switzerland) (figure 1). composite resin was applied in increments 1 - 2 mm thickness and carefully condensed with st instrument with plastic working end (optrasculpt, ivoclar vivadent, schaan, liechtenstein). each increment was light - polymerized for 40 s at 5 mm distance and an intensity of irradiation 130 mw / cm (futolux 2, carlo de gorgi, milano, italy). materials used in the study ceramic blocks were divided into 3 groups (n=6) according to surface treatment as follow : group 1 : airborne - particle abrasion (aa), using al2o3 (50-m aluminum oxide particles, pluradent, offenbach, germany) at 0.28 mpa for 13 s at a distance of 10 mm (ney blastmate ii, ney, ca, usa). zirconia ceramic blocks were ultrasonically cleaned in distilled water for 3 min and dried with oil - free air stream ; group 2 : silica coating (sc), airborne - particle abrasion using 50-m al2o3 particles at 0.28 mpa for 13 s at a distance of 10 mm followed by airborne - particle abrasion with 30-m sio2 particles (cojet sand, 3 m espe, seefeld, germany) at a pressure of 0.25 mpa for 20 s at a distance of 10 mm (ney blastmate ii). zirconia ceramic blocks were ultrasonically cleaned in distilled water for 3 min and dried with oil - free air stream ; group 3 silica coating and silane application (scsi), same as group sc followed by silane application (espe sil, 3-methacryloxyprophyltrimethoxysilane in ethanol, 3 m espe). each main group was divided into 3 subgroups (n=2) according to the following 3 luting agents, ketac cem plus, relyx unicem and multilink automix. bonding procedures were performed according to the manufacturers ' recommendations for each luting agent : ketac cem plus (gi) : self curing, radiopaque, fluoride - releasing, rmgic containing bisgma and hema (3 m espe). equal amounts of past a and b were extruded on waxed paper pad, mixed for 20 s using a plastic spatula until a uniform color was achieved. relyx unicem aplicap (un) : dual - cure, self - adhesive resin cement, containing phosphoric acid monomer and methacrylate monomers (3 m espe). the capsule was activated for 4 s, and then mixed in amalgamator (silver mix 80, carlo de giorgi, milano, italy) for 10 s. the capsule was inserted into the applier and cement was dispensed directly onto the intaglio surfaces of the zirconia ceramic blocks. multilink automix (ml) : self - curing, transparent, two - past adhesive resin cement, containing dimethacrylate and hema (ivoclar vivadent). equal amounts of adhesive resin cement were extruded, mixed for 20 s and applied to the intaglio surfaces of the zirconia ceramic blocks. the bonding assembly was kept under a static load of 40 n for 5 min (articolo 719/00, # 1l01 ; carlo de giorgi), combination of 3 surface treatments and 3 luting agents resulted in 9 test groups as follows : giaa, gisc, giscsi, unaa, unsc, unscsi, mlaa, mlsc and mlscsi. each block was then bonded with cyanoacrylate glue (uhu, batch 40267647, uhu, bhl, germany) to a metal base that was fixed to a cutting machine (isomet 1000, buehler ltd. each slice was rotated 90 and bonded to another metal base again. the first slice 0.5 mm was also disregarded. a total of 9 non - trimmed bar specimens (10 mm long and 10.1 mm) bonded surface area were obtained from each block. microscopic examination (wild makroskop m 420 ; heerbrugg, switzerland) at 20 magnification of the bar specimens revealed that only 15 specimens from group gisc and 14 specimens from group giscsi were free from microcracks. therefore for standardization of the test groups, only 14 specimens from each subgroup were used to complete the test. seven specimens from each subgroup were stored in water bath at 37c for 1 week. while the other 7 specimens were stored in water bath at 37c for one month followed by thermocycling (tc) for 7,500 cycles. each cycle was consists of 1 minute in 5c cold bath and 1 minute in 55c hot bath with a dwell time of 30 s. specimens were dried and glued parallel to the long axis of an adapted caliper using cyanoacrylate glue (uhu, batch 40267647, uhu). this apparatus was fixed to the universal testing machine (type 500, lloyd instrument, farnham, uk). specimens were loaded in tension to failure (figure 2) at a crosshead speed of 1 mm / min. microtensile bond strength tbs values were recorded for each specimen in mpa using the formula : =l / a, where ' l ' is the load at failure (n) and ' a ' is the bonded area 10.1 mm. debonded specimen glued to an adapted caliper and fixed to the universal testing machine statistical analysis was conducted using the spss statistical software, version 16.0 (spss inc, chicago, ill, usa). post hoc tukey - hsd test at =0.05 was performed to test statistical significance between the groups. the fractured interfaces of the debonded specimens were examined with a light microscope (wild makroskop m 420) at x20 magnification to determine the failure pattern, which was assigned to cohesive failure within resin cement or composite resin, adhesive at ceramic / cement interface or mixed adhesive / cohesive modes. representative specimens for each failure pattern were examined using a scanning electron microscope (sem ; xl 30 cp ; philips, eindhoven, netherlands) with an acceleration voltage of 15 kv and a working distance of 10 mm. ceramic blocks were divided into 3 groups (n=6) according to surface treatment as follow : group 1 : airborne - particle abrasion (aa), using al2o3 (50-m aluminum oxide particles, pluradent, offenbach, germany) at 0.28 mpa for 13 s at a distance of 10 mm (ney blastmate ii, ney, ca, usa). zirconia ceramic blocks were ultrasonically cleaned in distilled water for 3 min and dried with oil - free air stream ; group 2 : silica coating (sc), airborne - particle abrasion using 50-m al2o3 particles at 0.28 mpa for 13 s at a distance of 10 mm followed by airborne - particle abrasion with 30-m sio2 particles (cojet sand, 3 m espe, seefeld, germany) at a pressure of 0.25 mpa for 20 s at a distance of 10 mm (ney blastmate ii). zirconia ceramic blocks were ultrasonically cleaned in distilled water for 3 min and dried with oil - free air stream ; group 3 silica coating and silane application (scsi), same as group sc followed by silane application (espe sil, 3-methacryloxyprophyltrimethoxysilane in ethanol, 3 m espe). each main group was divided into 3 subgroups (n=2) according to the following 3 luting agents, ketac cem plus, relyx unicem and multilink automix. bonding procedures were performed according to the manufacturers ' recommendations for each luting agent : ketac cem plus (gi) : self curing, radiopaque, fluoride - releasing, rmgic containing bisgma and hema (3 m espe). equal amounts of past a and b were extruded on waxed paper pad, mixed for 20 s using a plastic spatula until a uniform color was achieved. relyx unicem aplicap (un) : dual - cure, self - adhesive resin cement, containing phosphoric acid monomer and methacrylate monomers (3 m espe). the capsule was activated for 4 s, and then mixed in amalgamator (silver mix 80, carlo de giorgi, milano, italy) for 10 s. the capsule was inserted into the applier and cement was dispensed directly onto the intaglio surfaces of the zirconia ceramic blocks. multilink automix (ml) : self - curing, transparent, two - past adhesive resin cement, containing dimethacrylate and hema (ivoclar vivadent). equal amounts of adhesive resin cement were extruded, mixed for 20 s and applied to the intaglio surfaces of the zirconia ceramic blocks. the bonding assembly was kept under a static load of 40 n for 5 min (articolo 719/00, # 1l01 ; carlo de giorgi), combination of 3 surface treatments and 3 luting agents resulted in 9 test groups as follows : giaa, gisc, giscsi, unaa, unsc, unscsi, mlaa, mlsc and mlscsi. each block was then bonded with cyanoacrylate glue (uhu, batch 40267647, uhu, bhl, germany) to a metal base that was fixed to a cutting machine (isomet 1000, buehler ltd., each slice was rotated 90 and bonded to another metal base again. the first slice 0.5 mm was also disregarded. a total of 9 non - trimmed bar specimens (10 mm long and 10.1 mm) bonded surface area were obtained from each block. microscopic examination (wild makroskop m 420 ; heerbrugg, switzerland) at 20 magnification of the bar specimens revealed that only 15 specimens from group gisc and 14 specimens from group giscsi were free from microcracks. therefore for standardization of the test groups, only 14 specimens from each subgroup were used to complete the test. seven specimens from each subgroup were stored in water bath at 37c for 1 week. while the other 7 specimens were stored in water bath at 37c for one month followed by thermocycling (tc) for 7,500 cycles. each cycle was consists of 1 minute in 5c cold bath and 1 minute in 55c hot bath with a dwell time of 30 s. specimens were dried and glued parallel to the long axis of an adapted caliper using cyanoacrylate glue (uhu, batch 40267647, uhu). this apparatus was fixed to the universal testing machine (type 500, lloyd instrument, farnham, uk). specimens were loaded in tension to failure (figure 2) at a crosshead speed of 1 mm / min. microtensile bond strength tbs values were recorded for each specimen in mpa using the formula : =l / a, where ' l ' is the load at failure (n) and ' a ' is the bonded area 10.1 mm. debonded specimen glued to an adapted caliper and fixed to the universal testing machine statistical analysis was conducted using the spss statistical software, version 16.0 (spss inc, chicago, ill, usa). post hoc tukey - hsd test at =0.05 was performed to test statistical significance between the groups. the fractured interfaces of the debonded specimens were examined with a light microscope (wild makroskop m 420) at x20 magnification to determine the failure pattern, which was assigned to cohesive failure within resin cement or composite resin, adhesive at ceramic / cement interface or mixed adhesive / cohesive modes. representative specimens for each failure pattern were examined using a scanning electron microscope (sem ; xl 30 cp ; philips, eindhoven, netherlands) with an acceleration voltage of 15 kv and a working distance of 10 mm. mean standard deviation, minimum and maximum tbs without and with thermocycling of test groups in mpa and p values are summarized in (table 1). tbs means were compared across all test groups using 3-way anova model including the following factors (luting agent, surface treatment, storage time and interaction). the overall f - test was highly significant (p<0.0001), indicating differences in mean tbs across at least one of the 3 factors. all individual factors were significant, (p<0.001).the interaction between surface treatment and luting agent and surface treatment and storage time were significant (p<0.001). however luting agent and storage time was not significant (p=0.208). meanstandard deviations (sd), minimum and maximum microtensile bond strength in mpa after 1 week and 1 month storage in water with thermocycling of test groups and p values ml : multilink automix aa : air borne particle abrasion scsi : silica coating and silane application multiple comparisons with post hoc tukey - hsd test at =0.05 revealed that, considering 3 surface treatment performed, scsi significantly increased tbs (p<0.05) compared to sc and aa. however, there was no statistically significant difference between sc and aa (p=0.26). considering 3 luting agents used, ml and un resin cements showed significantly higher tbs than the gic cement (p<0.001). however, there was no significant difference in the tbs of the 2 resin cements (p=0.37). considering aging condition, 30-day water storage and thermocycling significantly decreased tbs compared to 1-week water storage (p<0.05). after 1 week of storage in water, the debonded specimens showed mainly mixed failure pattern (figure 4). after 30 days of storage in water and thermocycling, the failure pattern was mainly adhesive with remnants of the luting cements still adhered to zirconia ceramic surface (figure 6). failure pattern of all test groups after 1 week and 1 month storage in water ml : multilink automix aa : air borne particle abrasion scsi : silica coating and silane application representative scanning electron microscope micrograph at 1,000x magnification showing mixed failure, adhesive at ceramic / cement interface and cohesive within the composite resin restorative material representative scanning electron microscope micrograph at 1,000x magnification showing cohesive failure within the composite resin restorative material representative scanning electron microscope micrograph at 1,000x magnification showing adhesive failure at ceramic / cement interface clinically, restorations are subjected to repeated thermal stress and mechanical fatigue due to masticatory forces. therefore, one limitation of this study is that specimens were subjected only to thermal stress without mechanical fatigue. in addition, storage in water during 1 month might be too short to allow water saturation of the luting cements. it is likely that hydrolytic effects might affect the bond strength negatively after longer time of storage in water. microtensile bond strength test is more accurate than shear and tensile. because the small dimensions and small interfacial bonding zone of the specimens result in a more uniform distribution of the applied stresses. airborne - particle abrasion and silica coating are surface treatments recommended by most of the manufactures of zirconia ceramics and luting agents to improve bonding to zirconia ceramics. a range of 10 - 13 mpa was suggested as the minimum range for acceptable clinical bonding. therefore the results of this study clearly indicated that, silica coating and silane application together with resin cements would ensure a durable resin bonding to this zirconia ceramic 15 - 16 mpa. considering surface treatment, airborne - particle abrasion produced an activated microroughened zirconia surface, increased the bonding area and modifying the surface energy and wettability. in case of silica coating, alumina particles modified with silica acid this tribochemical reaction produces a high temperature contact area that can hold the silica layer on the ceramic surface. these particles formed a base for micromechanical interlocking. because no silane was applied before bonding, bond strength was directly correlated to the quality of micromechanical interlocking with the silica coating layer. however, bond strength to zirconia ceramic after silica coating was not improved in comparison to airborne - particle abrasion. the results of this study are in agreement with the findings of several studies, which reported that bond strength to zirconia ceramics was not improved after silica coating compared to airborne - particle abrasion. (2009) and kern (2009) reported that airborne - particle abrasion of zirconia ceramics produces a certain roughness, but only limited undercuts were produced, thus not improving bonding to zirconia. (2006), after energy - dispersive x - ray analysis of silica - coated zirconia ceramic, reported that silica - coverage originating from the coating particles appears not to have become embedded onto the hard zirconia surface, consequently bond strength was not improved after silica coating. moreover, ultrasonic cleaning in distilled water might removed a significant amount of silica coating layer, consequently bond strength of resin cements to silica - coated zirconia ceramic was decreased, as reported by nishigawa,. however, the results of this study were contradicting to the results of other studies, which reported that silica coating improved bond strength to zirconia ceramics compared to airborne - particle abrasion. this difference in the results could be attributed to the fact that, atsu,. (2006) used 125-mm al2o3 for airborne - particle abrasion followed by 30-mm al2o3 particles modified by silica. panavia luting cement together with different ceramic primers were used for bonding. on the other hand lthy,. (2006) used the rocetac system for silica coating, which combines 110 mm al2o3 for airborne - particle abrasion followed by 110-mm al2o3 particles modified by silica. moreover both studies used shear test. in the present study, 50-mm al2o3 was used for airborne - particle abrasion followed by 30-mm al2o3 particles modified by silica and a tbs test were employed. silica coating and silane application significantly increased bond strength compared to airborne - particle abrasion or silica coating alone, as reported in several studies. silane coupling agent did not promote adequate bonding to zirconia ceramics, as these ceramics contain minimal or no silica content. therefore, silica coating of zirconia ceramics is a prerequisite for durable siloxane bonding, as it leave a physically and chemically active outer surface layer. silane coupling agent wets the adherent, increases its surface energy and makes it accessible for effective bonding. moreover, it is capable of forming covalent bond at silica coated ceramic / resin cement interface through formation of silanol groups. therefore, after silica coating and silanization, bond strength was based on both micromechanical interlocking plus chemical adhesion due to silane application. variations in chemical composition, wetting capacity, viscosity and mechanical properties for each luting cement could be responsible for variations in the bonding capacity to zirconia ceramics. according to the manufacturer, relyx unicem contains methacrylate monomers, adhesive phosphate monomer and silanated fillers in its chemical composition. adhesive phosphate monomer enhanced self bonding to zirconia ceramics especially after silica coating and silane application as reported in several studies. several studies have reported that bond strengths of bisgma containing resin cements were dramatically decreased after artificial aging due to their remarkable weak mechanical properties. the improved mechanical properties could be responsible for high bond strength results of multilink automix. (2009) found no significance difference in the mean bond strength of multilink automix 21.2 mpa and relyx unicem 23.1 mpa to zirconia ceramics after different surface treatments. according to the manufacturers flexural strength for relyx unicem (75 mpa) and multilink automix (70 mpa) are in the same range. however it is higher than that of ketac cem plus (31.6 mpa). therefore, variations in the mechanical properties of the 3 luting cements used could be another contributing factor for bond strength results as reported in other studies. in in vitro studies, water storage and thermocycling are two important factors that decrease the bond strength. therefore, 1 month storage in water and thermal cycling for 7,500 cycles was used as aging regime to simulate clinical conditions. after 30 days of storage in water and thermocycling, tbs were significantly decreased in all groups. this decrease in bond strength might be due to degradation of the luting cement itself and the hydrolytic effect of water at the luting cement / ceramic interface. moreover, mismatch between the coefficient of thermal expansion of the bonded specimens (zirconia ceramic, luting agent and composite resin) could result in hoop stress during thermocycling. another factor could be the fact that silanized surfaces were unstable in contact with moisture as reported by derand,. accumulation of negative effect of water, thermocycling and instability of silane could be responsible for the decrease of bond strength for all test groups. the initial high bond strength results were reflected on the failure pattern of debonded specimens as examined by optical reflection microscope and confirmed by scanning electron microscopy. all groups showed mainly mixed failure pattern or adhesive failure while cohesive failure was minimal. cohesive failure within composite resin could be due to the initiation of microcracks during cutting of the specimens. therefore, in this study, specimens with apparent microcracks under stereomicroscopic examination were discarded. after 30 days of storage in water and thermocycling, the failure pattern was mainly adhesive indicating a decrease in the bond strength due to the hydrolytic effect of water, hoop stress due to thermocycling and degradation of the luting resin itself. the general outcome of this study suggests that although conventional cements could be used for cementation of zirconia ceramics, resin cements are preferred for long - term bond durability. bonding techniques based on micromechanical interlocking and chemical adhesion using silanes are preferred than other techniques. within the limitations of this study, the following conclusions were drawn : silica coating and silane application significantly improved tbs compared to silica coating or airborne - particle abrasion. there was no significance difference in the bond strength after using self - adhesive resin cement or multistep adhesive resin cement. | objectivethis in vitro study aimed to evaluate the influence of different surface treatments, 3 luting agents and thermocycling on microtensile bond strength (tbs) to zirconia ceramic. material and methodsa total of 18 blocks (5x5x4 mm) were fabricated from zirconia ceramic (ice zirkonia) and duplicated into composite blocks (alphadent). ceramic blocks were divided into 3 groups (n=6) according to the following surface treatments : airborne - particle abrasion (aa), silica - coating, (sc) (cojet) and silica coating followed by silane application (scsi) (espe sil). each group was divided into 3 subgroups (n=2) according to the 3 luting agents used. resin - modified glass - ionomer cement (rmgic, ketac cem plus), self - adhesive resin cement (un, relyx unicem) and adhesive resin cement (ml, multilink automix) were used for bonding composite and zirconia blocks. each bonding assembly was cut into microbars (10 mm long and 10.1 mm2). seven specimens of each subgroup were stored in water bath at 37c for 1 week. the o ther 7 specimens were stored in water bath at 37c for 30 days then thermocycled (tc) for 7,500 cycles. tbs values were recorded for each specimen using a universal testing machine. statistical analyses were performed using a 3-way anova model followed by serial 1-way anovas. comparison of means was performed with tukey 's hsd test at (=0.05). resultstbs ranged from 16.8 to 31.8 mpa after 1 week and from 7.3 to 16.4 mpa after 30 days of storage in water and thermocycling. artificial aging significantly decreased tbs (p<0.05). considering surface treatment, scsi significantly increased tbs (p<0.05) compared to sc and aa. resin cements (un and ml) demonstrated significantly higher tbs (p<0.05) compared to rmgic cement. conclusionssilica coating followed by silane application together with adhesive resin cements significantly increased tbs, while thermocycling significantly decreased tbs. |
lupus anticoagulant - hypoprothrombinemia syndrome (lahps) is a very rare disease associated with transient viral infections, drug reactions, antiphospholipid syndrome, systemic lupus erythematosus (sle) and exists even in healthy individuals1,2,3). it is caused by antifactor ii antibody, which is usually counterbalanced by the prothrombotic effect of lupus anticoagulant (lac)1,2,3). corticosteroid is required to control the hemorrhage and correct the hypoprothrombinemia, and is effective in many cases3,4). however, some cases with fatal course showed poor response to steroid treatment1). we report a clinical case of a 15-year - old girl with sle, who was diagnosed with laphs and died due to uncontrolled pulmonary hemorrhage caused by factor ii deficiency in spite of supportive treatment including steroid and immunoglobulin a 15-year - old girl was admitted to the previous hospital due to easy bruising and prolonged bleeding after tooth extraction. after several days, she was transferred to our hospital for further evaluation for pancytopenia. she had no past or family history of any bleeding disorders or connective tissue diseases. the patient was stable in vital signs, and revealed no specific findings except for several bruises on both lower legs on physical examination. initial laboratory tests showed a leukocyte count of 1,560/l, hemoglobin 7.5 g / dl, and a platelet count 19,000 /l. the patient 's prothrombin time (pt) was 28.6 seconds (normal values, 10.1.14.0 seconds) and 23%, international normalized ratio was 2.65, and activated partial thromboplastin time (aptt) was more than 120 seconds (normal values, 21.0.38.0 seconds). the disseminated intravascular coagulation (dic) profile showed that 89% of antithrombin iii (normal values, 80%.120%), 0.47 mg / l of d - dimer (normal values, < 0.8 mg / l) and 1.08 g / ml of fibrin degradation products (normal values, < 5 g / ml), and these results suggested that she was not complicated with dic. the bone marrow biopsy, study for autoimmune disease and factor disease were planned to find out the cause of pancytopenia and prolongation of pt and aptt. the patient was given blood transfusion with packed red cells, fresh frozen plasmas (ffps), and single donor platelets. the chest radiography showed mild haziness with mild left pleural effusion, and she was started on intravenous antibiotics. peripheral blood still showed pancytopenia and there was no improvement of prolongation of pt and aptt in spite of blood transfusion including ffp. on hospital day 4, an antibody screening test for cold antibody and the direct coomb 's test were positive. the complement levels were c3, 18 mg / dl ; c4, < 1.0 mg / dl ; and ch50, < 10 u / ml. tests for antinuclear antibodies, antidouble stranded dna antibodies, lac antibodies, anticardiolipin antibodies immunoglobulin (ig) m, sle profile and syphilis reagin test were all positive. repeated complete blood count, pt and aptt showed no significant change in spite of continuous blood products transfusion. we diagnosed the patient with sle based on revised criteria of american college of rheumatology. the patient had no other symptoms except for purpura on lower extremeties, and radiographic finding showed no significant change compared to prior 2 days ago. on hospital day 7, the patient complained of dyspnea. evaluation of clotting factors revealed that the level of factor ii was 3.3% (normal values, 50%-150%). a 1:1 dilution of patient plasma with normal plasma partially corrected the pt and aptt. protein s activity was 147.4% (normal values, 65%-140%) and protein c activity, 76% (normal values, 70%-130%). we added high dose of intravenous methylprednisolone pulse therapy (20 mg / kg / day) but platelet count and prolongation of pt and aptt did not improve. on hospital day 10, the dyspnea was aggravated due to the pulmonary hemorrhage and she was moved into the intensive care unit. intravenous immunoglobulin (ivig) treatment (300 mg / kg / day) had no effect for correction of pt and aptt, but platelet count slightly increased. the blood test results showed a platelet count of 78,000 /l, pt of 42%, and aptt of 88.5 seconds (fig. however, levels of aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen and creatinine began to increase. we increased the dose of ivig up to 1 g / kg / day, but her chest condition deteriorated very rapidly and multiorgan failure got worse, too. finally she required intubation and mechanical respiratory support, and died due to aggravation of the pulmonary hemorrhage on hospital day 12. lac is an antiphospholipid antibody associated with hypercoagulable states and thromboembolic events1,2,3). in most children of young age, the presence of lac is transient, benign and postinfectious compared to autoimmune persisted type which mainly occurs in adolescence5). bleeding is a rare manifestation of lac, and when it occurs, it is nearly always due to thrombocytopenia or factor ii deficiency2,6). such bleeding results from the presence of antifactor ii antibody, which usually counterbalances the prothrombotic effect of lac1). and also, thrombosis was rare and strongly associated with persistently positive lac5). lac along with a specific nonphospholipid - dependent antiprothrombin antibody causes an acquired hypoprothrombinemia7). it occurs with viral infection and drug reactions in healthy individuals, or it appears mostly in young females with autoimmune disease like sle1,2,3,6). increasing incidence during adolescence and the gender distribution seventy - four cases of laphs has been reported worldwide according to a literature review retrieving the related articles published between 1960 and 2011. epidemiologic characteristics of these 74 cases shows that 43 patients (58%) were aged < 15 years at disease onset8). the mechanism suggested that antiphopholipid antibodies bound to prothrombin in circulation accelerate clearance rather than interfere with prothrombin function5). some authors suggest cross - reactivity between the antiphopholipid antibodies and phopholipid epitopes in the prothrombin molecules1). the clinical findings are generally asymptomatic, but there can be severe hemorrhagic symptoms such as gynecologic bleeding, macroscopic hematuria, gastrointestinal bleeding, brain hematoma, diffuse muscular hematoma and venous or arterial thrombosis2,8). despite the fact that about 50% of patients develop severe bleeding, the overall prognosis is quite good, with a mortality rate of less than 5%8). treatment of factor ii deficiency should be individualized, and the underlying cause should be found and treated. however, most of cases responded successfully to corticosteroid but not to vitamin k, ffp or blood transfusion3). most of patients previously reported in the literature have responded well to prednisolone treatment alone3,4). a few cases required additional immunosuppressive drugs such as cyclophosphamide, azathioprine or rituximab because the patients had symptom recurrences when drug therapy was tapered, but immunosupression may not work immediately1,3,9). therefore, corticosteroid therapy is the treatment of choice for lahps associated with sle1,2,3,4,6,8). it decreases the clearance of the prothrombin antibody complexes and normalizes abnormal coagulation times, including those of pt, aptt and clotting factors1,2,3,4,6). ivig treatment also has been shown to be effective in the setting of acute bleeding1). in our patient, coagulation studies improved significantly after administration of ivig but the patient died because of uncontrolled pulmonary hemorrhage. we think that the administration of ivig was started too late with an insufficient dose. our case shows that when bleeding occurs in sle patients, the possibility of hypoprothrombinemia should be considered and prothrombin time and factor study should be measured3). significant prolongation of the pt or aptt requires further evaluation for autoantibodies against coagulation factors10). but if factor ii levels are very low (under 10%), patients may experience severe life threatening bleeding such as uncontrolled pulmonary hemorrhage. moreover, the rapid progression of hemorrhage can be retarded by more aggressive treatments such as immunoglobulins and immunosuppressive agents. | lupus anticoagulant - hypoprothrombinemia syndrome (lahps), a very rare disease that is caused by the presence of antifactor ii antibodies, is usually counterbalanced by the prothrombotic effect of lupus anticoagulant (lac). patients with lahps are treated using fresh frozen plasma, steroids, immunosuppressive agents, and immunoglobulins for managing the disease and controlling hemorrhages. notably, steroids are the important treatment for treating hypoprothrombinemia and controlling the bleeding. however, some patients suffer from severe, life - threatening hemorrhages, when factor ii levels remain very low in spite of treatment with steroids. here, we report a case of lahps in a 15-year - old girl who experienced pulmonary hemorrhage with rapid progression. she was referred to our hospital owing to easy bruising and prolonged bleeding. she was diagnosed with lahps that presented with pancytopenia, positive antinuclear antibody, proloned prothrombin time, activated partial thromboplastin time, positive lac antibody, and factor ii deficiency. her treatment included massive blood transfusion, high - dose methylprednisolone, vitamin k, and immunoglobulin. however, she died due to uncontrolled pulmonary hemorrhage. |
tissue preparation : adult male mice (icr, weight : 40 5 g, age : 4 months 15 days) were decapitated under diethylether anesthesia. the basilar arteries were then gently isolated from the brain and transferred to ice - cold physiological saline (119 mm nacl, 4.7 mm kcl, 1.6 mm cacl2, 1.2 mm mgcl2, 25 mm nahco3, 1.2 mm kh2po4 and 10 mm glucose), ph 7.4, aerated with carbogen (95% (v / v) o2, 5% (v / v) co2). all experiments were performed in accordance with the kagoshima university guidelines for animal experimentation. reagents : we used the following reagents and final concentrations : na (1010 m), his hydrochloride (1010 m), diphenhydramine hydrochloride (1010 m), cimetidine (10 m), ang ii acetate salt (1010 m), losartan potassium (10 and 10 m), pd123319 ditrifluoroacetate salt (10 m), bk acetate salt (1010 m), des - arg- [leu]-bk (10 m), methoctramine hydrate (10 m), n-nitro - l - arginine (l - nna ; 10 m) and sodium nitroprusside (snp ; 10 m) (sigma - aldrich, st. 5-ht (serotonin)-creatinine sulfate (1010 m ; merck, darmstadt, germany), hoe140 (10 and 10 m ; peptide institute, osaka, japan), indomethacin (10 m ; nacalai tesque, kyoto, japan), ach chloride (1010 m ; daiichi sankyo, tokyo, japan), pirenzepine dihydrochloride (10 m ; santa cruz biotechnology, santa cruz, ca, u.s.a.), hexahydro - sila - difenidol hydrochloride, p - fluoro analog (pfhhsid, 10 m ; research biochemicals, natick, ma, u.s.a.) and u-46619 (10 m ; cayman chemical co., ann arbor, mi, u.s.a.). functional studies : two rings approximately 2 mm long were cut from each mouse basilar artery. each ring was mounted horizontally between two l - shaped stainless steel holders (outer diameter 0.02 mm), with one part fixed to an isometric force transducer (tb-611 t, nihon kohden kogyo, tokyo, japan), and immersed in a 4-ml water - jacketed micro tissue organ bath (umtb-1, unique medical co., ltd., tokyo, japan) containing oxygenated salt solution at 37c (ph 7.4). each suspended ring was left to equilibrate for at least 120 min under a resting tension of 0.03 g. this tension was chosen, because it allowed us to induce maximum contractions in the basilar artery. kcl (60 mm) was applied every 30 min until the amplitude of the contraction reached a constant value. changes in the kcl concentration of the physiological saline were compensated for by equimolar adjustment of the nacl concentration. the isometric tension was recorded with an amplifier (ap-621 g, nihon kohden kogyo, tokyo, japan), digitized with an analogue - digital converter (powerlab/8sp, adinstruments co., castle hill, nsw, australia) and stored on the hard disk of a personal computer. the cumulative concentration - response curve of each agonist was obtained by adding a solution of agonist directly to the fluid in the bath. the log concentration - ratio of ec50 values (i.e., concentration producing half - maximum response) in the absence or presence of antagonist was calculated and plotted against the logarithm of antagonist concentration to obtain the pa2 values. statistical analyses were performed by student s t - test or the bonferroni test after one - way analysis of variance (stat view j-4.5, abacus concepts inc., responsiveness to ach, bk, na, 5-ht, his and ang ii : we generated concentration - response curves for ach, bk, na, 5-ht, his and ang ii using isolated mouse basilar arteries (fig. 1.responsiveness of isolated mouse basilar artery to angiotensin ii (ang ii :), 5-hydroxytryptamine (5-ht :), histamine (his :), noradrenaline (na :) [a ], acetylcholine (ach :) and bradykinin (bk :) [b ]. relaxation in response to ach and bk was investigated under precontraction with u-46619 (10 m). contraction responses were compared with 60 mm kcl response, and relaxation responses were compared with 10 m snp response. absolute values of kcl - induced contraction and snp - induced relaxation were 0.038 0.004 g and 0.011 0.002 g, respectively. contractile response was measured under the resting tone of normal artery, whereas the relaxation response was measured under contraction with u-46619 (a thromboxane a2 analogue). responsiveness of isolated mouse basilar artery to angiotensin ii (ang ii :), 5-hydroxytryptamine (5-ht :), histamine (his :), noradrenaline (na :) [a ], acetylcholine (ach :) and bradykinin (bk :) [b ]. relaxation in response to ach and bk was investigated under precontraction with u-46619 (10 m). contraction responses were compared with 60 mm kcl response, and relaxation responses were compared with 10 m snp response. absolute values of kcl - induced contraction and snp - induced relaxation were 0.038 0.004 g and 0.011 0.002 g, respectively. responsiveness to l - nna and indomethacin : l - nna (a no synthase inhibitor, 10 m) induced contraction (13.4 1.8% to 60 mm kcl) under resting tension, and indomethacin (a cyclooxygenase inhibitor, 10 m) induced relaxation (5.8 0.5% to 10 m snp) under contraction with l - nna. maximal responses and pec50 values for ach, bk, na, 5-ht, his and ang ii : table 1table 1.pec50 values and maximal responses to agonistsagonistspec50max (%) bradykinin6.84 0.0965.5 4.3acetylcholine6.76 0.0646.4 4.4angiotensin ii6.81 0.0857.9 4.75-hydroxytryptamine6.64 0.1018.1 2.3histamine4.58 0.0241.6 2.4noradrenaline no responsea : relaxation induced by 10 m snp (0.011 0.002 g) was taken as 100%, b : contraction induced by 60 mm kcl (0.038 0.004 g) was taken as 100%, each point represents the mean sem of 812 mice. shows the maximal responses and pec50 values for the agonists examined bk was the most sensitive relaxing agent (pec50=6.84 0.09) and induced the most potent maximum relaxation (65.5 4.3%), whereas ang ii was the most sensitive contracting agent (pec50=6.81 0.08) and induced the most potent maximum contraction (57.9 4.7%) of the mouse basilar artery. a : relaxation induced by 10 m snp (0.011 0.002 g) was taken as 100%, b : contraction induced by 60 mm kcl (0.038 0.004 g) was taken as 100%, each point represents the mean sem of 812 mice. effect of endothelial denudation, l - nna, atropine, pirenzepine, methoctramine and pfhhsid on ach - induced relaxation : we investigated the effects of endothelial denudation, l - nna, atropine, pirenzepine (a m1 receptor antagonist), methoctramine (a m2 receptor antagonist) and pfhhsid (a m3 receptor antagonist) on the concentration - response curve for ach. atropine at 10 m and 10 m shifted the concentration - response curve for ach to the right and at 10 m largely abolished the ach - induced relaxation (fig. 2.effect of the non - selective muscarinic - antagonist atropine (, 10 m,, 10 m and, 10 m) on acetylcholine (ach)-induced relaxation () [a ] and effects of the endothelial denudation (), l - nna (, 10 m), m1 receptor antagonist pirenzepine (, 10 m), the m2 receptor antagonist methoctramine (,10 m) and the m3 receptor antagonist pfhhsid (, 10 m) on ach - induced relaxation () [b ] and schild plot of atropine [c ] for the isolated mouse basilar artery. the maximum relaxation induced by ach in the absence of antagonist was taken as 100%. cr : equieffective ach concentration ratio [concentration producing 50% maximal (ec50) in the presence of atropine / ec50 in the absence of atropine ].). the calculated pa2 value for atropine was 8.02 0.06 and its slope was 0.86 0.05, which was not significantly different from unity (fig. 2c). figure 2b shows the effects of endothelial denudation, l - nna, pirenzepine, methoctramine and pfhhsid on ach - induced relaxation under the contraction induced by u-46619. ach - induced relaxation was completely abolished in endothelial denudated artery and significantly inhibited by l - nna. none of the three antagonists had any significant effect on the ach - induced relaxation. effect of the non - selective muscarinic - antagonist atropine (, 10 m,, 10 m and, 10 m) on acetylcholine (ach)-induced relaxation () [a ] and effects of the endothelial denudation (), l - nna (, 10 m), m1 receptor antagonist pirenzepine (, 10 m), the m2 receptor antagonist methoctramine (,10 m) and the m3 receptor antagonist pfhhsid (, 10 m) on ach - induced relaxation () [b ] and schild plot of atropine [c ] for the isolated mouse basilar artery. the maximum relaxation induced by ach in the absence of antagonist was taken as 100%. cr : equieffective ach concentration ratio [concentration producing 50% maximal (ec50) in the presence of atropine / ec50 in the absence of atropine ]. effects of endothelial denudation, l - nna, indomethacin, and b1 and b2 receptor antagonists on bk - induced relaxation : endothelial denudation had completely abolished bk - induced relaxation, and the no synthase inhibitor l - nna partially inhibits it. the cyclooxygenase inhibitor indomethacin had no significant effect on bk - induced relaxation (fig. 3afig. 3.effects of endothelial denudation (), l - nna () (a nitric oxide synthase inhibitor) and indomethacin (, 10 m, a cycloxygenase inhibitor) on bradykinin (bk)-induced relaxation () [a ], effects of the b1 receptor antagonist des - arg-[leu]-bk (, 10 m) and the b2 receptor antagonist hoe140 (, 10 m and, 10 m) on bk - induced relaxation () [b ] and schild plot of hoe140 [c ] for the isolated mouse basilar artery. the maximum relaxation induced by bk in the absence of antagonist 2.). to characterize the bk receptor subtypes, the arteries were pretreated with b1 and b2 receptor antagonists. figure 3b shows the effect of des - arg-[leu]-bk (a b1 receptor antagonist) on bk - induced relaxation of the mouse basilar artery. des - arg-[leu]-bk (10 m) did not significantly affect bk - induced relaxation. this figure also shows the effect of hoe140 (a b2 receptor antagonist) on bk - induced relaxation. hoe140 shifted the bk - induced concentration - response curve to the right. the calculated pa2 value for hoe140 was 7.53 0.12 and its slope was 1.03 0.14, which was not significantly different from unity (fig. effects of endothelial denudation (), l - nna () (a nitric oxide synthase inhibitor) and indomethacin (, 10 m, a cycloxygenase inhibitor) on bradykinin (bk)-induced relaxation () [a ], effects of the b1 receptor antagonist des - arg-[leu]-bk (, 10 m) and the b2 receptor antagonist hoe140 (, 10 m and, 10 m) on bk - induced relaxation () [b ] and schild plot of hoe140 [c ] for the isolated mouse basilar artery. the maximum relaxation induced by bk in the absence of antagonist was taken as 100%. effect of 5-ht on isolated mouse basilar artery : 5-ht induced concentration - dependent contraction in 9 of 36 mouse basilar arteries, the second - time 5-ht response being significantly lower than the first (fig. 4.effect of repeated application of 5-hydroxytryptamine (5-ht) on the isolated mouse basilar artery (, 1st response,, 2nd response). the maximum contraction induced by the first application of 5-ht was taken as 100%. endothelial denudation or inhibition of no synthase by l - nna had no effect on this phenomenon (data not shown). effect of repeated application of 5-hydroxytryptamine (5-ht) on the isolated mouse basilar artery (, 1st response,, 2nd response). the maximum contraction induced by the first application of 5-ht was taken as 100%. effects of diphenhydramine and cimetidine on his - induced contraction : we investigated the effects of diphenhydramine (a h1 receptor antagonist) and cimetidine (a h2 receptor antagonist) on the concentration - response curve for his. diphenhydramine (1010 m) shifted the concentration - response curve for his to the right in parallel ; cimetidine (10 m) had no significant effect (fig. 5afig. 5.effect of the h1 receptor antagonist diphenhydramine (10 m, 10 m, 10 m, 10 m) and the h2 receptor antagonist cimetidine (10 m) on histamine (his)-induced contraction () [a ] and schild plot of diphenhydramine [b ] for the isolated mouse basilar artery. the maximum contraction induced by his in the absence of antagonist was taken as 100%. the calculated pa2 value for diphenhydramine was 6.62 0.11 and its slope was 0.81 0.19, which was not significantly different from unity (fig. effect of the h1 receptor antagonist diphenhydramine (10 m, 10 m, 10 m, 10 m) and the h2 receptor antagonist cimetidine (10 m) on histamine (his)-induced contraction () [a ] and schild plot of diphenhydramine [b ] for the isolated mouse basilar artery. the maximum contraction induced by his in the absence of antagonist effects of losartan and pd123319 on ang ii - induced contraction : we examined the effects of losartan (an at1 receptor antagonist) and pd123319 (an at2 receptor antagonist) on the concentration - response curve for ang ii (fig. 6.effect of the at1 receptor antagonist losartan (10 m, 10 m) and the at2 receptor antagonist pd123319 (, 10 m) on angiotensin (ang) ii - induced contraction () [a ] and schild plot of losartan [b ] in the isolated mouse basilar artery. the maximum contraction induced by ang ii in the absence of antagonist was taken as 100%. losartan (10 and 10 m) shifted the concentration - response curve for ang ii to the right in parallel. the calculated pa2 value for losartan was 8.12 0.10 and its slope was 0.79 0.03, which was significantly different from unity. effect of the at1 receptor antagonist losartan (10 m, 10 m) and the at2 receptor antagonist pd123319 (, 10 m) on angiotensin (ang) ii - induced contraction () [a ] and schild plot of losartan [b ] in the isolated mouse basilar artery. the maximum contraction induced by ang ii in the absence of antagonist was taken as 100%. to our knowledge, this is the first study to have demonstrated the responsiveness of the isolated mouse basilar artery to ach, bk, na, 5-ht, his and ang ii. although some of these vasoactive substances have been investigated by pressure myograph system previously, the receptor subtypes have not yet been described. ach is an endogenous substance producing endothelium - dependent vasorelaxation via no, prostacyclin and/or endothelium - derived hyperpolarizing factor (edhf). in the present study, ach - induced relaxation was completely abolished by endothelial denudation and significantly inhibited by l - nna as shown in fig. these results suggested that ach induces endothelium - dependent and no - mediated relaxation in mouse basilar artery. three types of muscarinic receptors appear to be involved in the relaxation or contraction of arteries, but in the present study, atropine shifted the concentration - response curve for ach to the right with a pa2 value of 8.02 as shown in fig. there is no report regarding the pa2 value of atropine in ach - induced relaxation on mouse artery or other tissues. but, our calculated pa2 value is similar to that reported for the rabbit aorta (8.14), but lower than that reported for the rat mesenteric artery (9.78). the differences might be due to the differences in artery and species of animals studied. these results differed from those obtained using chicken basilar arteries, where atropine (non - selective muscarinic receptor antagonist) and pfhhsid (selective m3 receptor antagonist), but not pirenzepine (selective m1 receptor antagonist) and methoctramine (selective m2 receptor antagonist), shifted the concentration - response curve for ach to the right. our results suggest that some other receptor subtypes apart from m1, m2 and m3 receptors might be responsible for ach - induced relaxation. molecular cloning studies have revealed the existence of five molecularly distinct ach receptor subtypes (m1-m5) [6, 30 ]. studies of the expression of the cloned m5 receptor gene in cultured mammalian cells have shown that the encoded receptor protein is functional and efficiently couples to g proteins of the gq family, similarly to the m1 and m3 receptor subtypes [5, 14 ]. as no m5 antagonist is commercially available, we were unable to characterize the receptor, although a previous study observed that ach - mediated dilation of cerebral arteries and microvessels was virtually abolished in m5 receptor - knockout mice. bk induced relaxation of mouse basilar arteries precontracted with u-46619, and this effect was abolished in arteries after endothelial denudation as shown in fig. >, who used light - dye or laser - dye techniques to show that cerebral vasodilator responses to bk in vivo were abolished after injury to endothelial cells in mice. pretreatment with l - nna partially shifted the concentration - response curve for bk to the right, and indomethacin had no significant effect on it. these results suggest that bk - induced relaxation might be partly mediated by no, but not by arachidonic acid metabolites. some other edhf might also be involved in bk - induced relaxation of mouse basilar artery. the dilative action of bk on small pial arteries is reportedly mediated by release of hydroxyl radicals in mouse and cat. in line with this, one previous study has verified that bk - induced relaxation of human forearm resistance vessels did not involve no or a vasodilator prostanoid, this effect being mediated by hyperpolarization of the vascular wall. thus, it seems that the vasorelaxing pathways involved in bk - induced relaxation vary depending on the vascular bed studied. in the present study, the relaxing effect of bk was significantly inhibited by hoe140, but not by the b1 receptor antagonist des - arg-[leu]-bk as shown in fig. these data indicate that the dilative effect of bk on the mouse basilar artery is mediated by the b2 receptor, and not by the b1 receptor. b1 receptor - mediated responses are generally not observed under normal physiological conditions. the calculated pa2 value of hoe140 is 7.53, which is identical to the value for the human isolated umbilical artery, i.e. 7.50. in the mouse basilar artery, this result was similar to that obtained by a previous study, which showed that na had no effect on the rat basilar artery. in mouse basilar artery, 5-ht induced contraction with an intensity of 18.1%, whereas in most species of animal, the intensity ranges from 40% to 100%. the frequency of 5-ht - induced contraction was 25% among all experimental cases (9 of 36), and furthermore second - time responses were significantly lower than the initial ones and absent in some cases. after first application, a possible desensitization or internalization and/or down regulation of the 5-ht receptor might interfere second - time responses. therefore, we were unable to carry out subsequent experiments to characterize the receptor subtypes involved in the 5-ht - induced contraction. this brings into question the usefulness of the mouse as a model for studies of migraine and stroke, conditions in which 5-ht is thought to play important roles [19, 31 ]. the h1 receptor antagonist diphenhydramine shifted the concentration - response curve for his to the right, whereas the h2 receptor antagonist cimetidine had no significant effect on the his - induced contraction as shown in fig. these results suggest that activation of the h1 receptor induces contraction of the mouse basilar artery. contraction of resting vascular tone in response to his has also been reported in guinea pigs, pigs, humans, horses and cattle. the calculated pa2 value for diphenhydramine was 6.62, which is lower than the values reported for bovine (7.61) and porcine (7.77) basilar arteries [17, 18 ]. a further study is needed to clarify the differences in pa2 values among species. the effects of ang ii occur via activation of two receptor subtypes, at1 and at2. the vasocontractile effects of ang ii are generally considered to result from activation of the at1 receptor. the selective at1 receptor antagonist losartan shifted the concentration - response curve for ang ii to the right, whereas pd123319 (an at2 receptor antagonist) had no effect as shown in fig. these results suggest that the ang ii - induced contraction in mouse basilar artery is mediated by activation of at1 receptors. a previous study showed that vasoconstriction of the cerebral artery in response to ang ii was markedly reduced in genetic at1a - deficient mouse. the calculated pa2 value for losartan is 8.12, which is similar to that reported in canine mesenteric (8.15) and pulmonary (7.96) artery. l - nna induced contraction and indomethacin induced relaxation through inhibition of no synthase and cyclooxygenase, respectively. these results suggest that the resting tone balance of the mouse basilar artery is also maintained by spontaneous release of no and thromboxane a2. small rodents, such as mice and rats, are frequently used in preclinical cerebrovascular research, mice being particularly useful, because an increasing number of transgenic models are becoming available. mice are often used as small animal models of brain ischemia, venous thrombosis or vasospasm, and alzheimer s disease. the majority of novel therapeutic approaches are tested in small animal models of human disease, especially those involving mice, prior to clinical testing. a variety of murine models of cerebrovascular disease are available, from which a number of molecular and structural elements of cerebral disorders have been clarified. in summary, we have investigated the responses of the mouse basilar artery to a number of pharmacological agents that are modulators of cerebrovascular circulation in both normal and pathophysiological states. we have demonstrated that the mouse basilar artery is responsive to ach and bk with relaxation, and to 5-ht, his and ang ii with contraction, but is unresponsive to na. | abstractwe investigated the responsiveness of the mouse basilar artery to acetylcholine (ach), bradykinin (bk), noradrenaline (na), 5-hydroxytryptamine (5-ht), histamine (his) and angiotensin (ang) ii in order to characterize the related receptor subtypes in vitro. ach and bk induced endothelium - dependent relaxation of precontracted arteries with u-46619 (a thromboxane a2 analogue). atropine (a non - selective muscarinic receptor antagonist) and n-nitro - l - arginine (a no synthase inhibitor, l - nna) shifted the concentration - response curve for ach to the right, whereas pirenzepine, methoctramine and pfhhsid (muscarinic m1, m2 and m3 antagonists, respectively) had no significant effect. l - nna and hoe140 (a b2 antagonist) shifted the concentration - response curve for bk to the right, whereas des - arg9-[leu8]-bk (a b1 antagonist) and indomethacin (a cyclooxygenase inhibitor) had no significant effect. na failed to produce any vasomotor action. his and ang ii induced concentration - dependent contraction. diphenhydramine (a h1 antagonist) shifted the concentration - response curve for his to the right, whereas cimetidine (a h2 antagonist) had no significant effect. losartan (an at1 antagonist) shifted the concentration - response curve for ang ii to the right, whereas pd123319 (an at2 antagonist) had no significant effect. these results suggest that the h1 and at1 receptor subtypes might play an important role in arterial contraction, whereas muscarinic receptor subtypes apart from m1, m2 and m3, and b2 receptors on the endothelium, might modify these contractions to relaxations. |
personalized medicine attempts to identify individual tailored treatments based on the susceptibility profile of each individual. precision medicine utilizes both conventional medicine and cutting edge technology to concur the disease proven to be resistant to conventional medical techniques. the borders of personalized medicine are defined by limitations in technology and our understanding of biology, and pathology of various conditions. current advances in technology have enabled us to uncover the molecular makeup of diseases and translating these findings to actionable targets has led to the development of small molecular inhibitors. monitoring disease outcome utilizing companion diagnostics has also assisted physicians in routine patient care. to date serious efforts are directed in increasing the efficacy of drug delivery to reduce the undesired side effects of medications (fig. 1). despite the current advances there are fundamental limitations on implementing personalized medicine into daily practice. here we lay out the steps from bench to bedside for personalized therapeutics in hematology and explore the complex problems at each steps. major discoveries utilizing these platforms will be discussed followed by summarizing the targeted inhibitors developed which are currently in clinical practice. next we will briefly discuss the advantages of small molecular inhibitors over existing chemotherapeutic regimens and explore conditions that affect the drug response to these inhibitors (pharmacogenomics and pharmacovigilance). finally we will discuss the drug delivery systems that could potentially enhance the outcome and limit the undesired effects of these medications. advances in human genetics have clearly demonstrated the contribution of specific genes to certain malignancies [13 ]. such genomic alterations are functionally manifested as dysfunctional proteins leading to aberrant signal transduction [47 ]. consequently, discovering genomic alterations underlying various conditions is a fundamental step in implementing precision medicine. selecting an appropriate screening technology is crucial for both discovery and diagnostics. in the era of genomic innovation, mass spectrometric genotyping, allele - specific pcr - based technologies, hybrid - capture massively parallel sequencing technologies, and whole - genome sequencing are among the available platforms. for discovery purposes, sequencing of the entire genome would be the preferred option, but, for diagnostic purposes, one must presently focus on cost - effective platforms that cover actionable cancer - associated mutations (massarrays) (fig. 2). though personalized medicine appears to bring us ever closer to a step away from the cure for cancer, the reality is far more complicated. despite current advances in genomics, there is still a long path to decoding all cancer - associated mutations, the signaling pathway of new and novel mutations which would be an additional area to explore. in both hematologic and solid tumors, a large fraction of affected proteins is represented by kinases, which are essential for physiological functions of cells, such as cellular growth and development [1618 ]. blocking these molecules usually drives the cells into developing compensatory mechanisms, and cancer cells eventually escape the inhibition, developing tumor resistance. another obvious challenge is excessive toxicity by nonselective inhibition of both mutant and wild - type proteins by some inhibitors [2124 ]. in particular, some genetic variations can alter the drug response of individuals, and this should be taken into consideration with drug dosing. therefore it is crucial to develop companion diagnostics by combining genomic information with proteomics as well as personal medical history and family history data to tailor the desired agent for targeting the neoplastic cells [2629 ]. consequently, it is essential to develop prognostic biomarkers to both screen the outcome of the treatment and screen for residual disease [3033 ]. it is essential to deliver the appropriate inhibitors to the affected cells ; however, advancement in the development of nanoparticles that can achieve selective cellular targeting remains limited. the use of nanoparticles has thus been restricted primarily to a reduction of drug toxicity, as evidenced by the success of doxil (liposomal doxorubicin), which decreases cardiotoxicity [3638 ]. fortunately, our understanding of the interactions between nanoparticles and living cells continues to improve. a conceptual understanding of biological responses to nanoparticles is essential for developing safer targeted drug delivery in the future. almost a decade after the completion of the first genome sequencing, genome research composes the main core of discovery in various cancers [3941 ]. the classical discovery platform used for sequencing the human genome was a capillary based electrophoresis (ce) system [4244 ]. although this system was developed by fredrick sanger in the late 70s, it was the most widely used technique for over two decades. the high cost of sample processing along with the restriction on clinical scalability led to the emergence of new technologies based on hybrid capture and massively parallel sequencing (mps) [4547 ], better known as next generation sequencings (ngs) [4850 ]. these profiling platforms enable the investigators to detect point mutations, copy number alterations, and chromosomal aberrations using a single run and a small amount of dna input. these platforms are highly sensitive (i.e. they detect genetic alterations in small allele fractions) and fairly scalable (i.e. they can be tuned for resolution and coverage). last but not least, these platforms are rather affordable and they have brought down the cost of the sequencing of the entire genome to 5000 usd per samples. it is suggested that these new sequencing instruments could sequence several samples in less than a day. for instance alk, pdgfr and fgfr are all discovered using sequencing platforms (sanger / ngs). it is well known that most hematologic malignancies are caused by genomic alteration (point mutation, chromosomal aberrations, copy number variations), and therefore complete understanding of these diseases can only be achieved by comprehensive screening of a large number of clinical samples. despite the fact that the cost of sequencing of the whole genome has dropped significantly in the past decade (from 3 billion usd to roughly 5000 usd), screening a large number of clinical samples could still impose economic challenges. also, most of the information provided by whole genome sequencing (wgs) can not be fully interpreted. despite the fact that there are over 800 new small molecules on developmental pipeline [5759 ], the number of drugable targets currently available in clinics is less than 30. the economic impact of a large scale application of wgs along with limited clinical applicability of information obtained from whole genome is suggestive for the utilization of alternative tools for diagnostic purposes. one of the first platforms developed for high throughput screening of clinical samples in oncology was a mass spectrometric base genotyping platform developed by garraway and colleagues for the detection of cancer - associated mutations. they relied on the fact that a large subset of cancer - associated deriver mutations affects hotspot amino acids. this led to the development of multiplex allele - specific pcr platforms [6163 ]. this platform enabled us to detect a braf - v600e mutation in langerhans cell histiocytosis (lch), a disease which, until this observation, was known as a reactive - inflammatory one [6466 ]. despite the high sensitivity of this platform, it had a very limited coverage and was biased to a subset of genes. it was also unable to detect chromosomal aberrations and large indels. to overcome these shortcomings, ngs platforms were adapted for enrichment of subsets of the human genome. by scaling these platforms and enriching a subset of genome, e.g. on exons or targeted genomic sequences for drugable targets and predictive biomarkers for drug response, several questions could be addressed for a fraction of the cost of wgs (table 2) [6871,132 ]. whole genome sequencing (wgs) enables identification of coding mutations and copy number alterations (amplifications and deletions), but its ability to detect chromosomal translocation in commercially available probes is rendered due to the lack of intronic sequences in capturing probes. stromal contaminations and genomic heterogeneity could also complicate the interpretation of data. on the other hand, targeted sequencing can achieve a larger depth of coverage and consequently higher sensitivity at a comparable cost this approach could be very useful for clinical samples with low preservation (samples derived from formalin fixed tissue) and high stromal contamination, or in the case of hematologic tumors, contamination by bystander cells [7375 ]. on the other hand, to fully understand the genetic background of a disease we must know the extent of gene expression as well. chromatin immunoprecipitations (chip - seq) could unravel the mutation and methylation statuses of gene regulatory sites and determine the activation status of genes, and consequently gene expression. transcriptome sequencing (rna - seq) captures the expressed genome of cancer cells enabling robust detection of deregulated genes, and gene fusions [7880 ]. combining expression data with genomic findings could shed light on the pathomechanisms of the disease and facilitate the design of targeted inhibitors (table 3). clinical applicability of these techniques is quite important and fda has recently approved several ngs instruments for clinical applications. targeted therapies or small molecular inhibitors block the proliferation of cancer cells by intercepting their specific target [8184 ]. since their range of action is smaller than general chemotherapy agents, the adverse effect caused by these inhibitors is smaller as well and they are better tolerated by patients [8588 ]. these inhibitors are not curative, and disease relapse remains a fairly common complication in these malignancies. several reasons could be attributed to disease relapse, among which is the escape of tumors cells which obtain new surviving mutations and the evolution of new neoplastic populations due to weakened immune response. on the other hand, there are obvious caveats in targeting cancer cells with very specific molecular inhibitors. first of all, most of the small molecular inhibitors currently available in clinics are targeting protein kinases [8991 ]. these are an essential cellular component and blocking these molecules could result in cellular compensation. this could manifest either as overproduction of the inhibited protein (upregulation on the gene level), or escalating alternative compensatory pathways for survival. second, the delivery of these molecules to the affected cells is limited by tissue vascularization and cellular uptake. this forces physicians to escalate the dose of inhibitor in compensation, leading to undesirable side effects. table 4 summarizes the current small molecular inhibitors in clinical practice [9296,130,131 ]. in the future we should drive our focus on enhancing the patient 's response based on their unique genetic makeup using appropriate companion diagnostics (pharmacogenomics) along with targeting the driver event. also, to maximize the benefits of small molecular inhibitors, we must deliver the targeted agents to a susceptible population based on individuals ' susceptibility profiles determined by companion diagnostics. eventually, a better outcome will be achieved by matching the right therapy to the right parties, taking us a step closer to a potential cure for these malignancies. last, but not least combining well designed collaborations between private sectors and academics will expedite the drug discovery process. pharmacogenomics is the science that studies the role of inherited and acquired genetic variations to drug response. it correlates gene expression and single nucleotide polymorphism (snp) with both drug toxicity and efficacy to optimize therapeutic regimens for each individual. one of the classical examples of pharmacogenomics is the effect of cytp450 variants on the dosing of warfarin [99101,111 ]. dose - related toxicities have traditionally been considered key end points of phase i trials and the maximum tolerated dose (mtd) was regarded as the optimal dose providing the best efficacy with manageable toxicity. recently, development of targeted inhibitors has challenged the paradigms used in cytotoxic chemotherapy trial design. in precision medicine pharmacokinetic (pk) and furthermore, molecular therapeutic agents usually result in prolonged disease stabilization and provide clinical benefit without tumor shrinkage, a characteristic seen with cytotoxic agents, therefore necessitating alternative measures of anti - tumor efficacy. these end points include biologically relevant drug exposures, pd biomarker measures of target inhibition, and intermediate end - point biomarkers, such as molecular biomarkers (fig., pharmacogenomics is complicated by the fact that two genomes are involved : the germline genome of the patient and the somatic genome of tumor, the latter of which is of primary interest. this genome predicts whether specific targeting agents will have a desired effect in the individual. on the other hand, germline pharmacogenetics can identify patients likely to demonstrate severe toxicities when given cytotoxic treatments. for example, germline snps in the gene encoding the enzyme thiopurine s - methyltransferase (tpmt) can result in increased sensitivity to mercaptopurine as a result of decreased drug metabolism, whereas the number of ta repeats in the promoter region of ugt1a1 can increase the toxic effects of irinotecan again as a result of decreased drug metabolism. therefore, understanding the variable response to drugs is quite pressing in oncology where cytotoxic agents have narrow therapeutic indices and severe side effects. table 5 summarizes the companion diagnostics developed by the fda for the treatment of hematologic malignancies. most of the affected individuals have unique profiles in their tumors in addition to the fact that every individual has a unique snp profile at a germline level. if a certain type of cancer carries several driver mutations then the choice of targeted therapy becomes complicated. in disseminated tumors, the picture would be further complicated by inter - tumor and intra - tumor heterogeneity of cancer [104107 ]. therefore, a greater understanding of the complexities of multiple gene modifiers of outcome, and the statistical challenge of understanding such data, will be needed before individualized therapy can be applied on a routine basis. if an individual carries several driver mutations which inhibitors should be prescribed ? what would be the appropriate dosing of each ? how will drug interactions affect the picture ? how can we increase the therapeutic index ? addressing these questions seems particularly pressing in the era of abundance of targeting inhibitors and the enormous economic pressures on healthcare providers. effective drug delivery could substantially increase the efficacy of small molecule inhibitors in cancer. currently, several nanoparticulate platforms are under investigation. a desirable carrier would be able to incorporate and release drugs with defined kinetics, should have stable formulation for extended shelf life, should be highly specific for its target, and should be bioinert. biological materials such as albumin, phospholipids, synthetic polymers, and even solid components can be used as substrates for nanoparticles (table 6). ideally, the particles could be readily conjugated with a targeting ligand to facilitate specific uptake by target cells. this would result in increased efficacy by increasing drug concentration in the intended target cells as well as in decreased systemic toxicity by reducing non - specific uptake. unfortunately several drug delivery matrix (nanoparticles) used by the pharmaceutical industry imposed risk to the patients. these toxicities varied depending on the surface properties of nanoparticles, chemical composition, their half life and distribution. among the in vivo side effects of nanoparticles, pulmonary inflammation (psp), pulmonary neoplasia (psp), immune response (polystyrene, cb, dep), and platelet aggregations (pm, latex - aggregate surface) are well established. in order to achieve enhanced delivery, reduced toxicity, and eventually enhanced therapeutic index, development of long - circulating and target - specific nanoparticles a conceptual understanding of biological responses to nanomaterials is necessary for development and safe application of nanomaterials in drug delivery in the future. furthermore, a close collaboration between those working in drug delivery and particle toxicology is necessary for the exchange of concepts, methods, and know - how to move this issue ahead. to date the most common vehicle used for targeted drug delivery is the liposomes. these molecules are non - toxic, non - hemolytic, and non - immunogenic even upon repeated injections. liposomes are currently used in cancer therapies (metastatic breast cancer, advanced melanoma, colorectal cancers) but their high cost creates severe limitations. currently, there are huge amounts of screening data available at the genomic level. one of the shortcomings is our limited understanding of the functional importance of these findings. it is curtailed to distinguish driver genomic events from passenger ones. today, there are over 800 new drugs (targeted inhibitors) in the development pipeline. at this point, our shortcoming is not the availability of targeted inhibitors but rather our limitations on the delivery of these molecules to the affected cells with a high degree of specificity. next, we must improve our ability to get the targeted inhibitors designed for malfunctioning cellular components into the affected cells. by increasing the efficacy of targeted drug delivery, we will both reduce the unwanted side effects of antineoplastic agents on healthy cells and increase their cytotoxicity on affected cells. an outstanding high cost will not be sustainable in the long term, so development of technologies for cost reduction should not be ignored. | personalized medicine is the cornerstone of medical practice. it tailors treatments for specific conditions of an affected individual. the borders of personalized medicine are defined by limitations in technology and our understanding of biology, physiology and pathology of various conditions. current advances in technology have provided physicians with the tools to investigate the molecular makeup of the disease. translating these molecular make - ups to actionable targets has led to the development of small molecular inhibitors. also, detailed understanding of genetic makeup has allowed us to develop prognostic markers, better known as companion diagnostics. current attempts in the development of drug delivery systems offer the opportunity of delivering specific inhibitors to affected cells in an attempt to reduce the unwanted side effects of drugs. |
mobility difficulties are common among older adults and are associated with poor quality of life, increased need for care, and are predictive of death [24 ]. understanding the processes that lead to disability is important in order to develop strategies to prevent or delay disability in older adults. recent studies suggest that advanced glycation end products (ages), which are active biomolecules formed by the non - enzymatic covalent binding of sugars with proteins and other molecules, may be related to muscle strength and physical performance [5, 6 ]. the western diet is high in ages, which are formed in high concentrations in foods that are prepared at high temperatures. ages are thought to be absorbed in the process of digestion, circulate in the blood, and can be deposited in different organs and tissues. sarcopenia, or loss of muscle strength and muscle mass, is an important factor underlying mobility difficulties such as slow walking speed in older adults. older adults have increased cross - linking of collagen and deposition of ages in skeletal muscle. in aging animals, cross - linking of collagen is associated with increased muscle stiffness, reduced muscle function [10, 11 ], and accumulation of ages. ages may also play a role in sarcopenia through upregulation of inflammation and endothelial dysfunction in the microcirculation of skeletal muscle through the receptor for ages, or rage. age - induced cross - linking of is collagen elevated in older adults and has been shown to increase the stiffness of human articular cartilage. the relationship between circulating ages and the development of disability has not been characterized in older adults. we hypothesized that women with elevated serum carboxymethyl - lysine (cml), an advanced glycation end product, had an increased risk of developing severe walking disability. to address this hypothesis, we examined the relationship between serum cml concentrations and severe walking disability among older women living in the community. subjects in this study were women who participated in the women 's health and aging study i (whas i), a population - based study designed to evaluate the causes and course of physical disability in older disabled women living in the community. whas i participants were recruited from an age - stratified random sample of women aged 65 years and older selected from medicare enrollees residing in 12 contiguous - zip - code areas in baltimore. women were screened to identify self - reported physical disability that was categorized into four domains. the domains of disability were ascertained in a 20- to 30- minute home interview that included questions related to (1) mobility and exercise tolerance, that is, walking for a quarter of a mile, walking up 10 steps without resting, getting in and out of bed or chairs ; (2) upper extremity function, that is, raising your arms up over your head, using your fingers to grasp or handle, lifting or carrying something as heavy as ten pounds ; (3) higher functioning tasks (a subset of instrumental activities of daily living, not including heavy housework, that is, using the telephone, doing light housework, preparing your own meals, shopping for personal items) ; (4) basic self - care tasks (a subset of nonmobility dependent activities of daily living, i.e., bathing or showering, dressing, eating, using the toilet). whas i enrolled the one - third most disabled women ages 65 and older, those with disability in two or more domains. of the 1409 women who met study eligibility criteria, 1002 agreed to participate in the study in 1992. there were no major differences in sociodemographic or reported health characteristics between eligible participants and those who declined to participate. at the 12-month follow - up visit (as baseline for the present study), 879 women returned for followup, of which 580 received a blood draw. 119 had severe walking disability, 412 did not have severe walking disability, and of whom 394 who had at least one follow - up visit thereafter were included in the present study. laboratory measurements of serum cml were done at the 12-month follow - up visit rather than at enrollment because of a greater availability of serum aliquots from this visit. race was assessed in a questionnaire as black, white, or other, current smoking as yes or no, and education as 08, 911, 12, or more than 12 years as the highest level of formal education achieved. two weeks later, a trained registered full - time study nurse practitioner examined each study participant in her home, using a standardized evaluation of physical performance and physical exam. approximately 75% of women also consented to phlebotomy performed during a separate visit by a trained phlebotomist who followed a standardized protocol. the definitions for the chronic diseases reported in this study were adjudicated by whas coinvestigators based on standardized algorithms that combined information from the questionnaire, physical examination, and physician contact. the mini - mental state examination (mmse) further details on the methods and sampling design of the whas studies are published elsewhere. the participant was asked to walk over a 4-meter course for each follow - up visit. participants were instructed to stand with both feet at the starting line and to start walking after a specific verbal command., the subject could use a cane, a walker, or other walking aid, but not the aid of another person. the times to complete the first meter and the entire path were recorded. the test was repeated three times, twice at the woman 's usual pace, and once at her fastest possible pace. the speed of the faster of the two usual - pace walks was used in the analyses. the length of the walk expressed in meters divided by the time in seconds was used to calculate the average walking speed. women were categorized as having severe walking disability based upon being unable to walk or having a walking speed < 0.4 m / sec. the 0.4 m / sec cut - off point was approximately at the top of the lowest quartile in the whas population at baseline and has been shown to predict functional dependence. demographic characteristics, self - rated health, and information about appetite and eating were measured in the whas questionnaires. chronic diseases were adjudicated by whas coinvestigators based on the questionnaire, physical examination, and physician contact. vital status was determined through matching with the national death index from the 12-month follow - up visit, 199396 through the end of 2000. the johns hopkins university institutional review board approved the study protocol, and written informed consent was obtained from all participants. nonfasting blood samples were obtained by venipuncture between 9 am and 2 pm. blood samples were delivered to quest diagnostics laboratories (formerly ciba - corning laboratories, baltimore, md) on the day of blood drawing for complete blood count and creatinine measurements. processing, aliquoting, and freezing were carried out at the core genetics laboratory of the johns hopkins university school of medicine following a standardized protocol. blood samples were stored continuously at 70c until the time of analyses of serum carboxymethyl - lysine (cml). cml is well characterized, circulating age, and one of the dominant ages in tissue proteins. cml was measured using a competitive elisa (age - cml elisa, microcoat, penzberg, germany). this assay has been validated, is specific, and shows no cross - reactivity with other compounds. measurements were performed in duplicate according to the protocol of the manufacturers, and the results were averaged. the within - assay and between - assay coefficients of variation for serum cml were 3% and 4%, respectively. body mass index (bmi) was categorized as underweight (< 18.5 kg / m), normal range (18.524.9 kg / m), overweight (2529.9 kg / m) and obese (30 kg / m). renal insufficiency was defined as estimated glomerular filtration rate of < 60 ml / min/1.73 m using the modification of diet in renal disease equation of levey and colleagues. plasma cml was divided into quartiles, and the cut - off between quartiles was 452.6, 558.8, and 689.1 ng / ml. grouped - time cox proportional hazards models were used to examine the associations between serum ages and the risk of developing severe walking disability because severe walking disability was determined at six - month intervals. the length of followup in longitudinal analyses was 30 months. women who died, or refused further participation, or were lost to followup after a follow - up visit were censored according to their severe walking disability status at their last visit in the study. during the 30 months following the original 12-month follow - up visit (as the baseline for the present study), there were 23 patients who died, 9 of them died after developing severe walking disability whereas 14 of them died with no severe walking disability until the last follow - up visit. cox proportional hazards models were used to examine the associations between cml and risk of developing severe walking disability. as a highly significant association between slow walking speed and death has been previously described, it is a reasonable assumption that some women developed severe walking disability prior to death. for those who died without developing severe walking disability, first, the multiple imputation technique was used to impute the data and then the complete data was analyzed ; second, the severe walking disability status known data only was analyzed with weighting by inverse probability weighting (ipw) on the data ; finally, two sensitivity analyses were applied to validate those two methods dealing with the incomplete data : (1) treating all missing data because of death as severe walking disability - free ; (2) treating the missing data as severe walking disability developed. the statistical program used was sas 9.1 (sas institute, cary, nc). there were 394 women who did not have severe walking disability at baseline had serum carboxymethyl - lysine (cml) measurements available, and had at least one followup involved in the present study. one hundred fifty - four (26.4%) of the women developed severe walking disability during followup with an overall rate of severe walking disability of 14.8 per 100 person - years. the characteristics of women who did and did not develop severe walking disability during followup are shown in table 1. women who developed severe walking disability were older and more likely to be affected by congestive heart failure, peripheral artery disease, and diabetes. there were no significant differences between women who developed and those who did not develop walking disability by race, education, current smoking, mmse score, body mass index, hypertension, coronary artery disease, stroke, depression, osteoarthritis, chronic obstructive pulmonary disease, and renal insufficiency. compared with the group of lower three quartiles of cml, the patients in the group of highest quartile of cml were more likely to develop severe walking disability. univariate cox proportional hazards models were used to examine the relationship between demographic and health characteristics and the development of severe walking disability (table 2). highest quartile of cml, older age, congestive heart failure, peripheral artery disease, and diabetes mellitus were associated with an increased risk of developing severe walking disability. multivariable cox proportional hazards model that adjusted for age, congestive heart failure, peripheral artery disease, diabetes mellitus, and renal insufficiency are shown in four scenarios (table 3). the present study shows that older community - dwelling women with elevated serum cml are at increased risk of developing severe walking disability. these findings are consistent with a previous, cross - sectional study which showed that elevated plasma cml was associated with slow walking speed in older community - dwelling men and women. the present study extends these findings and shows that elevated cml is an independent predictor of the development of severe walking disability.. increased ages may contribute to increased stiffness in muscle tissue and reduced viscoelastic properties of muscle and thus impair muscle function. collagen is the main structural component of the interstitial space of skeletal muscle and provides elasticity when muscles contract. aging muscle is characterized by an increase in pyridinium cross - linking of collagen, which is enzymatically derived, and the non - enzymatic accumulation of cross - linking ages. in adults without diabetes, the concentration of pentosidine, a major age in tissues, was found in skeletal muscle at greater than two times concentration in older compared with younger adults. tendons transfer the forces generated by muscles to bone and have elastic properties that affect the muscle tendon complex. ages are deposited in tendons and can affect their structural properties. in a rabbit model, cross - linking of collagen by nonenzymatic glycation increased the structural stiffness of achilles tendon. in humans, there was a sevenfold difference in concentrations of pentosidine in the patellar tendon in older compared with younger men. elevated ages are also associated with increased bone rigidity which is thought to occur through cross - linking of collagen. ages may also affect skeletal muscle and tendons through upregulation of inflammation via binding with rage. the carboxymethyl - lysine (cml) adduct of ages has been identified as a signal - transducing ligand for rage, both in vitro and in vivo. ligand binding with rage triggers the induction of increased reactive oxygen species, activation of nadph oxidase, and upregulation of inflammation through nf-b and other signaling pathways [36, 37 ]. inflammatory mediators that are upregulated through age and the nf-b pathway include tnf-, il-6, and c - reactive protein. a limitation of this study is that the results can not necessarily be extrapolated to men or to less disabled women in the community. there are many different ages, and in the present study, cml was the only age that was measured in serum. however, other studies have shown that circulating cml has a moderate correlation with other ages in the blood such as pentosidine and methylglyoxal derivatives. serum cml concentrations show a moderate correlation with dietary intake of ages in older adults. serum cml concentrations have been reported to vary with dietary intake of ages, but these findings have not been corroborated by others [41, 42 ]. older women with elevated plasma cml, an advanced glycation end product, had greater risk of developing severe walking disability. whether modification of dietary intake of ages can influence the pathway to disability in older adults is not known. | advanced glycation end products (ages) have been implicated in the pathogenesis of sarcopenia. our aim was to characterize the relationship between serum carboxymethyl - lysine (cml), a major circulating age, and incident severe walking disability (inability to walk or walking speed < 0.4 m / sec) over 30 months of followup in 394 moderately to severely disabled women, 65 years, living in the community in baltimore, maryland (the women 's health and aging study i). during followup, 154 (26.4%) women developed severe walking disability, and 23 women died. women in the highest quartile of serum cml had increased risk of developing of severe walking disability in a multivariate cox proportional hazards model, adjusting for age and other potential confounders. women with elevated serum cml are at an increased risk of developing severe walking disability. ages are a potentially modifiable risk factor. further work is needed to establish a causal relationship between ages and walking disability. |
leukemia is the most common cancer among children under 15 years of age, accounting for 32 % of all childhood malignancies [1, 2 ]. acute lymphoblastic leukemia (all) is the most common subtype of childhood leukemia, comprising ~80 % of total disease. the etiology of most cases of all in children is unclear, with few identified risk factors to date. a recent meta - analysis of epidemiological studies found that high birth weight (4,000 g) was associated with a modest but significantly elevated risk of childhood all (or = 1.23 ; 95 % ci, 1.151.32). other studies have probed this association with greater sophistication, taking into account gestational age, maternal height, and other factors to estimate the expected birth weight ; the results show that higher than expected birth weight is associated with increased risk of childhood all, even among children who do not have high birth weight [4, 5 ]. these findings suggest that accelerated fetal growth, rather than high birth weight itself, is associated with childhood all risk, pointing to a potential etiologic role for factors regulating growth during early life. among the most important regulators of fetal growth are the components of the insulin - like growth factor (igf) axis, including the growth factors igf1 and igf2, receptors igf1r and igf2r, and a number of binding proteins. while both igf1 and igf2 influence growth in utero, igf1 continues to affect growth postnatally as well. circulating levels of igf1 have been positively correlated with birth weight, birth length, and ponderal index (a measure of body leanness). regulators of energy intake and expenditure, which play a role in body size and growth throughout life, may also have a key role in leukemogenesis through modulation of growth during the perinatal period. specifically, neuropeptide y and ghrelin stimulate hunger, while the adipose - derived hormone leptin signals satiation. importantly, variants in genes encoding members of the igf axis and body size regulation pathway have been linked to birth weight [810 ]. in order to investigate the role of genes regulating fetal growth in childhood all risk, we utilized a haplotype - tagging approach to characterize variation in fetal growth and body size regulation genes in a population - based study of 377 all case children and 448 control children in northern and central california. to our knowledge, no previous studies have examined the role of fetal growth or body size regulation genes in the etiology of childhood all. the study was conducted among children participating in the northern california childhood leukemia study (nccls), a population - based case control study conducted since 1995. briefly, case children with incident childhood leukemia were ascertained via a rapid reporting system between the study office and participating hospitals. control children were randomly selected from the california birth records and matched to case children on date of birth, sex, race, and hispanic ethnicity (a child was considered hispanic if one or both parents reported hispanic ethnicity). data on demographic factors and potential risk factors were elicited in person from a biological parent (usually the mother) by trained interviewers using a structured questionnaire. this study was reviewed and approved by institutional review committees at the university of california berkeley, the california department of public health, and the participating hospitals. written informed consent buccal specimens were collected with cytobrushes from participating children at interview and processed by heating in the presence of 0.5 n naoh. isolated dna was later re - purified using an automated dna extraction system (autogen, holliston, ma, usa) and whole - genome amplified using genomeplex reagents (rubicon genomics, ann arbor, mi, usa). when buccal cytobrush dna was inadequate (26.6 % of subjects), dna was isolated from dried bloodspots (dbs) collected at birth and archived by the genetic diseases screening program of the california department of public health. after extraction (qiaamp 96 dna blood kit, qiagen, germany) all dna specimens were quantitated using human - specific alu - pcr to confirm a minimum level of amplifiable human dna. concordance of all genotypes in dna samples extracted from paired buccal cell and dbs specimens from 9 subjects was 98.9 %. we focused on genes in the igf axis, including igf1, igf2, igf1r, and igf2r, as well as other genes involved in modulation of body size, including leptin (lep), leptin receptor (lepr), ghrelin (ghrl), and neuropeptide y (npy). using haploview software in conjunction with single nucleotide polymorphism (snp) data from the 30 caucasian trios in the hapmap project (release 19, build 34, www.hapmap.org) and the 23 hispanics in the snp500cancer project (www.snp500cancer.nci.nih.gov), we applied the method of gabriel. to select haplotype - tagging snps (htsnps) that captured at least 80 % of the haplotype diversity for common haplotypes (> 5 % frequency) in either the caucasian or hispanic populations. because hispanics are a recently admixed ethnic group, and 42 % of our study population is hispanic (at least one parent reporting hispanic ethnicity), we placed special emphasis on capturing haplotype structures in hispanics. to maximize capture of potential regulatory regions, we included 10 kb stretches both up- and down - stream from the gene boundaries reported in the ucsc genome browser. finally, a set of ancestry informative markers (aims) was included for genotyping ; these have been previously identified to distinguish amerindian, african, and european populations, three populations that make up the genetic ancestry of hispanics. the selected htsnps were genotyped in 385 all cases and 456 controls using a custom illumina goldengate panel (illumina, san diego, ca, usa). we applied a gencall threshold of 0.25, and htsnp - wise and subject - wise call rate thresholds of 90 % and 95 %, respectively. htsnps with a minor allele frequency 10 %) over and above adjustment for self - reported race and ethnicity. thus, we used stratification or adjustment for the self - reported factors in our regression analyses. as a preliminary step prior to haplotype analysis, we tested for potential interactions of individual htsnps with hispanic ethnicity on a gene - by - gene basis using unconditional logistic regression and the likelihood ratio test at the 0.05 significance level, after adjusting for age, sex, and child s race. in haplotype analyses, we used a sliding window approach, as implemented in the haplo.stats package for r, which allows for adjustment for matching factors as well as other potential covariates, including birth weight. this approach examines sub - haplotypes using the full set of htsnp data for a given gene, with differently sized windows of adjacent alleles. this is an effective means of combining multi - locus data for hispanics and non - hispanics, as it is agnostic to potential ethnic differences in haplotype structure. accordingly, if none of the individual htsnps in a given gene interacted significantly with hispanic ethnicity, data for hispanics and non - hispanics were combined for sliding window analyses of that gene. otherwise, the sliding window analysis for that gene was conducted separately for hispanics and non - hispanics. rare haplotypes (5 % frequency among controls) were combined together for risk estimation. we utilized grasp to display and visualize sliding window results, and haplotype trend regression to estimate the magnitude of effect associated with risk haplotypes of the windows with the smallest global p values. the study was conducted among children participating in the northern california childhood leukemia study (nccls), a population - based case control study conducted since 1995. briefly, case children with incident childhood leukemia were ascertained via a rapid reporting system between the study office and participating hospitals. control children were randomly selected from the california birth records and matched to case children on date of birth, sex, race, and hispanic ethnicity (a child was considered hispanic if one or both parents reported hispanic ethnicity). data on demographic factors and potential risk factors were elicited in person from a biological parent (usually the mother) by trained interviewers using a structured questionnaire. this study was reviewed and approved by institutional review committees at the university of california berkeley, the california department of public health, and the participating hospitals. written informed consent buccal specimens were collected with cytobrushes from participating children at interview and processed by heating in the presence of 0.5 n naoh. isolated dna was later re - purified using an automated dna extraction system (autogen, holliston, ma, usa) and whole - genome amplified using genomeplex reagents (rubicon genomics, ann arbor, mi, usa). when buccal cytobrush dna was inadequate (26.6 % of subjects), dna was isolated from dried bloodspots (dbs) collected at birth and archived by the genetic diseases screening program of the california department of public health. after extraction (qiaamp 96 dna blood kit, qiagen, germany), these dna samples were amplified using repli - g reagents (qiagen). all dna specimens were quantitated using human - specific alu - pcr to confirm a minimum level of amplifiable human dna. concordance of all genotypes in dna samples extracted from paired buccal cell and dbs specimens from 9 subjects was 98.9 %. we focused on genes in the igf axis, including igf1, igf2, igf1r, and igf2r, as well as other genes involved in modulation of body size, including leptin (lep), leptin receptor (lepr), ghrelin (ghrl), and neuropeptide y (npy). using haploview software in conjunction with single nucleotide polymorphism (snp) data from the 30 caucasian trios in the hapmap project (release 19, build 34, www.hapmap.org) and the 23 hispanics in the snp500cancer project (www.snp500cancer.nci.nih.gov), we applied the method of gabriel. to select haplotype - tagging snps (htsnps) that captured at least 80 % of the haplotype diversity for common haplotypes (> 5 % frequency) in either the caucasian or hispanic populations. because hispanics are a recently admixed ethnic group, and 42 % of our study population is hispanic (at least one parent reporting hispanic ethnicity), we placed special emphasis on capturing haplotype structures in hispanics. to maximize capture of potential regulatory regions, we included 10 kb stretches both up- and down - stream from the gene boundaries reported in the ucsc genome browser. finally, a set of ancestry informative markers (aims) was included for genotyping ; these have been previously identified to distinguish amerindian, african, and european populations, three populations that make up the genetic ancestry of hispanics. the selected htsnps were genotyped in 385 all cases and 456 controls using a custom illumina goldengate panel (illumina, san diego, ca, usa). we applied a gencall threshold of 0.25, and htsnp - wise and subject - wise call rate thresholds of 90 % and 95 %, respectively. htsnps with a minor allele frequency 10 %) over and above adjustment for self - reported race and ethnicity. thus, we used stratification or adjustment for the self - reported factors in our regression analyses. as a preliminary step prior to haplotype analysis, we tested for potential interactions of individual htsnps with hispanic ethnicity on a gene - by - gene basis using unconditional logistic regression and the likelihood ratio test at the 0.05 significance level, after adjusting for age, sex, and child s race. in haplotype analyses, we used a sliding window approach, as implemented in the haplo.stats package for r, which allows for adjustment for matching factors as well as other potential covariates, including birth weight. this approach examines sub - haplotypes using the full set of htsnp data for a given gene, with differently sized windows of adjacent alleles. this is an effective means of combining multi - locus data for hispanics and non - hispanics, as it is agnostic to potential ethnic differences in haplotype structure. accordingly, if none of the individual htsnps in a given gene interacted significantly with hispanic ethnicity, data for hispanics and non - hispanics were combined for sliding window analyses of that gene. otherwise, the sliding window analysis for that gene was conducted separately for hispanics and non - hispanics. rare haplotypes (5 % frequency among controls) were combined together for risk estimation. we utilized grasp to display and visualize sliding window results, and haplotype trend regression to estimate the magnitude of effect associated with risk haplotypes of the windows with the smallest global p values. characteristics of the 377 all cases and 448 controls are shown in table 1. as expected due to the matched case control design of the nccls, the distribution of age, gender, race, and ethnicity was comparable. we examined a total of 108 htsnps in 8 genes : igf1, igf2, igf1r, igf2r, ghrl, lep, lepr, and npy. we found significant heterogeneity in the effects of htsnps in 3 genes (igf2, igf1r, and igf2r) between hispanics and non - hispanics. haplotype sliding window analyses for these 3 genes were stratified by hispanic ethnicity to permit detection of effects that might be apparent only by ethnicity, while those for the other 5 studied genes were conducted with both ethnic groups combined. results for genes with significant (p 0.05) haplotype effects that persisted through increasingly larger windows are presented in fig. 1. haplotype trend regression results estimating the magnitudes of effect for haplotypes with the lowest multi - htsnp p value in sliding window analyses are shown in table 2.table 1characteristics of genotyped childhood all cases and controls, ncclsvariablecasescontrolsn%n%total377100.0448100.0sex male20053.123752.9 female17746.921147.1age at diagnosis or reference (year) under 1123.2194.2 1524364.528262.9 6108522.59521.2 1114379.85211.6ethnicity hispanic22158.626960.0 non - hispanic15641.417940.0race white21456.825556.9 black154.0163.6 native american71.981.8 asian / pacific islander256.6357.8 mixed11029.213329.7 do nt know61.610.2fig. 1significant (p 0.05) haplotype sliding window results for fetal growth and body size regulation genes and childhood all. outlined blocks show a 6-snp haplotype association for igf1 (p = 0.002), a 3-snp haplotype association for igf2 (p = 0.037), and different but overlapping 3-snp haplotype associations for igf2r in hispanics and non - hispanics. the 6-snp haplotype association observed for igf2r among non - hispanics was driven by rare haplotypes among controls (< 5 % frequency) and therefore not considered furthertable 2haplotype trend regression results : fetal growth and body size regulation genes in childhood all risk, ncclsgenesnpsethnic grouphaplotypecontrol frequencycase frequencyor (95 % ci)p valueglobal pigf1(rs5742632, rs2195240, rs1019731, rs12821878, rs35767, rs35765)bothrare haplotypes0.0160.0369.90 (2.07, 47.26)0.0040.002a - a - c - a - g - c0.0580.0581.23 (0.51, 3.00)0.644g - g - c - g - a - c0.0760.1062.13 (1.04, 4.35)0.039a - a - c - g - a - a0.0910.0991.45 (0.72, 2.92)0.304a - a - a - a - g - c0.1380.1681.81 (1.02, 3.22)0.044g - g - c - g - g - c0.2000.1530.72 (0.41, 1.28)0.268a - a - c - g - g - c0.4200.3791.00 (ref)igf2(rs2585, rs734351, rs1003483)hisprare haplotypes0.0240.0499.34 (1.09, 80.14)0.0420.040g - g - c0.0530.0802.92 (0.72, 11.90)0.134a - g - a0.1480.1942.34 (1.00, 5.48)0.051g - a - a0.2260.2161.30 (0.57, 2.98)0.538g - a - c0.5490.4611.00 (ref)igf2r(rs635551, rs9457799, rs6455678)hispg - c - a0.0860.0871.25 (0.413.80)0.6970.051a - a - a0.0950.0480.29 (0.051.79)0.184a - c - g0.2710.3402.10 (0.984.53)0.058a - c - a0.5490.5251.00 (ref)igf2r(rs9457799, rs6455678, rs6920141)nhrare haplotypes0.0130.0399.69 (1.64, 57.34)0.0120.009a - a - g0.0950.1332.34 (1.02, 5.36)0.044c - g - g0.2010.1720.82 (0.44, 1.52)0.520c - a - a0.6910.6561.00 (ref)377 childhood all cases, 448 controlswald test ; bold type indicates significant resultsglobal test for haplotype association ; bold type indicates significant results includes any haplotypes with less than 5 % frequency among controls characteristics of genotyped childhood all cases and controls, nccls significant (p 0.05) haplotype sliding window results for fetal growth and body size regulation genes and childhood all. outlined blocks show a 6-snp haplotype association for igf1 (p = 0.002), a 3-snp haplotype association for igf2 (p = 0.037), and different but overlapping 3-snp haplotype associations for igf2r in hispanics and non - hispanics. the 6-snp haplotype association observed for igf2r among non - hispanics was driven by rare haplotypes among controls (< 5 % frequency) and therefore not considered further haplotype trend regression results : fetal growth and body size regulation genes in childhood all risk, nccls 377 childhood all cases, 448 controls wald test ; bold type indicates significant results global test for haplotype association ; bold type indicates significant results includes any haplotypes with less than 5 % frequency among controls among all subjects, igf1 showed significant haplotype associations that persisted through progressively larger htsnp windows (fig. 1). the selected six - htsnp haplotype window showed a stronger association (global p = 0.002) than any individual snp (table 2). in this window, haplotypes g - g - c - g - a - c and a - a - a - a - g - c were associated with significantly increased risk (or = 2.13 and p = 0.039, and or = 1.81 and p = 0.044, respectively). the 3-htsnps genotyped in igf2 showed a significant haplotype association among hispanics, and the a - g - a haplotype was significantly associated with an increased risk of all (or = 2.34, p = 0.051). igf2r showed significant haplotype associations in both hispanics and non - hispanics, but the haplotype windows showing the strongest associations differed between ethnic groups. among hispanics, the minimum p value among all haplotype windows was for a 6-htsnp window whose effects were driven by haplotypes with less than 5 % frequency among controls ; accordingly, this 6-htsnp window was not considered further. however, there was also a 3-htsnp window in igf2r that was significantly associated with childhood all risk among hispanics (global p value = 0.051). the a - c - g haplotype of this window showed a borderline significantly increased risk (or = 2.10, p = 0.058). among non - hispanics, a 3-htsnp haplotype window in which 2 htsnps overlapped with the window observed for hispanics was significantly associated with childhood all (global p = 0.009), and haplotype a - a - g was associated with a significantly increased risk (or = 2.34, p = 0.044). of the htsnps genotyped, four had putative function (non - synonymous variants, splice variants, variants in regulatory regions, or variants in strong linkage disequilibrium with functional variants) as listed in entrez gene. only one of these putative functional variants, rs35767 in igf1, was found to have a significant association with risk of childhood all. its minor allele was associated with a significantly increased risk of disease in both ethnicities combined (heterozygote : or = 1.48 ; 95 % ci, 1.101.99 ; homozygote : or = 1.74 ; 95 % ci, 0.883.45 ; ptrend = 0.005).table 3childhood all risk estimates for putative functional snps in fetal growth and body size regulation genes, ncclsgenesnpboth ethnicitiesn casen ctrlor (95 % ci)pplog addbpintcigf1rs35767(ld with functional microsatellite)gg2173001.00 (ref)0.0050.3190ag1401311.48 (1.1, 1.99)0.010 aa20161.74 (0.88, 3.45)0.111 igf2rrs629849(g1619r)gg3083561.00 (ref)0.3760.1920ag63780.94 (0.65, 1.35)0.720aa150.23 (0.03, 1.97)0.179hrlrs4684677(q78l)tt2733331.00 (ref)0.2310.8245at83991.03 (0.72, 1.47)0.880aa17102.11 (0.93, 4.79)0.074ghrlrs696217(l60m)cc3223701.00 (ref)0.3250.5753ac49680.83 (0.56, 1.24)0.365aa120.59 (0.05, 6.58)0.669wald test p value ; bold type indicates significant resultslog additive inheritance model ; bold type indicates significant results likelihood ratio test p value for interaction with hispanic status childhood all risk estimates for putative functional snps in fetal growth and body size regulation genes, nccls wald test p value ; bold type indicates significant results log additive inheritance model ; bold type indicates significant results likelihood ratio test p value for interaction with hispanic status in this population - based case control study, we examined the risk of childhood all associated with several genes involved in fetal growth and body size regulation, utilizing a haplotype - tagging approach to maximize capture of genetic variation. we identified haplotypes of several genes that were significantly associated with childhood all, including igf1, igf2, and igf2r. to our knowledge, no previous studies have expressly examined the role of genes involved in the igf axis or body size regulation in risk of childhood all. our findings support the hypothesis that factors involved in fetal growth and body size regulation, specifically components of the igf axis, play a role in mediating risk of childhood all. in this study, we found two haplotypes in the same 6-htsnp window of igf1 to be significantly associated with an increased risk of childhood all. the haplotype window of association here stretches across 25 kb, from the 5 promoter region into the second intron. igf1, also known as somatomedin c, is expressed by both the mother and the fetus during pregnancy and plays a critical role in modulating cellular proliferation, differentiation, and apoptosis. while igf1 controls growth directly in utero, it becomes subject to regulation by growth hormone in the postnatal period. we identified a significant 3-htsnp haplotype of igf2 associated with a markedly increased risk of childhood all. in contrast to igf1, igf2 is thought to play a more critical role in growth and development in utero versus postnatally. serological igf2 levels are higher during the in utero versus the postnatal period, and studies of igf2 knockout mice show stunted fetal growth but normal postnatal growth. although igf2 is an imprinted gene, meaning that only one allele is usually expressed, there are no consistent findings to support the hypothesis that loss of imprinting of igf2 is associated with an increased risk of childhood all. finally, we found significant associations of overlapping but distinct igf2r haplotype windows with risk of childhood all among hispanics and non - hispanics separately. interestingly, the two overlapping htsnps in each of these windows, rs9457799 and rs6455678, had significantly different single - htsnp effects in hispanics versus non - hispanics. the differential effect of these two htsnps was present in the haplotype analysis as well : the risk haplotype among hispanics included c - g at these two loci and was associated with a borderline significant increased risk, while among non - hispanics the haplotype including c - g at these two loci showed no association. conversely, the haplotype with a - a at these two loci was associated with a significantly increased risk among non - hispanics and null risk among hispanics. the igf2r gene product serves no signal transduction purpose ; rather, by binding igf2, it appears to serve mainly to regulate igf2 levels in utero [21, 22 ]. the putative functional igf1 snp (rs35767) found to be significantly associated with childhood all risk is in the igf1 gene promoter region. it is not functional itself but is in strong linkage disequilibrium with -841(ca)n, a common microsatellite that has been linked to circulating igf1 levels as well as birth weight and other measures of body size. this snp is part of the 6-htsnp haplotype window we found to be significantly associated with childhood all risk. these genes include lep, whose gene product leptin signals satiety and has been found to correlate with neonatal birth weight and size for gestational age, and lepr, whose gene product mediates the effects of leptin. ghrelin, encoded by ghrl and secreted primarily by the stomach, serves to stimulate appetite and promote adiposity and may have a compensatory effect on intra - uterine growth restriction, serving to boost growth in the postnatal period. neuropeptide y, encoded by npy, is a neurotransmitter that also leads to increased appetite and storage of energy as fat, as well as decreased physical activity. however, we found no significant haplotype associations between these genes and risk of childhood all. in this study, statistical adjustment for birth weight did not affect the associations observed for the significant risk haplotypes in igf1, igf2, and igf2r, or the single nominally significant (p < 0.05) putative functional snp in igf1. the independence of the observed associations from birth weight, coupled with the absence of direct associations of the haplotypes and htsnp in question with birth weight (data not shown), suggests that the effects of the risk haplotypes and alleles on childhood all risk are not directly mediated by birth weight. indeed, data from a recent meta - analysis of genome - wide studies did not identify birth weight associated loci in igf axis genes. because we did not begin collecting data on maternal height or pre - pregnancy weight until partway through recruitment of the study population, these data were unavailable for 49 % of study subjects, and therefore, we were unable to estimate percent optimal birth weight, a measure of appropriateness of fetal growth that takes into account maternal body size, gestational age, and other factors and may be more relevant to all risk than birth weight alone. maternal components of the igf axis are unable to cross the placenta ; accordingly, fetal levels of these factors are likely to be determined by fetal tissues, lending support to the notion that fetal genotypes are particularly relevant. however, maternal genotypes influencing maternal metabolism and growth may have an impact on fetal growth ; accordingly, investigation of maternal genotypes is warranted. hispanics, an understudied population with the highest reported rates of childhood leukemia in california. our htsnp selection strategy included elements designed to maximize capture of genetic variation in hispanics. we examined hispanics separately from non - hispanics where there was significant heterogeneity in between - group effects of individual htsnps in each gene. although this approach may have limited our ability to detect associations in the population as a whole, we felt it was necessary given that genetic susceptibility may be different in hispanics versus non - hispanics due to the hispanic population s relatively recent genetic admixture. results that differ between hispanics and non - hispanics may be due to differences in allele frequency and/or haplotype structure or may reflect underlying differences in exposures that modulate the effects of genes. regardless, if the results are not spurious, they represent potential risk loci, and we present them in either or both ethnic groups for replication and further follow - up. the limited size of racial / ethnic sub - populations within the non - hispanic group precluded further stratification of this group ; as such, genetic heterogeneity within this group might have obscured results. however, adjustment for calculated genetic ancestry did not markedly change the race- and ethnicity - adjusted results we present here, indicating that the potential impact of population stratification is minimal. two large genome - wide association studies on childhood all have been published to date (with 907 cases and 2,398 adult and child controls, and 317 cases and 17,958 adult controls, respectively) [28, 29 ]. these studies have identified a number of novel loci ; however, no significant associations were observed for genes in igf axis and body size regulation genes. this may be due to stringent multiple testing adjustment (at the p 1 10 level) to account for the large number of individual variants tested in genome - wide studies. in contrast to the agnostic approach to discovery used in such studies, our study focused on relatively few genes representing key elements of the igf axis and body size regulation pathways. it commenced prior to the recent publication of results linking birth weight to loci in two genes (adcy5 and ccnl1) ; as such, we were unable to investigate these genes. we concede that our study results may be due to chance and therefore must be replicated. however, the haplotype - tagging approach we adopted maximizes capture of total variation within each candidate gene, and the haplotype analysis increases statistical power to detect associations over analyses of individual variants. finally, although the haplotype - tagging approach does not pinpoint potential causal variants, it does localize risk - associated regions for additional study, including fine - mapping. recent studies showing correlations of umbilical cord serum igf1 levels with both cord stem cell levels and birth weight [30, 31 ] support the notion that increased fetal growth may increase cancer risk by either increasing pre - leukemic stem cell populations targeted for initiation, or promoting growth or survival of an initiated leukemic stem cell. together with this, our results are consistent with the notion that the igf axis influences the initiation or promotion of childhood all early in life., we set out to investigate the role of genes in the igf axis and body size regulation pathways in risk of childhood all. our results indicate that elements of the igf axis are associated with childhood all risk. these findings are consistent with the evidence that childhood all initiation and/or promotion may begin in utero, when lymphoid progenitor cells are not yet fully differentiated and therefore more susceptible to malignant transformation. the associations and interactions identified here should be considered targets for replication in additional studies with larger sample size and finer coverage of variants in the identified associated gene regions. | accumulating evidence suggests that childhood acute lymphoblastic leukemia (all) may be initiated in utero or early in the postnatal period. high birth weight (or rapid fetal growth) is associated with risk of all, but the mechanisms are not understood. in a population - based epidemiologic study of childhood all, we utilized a haplotype - based approach to assess the role of eight genes involved in fetal growth and body size regulation in 377 childhood all cases and 448 controls. we found significant haplotype associations with risk of childhood all for igf1 among non - hispanics and hispanics together (p = 0.002), for igf2 among hispanics (p = 0.040), and for igf2r among hispanics and non - hispanics (p = 0.051 and 0.009, respectively). no haplotype associations were observed for igf1r or the studied genes involved in body size regulation, including lep, lepr, ghrl, and npy. our study is the first to identify an association between the genes involved in the igf axis and risk of childhood all. these findings for childhood all emphasize the importance of fetal growth, when lymphoid progenitor cells are not yet fully differentiated and therefore more susceptible to malignant transformation. additional studies are needed to confirm these findings and identify specific causal variants. |
the number of people with diabetes is expected to rise to 300 million in 2025 in the world. hyperglycemia results from lack of insulin or inadequate insulin secretion following increased insulin resistance. in vivo and in vitro studies have found that deficiency of vitamin d results in reduction in insulin secretion which leads to hyperglycemia, increased hemoglobin a1c and insulin resistance.[23 ] insulin - dependent diabetes mellitus (iddm) and moderate to severe vitamin d deficiency in adults has frequently been shown to be associated with reduced bone mineral content. many previous experiments have demonstrated that the rate of osseointegration around dental implants was considerably reduced.[68 ] also, in the recent decade, dental implants have been considered as a well - accepted treatment modality to replace missing and lost teeth. in this study, the rats were rendered diabetic by means of alloxan (c4 h4 n2 o2).this drug is mainly used in treatment of pancreatic tissue cancers and also as a diabetes inducer drug in studies. intra - peritoneal injection of alloxan (in different dosages) results in increased blood glucose during 12 - 48 h in rats. the role of alloxan in induction of apoptosis is unknown but it may be due to free radicals of oxygen following its injection. vitamin d is fat - soluble, and consumed as ergocalciferol (d2) or cholecalciferol (d3) through dietary sources. the mechanisms whereby vitamin d may impact on the development and management of diabetes are : insulin secretion in isolated islets of langerhans is dependent upon vitamin d in animals.the presence of vitamin d receptors on beta cells of the islets of langerhans, and the ability of the islets to express 1-alpha hydroxylase activates 25 hydroxy vitamin d.an indirect effect of vitamin d on beta cell insulin secretion by means of increased parathyroid hormone (pth) which increases the intracellular calcium in the islet, resulting in inhibition of post - receptor binding action of insulin is also postulated.vitamin d receptors have also been identified on cells of the immune system. the presence of vitamin d receptors on beta cells of the islets of langerhans, and the ability of the islets to express 1-alpha hydroxylase activates 25 hydroxy vitamin d. an indirect effect of vitamin d on beta cell insulin secretion by means of increased parathyroid hormone (pth) which increases the intracellular calcium in the islet, resulting in inhibition of post - receptor binding action of insulin is also postulated. reported that treating with 1, 25 (oh)2 d3 (1 g / d:40 iu for 4 days) had no effect on fbs in diabetic patient. tanaka., reported that using 160 iu of vitamin d (sc) twice a week in rats with vitamin d deficiency did not show significant differences in perfusate insulin by 16.7 mm glucose. so, we increased the dose and decided to use 160 iu (4 g) vitamin d for 7 days to illuminate the effect of vitamin d supplement on the amount of osseointegration around tibial implants, as a marker of healing at 3 and 6 weeks.the hypothesis of this research was that the administration of vitamin d in diabetic rats could enhance the osseointegration of implants in rats tibia. forty eight healthy and vaccinated male wistar rats (24 weeks old ; weight range : 250 to 280 g) were used. the study protocol was approved by the committee on animal care of torabinejad dental research center, isfahan university of medical sciences, isfahan, iran and executed according to national animal law. each 5 rats were kept in a cage in an air conditioned environment (24 1c, 50% to 60% humidity), with a circadian light rhythm of 12 h day / dark. then the rats were rendered diabetic by iv (tail vein) injection of 35 mg / kg monohydrate alloxan (st. louis, mo, usa), freshly dissolved in physiological serum, by insulin syringe. blood glucose was measured in 24 h of alloxanisation by one - touch glucometer (bionime gmbh, heinrich wild strasse 202, ch-9435 heerbrugg, switzerland) and reflected by glycosuria, hyperglycemia, polyphagia, polydipsia and body weight loss. the rats which were showing fasting blood glucose levels between 130 and 200 mg/ dl were selected for the study. 1,76137 karl sruhe, germany), made of commercially pure titanium, 1.2 mm in diameter and 7 mm in length, were used in this study. the rats were anesthetized by intra - abdominal injection of equal mixture of 0.1 ml / kg of ketamine hydrochloride and xylazine. the skin on tibial bone was shaved and disinfected with 70% ethanol. a 15 mm incision was made and bone was exposed. after exposing the bone, a 0.8 mm pilot hole was drilled through the medial cortex, the medulla, and the lateral cortex of the tibia. subsequently, the hole was gradually widened by larger drills to the final diameter of the screws. the sterilized screws were placed in the proximal metaphyseal region of the tibia in all 48 rats. flunixin meglumine (flunex, razak, iran) (2.5 mg / kg body weight) was administered to reduce pain for 3 days. after implantation, the rats were randomly divided in two groups, and received 160 iu of vitamin d (3) or placebo orally each day for one week. five rats died within 3 weeks, including 2 rats of case group and 3 of control group ; nine rats in each group were sacrificed in 3 weeks of implantation and the others were remained for 6 weeks investigation because of the unexpected mortality. then tibiae were removed with trephine bur and embedded in methyl methacrylate (meliodent ; heraeus kulzer, berkshir, uk). then, implant body was cut axially using a microtome (denmark, copenhagen, stuers, 50-accutom) to prepare approximately 250 m thick non - decalcified grinded specimens, then polished to final thickness of approximately 100 m.the tissues were dyed with masson 's trichrome stain for microscopic observation [figures 1 and 2 ]. 36, stereo microscope image of case (3 weeks), bic:91.6% 100, polarizan microscope image of case (6 weeks), bic : 44% the amount of bone - implant contact and the changes in quantity of bone tissue around the implants were evaluated morphometrically by bone to implant contact (bic) parameter and total contact length around the implants. to perform the histomorphometric analysis, a millimeter grid transparent sheet was placed on the 20 25-cm amplified images. by counting the filled boxes, the proportion of osseous neo - formation areas stained by the marker was quantified. a total of 14 implants were dislodged during histologic processing, including 6 implants from case group and 8 from control group. the data were analyzed with spss 11.5 software and anova and t - test to discover the differences between the 2 groups at level of significance of p < 0.05. forty eight healthy and vaccinated male wistar rats (24 weeks old ; weight range : 250 to 280 g) were used. the study protocol was approved by the committee on animal care of torabinejad dental research center, isfahan university of medical sciences, isfahan, iran and executed according to national animal law. each 5 rats were kept in a cage in an air conditioned environment (24 1c, 50% to 60% humidity), with a circadian light rhythm of 12 h day / dark. then the rats were rendered diabetic by iv (tail vein) injection of 35 mg / kg monohydrate alloxan (st. louis, mo, usa), freshly dissolved in physiological serum, by insulin syringe. blood glucose was measured in 24 h of alloxanisation by one - touch glucometer (bionime gmbh, heinrich wild strasse 202, ch-9435 heerbrugg, switzerland) and reflected by glycosuria, hyperglycemia, polyphagia, polydipsia and body weight loss. the rats which were showing fasting blood glucose levels between 130 and 200 mg/ dl were selected for the study. 1,76137 karl sruhe, germany), made of commercially pure titanium, 1.2 mm in diameter and 7 mm in length, were used in this study. the rats were anesthetized by intra - abdominal injection of equal mixture of 0.1 ml / kg of ketamine hydrochloride and xylazine. the skin on tibial bone was shaved and disinfected with 70% ethanol. a 15 mm incision was made and bone was exposed. after exposing the bone, a 0.8 mm pilot hole was drilled through the medial cortex, the medulla, and the lateral cortex of the tibia. subsequently, the hole was gradually widened by larger drills to the final diameter of the screws. the sterilized screws were placed in the proximal metaphyseal region of the tibia in all 48 rats. flunixin meglumine (flunex, razak, iran) (2.5 mg / kg body weight) was administered to reduce pain for 3 days. after implantation, the rats were randomly divided in two groups, and received 160 iu of vitamin d (3) or placebo orally each day for one week. five rats died within 3 weeks, including 2 rats of case group and 3 of control group ; nine rats in each group were sacrificed in 3 weeks of implantation and the others were remained for 6 weeks investigation because of the unexpected mortality. then tibiae were removed with trephine bur and embedded in methyl methacrylate (meliodent ; heraeus kulzer, berkshir, uk). then, implant body was cut axially using a microtome (denmark, copenhagen, stuers, 50-accutom) to prepare approximately 250 m thick non - decalcified grinded specimens, then polished to final thickness of approximately 100 m.the tissues were dyed with masson 's trichrome stain for microscopic observation [figures 1 and 2 ]. 36, stereo microscope image of case (3 weeks), bic:91.6% 100, polarizan microscope image of case (6 weeks), bic : 44% the amount of bone - implant contact and the changes in quantity of bone tissue around the implants were evaluated morphometrically by bone to implant contact (bic) parameter and total contact length around the implants. to perform the histomorphometric analysis, a millimeter grid transparent sheet was placed on the 20 25-cm amplified images. by counting the filled boxes, a total of 14 implants were dislodged during histologic processing, including 6 implants from case group and 8 from control group. the data were analyzed with spss 11.5 software and anova and t - test to discover the differences between the 2 groups at level of significance of p < 0.05. the state of diabetes (130 mg / dl fbg levels 200 mg / dl) was predictably induced and monitored throughout the study. at 3 weeks, the control group (n = 5) reported a bic level at 44 19 and the vitamin d group (n = 7) at 57 20. at 6 weeks, the control group (n = 5) reported bic level at 70 29 and the vitamin d group (n = 10) at 65 22. the mean percentage of bic value was 66 23 [table 1 and figures 2 and 3 ]. comparison of bone to implant contact between the vitamin d and control groups at 3 mean percentage of bic value changes between vitamin d and control groups at 3 and 6 week twenty one samples were missed because of death and incorrect histologic processes. in this study, statistical significant difference of bic value was not observed between case and control groups (p = 0.703) and was not time dependent as well (p = 0.074). between group differences in bic in short term (3 weeks) also was not statistically meaningful. (p = 0.308) after 6 weeks this difference was not noticeable (p = 0.695) [table 1 and figures 2 and 3 ]. vitamin d regulates calcium homeostasis by influencing on intestinal calcium absorption, renal calcium re - absorption, and bone calcium metabolism. it was shown that moderate to severe vitamin d deficiency in adults was frequently associated with reduced bone mineral content, and the rate of osseointegration around dental implants was considerably reduced under vitamin d insufficiency. the positive effect of calcium and vitamin d supplements on improvement of bone healing around dental implants, was revealed by park., in 2007 in normal rats. then in september 2011, hong., approved this report by observing the healing process of surgically created alveolar sockets in dog model. she reported that vitamin d deficiency was associated with a decrease in bic value in the cortical area but no significant changes due to vitamin d depletion were noticed in the medullar compartment in comparison with control group that received 2400 iu/ kg vitamin d as a normal diet. vitamin d has an indirect effect on bic through an essential role on diabetes. in vivo and in vitro studies have found that deficiency of vitamin d resulted in reduction in insulin secretion and thus in hyperglycemia, increased hemoglobin a1c and insulin resistance. rabbits and mice with vitamin d deficiency were found to have impaired insulin secretion, however vitamin d supplementation corrected this defect. in the other hand, evidence indicates that persons with diabetes have lower serum concentrations of vitamin d and it plays an integral role in insulin sensitivity and secretion. our data is similar to witham 's data, because we did not see any noticeable changes in blood glucose after administration of vitamin d. in that study, vitamin d improved systolic blood pressure but not insulin resistance or glycosylated hemoglobin in patients with type 2 diabetes. insulin - dependent diabetes mellitus (iddm) has frequently been shown to be associated with reduced bone mineral content. alterations in microvascularization and wound healing associated with diabetes lead to diminished immune responses and a reduction in bone remodelling. some animal studies have demonstrated that diabetes mellitus could negatively interfere with the process of osseointegration before 8 weeks ; ottoni., in 2004 reported bic level of 39% in diabetic rats in comparison to 73% in control after 56 days. hideki., in 2008 demonstrated that bic levelwas 12% indiabetic group and 61% incontrol group at 4 weeks. a 2-fold difference remained at week 8, which is in agreement with a report on gk rats. this effect was also seen in our experiment, the mean percentage of bic in all groups showed reduction (60 23) but it was more than previous studies, because the lower range of blood glucose was chosen in our experiment. the consequence of diabetes on the healing process of soft tissue depends on the degree of glycemic control in the pre - operative period and the existence of chronic vascular complications. patients with poor metabolic control have their immune defense impaired and greater predisposition to infection of the wound. the microangiopathy arising as a complication of diabetes may compromise vascularization, thus delays healing and acts as a gateway for the infection of tissue. in our experiment, the lower blood glucose was chosen in this study to compare with previous studies (130 - 200 mg / dl). it may conceal the direct effect of vitamin d on diabetes. in the study of tempe., 120 mg / kg of alloxan was used to induce diabetes in rats, or luciane. considered the blood glucose level between 400 - 600 mg / dl as diabetic state., reported that treatment with 1,25 (oh)2 d3 (1 g / d:40 iu for 4 days) had no effect on fbs in diabetic patient. tanaka., reported that vitamin d (sc) supplementation for160 iu twice a week in vitamin d deficient rats did not show significant differences in perfusate insulin in response to 16.7 mm of glucose. the baseline and serum concentration of vitamin d is very important, so it may suggest that prescribing the 160 iu of vitamin d for one week was insufficient and resulted in low bioavailability of vitamin d. it is necessary to determine the total dose of vitamin d3 supplement to achieve optimal serum concentration. in human study, the relationship between diabetes and vitamin d deficiency may be affected by genetic variation in vitamin d receptors, and it may be an altered factor in this study. dose, age, body weight, race, sex and environmental factors were resembled. in the other word, it has been reported that females with type 2 diabetes have a high prevalence of hypovitaminosis d, and the review of the literature which was done in 2005, revealed that the age and gender of diabetic patients did not seem to influence implant survival, so we chose male rats, but we suggest that in the future studies larger sample sizes including both sexes with higher dosage of vitamin d or longer duration of supplementation be recruited to clarify the differences between groups. vitamin d has no effect on osseointegration of implants in diabetic rats (130 mg / dl blood sugar 200 mg / dl) at 3 and 6 weeks ; however, for better definition of the role of vitamin d in the bic, high - quality observational studies and rcts that measure blood 25-hydroxyvitamin d concentration and clinically relevant glycemic outcomes are needed. | background : diabetes has become the next most widespread disease after cancer. recent studies have found that diabetes and moderate to severe vitamin d deficiency are associated with reduced bone mineral content ; therefore administration of vitamin d may correct these conditions. the purpose of this research is to compare the effect of vitamin d administration on bone to implant contact in diabetic rats with control group.materials and methods : in this randomized placebo - controlled trial, 48 wistar rats were rendered diabetic (130 blood sugar 200 mg / dl) by iv injection of 35 mg / kg alloxan. implants were inserted in tibial bone ; then rats were divided into study and control groups and received oral vitamin d3 (160 iu) or placebo respectively for one week. bone to implant contact value was measured under light microscope at 3 and 6 weeks.results:analysis of data indicated that vitamin d had no significant effect on bone to implant contact (bic). at 3 weeks, the control group (n = 5) reported bic level at 44 19 and study group (n = 7) at 57 20. at 6 weeks, the control group (n = 5) reported bic level at 70 29, and study group (n = 10) at 65 22. twenty one samples were missed because of death or incorrect lab processes.conclusion:it seems that vitamin d supplement has no significant effect on bic in 130 mg / dl blood sugar 200 mg / dl (p = 0.703) andwas also not time dependent (p = 0.074). |
the hormonal form of vitamin d affects both adaptive and innate immune functions involved in the development of allergies. genetic factors of the vitamin d metabolism are involved in the development of allergic conditions showing a direct association between vitamin d receptor polymorphisms and atopic asthma. furthermore, studies on an animal model provided evidence for a link between early vitamin d supplementation and the later allergy where several vitamin d regulated genes seem to be involved. recently, low serum vitamin d levels were observed in association with airway obstruction and corticosteroid requirement in asthmatic patients influencing the asthma severity, atopy, or both [46 ]. vitamin d in the form of 1,25-dihydroxyvitamin d is a potent immune system modulator and at the molecular level has been shown to be involved in the suppression of dendritic cell maturation and consecutive th1 cell development [79 ]. in fact, vitamin d may suppress the production of il-12, thereby reducing the production of t helper type 1 (th1) cells and potentially leading to increased proliferation of allergy - associated t helper type 2 (th2) cells. additionally, studies in mice have shown that treatment with 1,25-dihydroxyvitamin d results in the reduced secretion of th1-type cytokines il-2 and ifn- and an increase in th2-type il-4. interleukin 33 (il-33) potently drives the production of proinflammatory th2-associated cytokines, including il-4, il-5, and il-13, by in vitro polarized th2 cells, mast cells, basophils and eosinophils [13, 14 ], and dendritic cells. furthermore, hematopoietic cells produce other inflammatory cytokines and chemokines, including il-6 and il-8, via il-33 stimulation. these activities suggest potential roles for il-33 in th2-associated immune responses, and thus il-33 is thought to be closely associated with allergic inflammatory diseases, including asthma. indeed, increased il-33 levels were observed in the bronchoalveolar lavage fluid from subjects with moderate asthma compared with mild asthmatics and controls without asthma. a single - nucleotide polymorphism in il-33 that showed a suggestive association with the circulating eosinophil count was also significantly associated with atopic asthma. interleukin 31 (il-31) is a t helper type 2 effector cytokine that plays an important role in the pathogenesis of atopic and allergic diseases. il-31, mainly produced by activated th2 cells, interacts with a heterodimeric receptor consisting of il-31 receptor a (il-31ra) and the oncostatin m receptor (osmr) constitutively expressed on epithelial cells and keratinocytes. plasma il-31 concentration was found to be significantly elevated in patients with atopic dermatitis (ad) compared to healthy individuals and positively correlated with disease severity. mainly, a crucial immunopathological role of il-31 and il-33 was observed in ad suggesting their inflammatory effect in atopic disease. the present study aims to investigate the plasma levels of 25(oh) vit d, il-31, and il-33 in children with allergic asthma and rhinitis. furthermore, since lower levels of 25(oh) vit d and high levels of il-31 and il-33 are associated with th2 immunity of allergic disease, we hypothesized a potential interaction between these markers and clinical and functional parameters in asthma and rhinitis. pediatric subjects (age between 8 and 13 years) were recruited among outpatients attending the pulmonology / allergy clinic of the national research council of italy in palermo. asthma diagnosis and assessment of severity ar diagnosis was performed at the study entry according to allergic rhinitis and its impact on asthma (aria) guidelines. the patients were divided in two groups : 35 children had concomitant intermittent to persistent allergic rhinitis and intermittent to moderate allergic asthma (aar), and 11 patients had intermittent to persistent allergic rhinitis (ar). the control group, having negative assessment of the atopic status and no asthma or rhinitis symptoms, was composed of 28 healthy children (hc). children stopped the treatment for asthma (ics) or rhinitis (antihistamines or topical steroid) 1 month before entering in the study. no patients had nasal polyposis or bronchial or respiratory tract infections or had a severe exacerbation of asthma resulting in hospitalization during the last month. within 2 days from blood samples, the study was approved by the ethics committee of the university hospitals of palermo and complied with the helsinki declaration (n7/2013). written informed consent was obtained from the parents of the patients enrolled in the study. skin prick tests were performed according to eaaci recommendations with a standard panel including dermatophagoides mix, grass mix, parietaria judaica, olive, dog and cat dander, alternaria, and blattella germanica, plus a positive (histamine 1%) and a negative (saline) control (stallergenes italia s.r.l., the reading was performed after 15 min : reactions were considered positive if the mean wheal diameter (computed as the maximum diameter plus its orthogonal divided by two) was 3 mm or greater, after having subtracted the wheal diameter of the reaction to the negative control. total and allergen - specific serum ige levels were determined by cap system (pharmacia - upjohn, uppsala, sweden). the total atopy index (degree of allergic sensitization) was also assessed, which is defined as the total number of allergens tested to which each subject had a positive response. the total allergen sige score (sum of classes of allergen specific ige test) was considered as a quantitative score able to reflect the levels of allergen exposure and the degree of multiple sensitizations in the patients. plasma 25(oh) vit d was assayed using an enzyme linked immunosorbent assay (elisa) kit (immunodiagnostic system ltd., boldon business park, boldon, tyne and wear, uk) according to the manufacturer instructions. the absorbance was read at 450 nm (reference 620 nm) by a wallac 1420 victor2 multilabel counter (perkin - elmer life sciences, turku, finland). this method had 100% and 75% specificity for 25(oh)d3 and 25(oh)d2, respectively, and assessed the overall vitamin d status but did not distinguish between these two forms. the limit of detection was 2 ng / ml. the intra- and interassay coefficients of variation were 5.3% and 4.6%, respectively. samples were run in duplicate with quality control samples to ensure day - to - day validity of results. plasma il-31 and il-33 protein levels were measured using the commercially available duoset elisa development system kits (r & d systems ; minneapolis, mn, usa) specific for human il-31 and human il-33. absorbance was measured at 450 nm (correction wavelength set at 540 nm) by a wallac 1420 victor2 multilabel counter (perkin - elmer life sciences, turku, finland). the detection limit was 15.6 pg / ml for il-31 and 4.0 pg / ml for il-33. statistical analysis was performed using kruskal - wallis test and the differences between groups were evaluated by nonparametric mann - whitney u test. the chi - square () analysis was performed to evaluate differences in frequency distributions of variables. we reported the demographic, clinical, and atopic characteristics of all patients included in the study in table 1. we found significant lower levels of 25(oh) vit d in ar (median ; range : 24.0 ng / ml, 22.528.3 ; p 135 nmol / l) levels. nevertheless, a significant but nonlinear relationship was found between 25-hydroxyvitamin d3 status and ige levels. it therefore appears that too little or too much vitamin d may predispose to the development of allergic immune responses. in this scenario, despite the controversy in the literature and despite our small sample of children with concomitant allergic asthma and rhinitis, we showed that the levels of 25(oh) vit d inversely correlated with total allergy sige score and with total atopy index, further demonstrating a negative effect of lower levels of vitamin d on respiratory allergic diseases. this finding clearly suggests that low levels of 25(oh) vit d are associated to the degree of multiple allergen sensitizations in the patients. finally, we showed that children with aar with positive sige to dust mites had lower levels of 25(oh) vit d. these findings further underline and support the concept that vitamin d might be necessary as an adjunct to subcutaneous allergen immunotherapy in asthmatic children sensitized to house dust mite as previously described. in conclusion, this study demonstrated the effect of vitamin d levels on the related outcomes of asthma and of allergic phenotypes of the disease, with a particular link with the host dust mite sensitization. since most allergies start in childhood, vitamin d deficiency or insufficiency in childhood might influence initiation of allergy targeting unclear and little known immunologic aspect of the disease. further clinical studies might clarify the link between vitamin d and asthma and allergic immunologic features. | low vitamin d is involved in allergic asthma and rhinitis. il-31 and il-33 correlate with th2-associated cytokines in allergic disease. we investigated whether low vitamin d is linked with circulating il-31 and il-33 in children with allergic disease of the airways. 25-hydroxyvitamin d [25(oh) vit d ], il-31, and il-33 plasma levels were measured in 28 controls (hc), 11 allergic rhinitis (ar) patients, and 35 allergic asthma with rhinitis (aar) patients. we found significant lower levels of 25(oh) vit d in ar and in aar than in hc. il-31 and il-33 plasma levels significantly increased in aar than hc. il-31 and il-33 positively correlated in ar and aar. 25(oh) vit d deficient aar had higher levels of blood eosinophils, exacerbations, disease duration, and total ige than patients with insufficient or sufficient 25(oh) vit d. in aar 25(oh) vit d levels inversely correlated with total allergen sige score and total atopy index. il-31 and il-33 did not correlate with 25(oh) vit d in ar and aar. in conclusion, low levels of 25(oh) vit d might represent a risk factor for the development of concomitant asthma and rhinitis in children with allergic disease of the airways independently of il-31/il-33 th2 activity. |
lipomas are most common benign neoplasms of mesenchymal tissues accounting for their 1520% of cases occurring in head and neck region, while only 14% cases seen intraorally. the etiology and pathogenesis of lipoma is not clear, but many factors such as mechanical, endocrine, and inflammatory influences have been reported inconclusively for their role. in oral cavity, lipomas are observed during routine intraoral examination and usually involve areas with fat accumulation. clinically, lipomas are slow growing asymptomatic painless nodular swellings with either yellow color or that of normal mucosa. intraorally, most commonly involved sites are buccal mucosa, tongue, floor of the mouth, buccal vestibule, and lips. we here present a rare case of lipoma occurring on mandibular mucogingival junction, an extremely rare intraoral site for lipoma with focus on differential diagnosis. a 32-year - old male patient reported with the complaint of painless swelling in left mandibular posterior region since 4 months that was gradually increasing. the patient had no history of dysphagia, difficulty in speaking, and no previous episode of swelling in the same region. the patient had not undergone any treatment for the swelling and not taken any medication. there was no significant family and medical history. no obvious facial swelling or lymphadenopathy was observed. the intraoral swelling measuring 12 mm 18 mm approximately was observed on the left mandibular mucogingival junction extending from the second premolar to third molar [figure 1 ]. on palpation, swelling was soft in consistency with smooth margins and not fixed to underlying deeper structures. intraoral periapical radiograph revealed normal trabecular bone structure and no pathological changes were observed in alveolar bone [figure 2 ]. based on the history and clinical examination, a provisional diagnosis of intraoral benign soft tissue tumor was given. the hematoxylin- and eosin - stained soft tissue specimen showed the presence of sheets of mature adipocytes in a distinct lobular arrangement with a thin fibrous capsule [figure 3 ]. intraoral swelling in left mandibular posterior region intraoral periapical radiograph revealing normal trabecular bone pattern photomicrograph showing sheets of mature adipocytes in a distinct lobular arrangement with a thin fibrous capsule, a = adipocyte, c = capsule. (h and e, 100, scale bar = 25 m) photomicrograph showing mature adipocytes demonstrating large clear cytoplasms and eccentric nuclei, a = adipocyte, represents blood capillary. lipomas are mesenchymal tumors of adipose tissue. they commonly involve trunk and proximal portions of extremities and are relatively uncommon in oral cavity the etiology is still not clear, but a role of various factors has been reported. according to hypertrophy theory these tumors occur in oral cavity due to obesity and inadvertent growth of adipose tissue. however, metaplasia theory states that aberrant differentiation ofmesenchymal cells into lipoblasts leads to the development of lipoma. it is thought that trauma and chronic irritation may trigger the proliferation of fatty tissue that can cause development of lipoma. mean age of occurrence of intraoral lipoma varies according to different studies, but they usually occur in fourth and fifth decades of life. their prevalence is similar in both the sexes, although a male and female predominance has also been recorded. the present case of lipoma was seen in a male patient with age of 32 years. most common intraoral sites are buccal mucosa, tongue, floor of the mouth, vestibule, and lip. in a study by taira., lipoma on gingiva was found in only 8.7% of the cases out of 207 cases of oral lipoma, suggesting it to be an uncommon site of occurrence. manor. could not document any case of gingival lipoma in an analysis of 58 cases of oral and maxillofacial lipomas. studart - soares. had documented gingiva to be the rarest intraoral site for lipoma in their extensive analysis of 450 cases of lipoma. oral lipomas clinically may present as slow growing solitary or multiple lesions which may be sessile or pedunculated. they present as asymptomatic, well - circumscribed soft encapsulated fluctuant masses or nodules with doughy consistency. grossly, the color may vary from that of normal mucosa to pink and some may present as yellowish masses. the diagnosis is not easy if the yellow color of the tumor is not visible through overlying thin mucosa. although the growth of oral lipomas is usually limited, they can reach great dimensions, interfering with speech and mastication, reinforcing the need for excision. in our case, the patient presented with a solitary soft sessile smooth mass measuring 12 mm 18 mm approximately. the complications typically associated with lipomas have been functional impairment due to giant size that they achieve occasionally. because of the similar clinical presentation, lesions such as oral dermoid and epidermoid cysts, oral lymphoepithelial cyst, benign salivary gland tumor, mucocele, benign mesenchymal neoplasm, ranula, ectopic thyroid tissue, and lymphoma are considered in its differential diagnosis. in our patient, the possibility of a dermoid or epidermoid cyst was also excluded as the location was not in favor. hemangioma, lymphangioma, rhabdomyoma, neuroma, or neurofibromas are the swellings to be ruled out when tumor is located on dorsal surface of tongue. although oral lipomas are well - circumscribed soft tissue lesions, rarely they give a radiographic impression of an intraosseous neoplasm within the mandibular canal. the gross specimen of lipoma when placed in a pot with water usually floats in it. it is difficult to distinguish lipoma from surrounding connective tissue when it is deeply placed. fine - needle aspiration biopsy (fnab) or ultrasound guided fnab sometimes can be used for aspiration in such cases. ultrasonography is a preferred technique as it is faster and inexpensive, and lipomas are hypoechoic with echogenic spots. a color doppler ultrasonography can also be done to evaluate the content of the lesion and its vascularity. the histopathological features constitute of a circumscribed aggregate of mature adipocytes which may be encapsulated. adipocytes show large clear cytoplasm in the absence of vascularity which serves as diagnostic feature of classic lipoma. all lipomas are usually well - vascularized, but the vascular network is compressed by the distended lipocytes and is usually not appreciable. the hematoxylin- and eosin - stained soft tissue specimen in our case showed the presence of mass of mature adipocytes arranged in lobules surrounded by a fibrous capsule. on the basis of microscopic features, lipomas can be classified as simple lipoma, fibrolipoma, infiltrating or intramuscular lipoma, angiolipoma, myxolipoma, spindle cell lipoma, pleomorphic lipoma, myolipoma, angiomyolipoma, chondroid lipoma, osteolipoma or ossifying lipoma, and salivary gland lipoma (sialolipoma). histopathologically, the differential diagnoses are normal soft fatty tissue, other histologic variants of lipoma and liposarcoma. other lesions which should be distinguished are schwannoma, myxoid neurofibroma, leiomyoma, nodular fasciitis, myxolipoma, fibrolipoma, malignant fibrous histiocytoma, myxoid liposarcoma, and myxoid solitary fibrous tumor. despite the close histological similarity to normal adipose tissue, lipomas, usually, have chromosomal aberrations such as translocations involving 12q13 - 15, locus interstitial deletions of 13q, and rearrangements involving 8q11 - 13 locus. immunohistochemistry has been used for differentiation between benign and malignant adipose tissue tumors with detection of ap2, a protein expressed by lipoblasts. immunocytochemical studies with cd34, bcl-2, 21, 24 assists in differentiating lipomas from other myxoid lesions. however, immunohistochemistry was not done in our patient due to financial constraints and the histopathological diagnosis was definite and in accordance with clinical findings. approximately, 58% of lipoma patients may present with multiple tumors which occur predominantly in upper half of the body and are three times more common in men than in women. the occurrence of multiple lipomas can be seen in association with cowden 's syndrome or multiple hamartoma syndrome, frohlich syndrome, proteus syndrome, and bannayan zonana syndrome. most common mucocutaneous lesions in the patients with cowden 's syndrome are small papular lesions in palate and gingiva, papillomatous and verrucous lesions of buccal mucosa, fissured tongue, and multiple cutaneous lipomas. frohlich syndrome also known as prune belly syndrome is defined by multiple lipomas, obesity, and sexual infantilism. proteus syndrome is marked by multiple lipomatous lesions, including pelvic lipomatosis, fibroplasia of feet and hands, skeletal hypertrophy, exostoses and scoliosis, and various pigmented lesions of skin. a case of congenital lipoma was described in a 7-year - old boy in upper labial frenum. zonana syndrome is characterized by congenital association of multiple lipomas, hemangiomas, and macrocephaly. the main treatment modality for intraoral lipomas and its histologic variants is simple surgical excision. recurrence rate described after excision is low, but infiltrative lipoma tends to recur after inadequate excision due to the fact that they are not encapsulated like simple lipomas. steroid injections are used when size of tumor is < 1 inch in diameter and their usage results in local fat atrophy and hence shrinks the size of tumor. malignant tumors are characterized by areas of lipoblastic proliferation, myxoid differentiation, cellular pleomorphism, increased vascularity, and mitosis. thus, lipomas should be excised completely as there is a rare possibility of malignant transformation. patients usually do not seek treatment for oral lipoma as it is mostly asymptomatic and may be observed during routine oral examination. although the features of lipoma are simple and straightforward, accurate clinical and surgical information along with a histopathological examination is the mainstay to its definitive diagnosis. the possibility of multiple lipomas must be considered and an accurate upper body examination must be done in patients with intraoral lipomas. clinicians should be aware about the possibility of lipomas in the oral cavity and should always consider it in their differential diagnosis of an inoffensive swelling in oral cavity. | lipoma is the most common tumor of mesenchymal tissues of body, but its occurrence in oral cavity is infrequent. buccal mucosa is the most common intraoral site of lipoma followed by tongue, floor of the mouth, and buccal vestibule. the involvement of mucogingival junction is rare. we present a unique case report of oral lipoma occurring on mandibular mucogingival junction with review of literature which has emphasis on differential diagnosis. |
a total of 123 patients with non - neoplastic colon (n=17), hyperplastic polyp (n=15), low grade tubular adenoma (n=22), high grade tubular adenoma (n=27), intramucosal crc (n=10), and invasive crc (n=32 ; 5 with t2 crc and 27 with t3 crc) treated at dongguk university gyeongju hospital between 2009 and 2012 were enrolled in this study. high grade tubular adenoma is defined as a mucosal change with cytological and architectural features of malignancy but without evidence of invasion into the stroma.18 we selected patients whose paraffin embedded tissues were relatively well preserved and whose medical records were complete. we excluded patients who underwent preoperative chemotherapy and emergency surgery, and patients who were diagnosed with mucinous adenocarcinoma. specimens were fixed in 10% formalin for 12 - 24 hours and embedded in paraffin blocks. tissue sections were sampled along the maximum tumor diameter and we included the deepest site of cancer invasion. differentiation, tumor depth and status of lymph node metastasis were assessed after reviewing each tumor slide. the stage was defined according to the tnm staging system of the american joint committee on cancer. the presence of budding was determined according to the criteria proposed by ueno.17 the authors defined an isolated single cancer cell and a cluster composed of fewer than five cancer cells as tumor budding. in invasive crc, tumor budding foci was examined in tissue sections immunohistochemically stained using the anti - e - cadherin antibody and the number of tumor budding (ntb) was counted in a field in which budding intensity was considered maximal at high power magnification. to identify the correlation between ntb and the expression level of cd10 and cd15 in invasive crc, our cases were divided into two categories based on the ntb : low ntb (0 - 10) and high ntb (> 10). tissue sections of 4 m thickness were made and were spread on poly - l - lysine coated slides. the paraffin sections were immersed in three changes of xylene and hydrated using a graded series of alcohol solutions. antigen retrieval was performed routinely by immersing the sections in 0.01 m citrate buffer (ph 6.0) in an autoclave for 15 minutes. endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 minutes, followed by incubation of the sections with primary antibody for two hours at room temperature ; the primary antibodies included anti - e - cadherin (1:1,000, transduction laboratories, lexington, ky, usa), anti - cd10 (1:100, novocastra laboratories ltd., newcastle, uk), and anti - cd15 (1:250, dako, santa barbara, ca, usa). immunohistochemical staining was performed using an envision kit (dako) and the color was developed with 3,3'-diaminobenzidine tetrahydrochloride (zymed laboratories, inc. mouse and rabbit igg isotypes, rather than the primary antibody, were used as negative controls. the immunoreactivity of cd10 was evaluated based on the extensity of tumor cells (tcd10), stromal cells (scd10) and infiltrating inflammatory cells (icd10). the immunoreactivity of cd15 was also determined on the extensity of tumor cells or epithelial cells (tcd15) and infiltrating inflammatory cells (icd15). positive tcd10 and tcd15 were defined as the presence of 10% or more positive cells in tumor cells or epithelial cells.10 in whole tissue section, the extensity of scd10, icd10 and icd15 was graded according to a 4-point scale based on the percentage of stained area : 0 (stained area, 0 - 10%), 1 (stained area, 11 - 20%), 2 (stained area, 21 - 50%), and 3 (stained area, > 50%). in invasive crc, the extent of scd10, icd10, and icd15 was evaluated in the tumor center and invasive front. for statistical analysis, our cases were divided into two groups : the negative group (0, 1) and the positive group (2, 3). a total of 123 patients with non - neoplastic colon (n=17), hyperplastic polyp (n=15), low grade tubular adenoma (n=22), high grade tubular adenoma (n=27), intramucosal crc (n=10), and invasive crc (n=32 ; 5 with t2 crc and 27 with t3 crc) treated at dongguk university gyeongju hospital between 2009 and 2012 were enrolled in this study. high grade tubular adenoma is defined as a mucosal change with cytological and architectural features of malignancy but without evidence of invasion into the stroma.18 we selected patients whose paraffin embedded tissues were relatively well preserved and whose medical records were complete. we excluded patients who underwent preoperative chemotherapy and emergency surgery, and patients who were diagnosed with mucinous adenocarcinoma. specimens were fixed in 10% formalin for 12 - 24 hours and embedded in paraffin blocks. tissue sections were sampled along the maximum tumor diameter and we included the deepest site of cancer invasion. differentiation, tumor depth and status of lymph node metastasis were assessed after reviewing each tumor slide. the stage was defined according to the tnm staging system of the american joint committee on cancer. the presence of budding was determined according to the criteria proposed by ueno.17 the authors defined an isolated single cancer cell and a cluster composed of fewer than five cancer cells as tumor budding. in invasive crc, tumor budding foci was examined in tissue sections immunohistochemically stained using the anti - e - cadherin antibody and the number of tumor budding (ntb) was counted in a field in which budding intensity was considered maximal at high power magnification. to identify the correlation between ntb and the expression level of cd10 and cd15 in invasive crc, our cases were divided into two categories based on the ntb : low ntb (0 - 10) and high ntb (> 10). tissue sections of 4 m thickness were made and were spread on poly - l - lysine coated slides. the paraffin sections were immersed in three changes of xylene and hydrated using a graded series of alcohol solutions. antigen retrieval was performed routinely by immersing the sections in 0.01 m citrate buffer (ph 6.0) in an autoclave for 15 minutes. endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 minutes, followed by incubation of the sections with primary antibody for two hours at room temperature ; the primary antibodies included anti - e - cadherin (1:1,000, transduction laboratories, lexington, ky, usa), anti - cd10 (1:100, novocastra laboratories ltd., newcastle, uk), and anti - cd15 (1:250, dako, santa barbara, ca, usa). immunohistochemical staining was performed using an envision kit (dako) and the color was developed with 3,3'-diaminobenzidine tetrahydrochloride (zymed laboratories, inc. mouse and rabbit igg isotypes, rather than the primary antibody, were used as negative controls. the immunoreactivity of cd10 was evaluated based on the extensity of tumor cells (tcd10), stromal cells (scd10) and infiltrating inflammatory cells (icd10). the immunoreactivity of cd15 was also determined on the extensity of tumor cells or epithelial cells (tcd15) and infiltrating inflammatory cells (icd15). positive tcd10 and tcd15 were defined as the presence of 10% or more positive cells in tumor cells or epithelial cells.10 in whole tissue section, the extensity of scd10, icd10 and icd15 was graded according to a 4-point scale based on the percentage of stained area : 0 (stained area, 0 - 10%), 1 (stained area, 11 - 20%), 2 (stained area, 21 - 50%), and 3 (stained area, > 50%). in invasive crc, the extent of scd10, icd10, and icd15 was evaluated in the tumor center and invasive front. for statistical analysis, our cases were divided into two groups : the negative group (0, 1) and the positive group (2, 3). as shown in fig. 1, cd10 was expressed in tumor cells, stromal cells and infiltrating immune cells. scd10 was mainly localized in subepithelial stromal cells in the periluminal region of the lamina propria in tubular adenoma and intramucosal crc. on the contrary, 2a, the positive rate of tcd10 expression was 0% (0 out of 17) in non - neoplastic colon, 0% (0 out of 15) in hyperplastic polyp, 14% (3 out of 22) in low grade tubular adenoma, 22% (6 out of 27) in high grade tubular adenoma, 40% (4 out of 10) in intramucosal crc and 44% (14 out of 32) in invasive crc. the positive rate of scd10 expression was 0% in non - neoplatic colon and hyperplastic polyp, 14% (3 out of 22) in low grade tubular adenoma, 41% (11 out of 27) in high grade tubular adenoma, 70% (7 out of 10) in intramucosal crc and 88%. the positive rate of icd10 expression was 47% (15 out of 32) in invasive crc and nearly 0% in other diseases. therefore, tcd10 expression level in invasive crc was significantly higher than that of non - neoplastic diseases and low grade tubular adenomas (p<0.05), and there was an insignificant difference in its expression level among high grade tubular adenoma, intramucosal and invasive crc. scd10 expression level was significantly higher in invasive crc compared to other diseases, except for intramucosal crc (p<0.05) and there was an insignificant difference in its expression level between high grade tubular adenoma and intramucosal crc. in addition, both tcd10 and scd10 in adenoma were not related to the degree of dysplasia. icd10 expression level was the highest in invasive crc compared to all other diseases (p<0.05). 2b, the positive rate of tcd15 was 0% in non - neoplastic colon, 13% (2 out of 15) in hyperplastic polyp, 23% (5 out of 22) in low grade tubular adenoma, 48% (13 out of 27) in high grade tubular adenoma, 50% (5 out of 10) in intramucosal crc and 44% (14 out of 32) in invasive crc. therefore, tcd15 expression level was significantly higher in high grade tubular adenoma, intramucosal and invasive crc compared to non - neoplastic colon (p<0.05), and there was an insignificant difference in its expression level among neoplastic diseases. the positive rate of icd15 was 6% (1 out of 17) in non - neoplastic colon, 0% in hyperplastic polyp, 23% (5 out of 22) in low grade tubular adenoma, 44% (12 out of 27) in high grade tubular adenoma, 30% (3 out of 10) in intramucosal crc and 59% (19 out of 32) in invasive crc. therefore, the icd15 expression level was significantly higher in invasive crc than in non - neoplastic diseases and low grade tubular adenoma (p<0.05) and there was an insignificant difference in its expression level among high grade tubular adenoma, intramucosal and invasive crc. as shown in fig. there was a positive correlation between the expression level of icd10 and icd15 in whole tissue sections, the tumor center and invasive front of invasive crc (r=0.815, r=0.730, and r=0.776, respectively ; p<0.05). in invasive crc, we compared the expression of scd10, icd10 and icd15 in the tumor center to that in the invasive front and examined the relationship between their expression levels in the invasive front and ntb. 3, the positive rate of scd10 was 72% (23 out of 32) in the tumor center and 69% (22 out of 32) in the invasive front, that of icd10 was 16% (5 out of 32) in the tumor center and 41% (13 out of 32) in the invasive front, and that of icd15 was 22% (7 out of 32) in the tumor center and 53% (17 out of 32) in the invasive front. therefore, the expression level of icd10 and icd15 was significantly higher in the invasive front compared to the tumor center (p<0.05). in invasive crc, there were 11 tumors with low ntb and 21 tumors with high ntb. 4, the positive rate of icd15 was 18% (2 out of 11) in invasive crc with low ntb and 71% (15 out of 21) in invasive crc with high ntb. there was a significant correlation between ntb and icd15 expression of the invasive front (p<0.05). as shown in table 2, the positive rate of td15 in whole tissue sections was 0% (0 out of 5) in t2 crc and 52% (14 out of 27) in t3 crc. the expression level of tcd15 was significantly associated with tumor depth (p<0.05). however, any other clinicopathologic parameters did not show significant association with the expression of cd10 and cd15. in addition, the expression of cd10 and cd15 in the tumor center and invasive front, respectively, was not significantly associated with any clinicopathologic parameters (data not shown). cd10 is an important molecule capable of integrating signals from either the cell environment or the intracellular compartment by cleaving peptides through enzymatic activity and through intracellular signaling pathways that interfere with other major signaling pathways.2 therefore, the deregulation of cd10 expression leads to the accumulation or loss of peptides, disturbing the regulation of cellular proliferation and differentiation, and altering intracellular signaling pathways.2 it is thus obvious that the derangement of cd10 expression is associated with the development or progression of a variety of tumors. in the current study, cd10 was expressed in stromal cells, inflammatory cells, and tumor cells during crc development, which is consistent with previous publications.7 - 11 furthermore, its expression level in each cell was progressively increased from non - neoplastic colon to crc. these findings suggest that cd10 expression in the tumor microenvironment is involved in crc development. in the current study, tcd10 expression was significantly higher in invasive crc than in non - neoplastic diseases and low grade tubular adenoma, and did not show significant difference among high grade tubular adenoma, intramucosal and invasive crc. scd10 expression was significantly higher in invasive crc than other diseases except for intramucosal crc and there was an insignificant difference in its expression level between high grade tubular adenoma and intramucosal crc. these findings indicate that scd10 expression is more important in cancer cell invasion than tcd10 expression and the biologic behavior of tcd10 and scd10 is similar between high grade tubular adenoma and intramucosal crc. a previous study suggested that scd10 expression may be induced by the local inflammatory process in malignant tumors.19 cd10 can create a tumor microenvironment to facilitate cancer cell invasion and metastasis due to its structural similarity to mmps.2 in addition, a recent in vitro study has demonstrated that scd10 strongly interacted with cd133-positive colon cancer cells and enhances its invasion.20 moreover, a previous study suggested that scd10 had an important role in invasion and metastasis in the tissues of crc.9 on the other hand, some reports have shown that tcd10 expression in crc was closely related to liver metastasis and high clinical stages.7,8 mucosal high grade neoplasia in gastrointestinal epithelial neoplasia is composed of four diagnostic terms such as high grade adenoma, carcinoma in situ, suspicious for invasive carcinoma and intramucosal carcinoma.21 the rationale for putting together these diagnoses under one category is that their clinical behavior is nearly identical and they can not be reproducibly diagnosed. based on the studies that scd10 and tcd10 are involved in cancer cell invasion and metastasis, the current result that the expression of scd10 and tcd10 is similar between high grade adenoma and intramucosal crc supports the notion that the clinical behavior is the same among the four diagnoses categorized under mucosal high grade neoplasia. our study showed that there was no significant correlation between expression of tcd10 and scd10 and prognostic factors such as nodal metastasis and tumor depth in invasive crc. this may be due to so the low number of crc cases analyzed. in the current study, cd10 was also expressed in inflammatory cells, which revealed cd15 expression and showed morphology similar to neutrophils. recent studies have demonstrated that most cd10-positive immune cells are derived from myeloid cells and are neutrophil granulocytes based on the results showing the coexpression of cd11b and cd15, and morphology similar to neutrophils.10,11 the present study showed that icd15 expression level was higher than the icd10 expression level during crc development. this indicates that tumor - infiltrating neutrophils were divided into a high cd10-expressing group and a low cd10-expressing group. granulocyte - macrophage colony - stimulating factor (gm - csf) can enhance cd10 expression in normal neutrophils in vitro.22 its serum level was significantly higher in crc than in normal persons.23 the infiltration of neutrophils with high cd10 expression was correlated with a poor prognosis, while low expressing neutrophils showed a favorable prognosis in crc.11 however, the present study did not show any correlation between the icd10 expression level and prognostic factors such as nodal metastasis and tumor depth. the possibility that other pathophysiological functions of neutrophils may be closely associated with the prognosis of crc also can not be excluded. recently, accumulating evidence indicates that neutrophils play an important role in malignant transformation, tumor progression, angiogenesis and the modulation of the antitumor immunity in several tumors.24 neutrophils regulate the invasion at multiple levels. neutrophils can facilitate tumor invasion by directly degrading ecm through the release of mmp-9.25 tumor - derived cytokines such as tnf - alpha and gm - csf induce the release of hepatocyte growth factor and oncostatin m by neutrophils, which promote the invasion and migration of tumor cells.26 neutrophils may prepare tumor cells to degrade ecm and enhance the motility of tumor cells through the activation of mmp-2 and rho kinase, and by phosphorylating focal adhesion kinase and paxillin.27 in the current study, icd15 and icd10 were expressed more in the invasive front than in the tumor center. in addition, the icd15 expression level in the invasive front was closely associated with the degree of tumor. these findings suggest that tumor - infiltrating neutrophils are involved in tumor invasion and its mechanism can be explained by the above mentioned pathophysiological function as well as cd10 function. cd15 is expressed in neutrophils and it binds to the e - selectin of endothelial cells, which leads to neutrophil migration into tissue.12 cd15 expression in tumor cells was also involved in the adhesion of tumor cells to endothelial cells and its expression level in colon cancer cells was correlated with the capacity of invasion and metastasis.13,14 therefore, previous publications have reported that high tcd15 expression was related to liver metastasis tumor depth and disease recurrence in crc.16,28 at this time, the current study could not confirm the role of tcd15 expression on disease recurrence and metastasis. however, our result that tcd15 expression was associated with tumor depth could verify the biologic function of tcd15 expression related to tumor invasion. in addition, a recent publication has reported that cd15 expression in tumor or epithelial cells was observed in 7% of healthy tissue, 27% of adenomas and 75% of crc.15 in that report, its expression level in adenoma was significantly associated with the degree of dysplasia. however, the current study showed that tcd15 expression was higher in high grade tubular adenoma and crc than in non - neoplastic colon, but there was an insignificant difference in its expression level among neoplastic diseases. this discrepancy in the role of tcd15 during crc development is to be confirmed by more extensive studies. in summary, the present study showed that cd10 was expressed in tumor cells, stromal cells and inflammatory cells, and cd15 was also expressed in tumor cells and inflammatory cells. icd10 expression level was significantly correlated with icd15 expression level in invasive crc. icd10 and icd15 were expressed more in the invasive front than in the tumor center. moreover, the icd15 expression level of the invasive front was significantly correlated with the degree of tumor budding. tcd15 expression level was closely associated with the tumor depth of invasive crc. in conclusion, the expression of cd10 and cd15 are associated with the development and progression of crc. | backgroundthe aim of this study was to examine the expression of cd10 and cd15 in tumor cells, stromal cells and infiltrating inflammatory cells during colorectal carcinoma (crc) development and to investigate their expression levels between the tumor center and invasive front and compare them to clinicopathological parameters in invasive crc.methodswe performed immunohistochemical staining for cd10, cd15, and e - cadherin in 42 cases of crc, 49 of tubular adenoma, 15 of hyperplastic polyp, and 17 of non - neoplastic colon.resultscd10 was expressed in tumor cells (tcd10), stromal cells (scd10) and infiltrating inflammatory cells (icd10), and cd15 was expressed in tumor cells (tcd15) and infiltrating inflammatory cells (icd15). their expressions were progressively increased during crc development and the icd10 expression level was significantly correlated with the icd15 expression level in invasive crc. invasive front revealed a higher expression level of icd10 and icd15 than the tumor center. moreover, the icd15 expression level of invasive front was significantly correlated with the degree of tumor budding and tcd15 in whole tissue sections was closely associated with tumor depth.conclusionsthe present study suggests that the expression of cd10 and cd15 is associated with the development and progression of crc. |
the initial formation of many major structures of the human embryo takes place during a very narrow window of time in the first weeks after conception, often before a woman even knows she is pregnant. any change in the early embryonic environment, including nutrient availability, has the potential to adversely or positively affect the developing embryo. evidence is evolving that specific components of maternal diet can play a critical role in modifying the risk for birth defects. the best example of the influence of maternal diet on birth defect risk is the intake of folic acid for prevention of neural tube defects (ntds). randomized controlled folic acid intervention trials demonstrating the effectiveness of folic acid for ntd risk reduction have led to the current public health recommendation, as well as folic acid fortification of the food supply. prevention of ntds with supplemental folic acid is likely to involve a complex interaction between folic acid and multiple genetic factors ; this is the focus of ongoing investigations. the success of folic acid supplementation in reducing ntds has led researchers to evaluate the association of other dietary components, such as fat, with a risk for birth defects. researchers have found that pre - pregnancy diabetes and obesity are associated with an increased risk for numerous birth defects (for example, specific heart defects, ntds and cleft palate). a recent meta - analysis revealed that certain aspects of preconception care reduced the risk for some birth defects among women with diabetes : the risk reductions were associated with changes in behavior, such as increased folic acid use or improved glycemic control, or both (depending on the study). both diabetes and obesity are related etiologically to high - fat diets, although the mechanisms by which these conditions are or might be teratogenic during pregnancy are unclear. a high - fat western diet has been shown in a retrospective case - control study to be associated with a more than twofold increased risk for cleft lip, with or without cleft palate. the mechanisms by which dietary fat intake might influence the risk for birth defects remain under investigation. one of the strongest risk factors for many birth defects is a family history of the same defects ; this reflects both genetic and environmental risks for conditions with complex inheritance patterns, and, as a result, it has been hypothesized that many birth defects have both genetic and environmental influences. a recent paper from bentham. described the interaction of a high - fat maternal diet and the embryonic cited2 genotype in increasing the penetrance of left - right patterning birth defects in mouse embryos. this variable penetrance results in left - right patterning defects that include heart defects (septal, outflow tract, and abnormal ventricular topology with sinistral looping) and right atrial and pulmonary isomerisms. the evidence of increased penetrance (but not complete) in left - right patterning defects in certain mouse strains suggests a role for genetic modifiers of the phenotype, as well as a role for environmental influences. bentham. also examined the possible role of the fat content of the maternal diet on the phenotype. they used a diet designed to mimic the high - fat, calorie - dense diet associated with the current obesity epidemic in the usa. it contained lard in place of starch, which has been shown in previous studies to be associated with weight gain and fasting hyperinsulinemia. the experiment showed that the high - fat maternal diet increased the penetrance of left - right patterning defects in mouse embryos (60% of cited2-/- on the high - fat diet versus 24% of cited2-/- on the control diet). the severity of cardiac defects was greater in heterozygous embryos (cited2+/-) from the high - fat diet group. additionally, embryos on the high - fat diet exhibited a cleft palate defect not reported previously for cited2-/-. further studies revealed that there were greater than twofold decreases in pitx2c expression in the embryos from the high - fat diet group with the cited2-/- genotype. a high - fat diet increases adiposity and glucose intolerance, as well as fasting hyperinsulinemia. however, it is unclear what aspect of the high - fat diet contributes to the increased penetrance and severity of the cited2 genotype. the role of cited2 in malformations in mice is clear ; however, among humans, there have been limited numbers of studies to explore the effects of cited2 mutations. examined a group of 392 human study participants with sporadic non - syndromic congenital heart disease, and seven new mutations in cited2 were found among eight of these participants. additionally, a recent publication by yang. found three new mutations in cited2 among 120 human study participants with congenital heart defects with a variety of cardiac lesions. consistent with the variable mouse phenotype, there were no clear groupings of the types of cardiac defects in the human studies associated with the cited2 mutations, and even the same deletion resulted in different cardiac defects. mouse models can play a critical role in the discovery of disease pathology because they allow control over variables that are uncontrollable among humans. however, results in mice can lead researchers astray, and results need to be confirmed carefully through use of human studies. for example, we know that folic acid food fortification reduces ntd rates among human populations by 19% to 32%, and that the dietary supplementation of women of childbearing age with 400 g folic acid per day reduces ntd rates up to 85% among humans. however, there are over 240 mouse models of ntds, and only a minority of these models implicate folic acid metabolism through mutations in folate pathway genes or have been shown to be responsive to folic acid supplementation. so, it is unlikely that mouse models alone would have led the scientific community to what has become an effective public health intervention. the presence of cited2 mutations in more than 2% of human study participants with congenital heart defects examined to date suggests that cited2 might have a role in human malformations. interestingly, for some of the same types of defects (for example, heart defects, ntds, cleft palate and heterotaxia) seen in mice with cited2 mutations fed on a high - fat diet, associations with diabetes or obesity, or both, have been suggested in some human studies of birth defects. significant additional work is needed to determine if a similar interaction of genotype and diet is applicable to human embryogenesis. larger population - based studies are needed to determine the effects of mutations in cited2 on the burden of specific types and constellations of malformations among humans, and to show whether these mutations have increased penetrance in the presence of high - fat maternal intake. a better understanding of the interface of gene - environment interactions has the potential to increase our knowledge of the etiology of birth defects and lead to better targeted prevention programs. interventions at the maternal - fetal interface are complex because of the environmental interaction with both the maternal and fetal genomes during critically limited windows of time. before any changes in dietary recommendations can be made, additional large - scale studies are needed to define the ' at - risk ' population, followed by randomized controlled trials to determine whether changes in diet can modify the risk and reduce the burden of birth defects. the authors declare that they have no competing interests. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. ksc and lbb were involved in the drafting of the manuscript and have reviewed and approved the final version. ksc is a geneticist at the national center on birth defects and developmental disabilities, centers for disease control and prevention, and lbb is human nutrition professor, food science and human nutrition department, university of florida. | the risk of certain birth defects can be modified by maternal diet. a high - fat maternal mouse diet has recently been reported to substantially increase the penetrance of birth defects known to be associated with a deficiency of transcription factor cited2 as well as induce cleft palate. these effects were associated with a more than twofold reduction in embryonic expression of pitx2c. this investigation suggests the need to further explore this provocative gene - diet interaction in human studies. |
a translational roadmap for a vast majority of the discovery - oriented immunology research community is currently uncharted and thus nearly impossible to follow. researchers in academia are woefully unprepared and untrained for the complex, arduous, and difficult process of translating basic immunology to the clinic, as trainees are taught to focus on scientific discovery. the academic immunology research community has been successful in creating the infrastructure to nurture and reward discovery research. however, upon making a discovery with therapeutic potential, it is rare for an academic investigator to think about or have access to the resources necessary to nurture translational opportunities. most senior immunologists can cite examples of lost opportunities in which potential clinical advances were derailed by a failure to pursue early partnerships with industry and legal experts, or by unskilled negotiations that created unnecessarily complicated agreements or royalty arrangements and hindered progress. yet knowledge of the legal parameters involved in these collaborative ventures is only one piece of the puzzle. it is perhaps more important to develop a culture in which translational goals are a priority, so that the motivation to seek out clinical colleagues, to reach fair compromises between academic, industrial, and regulatory entities, and to establish a collegial relationship with institutional intellectual property officers is a reasonable expectation, rather than a rare exception. academic institutes are not well - suited for the collaborative therapeutic development that is needed for translational research in autoimmunity. the key issues are well - illustrated by attempts to induce antigen - specific tolerance in patients with type 1 diabetes. the major work in this area is underwritten by the national institutes of health (nih) and nongovernment organizations such as the juvenile diabetes research foundation (jdrf), with a relative absence of industry support. as a consequence, the approaches are largely investigator initiated and are sometimes uncoordinated, with no overarching prioritization. grants of short duration (typically 3 years) that are reviewed by discovery - oriented basic investigators are simply not adequate, as it may take 5 years or more to apply ideas from mouse models to testing in humans. yet short - term grants remain the principal mechanism for funding investigator - initiated translational work. several important attempts are underway to address this fundamental problem, including a new prioritization review system within the trialnet consortium (http://www.niddk.nih.gov/fund/diabetesspecialfunds) sponsored by the national institute of diabetes and digestive and kidney diseases, a strategic planning process within the immune tolerance network, and a refocusing of the funding portfolio of the jdrf toward applied research goals. nevertheless, allocating scarce research funds for infrastructure at the expense of basic research continues to be unpalatable for a field in which industry partners the traditional source for clinical trials and product development have not materialized. it is also fair to ask whether academically directed clinical research is the best approach. some therapeutic avenues, such as cell - based therapies, are inherently expensive and would benefit from industrial partnerships that could help cover early development costs. industry is also generally more efficient in dealing with the regulatory issues that surround clinical trials. in the case of diabetes immunotherapy, complicated quality of life issues also arise, as the short - term benefits of replacing insulin therapy are currently used as a clinical endpoint, whereas the real medical benefit lies in the prospect of decreasing long - term diabetic complications. these and other complex issues necessitate close interactions with the food and drug association (fda) and other regulators during trial development a specific issue for immune - based interventions and tolerance induction in autoimmunity is the need to develop surrogate markers for therapeutic efficacy (which again requires fda involvement) in order to optimize assays to measure alterations of autoimmune responses in blood samples and to define the clinical indicators of successful or deleterious outcomes. how many academic immunologists think about (or are even aware of) these issues ? it will therefore be important to allocate funds to create infrastructure within academic institutes to facilitate interactions with regulatory bodies. the historical hand - off juncture between basic and clinical immunology has been the demonstration of therapeutic efficacy in animal models of autoimmunity. but in many animal models, translating these findings to humans may be a huge leap for clinicians. one problem is that cells traveling within the human blood, which is our main source of readily accessible autoantigen - specific immune cells, might not accurately reflect the events taking place in a given autoimmune target organ, such as the brain or pancreatic islets of langerhans. this simple observation pinpoints an important problem with our scientific approach. when studying animal models, we generally recover cells from organs and tissues that we think will provide the most reliable answer to the question asked. in the case of diabetes, these include the pancreatic draining lymph node, the islet cells themselves and sometimes the spleen all sites that are not readily accessible in humans. although this strategy gives us good answers in animal models, it misses a crucial step in translating the discovered mechanistic insight to clinical trials : how to test for the establishment of the same protective mechanism, such as the development of antigen - specific tolerance, in humans. for example, the deletion of aggressive t cells or induction of regulatory t (t reg) cells commonly used approaches for ameliorating autoimmunity in mice is reflected in the lymph nodes or spleen but not in the peripheral blood of rodents, suggesting that monitoring peripheral circulating cell populations in humans might not be fruitful. another difficulty that arises in the design and monitoring of clinical trials is determining the correct antigen dose for therapies, which can not necessarily be scaled up directly from mouse to human. in a recent diabetes prevention trial (1), oral insulin was given to prediabetic patients based on overwhelming evidence from animal models that oral insulin can induce t reg cells that suppress aggressive antiislet responses in the pancreatic lymph node and thus prevent disease. it was clear that, in mice, this autoantigen - induced suppressive effect was dose dependent, as very low or very high oral insulin dosages did not have the desired effect (2,3). when designing the human trial, the investigators were faced with the difficult question of how to translate a rather large insulin dose (1 mg p.o. 2/week) in the mouse for human application. they chose to emphasize safety rather than simply scale up from the animal model a cautious and logical choice and used a comparatively low dose (7.5 mg), based on gut size and body weight. interestingly, a partial response reflected by delayed disease progression was noted only in a subgroup of patients who already had significant signs of antiislet autoimmunity (as assessed by autoantibodies). since we have no reliable way to correlate a given stage in the development of autoimmunity in animal models to the course of human diabetes, there is really no straightforward way to apply the enormous body of animal data to the rational determination of antigen dose in future human trials. another example of the difficulty in translating antigen - specific approaches from mice to humans is the failed clinical trials using altered peptide ligands of myelin basic protein (mbp) in patients with multiple sclerosis. the peptides chosen for the human therapeutics were based on inbred mouse models of experimental autoimmune encephalomyelitis, in which disease is induced by mbp peptide administration. the human trial was quickly terminated when it became evident that some patients were responding to the peptide as an agonist, with deleterious clinical consequences (4,5). in retrospect, this adverse response might have been predicted if human in vitro studies using polyclonal cell populations or mouse studies using genetically diverse animal models had been performed, rather than relying on a single animal model. indeed, the genetic diversity among human subjects may necessitate individual predictive testing of peptide responsiveness, analogous to provocative allergen testing before desensitization therapy, as a necessary component of future trials. in this manner, it may be possible to distinguish which patients are likely to have a deleterious response to the peptide therapeutic (such as an agonist response or augmentation of autoaggressive type 1 helper or cytotoxic t cell responses), and which patients are likely to benefit from such treatment. human genetic diversity is another monster in the immunological closet, which is becoming hard to ignore. the more we know about the impact of genetic diversity on immune function, the more variable phenotypes we begin to recognize, even among subjects with the same clinical diagnosis. yet most animal models of autoimmunity use a very small number of highly inbred mouse strains. type 1 diabetes research is a flagrant example of this, as it is heavily reliant on the nonobese diabetic (nod) mouse model, despite the fact that the nod strain has a number of unique immunological defects (6) that are not found in other mouse strains and likely not in most human patients with diabetes, either. clinical immunologists would be reluctant to draw major conclusions based on the study of a single individual, but basic immunologists do this routinely by testing what are essentially multiple copies of a single animal. it is impossible to argue with the major insights into antigen - specific tolerance that have been gained from testing in well - defined inbred mouse models of autoimmunity. however, we may need to acknowledge a new set of criteria for translation, in which validation of a finding made in genetically diverse models is a required component (7). genetically diverse models will not only be critical for assessing disease manifestations themselves, but will be even more important for evaluating the response to a particular therapy. an alternative approach that offers some possibilities for modeling human genetic diversity is the utilization of partially humanized mice (8,9,10). even in these models, however, there are limitations to the diversity that can be modeled. for example, autoimmune disease models in human hla - transgenic mice show major differences in antigen epitope recognition by t cells and in disease penetrance, depending on whether they express a single hla molecule or two different hla molecules. since every human expresses between 8 and 12 hla molecules, it is doubtful whether it will be possible to create an animal model that faithfully represents this important immunologic control element. however, these mice can still be seen as multiple copies of a single genetic individual, and findings in these mice are not likely to be translate into useful immunotherapies for the human population in general. indeed, is it too uncomfortable to ask whether, in some cases, in vitro human studies are more likely to be informative than in vivo mouse work ? if no mouse model is perfect, at what point is it no longer cost effective to utilize scarce resources to create additional mouse models ? rather, important and promising observations made in mouse models could be solidified by proof - of - principle in vitro studies using human blood cells. this strategy might also have an added benefit if the goal is the induction of antigen - specific regulation or tolerance. as we are still in the process of establishing reliable in vitro assays that accurately reflect the induction of antigen - specific tolerance in vivo, such investigations might be a useful step in reaching this goal. some of the structural barriers to translational research are deeply entrenched in the academic system, and are beyond the scope of this discussion, which is centered on immunologic tolerance. tenure, promotion, and funding decisions that constrain the training and performance of translational and clinical immunologists are certainly issues needing redress. however, with respect to the opportunities that now exist in the immunological community, we are blessed with a large array of potential immunotherapeutic strategies that are in need of testing and translation (11). substantial changes need to be made if we are to avoid a piecemeal approach in the near term, and failed or lost therapeutic opportunities in the future. some initiatives are now underway, such as the focis centers of excellence program, which provides a structure in which translational and clinical immunologists at collaborating institutions can self - organize in order to garner local resources more effectively. consortia such as the immune tolerance network and trialnet have a key role to play on the national level, where firm nih support is essential. constructing a useful translational roadmap for academic researchers to follow, like that proposed in fig. 1, will require a substantial effort that is compelled by the prospect of therapeutic interventions in a vast array of human diseases. immediately after a new discovery has been made, it will be essential for the academic institute to support a rapid evaluation of its therapeutic potential and the securing of intellectual property. even at this early stage, designated skilled officials should contact potential industry partners and establish relationships that will be crucial for translating the invention to the clinic. indeed, academics and industry have diverging goals for follow - up experimentation (mechanistic insights and publications versus toxicology, safety, and reproducibility), and financing will be needed to satisfy both groups. we also believe that the individual investigator should remain involved throughout the discovery and development process and that industry partners must accept the possibility that the new intervention may have undesirable as well as desirable effects. a translational roadmap for academic researchers. the potential impact of immunologic therapies on human health is enormous, and the opportunity to advance our knowledge of immunology in concert with clinical experience is equally large. for this vision of interventional immunology to realize its full potential, all parts of this enterprise funding agencies, regulators, investigators, industry, and academic institutions need to recognize that the cost of lost opportunity is high. | induction of selective, autoantigen - specific tolerance is the holy grail for the treatment and prevention of autoimmune diseases. despite successes in many differential murine models, rational and efficient translation to the clinic has been difficult. during the 5th annual federation of clinical immunological societies (focis) meeting, may 1216, 2005, in boston, ma, a kirin - sponsored ideashop was convened to discuss this theme amongst scientists, clinicians, industry partners, and funding agencies. |
serotonin toxicity (st) is a rare clinical state, which may be serious and life - threatening. the most common reasons are usage of multiple serotonergic drugs together or combination of one serotonergic agent and one monoamine oxidase (mao) inhibitor. isoniazid (inh) is a weak mao inhibitor and also may affect cytocrome enzyme system. st due to combination of isoniazidand essitalopram was rarely reported before. to suspect from the state and history of serotonergic drug usage are main factors for diagnosis. although clinical signs of st on nervous system are well - known, no specific electrophysiological finding was demonstrated before. here, we report a case of serotonin toxicity due to the combination of essitalopram and isoniazid. we also observed transient electromyographic findings, which may probably be due to serotonergic activity. a forty - three - years - old female developed acute onset fever and confusion. she was receiving corticosteroids with the diagnosis of takayasu arteritis for the recent three years. she was also taking isoniazid cause of a suspicious positive result in tuberculin skin test (ppd) for the last three months. neurological examination revealed fever, rigidity, clonus, bilateral positive babinsky and hoffman signs, and hyperactive deep tendon reflexes. cranial and spinal magnetic resonance imaging (mri) showed no abnormalities nor contrast enhancement. nerve conduction studies were normal, but needle electromyography (emg) demonstrated spontaneous neurogenic changes (fibrillation and positive waves) in her extensor digitorum brevis (edb), tibialis anterior (ta), and gastrocnemius muscles. we learned that she had been taking essitalopram 20 mg / day for last month from her relatives. with the help of this medical history, she was diagnosed as serotonergic toxicity, essitalopram was stopped, and the patient was hydrated. in her follow - up, she started to get better on the third day of drug - free period. nowadays, the term serotonin toxicity (st) is often referred to serotonin syndrome (ss). there is no specific laboratory or diagnostic test, so the diagnosis is mostly based on the clinical aspects. a history of serotonergic agent or increment in the dosage, four major or three major + two minor symptoms, exclusion of psychiatric, toxic, metabolic and endocrine diseases, and neuroleptic usage were needed for diagnosis. our case fulfilled all criteria and revealed six major symptoms like myoclonus, tremor, rigidity, hyperreflexia, fever, and confusion. although citalopram is widely used because of its favorable safety profile and lack of cytochrome p450 inhibition, it has been reported to cause serotonin toxicity, usually in the setting of an overdose. citalopram is a substrate for cyp2c19 and 3a4, and isoniazide may inhibit cytochrome p450 (cyp450) isoforms, potent inhibition of cyp2c19 and cyp3a. inh is also a mao inhibitor, so may affect the metabolism of anti - depressant drugs. general management measures include removal of the precipitating serotonergic agent and supportive strategies to control the state. in our case, general supportive treatment to stop essitalopram improved the clinical symptoms and signs of the patient. ss is a generalized toxic syndrome ; therefore, both cognitive and neuromuscular findings may be observed. but, in most of the cases, clinical symptoms are more prominent in lower and distal part of extremities, which also may be a clue for a spinal process. as to our best knowledge the spontaneous denervation potentials that we observed during the etiological investigations of the patient may be due to a coincidental disease. nerve conduction studies were normal while there were acute denervation potentials, which lead to the idea of a pathology located more proximal to spinal ganglia. moreover, disappearance of spontaneous denervation potentials after treatment of serotonergic syndrome would be an evidence for toxic effects of serotonin on spinal cord cells. as a result, usage of inh inhibited the metabolism of serotonin and induced serotonergic toxicity in our case. interaction of inh and essitalopram was rarely reported before ; however, our case is the first to report probable electrophysiological findings due to serotonergic toxicity. | here, we report a case of serotonergic toxicity due to combination of essitalopram and isoniazid, which was rarely reported before. moreover, we observed transient neurogenic denervation potentials in needle electromyography, which disappeared with the treatment of serotonergic toxicity. as to our best knowledge, this is the first case, reporting transient electromyographic changes probably due to serotonergic toxicity. |
the top priority of the government, in regards to medical marijuana, should be to reclassify the drug as a schedule ii drug. this would enable dispensaries, clinics, pharmacies and physicians to provide patients with standardized, unadulterated forms of marijuana. if marijuana continues to be unregulated, patients will be forced to seek black - market marijuana, and risk possible legal repercussions to alleviate their condition. utilizing the proper legal and medical controls can provide an effective strategy to identify and reduce health hazards associated with smoked marijuana, as well as help to reduce legal prosecution faced with unregulated marijuana. the main psychoactive compound in the drug is thc, which controls the strength or potency. thc concentration in black - market marijuana can vary greatly, which can lead to adverse effects for patients who may seek alleviating effects for their condition. to minimize such health risks, the federal government, specifically the fda, must monitor marijuana produced for medical purposes. recently, there have been numerous crackdowns on people who grow marijuana for medical uses. if the fda was to intervene and oversee the production of marijuana, this would reduce the number of questions surrounding the growing of marijuana and the arrests that follow, as well as control the hazardous aspects of marijuana. if fda regulation is present in medical marijuana production, the thc concentration and concentration of other hazardous compounds in marijuana can be controlled, thus reducing the harmful effects that impact the health of numerous patients. the medical community has provided studies proving the efficacy of marijuana in treatment of patients who have not responded to other treatments. specifically, these studies have shown the therapeutic value of marijuana in controlling pain, alleviating nausea and vomiting, as well as alleviating symptoms of multiple sclerosis (ms) and aids. in 2011, a randomized controlled trial of cannabinoids treatment of chronic non - cancer pain also demonstrated positive outcomes. significant analgesic effects were seen in treating neuropathic pain, fibromyalgia, and rheumatoid arthritis. the most effective cannabinoid available to patients is smoked marijuana, however due to varying thc concentrations and the fact that the mode of ingestion is inhaled smoke, there are also adverse effects. two options that may help to reduce these adverse effects are more pure forms of smoked marijuana and cost effective alternatives. a more pure form of smoked marijuana (i.e. less toxic compounds) would reduce the harmful effects of smoked marijuana, and therefore increase the benefits. cannabinoid alternatives reduce the amount of these harmful compounds in marijuana. such alternatives like marinol, nabilone, and sativex do exist, however the two concerns that these alternatives pose are efficacy and cost. smoked marijuana continues to be substantially more effective than these alternatives, and the cost of smoked marijuana is significantly less. in order to improve these alternatives and create new options, more research medical marijuana research is contingent upon national institutes of health (nih) funding. for 2011, the nih has allocated only $ 2 million in the form of 45 grants for research in marijuana. in order to properly research safer and cost effective alternatives, more nih funding is necessary, and must be done to provide suffering patients with a beneficial treatment. to advance the development of new marijuana treatment alternatives, pharmaceutical companies should be given incentives to continue to explore new avenues for suffering patients. one such company that has begun development on a medical marijuana patch is medical marijuana delivery systems (mmds) llc. in february 2011, mmds announced that they had acquired united states patent rights to develop a marijuana patch for medical use. walter cristobal, the patch inventor, is working with mmds to develop the patch - based delivery, as well as other delivery systems like creams and gels. another recent development in the marijuana industry has come from the pharmaceutical company medicinal genomics. as of august 2011, the company has successfully sequenced the entire genome of the cannabis plant, a breakthrough which has the potential to grow the number of treatment options available to patients. ethically speaking, denying physicians the right to prescribe a therapy that relieves pain and suffering to their patients is a violation of the physician - patient relationship. patients are entitled to full disclosure of all possible treatment options from their physician in order to make an informed medical decision regarding their health. it is the medical responsibility of a physician to offer adequate relief from pain for their patients so that the patient may have an acceptable quality of life. failure to provide an available therapy that has been proven effective would violate the basic ethical principle of beneficence, which is the obligation of physicians to seek the well - being or benefit of the patient. under beneficence, a physician s duties include preventing and removing harm, as well as promoting the good of their patient. to allow a patient to suffer when an effective treatment is available overall, all people, especially in the federal government and the medical field, should be concerned over the quality of life of those suffering from neurological and movement disorders, cancer, wasting syndrome attributable to aids, etc. a 2010 gallup poll of americans has shown significant support for making marijuana legally available for doctors to prescribe for patients. the poll found that seventy percent of americans are in favor, as negative feelings continue to decline. medical marijuana has proven invaluable in the battle against terminal illnesses ; however, unless the federal government publically acknowledges this fact, numerous terminal patients will continue to suffer needlessly. the fight against drug abuse is important because may lives are lost to drug addiction, but the effects of devastating illnesses impacts a substantially greater number of americans. medical marijuana can be an important treatment for physicians to confront the challenges of patients pain and suffering. the apparent political motivations present in the federal government must be eliminated because the quality of numerous american lives hangs in the balance. the dignity and respect of all persons must be a priority for the obama administration. it is time to voice support for the most vulnerable and reclassify medical marijuana as a schedule ii drug, because for many patients it is truly a medical necessity. | summarymarijuana is classified by the drug enforcement agency (dea) as an illegal schedule i drug which has no accepted medical use. however, recent studies have shown that medical marijuana is effective in controlling chronic non - cancer pain, alleviating nausea and vomiting associated with chemotherapy, treating wasting syndrome associated with aids, and controlling muscle spasms due to multiple sclerosis. these studies state that the alleviating benefits of marijuana outweigh the negative effects of the drug, and recommend that marijuana be administered to patients who have failed to respond to other therapies. despite supporting evidence, the dea refuses to reclassify marijuana as a schedule ii drug, which would allow physicians to prescribe marijuana to suffering patients. the use of medical marijuana has continued to gain support among states, and is currently legal in 16 states and the district of columbia. this is in stark contrast to the federal government s stance of zero - tolerance, which has led to a heated legal debate in the united states. after reviewing relevant scientific data and grounding the issue in ethical principles like beneficence and nonmaleficence, there is a strong argument for allowing physicians to prescribe marijuana. patients have a right to all beneficial treatments and to deny them this right violates their basic human rights. |
entamoeba histolytica is the enteric protozoan parasite responsible for human amoebiasis that affects 50 million people around the world, causing colitis and liver abscesses. in this organism, phagocytosis and vesicular trafficking vacuolar protein sorting (vps) factors and some proteins that participate in vesicle transport in eukaryotes have been identified in e. histolytica [24 ]. in eukaryotic cells, endocytosis consists in phagocytosis, micropinocytosis and pinocytosis. particularly, phagocytosis involves the ingestion of particles of varying size into phagosomes, which sequentially fuse with early and late endosomes forming multivesicular bodies (mvb), as well as with lysosomes to form phagolysosomes. additionally, mvb are critical for cell receptors down - regulation, retroviral budding and other processes [68 ]. mvb formation is driven by the assembly of endosomal sorting complexes required for transport (escrt), which result from the interaction of different class e vps proteins [9, 10 ]. the mvb - sorting process initiates with the association of vps27 and hse1 proteins to form the escrt-0 complex, which is targeted to endosomal membrane domains that bind ubiquitinated cargo proteins (reviewed in [6, 7 ]). then, escrt-0 recruits escrt - i formed by vps23, vps28, and vps37 proteins, as well as an additional subunit called mvb12 [11, 12 ]. later, escrt - ii, composed by vps22, vps25, and vps36 proteins, is activated by escrt - i. ubiquitinated cargo proteins are recognized by escrt - i via vps23 and by escrt - ii via vps36. then, escrt - iii, formed by vps20, vps32, vps2, and vps24, interacts with escrt - ii components to complete escrt formation. escrt - iii is required for cargo molecules concentration into mvb vesicles and it coordinates the association of bro1 protein and doa4-deubiquitinating enzyme [1517 ]. finally, vps4 protein catalyzes the atp - dependent dissociation of escrt complexes from endosomes to initiate new rounds of vesicle formation and cargo molecules transport [6, 7 ]. particularly, it has been reported that substitution of the conserved e amino acid residue by a q residue in the vps4 atpase motif impairs atp hydrolysis activity [18, 19 ], resulting in an inefficient protein transport from endosomal compartments to vacuoles or lysosomes [2022 ], which evidenced the critical role of vps4 in protein transport and vesicle trafficking. in e. histolytica, experimental evidence suggests that mvb - like structures are formed by fusion of inward budding membranes with phagosomes. ubiquitin, deubiquitinating enzymes and a snf7 homologue (vesicular trafficking protein) have been identified in isolated phagosomes by proteomic analysis, suggesting that a mechanism similar to mvb formation could be present in e. histolytica [2325 ]. additionally, the bro1 domain - containing ehadh112 protein, which forms part of the e. histolytica ehcpadh complex involved in parasite virulence, is located in mvb - like structures in trophozoites [26, 27 ]. however, molecular mechanisms regulating mvb formation in e. histolytica remain poorly understood. here, by in silico analysis from parasite genome databases, we identified 20 escrt protein - encoding genes in e. histolytica and showed that most of them were transcribed in trophozoites. since vps4 has been described as a key molecule to complete the disassembly of escrt and associated factors in other systems, we initiated the study of escrt machinery in e. histolytica by cloning and characterizing the ehvps4 protein. interestingly, by using trophozoites overexpressing the wild type and a mutant version of ehvps4, we provide data supporting a role for this protein in phagocytosis and virulence. sequence similarity searches for escrt genes were performed in e. histolytica genome database (http://pathema.jcvi.org/cgi-bin/entamoeba/pathemahomepage.cgi) by blast using human and yeast escrt protein sequences as queries. putative e. histolytica escrt homologous proteins were selected using the following criteria : (i) at least 20% identity and 35% similarity to the query sequence ; (ii) e - value lower than 0.002 ; and (iii) absence of stop codons in the coding sequence. predicted amino acids sequences were aligned by clustalw software (http://www.ebi.ac.uk/clustalw/). functional and structural domains were predicted using prosite (http://www.expasy.org/tools/scanprosite/) and pfam (http://www.sanger.ac.uk/software/pfam/) databases. phylogenetic relationships among putative escrt proteins from e. histolytica and other organisms were analyzed using the neighbor - joining distance method as implemented in the mega package version 3.1. phylogenetic trees were generated for each putative e. histolytica escrt protein aligned with homologues from different species. robustness of phylogenetic inferences was tested by bootstrapping method, involving 1000 replications of the data based on the criteria of 50% majority - rule consensus. for 3d modeling of mit and aaa domains of e. histolytica ehvps4, predicted tertiary structures were obtained with the phyre server (http://www.sbg.bio.ic.ac.uk/phyre/), using crystal data from yeast vps4 mit (2v6xa) and aaa (2qpab) domains as templates. trophozoites of e. histolytica clone a (strain hm1 : imss) were axenically cultured in tyi - s-33 medium at 37c and harvested during exponential growth phase. medium for transfected trophozoites was supplemented with 40 g / ml g418 (gibco). cell viability was monitored by microscopy using trypan blue dye exclusion test. using trizol reagent (invitrogen), total rna was extracted from 10 trophozoites grown in tyi - s-33 medium or 5 minutes after red blood cells (rbc) ingestion.semiquantitave rt - pcr was performed using 1 g of dnase i - treated total rna that was reverse transcribed using superscript ii (invitrogen) for 2 hours at 42c. cdna samples were subjected to pcr amplification using specific internal primers for distinct e. histolytica putative escrt genes(see table t1 in supplementary material available on line at doi : 10.1155/2010/890674).briefly, pcr consisted in an initial denaturation step at 94c for 5 minutes followed by 25 cycles of 35 s at 94c, 30 s at tm calculated for each gene(supplementary data table t1),1 minute 30 s at 72c and a final extension step at 72c for 7 minutes. as a control, we amplified a eh25s rrna gene internal sequence which was used to normalize densitometric data. products were separated by 1% agarose gel electrophoresis, stained with ethidium bromide and visualized by uv light in a gel doc 1000 apparatus (biorad). statistical significance was determined by t student test. the full - length ehvps4 gene (1260 bp) reported at locus ehi_118900 in e. histolytica pathema database was pcr amplified from e. histolytica genomic dna using the ehvps4-forward (5-cccccggatccatgacatcgttacttgataaagg-3) and ehvps4-reverse (5-cccccctcgagttatccatcttgtccaaattgttc-3) primers in the presence of 0.2 u pfx dna polymerase (invitrogen). briefly, pcr consisted in an initial denaturation step at 94c for 5 minutes followed by 28 cycles (30 s at 94c, 35 s at 55c, and 1 minute 30 s at 72c) and a final extension step at 72c for 7 minutes. the pcr product was cloned into topo vector (invitrogen) yielding the topo - ehvps4 plasmid. using the quikchange mutagenesis kit (stratagene), we generated a point mutation in the ehvps4 atpase domain at amino acid 211 to replace glutamic acid (e) by glutamine (q) to obtain the topo ehvps4-e211q plasmid. then, ehvps4 and ehvps4-e211q genes were pcr amplified from topo plasmids and subcloned in the pgex-6p1 expression vector (amersham biosciences) to generate the recombinant pgex-6p1-ehvps4 and pgex-6p1-ehvps4-e211q plasmids, respectively. escherichia coli bl21 (de3) plyss (invitrogen) bacteria were transformed with pgex-6p1-ehvps4 or pgex-6p1-ehvps4-e211q plasmids to produce the gst - tagged rehvps4 and rehvps4e211q proteins. bacteria were grown at 37c in 2-ty medium containing 100 g / ml ampicillin and 34 g / ml chloramphenicol. rehvps4-gst expression was induced by 1 mm isopropyl beta - d - thiogalacto pyranoside (iptg) for 3 hours at 37c. cells were harvested, resuspended in ice cold pbs buffer (140 mm nacl, 2.7 mm kcl, 10 mm na2hpo4, and 1.8 mm kh2po4 ph 7.3) and lysed by sonication at 4c in the presence of lisozyme (1 mg / ml). rehvps4-gst and rehvps4-e211q - gst polypeptides were purified near to homogeneity through glutathione affinity chromatography, according to manufacturer recommendations (amersham biosciences). to further use the recombinant protein as antigen, the gst tag (25 kda) was removed from rehvps4-gst protein by incubation with 2 u prescission protease (amersham biosciences) during 12 hours at 4c. cleaved rehvps4 protein was dialyzed into pbs ph 7.4. identity and integrity of purified rehvps4-gst, rehvps4-e211q - gst and rehvps4 proteins were confirmed by 10% sds - page and western blot assays using anti - gst, (ge - healthcare, 1 : 5000 dilution) and anti - rehvps4 (1 : 15000 dilution) antibodies, respectively, and the ecl - plus western blotting detection system (amersham biosciences). purified rehvps4 without gst tag was submitted to preparative 10% sds - page and electroeluted from coomassie stained gels. rehvps4 (200 g) in complete freund 's adjuvant (sigma) was subcutaneously inoculated into a new zealand male rabbit, and then, three doses of 100 g rehvps4 in incomplete freund 's adjuvant were injected at 15 days intervals. the atpase activity assay procedure was based on that previously described using the piper phosphate assay kit (invitrogen), with minimal modifications. briefly, amplex red was diluted in dmso to a 10 mm final concentration. maltose phosphorylase, maltose, glucose oxidase, and horseradish peroxidase were diluted in enzyme buffer (0.1 m tris - hcl, ph 7.5) to a final concentration of 200 u / ml, 40 mm, 200 u / ml, and 100 u / ml, respectively. assay buffer (100 mm tris, 20 mm kcl, and 6 mm mgcl2, ph 7.4) was added to a 96-well plate. the volume corresponding to 3.5 g and 5 g of purified and dialyzed rehvps4-gst, rehvps4-e211q - gst, and rgst proteins was added to wells and enzyme buffer was added up to 27 l. 50 l of working solution (2 u / ml glucose oxidase, 4 u / ml maltose phosphorylase, 0.4 mm maltose, 100 m amplex red, 0.4 u / ml hrp) were added to wells. fresh atp was diluted in assay buffer to a concentration of 2.5 mm and added to each well to a final volume of 100 l. then, reactions were kept at 25c for 30 minutes and protected from the light. the absorbance was measured at 562 nm at 30 minutes and data were documented. assays were performed twice by triplicate. the mutant ehvps4-e211q gene and the wild type ehvps4 gene were pcr amplified from topo ehvps4-e211q and topo - ehvps4, respectively, using the ehvps4-s - kpni (5-cccccggtaccatgacatcgttacttgataaagg-3) and ehvps4-as - flag - bamhi (5-cccccggatccttacttatcgtcgtcatccttgtaatctccatcttgtccaaattgttc-3) primers and cloned into pneo vector for further transfection assays. the underlined nucleotide sequence corresponds to the flag epitope (1 kda) that was added to the carboxy terminus of recombinant proteins to allow their specific immunodetection in transfected trophozoites. trophozoites were transfected with 200 g of pehvps4-e211q, pehvps4 and control pneo plasmids by electroporation as described. briefly, amoebae (10) were washed twice in cold pbs and once in cold complete cytomix buffer. electroporation was performed with the bio - rad gene pulser using 1200 v / cm and 25 f, with a constant time of 0.4 ms. electroporated cells were transferred into fresh culture medium for 48 hours before selecting them with 10 g / ml g418. in vitro virulence of nontransfected and transfected trophozoites (10) was measured on mdck cells (6 10) as described [35, 36 ]. rate of erythrophagocytosis was evaluated using trophozoites and rbc in a 1 : 100 relation, at 5, 10, and 15 minutes. in vivo virulence of transfected trophozoites was evaluated as described. briefly, under sterile conditions and intraperitoneal anaesthesia with 0.2 mg anesthesal (pfizer), three groups of six hamsters each were laparotomized and intraportally challenged with pehvps4-e211q, pehvps4, or pneo transfected trophozoites (2.5 10) in a volume of 0.1 ml pbs using a 29-gauge needle. seven days after challenge, animals were anaesthetized and livers were removed to evaluate hepatic damage. western blot assays were performed using 30 g of total proteins from nontransfected or pneo, pehvps4, and pehvps4-e211q transfected trophozoites. we used anti - flag monoclonal (1 : 700) or anti - rehvps4 polyclonal (1 : 15000) antibodies and rabbit anti - mouse and goat anti - rabbit igg horseradish peroxidase - labeled secondary antibodies (zymed ; 1 : 10000), respectively. immunodetected proteins were revealed with the ecl plus western blotting system (amersham biosciences). as an internal control, we used monoclonal antibodies raised against e. histolytica actin (1 : 1000) and goat anti - mouse igg horseradish peroxidase - labeled secondary antibodies (zymed ; 1 : 10000). for confocal microscopy assays, nontransfected and transfected trophozoites were grown on coverslips, fixed with 4% paraformaldehyde at 37c for 1 hour, permeabilized with triton x-100 and blocked with 1% bovine serum albumin in pbs. then, nontransfected and transfected cells were incubated with polyclonal anti - rehvps4 (1 : 5000) or monoclonal anti - flag primary antibodies (sigma ; 1 : 500), respectively, at 37c for 2 hours, followed by incubation with anti - rabbit or anti - mouse fluoresceinated (zymed ; 1 : 100) monoclonal antibodies, at 37c for 1 hour. next, trophozoites were washed three times with pbs 1x at room temperature and dna was counterstained with 4, 6-diamidino-2-phenylindole (dapi) (5 g / ml) for 5 minutes. to immunolocalize ehvps4 during erytrophagocytosis, pehvps4 transfected cells were incubated with rbc (1 : 20) for 10 minutes. ingested rbc were stained with diaminobenzidine (0.84 mm 3,3-diaminobenzidine, 0.048% h2o2 and 50 mm tris - hcl, ph 9.5) for 5 minutes and cells were incubated with polyclonal anti - rehvps4 (1 : 5000) to be processed for immunofluorescence as described above. light optical sections (1.5 m) were obtained through a leica inverted microscope attached to a laser confocal scanning system tcs - sp2 (leica). to investigate the presence of escrt - related genes in e. histolytica, we surveyed the parasite genome sequence at pathema database using the amino acid sequences of yeast and human escrt proteins as queries. we detected a set of 20 putative escrt protein - encoding genes, which exhibited significant e - values (1.1e 114 to 0.00032) and high similarity (20 to 62%) to yeast and human escrt (escrt 0-iii and associated factors) orthologues (see table 1). however, we did not find homologues for yeast mvb12, and bul1p protein involved in protein ubiquitination, neither human vps37-b and vps37-c proteins. phylogenetic inference revealed that e. histolytica escrt predicted proteins are closely related to homologous proteins from other protozoa, such as giardia lamblia, tetrahymena thermophila, trichomonas vaginalis, plasmodium vivax, cryptosporidium homini, and trypanosoma cruzi (data not shown). bioinformatics analysis showed that e. histolytica escrt predicted proteins have the most characteristic functional domains (see supplementary data figure s1) and tertiary structure folding described for yeast and human escrt factors (data not shown). to determine whether the putative e. histolytica escrt genes were expressed, we performed rt - pcr assays for the 16 most conserved genes. in basal culture conditions, 15 genes were transcribed, but the ehvps22 transcript was not detected in these experiments (see figures 1(a) and 1(b)). we also performed rt - pcr assays using rna obtained from trophozoites incubated with rbc for 5 minutes to investigate whether erythrophagocytosis has an effect on escrt gene expression (see figures 1(a) and 1(b)). in these assays, we found that all genes were expressed during erythrophagocytosis at similar levels as in basal conditions. interestingly, ehvps23 and ehadh112 were 2- and 3-fold up - regulated, respectively. by densitometric analysis, the band given by the amplified eh25s rrna product in both conditions was taken as 100% and used to normalize escrt genes data. since vps4 orthologues have been described as key molecules for the dissociation of escrt, we first focused on the characterization of the e. histolytica ehvps4 protein. the predicted ehvps4 contains the n - terminal mit (microtubule interacting and transport), aaa (atpase associated with a variety of activities) and vps4 c - terminal domains (see figure 2(a)) that are characteristic and essential for vps4 proteins biological functions [39, 40 ]. phylogenetic trees revealed that ehvps4 is more related to protozoan vps4 than orthologous proteins from higher eukaryotes (see figure 2(b)). interestingly, 3d modeling of ehvps4, using the crystal structure of mit and aaa domains from yeast vps4p and human vps4b as templates (see figure 2(c)), showed that ehvps4 region spanning the n - terminal mit and aaa atpase domains exhibited a similar folding with the conserved vps4 -helices. we produced and purified the recombinant ehvps4 protein as a gst - tagged fusion polypeptide (rehvps4-gst) in e. coli (see figure 3(a), lanes 3, 4). by western blot assays using commercial anti - gst monoclonal antibodies, we immunodetected the purified rehvps4-gst as a 72 kda protein in iptg - induced bacteria (see figure 3(b), lane 2) but not in protein extracts from noninduced bacteria (see figure 3(b), lane 1). the rehvps4-gst was digested with the prescission protease to obtain the rehvps4 untagged protein (47 kda) (see figure 3(c), lanes 2 and 3), which was also purified (see figure 3(c), lane 4) and used to generate rabbit polyclonal antibodies. anti - rehvps4 serum recognized the untagged rehvps4 protein, confirming the antibodies specificity (see figure 3(d)). in addition, the same antibodies detected a single 47 kda band in e. histolytica protein extracts (see figure 3(e), lane 1), which corresponds to the expected molecular weight for the predicted ehvps4 polypeptide amino acid sequence. antibodies did not detect any other aaa atpases predicted in the e. histolytica genome. pre - immune serum, used as a control, did not recognize any band in trophozoite extracts (see figure 3(e), lane 2). through immunofluorescence and laser confocal microscopy assays, the specific rabbit polyclonal antibodies revealed ehvps4 as abundant small dots dispersed in the cytosol (see figure 3(f)). to investigate whether the recombinant protein exhibited atpase activity we used the rehvps4 purified protein and a mutant version (ehvps4-e211q) in which we changed the e211 residue of the aaa motif, essential for enzyme activity, by a q residue (see figure 4(a)) [18, 42 ]. reactions were carried out using the piper phosphate assay kit (invitrogen) in the presence of 500 m atp. inorganic phosphate (pi) release was measured spectrophotometrically at a562. at zero time, absorbance values of samples containing rehvps4-gst, rehvps4-e211q - gst and control rgst purified proteins (see figure 4(b)) were similar and they were taken as background. at 30 minutes, a562 values increased to 0.115 0.008 and 0.162 0.022, using 3.5 g and 5 g of rehvps4-gst protein, respectively. no significant a562 increase was detected when we used rehvps4-e211q - gst or rgst proteins at 30 minutes (see figure 4(c)). these results showed that rehvps4-gst has atpase activity and that the aaa motif is essential for atp hydrolysis. to investigate the role of ehvps4 in e. histolytica, we generated transfectant trophozoites that overexpress the ehvps4 and the mutant ehvps4-e211q flag - tagged proteins (see figure 5(a)). viability of transfected trophozoites was up to 90% as determined by trypan blue exclusion assays. rt - pcr assays, using specific primers for ehvps4 gene, showed that trophozoites transfected with pehvps4 or pehvps4-e211q plasmids expressed a higher amount of the ehvps4 transcript in comparison to pneo transfected cells (see figure 5(b), upper panel, lanes 1, 2, and 3). as control, we amplified the eh25s rrna transcript, which showed minimal changes among the different transfectants studied (see figure 5(b), lower panel). by western blot assays, using the anti - flag antibodies, we detected the ehvps4-flag and ehvps4-e211q - flag proteins in pehvps4 and pehvps4-e211q transfectant cells, whereas no signal was detected in pneo transfectantes, as expected (see figure 5(c), upper panel). however, specific anti - rehvps4 antibodies recognized endogenous ehvps4 protein in pneo transfectants (see figure 5(c), middle panel, lane 1), meanwhile signal increased 2.8- and 9.2-fold in pehvps4 and pehvps4-e211q transfected cells, respectively (see figure 5(c), middle panel, lanes 2, and 3). anti - actin antibodies detected similar amounts of protein in the different transfectants (see figure 5(c), lower panel). for densitometric analysis, actin band was taken as 100% in each lane and used to normalize ehvps4, ehvps4-flag, and ehvps4-e211q - flag data (see figure 5(d)). to investigate the cellular localization of exogenous wild type ehvps4 and mutant ehvps4-e211q flag - tagged proteins in transfected trophozoites, we performed immunofluorescence assays using anti - flag antibodies (see figure 6). in pehvps4 transfected trophozoites, anti - flag antibodies detected the overexpressed exogenous protein diffuse in the cytoplasm and at the plasma membrane (see figure 6(d)). in contrast, in mutant pehvps4-e211q transfected cells, signal was not observed at the plasma membrane, but it was present as cytoplasmic structures of varying sizes (see figure 6(f)). as expected, no signal was detected by anti - flag antibodies in pneo transfected cells (see figure 6(b)). control assays without anti - flag primary antibodies did not show any signal in transfected trophozoites (see figure 6(h)). to initiate the phenotypical characterization of trophozoites transfected with pehvps4 or pehvps4-e211q constructs after 1 hour interaction of mdck cells with trophozoites of clone a, pneo or pehvps4 transfected trophozoites, monolayers presented in mean 86.8 13.2% destruction. interestingly, pehvps4-e211q transfected cells were less efficient to destroy mdck cell monolayers, since they only produced 46.4 6.4% cell destruction (see figure 7(a)). the rates of erythrophagocytosis of non - transfected clone a, pneo and pehvps4 transfected cells were similar, at each time tested. in contrast, pehvps4-e211q transfected trophozoites showed 60, 55%, and 57% decrease in erythrophagocytosis rate at 5, 10, and 15 minutes, respectively, when compared with the mean rate from the other trophozoites (see figure 7(b)). then, we investigated the capacity of pehvps4 and pehvps4-e211q transfected trophozoites to induce hepatic abscesses formation in hamsters. none of the six hamsters that were infected with pehvps4-e211q transfected trophozoites presented hepatic damage (see figures 7(c)), whereas the six animals infected with pneo and pehvps4 transfected trophozoites developed extensive abscesses. to determine the localization of ehvps4 in pehvps4 transfected trophozoites during erythrophagocytosis, we performed immunofluorescence assays with anti - rehvps4 serum. antibodies detected ehvps4 as a signal surrounding ingested erythrocytes, suggesting that this protein may be involved in phagocytosis (see figure 8). in e. histolytica trophozoites, phagocytosis, and vesicular trafficking are important events for parasite nutrition and destruction of target cells. as in other eukaryotes, the endocytosis and phagocytosis pathways involve the formation of endosomes, mvb and lysosomes [5, 43 ]. indeed, mvblike structures have been detected in trophozoites [26, 27 ] and within isolated phagosomes, but proteins participating in their formation have not been identified. here, by mining the parasite genome sequence, we demonstrated the presence of 20 genes that encode putative escrt machinery components, which could participate in endosomal transport and mvb formation in e. histolytica. most predicted proteins have the conserved functional domains and share high similarity and phylogenetic relationship with homologous proteins from other eukaryotes, strongly suggesting that they have similar functions. rt - pcr assays evidenced that 15 out of 16 escrt components tested are transcribed, which is consistent with the activity of endosome formation and vesicle trafficking exhibited by e. histolytica trophozoites (reviewed in [5, 43 ]). except for the ehadh112 and ehvps23 genes, transcript amount was not significantly modified after 5 minutes of rbc interaction. analysis of published e. histolytica microarrays data confirmed that most escrt genes are transcribed without any significant change under distinct experimental conditions [44, 45 ]. additionally, in dictyostelium discoideum, microarrays assays evidenced that expression of most genes involved in intracellular vesicle traffic is not significantly changed during phagocytosis. in mouse macrophages, phagocytosis causes cellular redistribution of hrs protein (the homologue of yeast vps27p), but the protein amount remains unchanged. it is possible that trophozoites could have enough escrt and associated proteins to perform erythrophagocytosis, if, as we think, these proteins are involved in phagocytosis in e. histolytica. however, changes in the gene expression of e. histolytica escrt members at shorter or larger times can not be discarded. in yeast and mammal cells, vps4 is a key component for disassembly of escrt complexes from the endosomal membrane at the mvb invagination pathway [42, 48, 49 ]. we focused on ehvps4 protein to investigate its role in endocytosis, specifically in one of the professional function of e. histolytica trophozoites : phagocytosis. as in yeast, e. histolytica has only one vps4 gene, while higher eukaryotes have two vps4 genes [21, 50, 51 ]. by confocal microscopy, ehvps4 was immunodetected as abundant small dots dispersed in the cytosol. in yeast, mammals, plants, and the protozoan leishmania major, similar structures have been identified as mvb formed during endocytosis and vesicular trafficking [22, 42, 51, 52 ]. yeast vps4p protein exhibits atpase activity, which depends on the e233 residue present within the aaa domain [18, 42 ]. in this work, we showed that ehvps4 is an atpase, which conserves the characteristic domains and folding of vps4 homologues. this activity depends on the aaa domain since the mutant rehvps4-e211q - gst, in which e211 amino acid residue was substituted by q residue, did not exhibit detectable atp hydrolysis, as reported in yeast [18, 42 ]. we further investigated the biological relevance of ehvps4 by generating trophozoites that overexpress wild type ehvps4-flag and mutant ehvps4-e211q - flag protein. as the endogenous protein, exogenous ehvps4-flag was located in abundant punctuate structures in the cytosol as reported in other organisms [42, 50, 52, 53 ]. however, overexpressed ehvps4-flag was also detected at plasma membrane. in transfected mammal cells that overexpress sendai virus protein c and wild type vps4, alix / aip1 proteins are recruited at the plasma membrane to facilitate the budding of virus - like particles. ehadh112, the e. histolytica homologue of alix, has been detected at the plasma membrane. experiments currently in progress will help us to define the role of vps4 at the membrane of trophozoites. erythrophagocytosis and cytopathic activities were not improved in trophozoites overexpressing wild type ehvps4-flag, probably because other escrt proteins are limiting factors for cell destruction and rbc intake [42, 52, 56 ]. however, the dominant negative effect of mutant ehvps4-e211q - flag protein in mdck cells destruction, the rate of rbc intake, and liver abscesses formation in hamsters, suggest a role for ehvps4 in e. histolytica virulence properties., we showed that e. histolytica has an escrt machinery, which is transcribed in trophozoites. particularly, we presented evidence that the conserved ehvps4 is an atpase that could participate in cytopathic activity and erythrophagocytosis, as well as in hepatic damage in hamster. work currently in progress is focusing on the identification of cytoplasmic structures containing ehvps4, as well as its interaction with other components of the e. histolytica escrt machinery. | eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (escrt). here, we showed the presence and expression of homologous escrt genes in entamoeba histolytica. we cloned and expressed the ehvps4 gene, an escrt member, to obtain the recombinant ehvps4 and generate specific antibodies, which immunodetected ehvps4 in cytoplasm of trophozoites. bioinformatics and biochemical studies evidenced that rehvps4 is an atpase, whose activity depends on the conserved e211 residue. next, we generated trophozoites overexpressing ehvps4 and mutant ehvps4-e211q flag - tagged proteins. the ehvps4-flag was located in cytosol and at plasma membrane, whereas the ehvps4-e211q - flag was detected as abundant cytoplasmic dots in trophozoites. erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing ehvps4-flag. in contrast, ehvps4-e211q - flag protein overexpression impaired these properties. the localization of ehvps4-flag around ingested erythrocytes, together with our previous results, strengthens the role for ehvps4 in e. histolytica phagocytosis and virulence. |
depression is a significant health problem affecting 14.7% to 30% of all older persons [1, 2 ]. depression rates are typically higher among senior citizens who live in nursing homes, as compared to those who live in community settings [35 ]. between 30% and 40% of older persons living in nursing homes and other institutional settings have been reported as suffering from some degree of depression. depression is often simply thought of as a constellation of negative feelings such as hopelessness, sadness, anger, and irritability. depression is much more than an emotional response, however, as depression affects behavior and both cognitive and physical health. depression is now clearly linked with an increased susceptibility for acquiring other serious health problems [8, 9 ], and with a higher risk of death from suicide and other causes. in spite of the common perception that depression is a natural or perhaps even inevitable aspect of the aging process, depression among older persons is not normal and it should be treated as an illness that can be cured or at least moderated. typical interventions for depression are psychotherapy and pharmaceuticals, or a combination of the two. unfortunately, research is demonstrating that drug therapy outcomes are not always optimal for older persons and that nonpharmacological treatments should be increasingly considered [1, 9 ]. some studies have demonstrated the benefits of alternative therapies such as exercise, light therapy, and herbs. the experimental study presented here tested the effects of a four - week hope program for depressed older persons living in a large nursing home in the western canadian province of alberta. this small pilot study primarily involved a between and within groups comparison of data collected through the geriatric depression scale short form and the herth hope index. older persons who live in institutions such as nursing homes are highly vulnerable to depression, in part because they have become isolated from familiar surroundings, neighbors, and family members [15, 16 ]. an episode of severe illness, a decline in health, and the passing of a spouse often precedes their admission to a care facility, with this transition in health, wellbeing, family connections, and/or living arrangements contributing to depression. depression may have also been present earlier in the person 's life, with episodes of moderate to severe depression potentially necessitating treatment. a decline in personal strength is another possible factor for depression among nursing home residents, with this decline thought by some to be an outcome of aging and the many losses that can accompany old age. clearly, given the high rate of depression among institutionalized older persons, efforts to reduce depression are important to explore. the therapeutic value of hope to health, healing, wellbeing, and quality of life is now well documented. in the last three decades, interest in hope has intensified, as shown by a surge in qualitative and quantitative studies many of which having occurred in healthcare settings. to date, this body of research has linked hope and healing, hope and coping [2022 ], hope and goal setting, hope and finding meaning in suffering and illness [2, 24, 25 ], and hope as an antidote to hopelessness. among healthcare professionals, hope has been identified as a fundamental necessity, being vital to both life and living, and an essential human need particularly among elderly persons. research has emphasized the relational nature of hope, exploring in particular the centrality of caregivers, family, and friends to fostering hope. nurses have also been found to be in a unique position to offer hope to their patients or clients [18, 24, 29, 30 ]. spirituality and faith religious beliefs, including faith in a higher power, life after death, and the power of prayer, can raise hope. davis 's study involving people aged 6089 confirmed positive correlations between hope and spirituality, hope and well - being, and spirituality and well - being. the research to date on hope has thus revealed it as a life force, something that individuals can benefit from in times of crisis, tragedy, or illness. exploring the potential for hope to be instilled, nurtured, and/or encouraged among depressed older persons living in nursing homes is perhaps one of the most important steps now for researching the therapeutic value of hope. several studies have already explored levels of hope among older persons [21, 30, 32, 33 ]. others have studied hope among persons with specific afflictions such as cancer [34, 35 ] and stroke. one such study used the herth hope index (hhi) as a measurement tool in a pre - post test design involving two hope interventions (a 20-minute hope video and a daily reflective journal) for persons receiving or providing palliative care. no studies on hope as an intervention for depressed older persons in nursing homes or other institutional care settings appear to have been conducted. a pilot study was planned and conducted in one canadian nursing home to discriminate the effects of a four - week hope program on depression and hope. this nursing home was convenient to the researchers, and with a one - site study planned to control for intervening effects that are likely to occur with multiple study sites. a single study site was also considered an ethical option, as the hope program had been newly developed by a multidisciplinary facility team and its effects are untested. this hope intervention program was designed after the hope literature had been reviewed and local hope experts consulted. two established measurement tools, the hearth hope index (hhi) and the geriatric depression scale short form (gdssf), were selected to gather comparative data among subjects. the hhi, a shorter (12-item) version of the original hearth hope scale, was designed specifically for use in clinical settings. the hhi employs a four - point likert scale per item to measure dimensions of hope, with total potential tool scores ranging from 12 to 48. this tool is easy to understand and quick to administer, with the developer indicating it is as effective as the original longer herth hope scale. construct validity, internal consistency, and test - retest correlations have continued to establish the efficacy of both herth hope tools. the gdssf is a 15-item tool that was developed as a short form of the original geriatric depression scale long form, with this shorter tool also identified as valid and reliable [38, 39 ]. the gdssf is easy to understand and quick to administer in clinical settings, with this tool specifically chosen for this study because the target population was depressed older persons living in a nursing home. this study was approved by the administrators of the nursing home and also by the university of alberta 's health research ethics board, after one major change to the experimental design was made as mandated. this change was that the persons randomly assigned to the control group would receive a brief friendly weekday greeting from the research assistant instead of their only receiving their usual daily care. the nursing home where the study was conducted has 436 continuing care beds and thus 436 potential subjects, as 100% bed occupancy is common. the inclusion criteria for this study, however, were that all subjects had to be 65 years of age or older, diagnosed with and continuing to suffer from depression, able to voluntarily sign an informed consent form, could speak and understand english, had resided in this nursing home for three or more months, had no changes in their depression drug therapy in the three months prior to the initiation of this study, and also no changes in their depression drug therapy throughout the four - week study. a total of 58 potential subjects were identified by the facility 's nursing managers as meeting all inclusion criteria. the charts of these 58 persons were reviewed for inclusion criteria, with all 58 remaining as potential research subjects. a flyer describing the study was then circulated to these 58 residents, with a member of the research team approaching each later that week to inform them about the study. each potential subject, who agreed to be involved in the study, was then tested for mental competency using the mds cognitive performance scale (cps), a tool often administered in nursing homes to determine mental competency. this test is highly correlated with the mini - mental status examination. all persons who scored 2 or less were retained in the potential subject pool, as a cps score of 2 or less indicates mild or no cognitive impairment. this testing was done to ensure each person was cognitively able to provide informed consent. the goal for this experimental study was to recruit at least 40 subjects, with 20 randomized to the control group and 20 the intervention or treatment group, in order to achieve statistically meaningful findings. however, among the 58 possible subjects, 8 lacked cognitive capacity to make decisions and 1 refused to take the cps test ; these 9 were excluded from the study. of the remaining 49, 19 were not currently depressed as they were found to have a score of 3 or more on the depression rating scale (a tool developed and routinely used in this nursing home to identify clinically depressed residents). another 13 were excluded due to low comprehension of the english language or lack of interest in the study. the remaining 17 participants were randomly assigned to the control group or the intervention group, with 9 becoming controls and 8 intervention group members. the 8 persons who were randomly assigned to the treatment group received a brief weekday hope intervention from a research assistant over four consecutive weeks. the 9 persons randomly assigned to the control group received a brief friendly weekday greeting from the research assistant over the same time period. immediately prior to the study, all 17 subjects were administered the gdssf and hhi tests to gather baseline hope and depression data. in addition, select sociodemographic data were collected from each chart to enable a description of the participants and to permit a comparison of control and treatment subjects. the research assistant also began to keep field notes to record her perceptions of the effect of her greeting on control subjects and the effect of the various hope interventions on treatment subjects. over the four weeks, the control and intervention group subjects continued to receive their usual prescribed medications and all care normally provided to them. as indicated above, each subject in the control group was also briefly visited each weekday by the research assistant, a daily visit mandated by the health research ethics board to ensure their ongoing willingness and ability to continue in the study, and to prevent them from feeling neglected while taking part in a research study that provided other residents in the nursing home with visible hope interventions. subjects who received the hope intervention were also visited each weekday over the same four - week period, although the focus of each visit was to provide them with that day 's hope intervention. during the first week, a hope card was delivered each weekday. the message was read to the participant and the card was left for them to keep. over the second week, in addition to receiving a new hope card each weekday, the participant was asked to recall a time in the past when they had experienced hope and to share this experience with the research assistant. in the third week, different hope pictures were shown to each subject every weekday. these pictures had an accompanying message that was read to the subject by the research assistant. the research assistant added this goal to a card that had been prepared in advance with the words today, my goal is in addition, in week three, a short qualitative interview not lasting more than 30 minutes took place. this interview was designed to gain information associated with two questions : (a) can you recall a time when you had hope ? and (b) would you please tell me about that time ? in the fourth and final week, each subject was given a choice of one picture or image that represented hope from among four presented to them each weekday. participants were asked to say why the chosen picture or image represented hope to them. this picture was then left with the participant. at the end of the four - week period, the hhi and gdssf were readministered to the 15 remaining participants, with two subjects in the intervention group having dropped out of the study by then. the data from the two tools collected at both intervals (pre- and poststudy) and the sociodemographic data were entered for all 15 subjects into an spss spreadsheet. the hope and depression data were compared between and within groups using t - tests, a test appropriate for the data variables and the specific purpose of this experimental study to test the efficacy of the hope intervention on hope and depression. the qualitative data recorded over the course of this study by the research assistant were also reviewed by the research team. this review of the field notes was mainly undertaken to help the research team understand the findings. as indicated previously, 9 subjects were initially randomized to the control group and 8 to the intervention group. the two participants in the intervention group who dropped out did so because of a negative reaction to the hope interventions. one indicated that she was too tired to take part in the study, as it took too much out of her, and the other said she did not like the study. no subjects in the control group dropped out, and no negative reactions to this study among these subjects were noted by the research assistant. instead, consistent positive reactions among the control subjects were noted, with some openly stating that they looked forward to the brief weekday visit from the research assistant. these findings represent the 15 subjects who completed this study, as replacements for the two subjects who withdrew were not possible. the average age, average length of nursing home stay, average cognitive performance scale (cps) score, and average depression rating scale (drs) score did not differ significantly between the control and intervention subjects. as such, the control and intervention group subjects were relatively similar in age, length of stay in the nursing home, cognitive ability, and degree of depression. table 1 also outlines the average pre- and poststudy gdssf and hhi scores for the control group and the treatment group. no difference in the average prestudy gdssf or the average prestudy hhi scores between the control and intervention groups was found, findings that indicate that these two groups were initially quite similar to each other with regard to their degree of depression and their level of hope. table 1 also shows that the average poststudy gdssf and hhi scores for both the control and intervention group subjects continued to be similar to each other after the four - week study ended. these findings indicate that there was no difference in the effect of the four - week hope intervention program as compared to the brief informal visits by the research assistant. table 2 outlines the average pre- and poststudy gdssf and hhi scores for the control group and the average pre- and poststudy gdssf and hhi scores for the treatment group. the intervention group had a statistically significant drop in their average hope scores (from 33.0 to 32.0), a finding that indicates their level of hope declined despite their having received a month - long hope intervention program. in contrast, the measured level of hope among the control subjects increased, although this increase was not statistically significant. another key finding was that the depression scores among the control group subjects declined to a near significant level, while the intervention group 's depression scores declined only slightly, indicating that the control subjects had improved more by having less depression as compared to the intervention subjects. as indicated, the purpose of this experimental study was to test the efficacy of a relatively simple and inexpensive month - long hope intervention program for depressed older persons living in a nursing home, with the ultimate goal of this study to gain research evidence relevant to alleviating or reducing depression among nursing home residents through nonpharmacological methods. psychotherapeutic and pharmacotherapy are common approaches to treating depression, while hope treatments for depression (such as the one used in this study) have not previously been examined for their effects. the findings of this pilot study are both surprising and revealing. although it was anticipated that the hope intervention program would relieve depression and improve hope among the treatment group subjects, subjects in the control group appeared to benefit more through their involvement in this study. the control group had a nearly significant decrease in depression while the treatment group only showed a slight decline in depression. this finding could be a result of sampling or testing issues, with other hope interventions perhaps more helpful for reducing depression. it is important to note, however, that the participants in both groups were not as depressed after the four weeks, a finding that suggests depression among nursing home residents is potentially reducible through nonpharmacological interventions. another important finding was the unanticipated reduction in hope among the treatment group subjects, particularly as compared to the control group where an increase in hope was found. although the small number of intervention and control group subjects could explain this finding, it is also possible that this specific hope intervention program was not designed appropriately for depressed older persons. other hope interventions could perhaps have more efficacy for improving hope and reducing depression among nursing home residents. another possible rationale for the lack of expected treatment effect could be that 30 days is not enough time to instill hope or significantly reduce depression among nursing home residents. nursing home residents, who are advanced in age and dependency requiring nursing home level care, may not be as responsive to this or other therapies particularly if they have suffered from long - standing depression. another possible explanation for the unexpected findings of this study is that the subjects in the control group responded more positively to the friendly weekday greeting by the research assistant as compared to the less personal and more structured or purposive weekday visit. a growing body of literature is revealing the importance of visitors to help people recover from illnesses, with other positive outcomes also having been identified to date. as such, the unexpected findings of this pilot study suggest that paid or volunteer visitors could potentially relieve depression among institutionalized older persons. furthermore, these findings suggest care staff should be educated to be outwardly friendly when approaching and while working with depressed older persons. the use of humor by caregivers or visitors could also be considered. humor is important for building therapeutic relationships and for other therapeutic effects among ill persons. given the high rate of depression among older persons, particularly those living in nursing homes, much more should be done to alleviate it. although this study found that a simple cheerful greeting was more effective for relieving depression and instilling hope than a four - week program of carefully planned and constructed hope interventions, more research is needed to substantiate or refute this conclusion. the surprising yet revealing findings of this study are not generalizable as they are limited by the small sample size, but they provide a starting point for other studies. studies with a standard control group, larger samples, and multiple nursing homes are encouraged to test the efficacy of hope interventions. other experimental studies should test the effect of brief social visits for relieving depression in nursing homes. qualitative or mixed - methods studies are also encouraged to gain needed insight into nonpharmacological ways of relieving depression. | depression is common among older persons. an experimental study was undertaken to test the impact of a four - week hope program on depressed nursing home residents. residents aged 65 or older, who met the criteria for this pilot study and agreed to participate, were randomly assigned to (a) an intervention group, and provided with weekday hope interventions mainly involving positive messages and pictures or (b) a modified control group, and provided with a friendly weekday greeting. the structured hope intervention was not proven effective for reducing depression or raising hope. instead, a significant reduction in depression among the control subjects was found, as well as a nonsignificant increase in their level of hope. although these findings suggest friendly visitors may be a more efficacious nonpharmacological approach for reducing depression, further investigations are needed to confirm this and to explore the impact of other hope interventions. |
osteoarthritis (oa) is the most common chronic progressive joint disease in the elderly and has a significant impact on quality of life as functional status decreases as a result of the pain. furthermore, difficulties in carrying out activities of daily living have detrimental effects on psychological well - being. in general, the goals of clinically managing knee oa are to provide pain relief and to maintain or to improve functionality. a diverse range of pharmacological, non - operative and surgical options are available for treating oa, yet each therapeutic modality has its respective limitations and side effects1. short - wave diathermy, transcutaneous electrical nerve stimulation, ultrasound (us) therapeutic acoustic radiation, and applying hot packs are all commonly utilized noninvasive modalities to control both acute and chronic oa pain2. therapeutic acoustic radiation is transmitted into target tissues by us via high - frequency pressure waves that are generated by piezoelectric crystals in the transducer. heat is generated by the pressure waves, and it has been shown that low - intensity us pulses stimulate cellular metabolism that enhance tissue regenerative capacity3,4,5,6. this relatively new treatment modality has been shown to promote the repair of full - thickness tears in articular cartilage. specifically, it has been found that the pressure waves induce stromal cell and chondrocyte proliferation in addition to mesenchymal stem cell differentiation at the lesion site7,8,9. however, the therapeutic effects of us are more pronounced in the joint spaces, such as the knee joints, because synovial fluid has high water content10. it is the one of several physical therapy modalities suggested for the management of pain and loss of function due to oa and can be used as part of an over all rehabilitation program11. several studies compare us and physical therapy versus placebo for patients with knee oa, but the clinical efficacy of us remains controversial2, 12,13,14,15,16,17,18,19,20,21. despite these results, us therapeutic acoustic radiation is still very popular for the treatment of musculoskeletal disorders and it has been asserted that it reduces edema, relieves pain, and increases range of motion. the aim of this prospective randomized single - blinded trial was to evaluate whether an 8-minute regimen of us therapeutic acoustic radiation is superior to a 4-minute regimen for the treatment of knee oa. this prospective randomized single - blinded trial was conducted at the istanbul physical medicine and rehabilitation training hospital in turkey. a total of 100 patients between the ages of 4070 years diagnosed with knee oa and a kellgren - lawrence grade of 2 or 3 according to american college of rheumatology criteria were enrolled in the study22, 23. exclusion criteria from the study included : any contraindications against performing physical therapy, dermatological problems, any systemic disease, abnormal laboratory test results, previous history of knee surgery or arthroplasty, previously participating in a physical therapy program, previous history of hyaluronic acid or steroid intraarticular injections, or having us therapeutic acoustic radiation within the last 6 months. patients were evaluated at baseline and after the entire treatment regimen lasting 2 weeks by a certified physician who was blinded to patient treatment condition. informed consent was obtained from all subjects after the nature of the study had been fully explained. the study was approved by our institution s ethics committee and was carried out in accordance with the principles of the declaration of helsinki. patients were asked to rate their pain level via a visual analogue scale (vas) at rest and during activity. scores ranged from 0 to 10, with a score of 0 indicating no pain and 10 indicating extremely severe pain22. the turkish translated version of the western ontario and mcmaster universities osteoarthritis index (womac) was utilized to assess oa patient symptoms23. the 24-item womac is a self - administered questionnaire that is divided into 3 subscales including pain (5 questions, score range : 020), joint stiffness (2 questions, score range : 08), and physical functionality (17 questions, score range : 068)24. previous studies demonstrated that this scale is both a valid and reliable tool to evaluate patients with knee oa25, 26. higher scores on the womac indicate more severe oa symptoms and so translate to more profound physical limitations. the extent of patient disability was evaluated with the lequesne (leq) index27. the questionnaire is comprised of 11 items regarding knee discomfort, ability to endure ambulation, and difficulties in carrying out activities of daily living. the beck depression inventory (bdi) was utilized to measure the intensity and severity of patient depressive symptoms. specifically it is composed of 21 questions and a score of 9 and above was considered to be a diagnosis of depression28). the first group (g1) received 4-minute us therapeutic acoustic radiation treatments, performed exercise, and received transcutaneous electrical nerve stimulation. the second group (g2) received an identical treatment, but instead these patients were treated with 8-minute regimens of us therapeutic acoustic radiation. continuous ultrasonic waves were delivered at a 1 mhz frequency and at an intensity of 1.5 watt / cm via a transducer with a diameter of 5 cm (chattanooga, usa). the patient maintained a supine position with both knees fully extended throughout treatment while us therapeutic acoustic radiation was directed around the knee joint. aqueous gel was utilized as a coupling medium to facilitate the transmission of the high pressure waves. the transducer was moved in a circular fashion at a 90 degree angle to the knee surface during treatment. every patient received 20 minutes of superficial heating therapy delivered via infrared radiation, 20 minutes of tens, and 15 minutes of isometric quadriceps exercises for both knees. patients in each group received treatments five times weekly for a total of 2 weeks. nonsteroidal anti - inflammatory drugs and antidepressants were not permitted throughout the course of treatment, but medications for the treatment of comorbid diseases were permitted during the study. data were analyzed using the statistical package for social sciences (spss) version 21.0 for windows (spss inc., demographic data were presented as the mean plus or minus one standard deviation (sd). the kolmogorov - smirnov test was utilized to determine whether continuous variables followed a normal distribution. the mann - whitney u - test and independent samples t - test were used to perform quantitative analyses of the data. the wilcoxon test was used to assess whether sample means differ over repeated measurements. the mean ages of groups 1 and 2 were 56.449.35 years and 56.7210.35 years, respectively. there were no statistically significant differences between sociodemographic data between each group (p>0.05 ; table 1table 1.demographic featuresgroup 1group 2meansd / n-%meansd / n-%genderfemale29 58.0%37 74.0%male21 42.0%13 26.0%age (years)56.44 9.34656.72 10.306bmi (kg / m)30.24 4.41231.01 4.633marital statusmarried37 74.0%38 76.0%single0 0.0%2 4.0%divorced13 26.0%10 20.0%professionhouse wife17 34.0%15 30.0%working14 28.0%17 34.0%retired19 38.0%18 36.0%duration (years)4.66 2.4464.50 2.375). there were no significant differences between baseline vas, leq, bdi and womac values between each group (p0.05). following treatment, vas, womac, leq, and bdi values decreased for both treatments groups (tables 2 and 3table 2.womac valuesgroup 1group 2meansd / n-%meansd / n-%womac painbefore11.704.17611.104.423after7.543.0986.623.356difference4.162.0344.481.854womac stiffnessbefore1.522.0532.222.207after0.921.4691.121.272difference0.600.7281.101.460womac functionbefore38.0611.13436.608.760after27.6210.85123.966.755difference10.444.34312.645.244womac totalbefore51.2815.11549.8813.055after36.0814.04531.429.712difference15.205.91418.467.083womac : western ontario and mcmaster universities osteoarthritis index). no statistically significant differences were observed for vas at rest values, womac pain and stiffness scores, and bdi values after treatment between g1 and g2 (p0.05). womac : western ontario and mcmaster universities osteoarthritis index vas in activity values and leq index values were significantly lower in g1 than g2 (table 3table 3.vas, leq and bdi valuesgroup 1group 2meansd / n-%meansd / n-%vasbefore2.582.3572.282.129after1.441.5541.001.262difference1.140.9481.281.310vas in activitybefore6.961.8956.701.344after4.461.7753.481.389difference2.501.0743.221.130leqbefore13.323.68912.443.715after9.503.6276.983.172difference3.821.8265.462.062bdibefore14.389.35212.728.461after11.347.6129.847.366difference3.042.0202.881.560vas : visual analog scale, leq : lequesne index, bdi : beck depression index). vas : visual analog scale, leq : lequesne index, bdi : beck depression index in this study we investigated the relative effectiveness of us therapeutic acoustic radiation for patients with knee oa over 8-minute and 4-minute treatment sessions. patients also received physical therapy sessions and transcutaneous electrical nerve stimulation throughout the study and significant improvement was noted in self - ratings of pain intensity, physical functionality, and depressive symptoms. yet, patients randomly assigned to the 8-minute us group achieved better results in their pain and ability to perform activities of daily living in comparison to patients receiving 4-minute treatments. there are conflicting data regarding the efficacy of us therapeutic acoustic radiation treatment for knee oa29,30,31,32,33,34. specifically, in 2010 the international osteoarthritis research society recommended that the optimal management for hip and knee oa requires a combination of non - pharmacological and pharmacological modalities including physical therapy, but does not specifically mention us therapeutic acoustic radiation as an adjunct35. several authors have researched the effectiveness of us therapeutic acoustic radiation treatment, but its clinical efficacy remains controversial2, 15,16,17,18,19,20. however, our findings indicate that us therapeutic acoustic radiation treatment may be more effective in treating knee oa than previously thought. in fact a study that ozgonenel.16 compared us with sham us in the treatment of knee oa revealed that us was superior to placebo, yet there were further oa treatments provided that may have obscured whether or not the effect was due to us alone. the intensity, the size of the area treated, and the duration of us therapeutic acoustic radiation treatment used in previously published studies varies considerably and there is little guidance in the literature as to what may be the optimal dosage to treat knee oa. yet, it has been recommended that for degenerative arthritis, us should be provided at a constant intensity from 0.5 to 2 watts / cm for 5 to 10 minutes36. guided by these recommendations, we utilized an intensity of 1.5 watts / cm over 4 or 8 minutes in our study. it has been reported that us therapeutic acoustic radiation provides pain relief and improvement in functionality for patients with knee oa15, 16,17,18,19. in our study, vas pain scores improved and womac scores were better after treatment for both treatment groups. yet, ulus.21 demonstrated no increase in womac functionality scores for patients receiving us treatment versus those receiving sham us treatment. in sale.37 study, general depression is commonly observed in patients who suffer from chronic pain. ozcetin.38 showed the prevalence of depressive symptoms is high among the elderly with oa. axford.39 has identified an association between depression, disability, pain, and symptom severity in patients with knee oa. in our study, the bdi was used to assess the degree of depressive symptoms and a decrease in depressive symptoms was seen in both treatment groups such that their self - ratings of depression did not differ significantly. similarly, ulus.21 studied the effects of us therapeutic acoustic radiation on patient psychological well - being and there were no differences between groups after receiving treatment as well. huang.18 showed that patient perception of impairment and disability due to oa are important factors in patient ratings of disease severity. deniz.40 studied effectiveness of pulsed and continuous diclofenac gel phonophoresis with topical diclofenac gel treatment in knee osteoarthritis. they showed that both continuous and pulsed ultrasound diclofenac gel phonophoresis is more effective for pain and functional status of patients with knee osteoarthritis than topical application of diclofenac gel. yldrm.41 compared the efficacy of ultrasound treatments of 4- and 8- minute durations for patients with subacromial impingement syndrome. eight minutes of ultrasound treatment was shown to be more effective than 4 minutes of ultrasound treatment. in this study, furthermore, the 8-minute treatment group reported a significantly better improvement in physical functionality than the 4-minute treatment group. these results suggest that longer us therapeutic acoustic radiation treatment durations may confer greater improvement in patient functionality and ability to perform activities of daily living. in all, our findings indicate that us therapeutic acoustic radiation treatment has true potential in improving symptoms for patients with knee oa. we recommend that further studies are performed in the future to develop a standardized treatment protocol to optimize the therapeutic benefit of this modality for patients with oa. | [purpose ] the aim of study was to compare different durations of ultrasound in patients with knee osteoarthritis. [subjects and methods ] one hundred patients diagnosed with bilateral knee osteoarthritis (oa) were enrolled in this study. patients were divided into two groups. the first group (g1) received 4 minutes of ultrasound. the second group (g2) received the exact same treatment, but the duration of ultrasound was longer at 8 minutes. patients in both groups underwent a total of 10 ultrasound over 2 weeks. following treatment, all patients provided self - evaluations of pain via the visual analog scale (vas), overall physical function with womac, disability via the lequesne index (leq), and depressive symptoms with the beck depression index (bdi). [results ] there were no significant differences in vas, womac leq, and bdi values between groups 1 and 2. after treatment, vas, womac, leq, and bdi values improved for both treatment groups. however, following treatment, g2 had significantly greater values for womac functional and total scores than g1. no statistically significant differences were observed for vas scores while inactive, womac pain and stiffness scores, and bdi values after treatment between both groups. vas pain scores while active and leq index values were significantly lower in g1 than g2. [conclusion ] patients in both groups demonstrated improved functionality, pain and psychological status following a consistent, 2-week regimen of 4-minute or 8-minute treatments with ultrasound. yet, patients that experienced longer treatment durations of 8 minutes demonstrated better outcomes in pain and the ability to carry out activities of daily living. |
table 1 shows that the sample consisted of 52 young male adults (17 - 22 years) from southern and central part of india who volunteered for the study involving empathy, adjustment and personality. participants mean age was 18.12 years (sd=0.74), and 45% were in senior secondary school and 55% were pursuing graduation at bhopal (central india) and bangalore (south india). the main inclusion criteria were higher secondary education level. family psychopathology and psychiatric problems were ruled out. correlation between empathy, adjustment and personality traits the empathy quotient (eq) that is a self - administered forced choice scale comprising of 60 items, 40 assessing empathizing and 20 filler items. approximately, half the items are worded to produce a disagree response and half the internal consistency of the empathy quotient, measured by cronbach 's alpha coefficient was 0.78. the test retest reliability was good as measured by pearson 's r correlation coefficient was r=0.84 (p<0.001). moderate associations were found between the empathy quotient and interpersonal reactivity index subscales, suggesting concurrent validity. the jackson personality inventory (jpi) is widely considered to be one of the most psychometrically sound measures of personality. the jpi - r provides valid and reliable insight into an individual 's personality and ability to function in a wide range of settings from work, organizational, leadership and social situations. in one convenient form, the jpi provides a measure of personality consisting of 16 subscales that reflect a variety of social, cognitive and value orientations, which affect an individual 's functioning. reliability as median internal consistency (bentler 's theta) were 0.90 and 0.93 and item - total correlations indicate that items correlate higher on average with their own scale 's total score than they do with the total score on any of the other 14 scales. correlations between the jpi - r and several well - known measures of personality provide evidence of convergent and discriminate validity. it consists of 140 items, which are to be answered in yes, no or question mark (?), pertaining to four adjustment domains, that is, home, health, social and emotional. they were provided with necessary information about the study and were ensured about the confidentiality. the order of the questionnaires was changed for different groups to check the potential order effects. table 1 shows that the sample consisted of 52 young male adults (17 - 22 years) from southern and central part of india who volunteered for the study involving empathy, adjustment and personality. participants mean age was 18.12 years (sd=0.74), and 45% were in senior secondary school and 55% were pursuing graduation at bhopal (central india) and bangalore (south india). the main inclusion criteria were higher secondary education level. family psychopathology and psychiatric problems were ruled out. the empathy quotient (eq) that is a self - administered forced choice scale comprising of 60 items, 40 assessing empathizing and 20 filler items. approximately, half the items are worded to produce a disagree response and half agree response. the internal consistency of the empathy quotient, measured by cronbach 's alpha coefficient was 0.78. the test retest reliability was good as measured by pearson 's r correlation coefficient was r=0.84 (p<0.001). moderate associations were found between the empathy quotient and interpersonal reactivity index subscales, suggesting concurrent validity. the empathy quotient (eq) that is a self - administered forced choice scale comprising of 60 items, 40 assessing empathizing and 20 filler items. approximately, half the items are worded to produce a disagree response and half agree response. the internal consistency of the empathy quotient, measured by cronbach 's alpha coefficient was 0.78. the test retest reliability was good as measured by pearson 's r correlation coefficient was r=0.84 (p<0.001). moderate associations were found between the empathy quotient and interpersonal reactivity index subscales, suggesting concurrent validity. the jackson personality inventory (jpi) is widely considered to be one of the most psychometrically sound measures of personality. the jpi - r provides valid and reliable insight into an individual 's personality and ability to function in a wide range of settings from work, organizational, leadership and social situations. in one convenient form, the jpi provides a measure of personality consisting of 16 subscales that reflect a variety of social, cognitive and value orientations, which affect an individual 's functioning. reliability as median internal consistency (bentler 's theta) were 0.90 and 0.93 and item - total correlations indicate that items correlate higher on average with their own scale 's total score than they do with the total score on any of the other 14 scales. correlations between the jpi - r and several well - known measures of personality provide evidence of convergent and discriminate validity. it consists of 140 items, which are to be answered in yes, no or question mark (?), pertaining to four adjustment domains, that is, home, health, social and emotional. they were provided with necessary information about the study and were ensured about the confidentiality. the order of the questionnaires was changed for different groups to check the potential order effects. empathy scores correlated positively with the adjustment scores on the dimensions of home adjustment (r=0.18) and health adjustment (r=0.05) that is not significant. but empathy positively and significantly correlated with the social adjustment dimensions of adjustment scores (r=0.29) ; on the contrary empathy was negatively correlated emotional adjustment (r=0.26) that was also not significant. it also emerged that home adjustment was positively and significantly correlated with interpersonal affect dimension of personality (r=0.33, p<0.05) where as intercorrelation between health adjustment and different personality dimensions were not significant except with anxiety (r=0.33, p<0.01). the score on social adjustment (c) were correlated positively and significantly with the anxiety (r=0.29, p<0.05), breadth of interest (0.37, p<0.01), complexity (0.29, p<0.05), conformity (0.34, p<0.05), interpersonal affect (0.46, p<0.01) and value orthodoxy (0.35, p<0.05) dimensions of personality. the scores on emotional adjustment was positively and significantly correlated with only two dimensions of personality, namely anxiety (0.59, p<0.01) and conformity (0.34, p<0.05). stepwise multiple regression analyses were used to evaluate the unique contribution of personality traits and empathy on four domains of adjustment. multiple regression summaries of social adjustment multiple regression summaries of home adjustment multiple regression summaries of health adjustment multiple regression summaries of emotional adjustment model 1 shows multiple regression summary of empathy and social adjustment, wherein correlation between the two was found to be 0.29 (sig=0.05). it shows that higher the score on empathy, the better will be social adjustment. model 2 shows multiple regression summary of anxiety (anx), breadth of interest (bdi), complexity (cpx), conformity (cny), interpersonal affect (iaf) and value orthodoxy (vol) dimensions of personality and social adjustment. it shows that these six dimension of personality combined together are a significant predictor of social adjustment. table 3 shows multiple regression summary of iaf dimension of personality and home adjustment, wherein correlation between the two was found to be 0.33 (sig=0.05). further it reveals that iaf dimension of personality is a significant predictor of home adjustment. it shows that higher the score on iaf, the better will be home adjustment. table 5 shows multiple regression summaries of anx and cny dimensions of personality and emotional adjustment. level of empathy that emerged from the sample of young adults is above average as the average score is 46.2 (mean=46.2, sd=10.6) that indicates the good ability for understanding how other people feel and responding appropriately and they know very well how to treat people with care and sensitivity. low scores on bdi indicate that young adults had narrow range of interests, remain uninterested when exposed to new activities and also had few hobbies. sample showed low scores on complexity, which indicates that they prefer concrete to abstract interpretations, avoid contemplative thought and uninterested in probing for new insight. further, anx, bdi, cpx, cny, iaf and vol were also significantly correlated with social adjustment (c). studies have found that stress is associated with depressive symptoms, anxious symptoms and greater suicide ideation. finally, a number of studies have shown that stress often mediates the link between personality process variables and psychological adjustment. for example, recent studies have shown that stress partially mediates the relationship between perfectionism and psychological adjustment. anxiety accounted for a great variance in adjustment of those children whose one parent was suffering from cancer. in another study, social adjustment of the medical cadets was found to be positively correlated with personality, emotional stability, sensitivity, group conformity, social boldness, self - control, etc. the home adjustment score was found to be high average that indicates unsatisfactory home adjustment among young adults. this might be because of high pressure of parents as competitive environment prevailing in the society. studies have found that one - third to one - half of adolescents struggle with low self - esteem, especially in early adolescence. the results of low self - esteem can be temporary, but in serious cases can lead to various problems including depression, delinquency, self - inflicted injuries, suicide and anorexia nervosa. theorists argue and found support for the idea that negative emotionality is closely linked to the experience of prosocial emotion such as empathy and guilt. emotion has also been associated with low level of aggression and high level of prosocial behavior. empathy plays a crucial role in the development of prosocial behavior, which in turn helps inhibit aggression toward others. however, this study hinted at the idea that anxiety and interpersonal affect can be used to predict adjustment in young adults. it would be premature to consider this result as proof for a specific adjustment theory. however, it can be assumed that within an academic contest this notion might be more valuable. the study was conducted on males only and the results could not be compared with females and makes the gender differentiation difficult. the sample size is relatively small and future studies with larger sample may reveal generalized findings on the predictors of adjustment. since only the urban and semiurban adults who volunteered to be a part of this study were selected, the rural population could not be studied. sample from both the parts of india was pooled, so the difference in the performance of these geographically and culturally diverse samples could not be analyzed. | background : although adjustment has been studied in relation with a host of variables, the relevance of empathy and its importance in the process of adjustment has received little attention. it is a well - known fact that personality plays a very important role in our interactions and dealings and also that empathy facilitates this process.settings and design : this study evaluated whether these two things combined together affect or predict adjustment. a random sample of 52 young male adults volunteered for this study.materials and methods : these 52 male participants filled up questionnaires related to personality (the jackson personality inventory), empathy. the empathy quotient) and adjustment (bell adjustment inventory).analysis and results : the data were analyzed using correlation and regression analysis. personality traits like interpersonal affect, conformity facilitated the process of adjustment, whereas traits like anxiety worked in the opposite direction. empathy also emerged as a significant contributor to the social adjustment.conclusions:results showed that both empathy and personality traits accounted for unique variance in adjustment. results are discussed in terms of empathy, personality traits and their role in adjustment. |
there several liver conditions that cause chronic or continuing liver inflammation, and the most common causes of end - stage liver disease are chronic viral hepatitis b and c, alcohol - related liver disease, autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis, steatohepatitis, liver disorders inherited or present at birth, and drug - induced liver damage. in brazil, since 2006, donor liver allocation has been based on the model for end - stage liver disease (meld) scoring system. at present, the meld has been validated on a broad series of patients with liver diseases of various etiologies and severity. the lab values used in the meld calculation are serum bilirrubin, international normalization ratio (inr) and serum creatinine (figure 1). model for end - stage liver disease (meld) calculation the relationship between the introduction of the meld allocation system in brazil and a reduction of donor organ waiting list mortality is controversial. however, the number of recipients of liver transplants has grown dramatically in the last few years and is likely to continue to do so in the future. the improved survival rates and transplant outcomes is predicated upon proper screening and evaluation, as well as breakthroughs in surgical techniques and immunosuppressive therapies. despite these breakthroughs, infection continues to be a leading cause in graft loss or death of transplant recipients. although evaluation for chronic systemic infections in transplant recipients is standard, this can not be said of pre - transplantation dental evaluation. a survey of united states organ transplant centers, conducted between 2003 and 2004, found that among the 294 respondents, 28 (9%) of organ transplant centers reported one or more incidents of sepsis of dental origin in transplant recipients. in this same survey, 34 (11%) transplant centers reported experiencing one or more episodes of a dental infection prior to transplantation that needed cancellation or postponement of the surgery. a study performed by the starzl transplant institute involving 300 candidates for liver transplantation conducted between january 2004 and march 2005, reported that oral health attributes (i.e., gingivitis, dental plaque, dental caries, periodontal disease, edentulism, and xerostomia) were similar to those seen in the general population. candidates for liver transplantation who have not had a dental evaluation for more than 1 year were significantly more likely to present with neglected oral health, untreated dental conditions or habits (e.g., smoking) that were potential precursors and risk factors for dental disease. providing oral health care pre- and post - liver transplantation is essential for a better prognosis and quality of life of transplant recipients. the main concern before the transplantation is to eliminate oral foci of infection, such as those of periapical and periodontal origin. during the post - transplant period, the dental professional must be aware of the increased susceptibility to infection in the patient, and the risk for organ rejection, both of which emphasize the importance of vigilant oral health maintenance. patients are severely immunocompromised and must be well - educated and treated for the rest of their post - transplant lives. the objectives of this study were to document the prevalence of oral disease and abnormalities in patients on a liver transplant waiting list presenting to an urban dental school clinic, discuss the appropriated dental treatment, according their systemic conditions and compare the oral manifestations with those of healthy individuals. this study was approved by the research ethics committee of the school of dentistry of the university of so paulo. a pilot study was conducted involving 16 end - stage liver disease individuals (study group - sg) attending the special care dentistry center of the university of so paulo and 16 control individuals (control group - cg) with no liver diseases, who were receiving dental care at the dental school of the university of so paulo. sg was formed by all patients with end - stage liver disease that had been referred to the special care dentistry center for routine dental treatment. medical history review and physical examination, with data recorded on a form specifically designed for this study, were compiled in a special form for all the patients. all patients were questioned about variables such as demographics and history of liver disease. coagulation studies [i.e., prothrombin time (pt)/international normalization ratio (inr), activated partial thromboplastin time (aptt), and platelet count ] were also performed on all patients. the dental status of each patient was evaluated at the initial visit by two trained oral medicine dentists. the patient evaluation consisted of a clinical examination of the hard and soft oral tissues and a radiographic examination which consisted in panoramic and periapical radiography. all abnormalities detected in each patient were recorded using a special form designed for this study. based upon each patient 's dental treatment indications and needs, a dental treatment plan was formulated, and specific dental management recommendations for each patient were presented. among 16 individuals from sg, 13 were male and 3 were female, ranging in age from 37 to 68 (median age of 51 years). of the 16 individuals from cg, 13 were male and 3 were female, ranging in age from 34 to 70 (median age to 50 years). the patients with end - stage liver disease presented hepatitis c and alcohol related liver disease (5/16 ; 31.25%), hepatitis c (5/16 ; 31.25%), alcohol - related liver disease (3/16 ; 18.75%), hepatitis and alcohol related liver disease b (2/16 ; 12.5%) and wilson disease (1/16 ; 6.5%) (figure 2). characteristics of the liver transplant candidates abnormal coagulation test results, reflecting an increased risk of bleeding, were found in 15 patients from sg (15/16 ; 93.75%). the most common finding was an abnormally low platelet count (56 to 96x10/l) found in 11 patients (11/16 ; 68.75%), followed by prolonged pt (10 to 52 s) in 13 patients (13/16 ; 81.25%), prolonged aptt (52 to 46.9 s) in 5 patients (5/16 ; 31.25%), and increased inr (> 1.3) in 7 patients (7/16 ; 43.75%). data collected from oral and radiographic examinations showed that all patients from sg (100%) were diagnosed with least one oral disease or condition that would require dental treatment prior to liver transplantation. caries was diagnosed in 13 patients (13/16 ; 81.25%) ; periodontal disease in 11 patients (11/16 ; 68.75%) ; petechiae in 3 patients (3/16 ; 18.75%) ; oral candidiasis in 2 patients (2/16 ; 12.5%) ; and gingival overgrowth, ulceration caused by cytomegalovirus (cmv), xerostomia and angular cheilitis were diagnosed 1 patient (1/16 ; 6.25%). the patients from cg presented caries (7/16 ; 43.75%), periodontal disease (10/16 ; 62.5%) and oral candidiasis (1/16 ; 6.25%). the individuals from sg exhibited a higher incidence of oral diseases compared with those from cg (p=0.0327). figure 3 summarizes the oral conditions found in all individuals enrolled in this study. oral manifestations of study group and control group patients cmv = cytomegalovirus oral manifestations and coagulation abnormalities legend : pd = periodontal disease ; go = gingival overgrowth ; oc = oral candidiasis ; ptc = petechiae ; ac = angular cheilitis ; cmv = ulceration caused by cytomegalovirus ; aptt = activated partial thromboplastin time ; pt = prothrombin time ; inr = international normalization ratio after the initial exam and diagnosis of oral diseases, indicated dental treatment procedures were performed on all patients (sg and cg) by the same dentists and included 20 restorations, 21 periodontal scaling and root planning procedures, 6 extractions, 1 incisional biopsy, 3 brush (exfoliative) cytology exams, 2 prosthetic rehabilitations (1 removable prosthesis and fixed prosthesis) and 2 endodontic treatments. although the majority of patients showed abnormal coagulation values, none of them exhibited critical values that would represent a contraindication for invasive dental treatment. for this reason, procedures that involve bleeding, such as dental extraction, biopsy and periodontal treatment, were conducted using hemostatic local measurements, such as tranexamic acid paste and absorbable gelatin sterile sponge, as recommended by ramstrom,. transplant centers are very specialized facilities that are usually located at university teaching hospitals or large medical centers. they require a large staff of surgeons and other professionals to evaluate and select patients, and perform surgery and follow - up care. however, even though patients are on waiting lists for matching donor livers for time enough to receive definitive dental treatment, this is not a major priority on the protocols of some transplant centers in our city. the individuals from sg exhibited a higher incidence of oral manifestations compared with cg (p=0.0327) and all of them were diagnosed with at least one oral abnormality that required dental treatment prior liver transplantation in order to avoid potential complications due to infection after transplantation secondary to drug - induced, anti - organ rejection immunosuppression. the major concern for dental management of patients with end - stage liver disease is the occurrence of bleeding disorders. routine dental procedures such as dental prophylaxis, tooth extractions, minor periodontal surgeries, and even administration of local anesthetic for restorations can result in serious complications if the dentist is not aware of potential risk for impaired hemostasis and increased bleeding in these patients. it is generally accepted that minimally invasive procedures such as simple restorations, supra - gingival prophylaxis, and prosthodontic procedures do not usually involve a considerable risk of bleeding when compared with significantly more invasive procedures such as periodontal surgery and multiple extractions. the difficulty lies is assessing bleeding risk for procedures with a moderate level of invasiveness such as subgingival scaling (especially in the presence of more severe periodontal disease) and extensive (e.g., subgingival) restorations. in patients with mild to moderate hemostatic impairment, (i.e., inr not greater than 3.5, or a platelet count not less than 50x10 l) careful surgical technique, including an attempt at obtaining primary wound closure and use of local hemostatic measures will usually suffice. however, these materials also carry a risk of infection and may delay healing and should therefore be avoided in immunosuppressed patients. topical thrombin is an effective agent when applied directly on the bleeding wound as it converts fibrinogen to fibrin and allows rapid hemostasis in a wound. topical fibrin glue can reduce the amount of factor replacement needed when used along with antifibrinolytic agents. oxidized cellulose tranexamic acid rinses, astringents (e.g., aluminum chloride), microfibrillar collagen, thrombin - soaked gauze, fibrin sealant and adhesive, electrocautery, absorbable gelatin sponges, and amino - caproic acid (eaca) to prevent clot lysis, patients with liver failure from any cause are difficult to evaluate from the standpoint of risk for oral bleeding. a relatively small elevation of the pt test (e.g., pt=13 - 15 s) suggest significant (i.e., greater than 50%) liver damage. there are various systemic methods that can be used to help compensate for the coagulopathies seen in these patients and decrease the risk of prolonged bleeding from invasive dental procedures. vitamin k (at doses of 10 mg i m) corrects hypoprothrombinemia from malnutrition and biliary obstruction, but not when due to intrinsic liver disease. fresh frozen plasma will lower the pt, and platelet transfusion addresses both quantitative and qualitative platelet defects. in the case of more significant coagulopathies, the approach to an invasive procedure may need to be adjusted accordingly, and the patient should be observed postoperatively in the surgery for a longer time period than usual. care should be exercised with the use of narcotic analgesics (e.g., morphine) and sedatives (e.g., diazepam), as their duration of action may be prolonged in patients with decompensated liver disease. as with chronic renal failure, reduced doses should be used for drugs that are metabolized by the liver. in particular, avoid the use of paracetamol in the presence of liver failure and alcoholism, although doses of up to 4 g / day for up to 2 weeks are allowable. patients should be strongly advised not to take alcohol during the period when they are using acetaminophen. careful oral examination and evaluation of the patient, including laboratory tests, will ensure correct oral preparation and control of oral disease prior to liver transplantation. patients with chronic liver diseases may present at any dental office for treatment and therefore general dentists should be aware of the unique concerns involving their assessment, education, treatment and maintenance of oral health. the patients with chronic liver diseases evaluated in this study exhibited a higher incidence of oral manifestations compared with the healthy control group and had at least one oral disease or abnormality that required dental treatment prior to liver transplantation. | liver transplantation has become a standard treatment for end - stage liver disease and the number of recipients has grown rapidly in the last few years. dental care during pre - transplant workup is important to reduce potential sources of infection in the drug - induced immunosuppression phase of liver transplantation.objectives the objectives of this study were to document the prevalence of oral abnormalities in patients on a liver transplant waiting list presenting to an urban dental school clinic, discuss the appropriate dental treatment according their systemic conditions and compare their oral manifestations with those of healthy individuals. material and methods a pilot study was conducted involving 16 end - stage liver disease individuals (study group- sg) attending the special care dentistry center of the university of so paulo and 16 control individuals (control group- cg) with no liver diseases, receiving dental care at the dental school of the university of so paulo. these individuals were assessed for their dental status (presence of oral disease or abnormalities), coagulation status, and dental treatment indications. results the patients from sg exhibited a greater incidence of oral manifestations compared with cg (p=0.0327) and were diagnosed with at least one oral disease or condition that required treatment. coagulation abnormalities reflecting an increased risk of bleeding were found in 93.75% of the patients. however, no bleeding complications occurred after dental treatment. conclusions the patients with chronic liver diseases evaluated in this study exhibited a higher incidence of oral manifestations compared with the control group and had at least one oral disease or abnormality which required dental treatment prior to liver transplantation. careful oral examination and evaluation of the patient, including laboratory tests, will ensure correct oral preparation and control of oral disease prior to liver transplantation. |
human immunodeficiency virus (hiv) is the most devastating plague facing us as the 21st century begins. in india, nearly 22.7 lakh people are living with hiv infection. progressive destruction of immune system by chronic hiv infection leading to progressive fall in the level of cd4 cells (< 200/l to < 50 / l) is known to be responsible for the occurrence of infections by a variety of opportunistic microorganisms. this is also responsible for the recurrent, prolonged, intractable and severe nature of infection in hiv seropositive individuals. knowledge of the pattern of opportunistic infections can often guide therapy when resource limitations hamper the exact diagnosis of the etiological agent. hiv / aids may not be curable but most of the opportunistic infections can be effectively treated. prophylaxis against some of these infections will not only prolong the life of an hiv infected individual but also improve the quality of life. national aids control organization (naco) data reveal that tuberculosis is the commonest infection in aids patients, followed by candidiasis, cryptosporidiosis and others. considering this fact, the present study was conducted to determine the prevalence of bacterial, parasitic, fungal infections in hiv seropositive patients. the present study was carried out on 100 hiv seropositive patients in department of microbiology, s. s. g. hospital, vadodara, during the period from jan 2006 to jan 2007. randomly selected 100 hiv seroreactive patients from in - patients and out - patients department of the hospital were included. their hiv status was confirmed by three tests with different principles / antigens (enzaids elisa, i.e. enzyme - linked immunosorbent assay, combaids - rs, hiv tridot) in the integrated counselling and testing centre attached to the department as per naco guidelines. antibodies against immunodominant regions of hiv 1 (gp 120, gp 41) and hiv 2 (gp 36) and p24 antigens were detected. necessary pre and post - test counseling of the patients was carried out and detailed history was taken. sputum samples, stool samples, oral swabs and cerebrospinal fluid (csf) from patients were collected taking all aseptic precautions. (saline and iodine) and wet preparation from formal ether concentrated samples were used for the detection of protozoan trophozoites, cysts and helminthic eggs and larva. for the detection of coccidian parasites, smears of stool samples were prepared and stained with modified acid fast method. early morning expectorated sputum samples were collected in sterile containers. for the detection of bacterial pathogens, samples were processed for gram 's stain, zeihl and nelson (zn) stain and aerobic culture on selective and enrichment media (lowenstein jenson media, chocolate agar, macconkey agar) as per the standard methods. one swab was used for wet mount (10% koh, i.e. potassium hydroxide) and gram stain. the other swab was inoculated on sabouraud 's dextrose agar (sda) with antibiotics. the growth obtained was identified for yeasts as per the standard protocol including germ tube test, chlamydospore formation, sugar assimilation, sugar fermentation and urease test. india ink preparation, wet mount, gram stain and culture on sda, chocolate agar and macconkey agar were carried out in csf samples. randomly selected 100 hiv seroreactive patients from in - patients and out - patients department of the hospital were included. their hiv status was confirmed by three tests with different principles / antigens (enzaids elisa, i.e. enzyme - linked immunosorbent assay, combaids - rs, hiv tridot) in the integrated counselling and testing centre attached to the department as per naco guidelines. antibodies against immunodominant regions of hiv 1 (gp 120, gp 41) and hiv 2 (gp 36) and p24 antigens were detected. necessary pre and post - test counseling of the patients was carried out and detailed history was taken. sputum samples, stool samples, oral swabs and cerebrospinal fluid (csf) from patients were collected taking all aseptic precautions. saline and iodine) and wet preparation from formal ether concentrated samples were used for the detection of protozoan trophozoites, cysts and helminthic eggs and larva. for the detection of coccidian parasites, smears of stool samples were prepared and stained with modified acid fast method. early morning expectorated sputum samples were collected in sterile containers. for the detection of bacterial pathogens, samples were processed for gram 's stain, zeihl and nelson (zn) stain and aerobic culture on selective and enrichment media (lowenstein jenson media, chocolate agar, macconkey agar) as per the standard methods. one swab was used for wet mount (10% koh, i.e. potassium hydroxide) and gram stain. the other swab was inoculated on sabouraud 's dextrose agar (sda) with antibiotics. the growth obtained was identified for yeasts as per the standard protocol including germ tube test, chlamydospore formation, sugar assimilation, sugar fermentation and urease test. india ink preparation, wet mount, gram stain and culture on sda, chocolate agar and macconkey agar were carried out in csf samples. out of the total 100 patients in the study, majority were males (75%, 75/100). the preponderant age group affected from hiv was 20 - 29 years (61%, 61/100). other routes included homosexual contact (1%, 1/100), blood transfusion (3%, 3/100) and vertical transmission (2%, 2/100). fever, cough, weight loss, diarrhea and oral thrush were the most common symptoms. candida (32.67%, 32/101) was the commonest pathogen identified, followed by mycobacterium tuberculosis (22.77%, 23/101) and cryptosporidium parvum (19.8%, 20/101). out of the four patients who presented with meningitis, cryptococcus neoformans was isolated in two patients and in the remaining two patients no fungal pathogen was identified. clinical presentation of study population microbial profile of hiv seropositive patients polymicrobial profile of hiv seropositive patients (n=30) in the present study, out of 100 patients, majority were in the reproductive age group, i.e. 2029 years (61%, 61/100), with a male preponderance (75%, 75/100). rangnathan and his colleagues reported 77.4% male patients with 81% patients in the age group of 2140 years in their study done at south india. reported the highest number of hiv patients to be in the age group of 2130 years (63%) with majority of male patients (94%) in their study done at manipur. majority of the patients in the study group were laborers (42%), followed by truck drivers (18%). majority of them were migrants staying away from home, thereby increasing the risk of hiv infection. heterosexual contact (95%) was the commonest route of transmission in our study which correlates well with other studies. more than 80% rate of heterosexual transmission was reported by naco (87.1%) and rangnathan (95%). in the present study, majority of patients had more than one symptom and 27% patients had multiple system involvement. fever and weight loss were the predominant symptoms, followed by cough, oral lesions and diarrhea. the clinical profile reported in the present study was similar to that reported in lucknow by ayyagari. and in amritsar by aruna aggarwal. polymicrobial etiology in 30 of the hiv reactive patients is a significant finding, indicating severity of the infection in this group. in the present study, 43% (43/100) diseases like bacterial pneumonia occur at a rate many times higher in the hiv infected population than in the general population. m. tuberculosis (60.5%, 23/38) was the commonest organism among the bacterial respiratory pathogens, followed by klebsiella pneumoniae (15.7%, 6/38), pseudomonas aeruginosa (10.5%, 4/38), staphylococcus aureus (7.8%, 3/38) and escherichia coli (1.9%, 2/38). aruna aggarwal. in their study at amritsar reported m. tuberculosis (32.75%) as the commonest bacterial respiratory pathogen, followed by klebsiella spp. (23.8%), p. aeruginosa (12.69%) and s. aureus (12.69%). reported m. tuberculosis (28%), followed by k. pneumoniae (10%), streptococcus pneumoniae (8%) and s. aureus (4%). this difference may be due to geographical differences and also possible hospital acquired superadded infections. oral lesions were present in 40% (40/100) of patients in the present study. out of 33 isolates from oral lesions, 27 were candida albicans (81.8%) and 6 were non - albicans candida spp. 23.08%). oral and esophageal candidiasis (57.5%) is reported as the second most common opportunistic infection among hiv patients from india. it is the second most common fungal infection and the fourth commonest cause of life - threatening illness in aids patients in india. singh. reported 7% rate of cryptococcal meningitis in their study done at manipal. an incidence of 3% of cryptococcal meningitis has been reported in hiv infected patients in india. in the present study parvum (71.4%, 20/28) was the commonest parasite found, followed by isospora belli (10.7%, 3/28), giardia lamblia (7.1%, 2/28) and entamoeba histolytica (7.1%, 2/28) parvum was reported as the most common enteric parasite by aruna aggarwal (51.42%) and singh (43%). i. belli was found as the commonest parasite by gupta at jamnagar (17.24%) and kumar at chennai (18%). differences in the incidence of intestinal parasitic infections can be attributed to the differences in geographical distribution of parasites, sanitary practices and different selection of cases. it is concluded that oral candidiasis, pulmonary tuberculosis and cryptosporidiosis are the commonest opportunistic infections in the hiv seropositive patients in the present study group. improved hygienic practices, regular examination and appropriate antimicrobial prophylaxis can reduce the substantial morbidity and mortality caused by opportunistic infections in patients with hiv infections. with better knowledge and diagnosis of the opportunistic infections, | objectives : the present study was undertaken to determine the prevalence of respiratory, gastrointestinal and other pathogens in 100 human immunodeficiency virus (hiv) seropositive patients.settings:this study was carried out on randomly selected 100 hiv seropositive patients from s. s. g. hospital during the period from jan 2006 to jan 2007.materials and methods : sputum samples, stool samples and oral swabs were collected from all the patients and cerebrospinal fluid (csf) was collected from symptomatic patients and processed as per the standard protocol. sputum samples were examined by microscopy and cultured for bacterial respiratory pathogens. stool samples were concentrated and examined by microscopy for enteric parasites. oral swabs and csf were also examined microscopically and cultured for fungal pathogens. csf was also examined for bacterial pathogens.results:a total of 101 pathogens were detected in 60 patients. more than one pathogen was observed in 30 patients. candida was the commonest isolate (32.67%), followed by mycobacterium tuberculosis (22.71%) and cryptosporidium parvum (19.8%).conclusions : since opportunistic infections are a major cause of mortality and morbidity in hiv seropositive patients, an early diagnosis and effective treatment are required to tackle them. the type of pathogens infecting hiv patients varies from region to region, and therefore such patients should be constantly screened for these pathogens. |
in 2010, it is estimated that prostate cancer will account for 28% of newly diagnosed cancers among males in the united states.1 a large majority of these cases (approximately 92%) will be diagnosed at a local or regional stage, with 5-year survival rates approaching 100%. however, for individuals who are diagnosed with (or subsequently develop) metastatic prostate cancer the prognosis remains limited.2 until recently, the treatment algorithm for metastatic disease remained relatively simple. observations by huggins in 1941suggested that castration could induce regression of prostatic tumors.3,4 thereafter, permutations of synthetic luteinizing hormone releasing hormone (lhrh) agonists and antiandrogen therapy supplanted surgical intervention.58 upon failure of these therapies, further options were limited until recently. two large, randomized phase iii trials demonstrated an overall survival (os) advantage with docetaxel compared to mitoxantrone - based regimens.9,10 beyond docetaxel, strategies such as crossing over to mitoxantrone - based regimens appear to be of limited efficacy.11,12 clinical data have amassed over the past several years that now position several agents in either the pre - or postdocetaxel space (and potentially both) in the prostate cancer treatment paradigm.13 the phase iii impact trial assessed sipuleucel - t, an autologous cellular vaccine, in a largely chemotherapy - nave cohort of patients with castration - resistant prostate cancer (crpc).14 relative to placebo, sipuleucel - t significantly prolonged os (25.8 vs 21.7 months, p = 0.04), leading to food and drug administration (fda) approval of this agent. as an alternative, several novel endocrine therapies have shown substantial efficacy in the setting of crpc. promising phase ii data for abiraterone, mdv3100, and tak700 have led to the design of large, randomized trials.1521 notably, a placebo - controlled, phase iii study enrolling patients with docetaxel - refractory crpc demonstrated a survival advantage with abiraterone therapy.22 just as these novel therapies challenge the paradigm of castration resistance in the setting of crpc (figure 1), clinical data for the novel taxane cabazitaxel suggest that a chemotherapeutic strategy may be effective even after failure of docetaxel. whereas vinca alkaloids inhibit incorporation of tubulin into microtubules, the taxanes appear to inhibit microtubule disassembly.2325 although the microtubular binding mechanism of cabazitaxel does not appear to be distinct from docetaxel or paclitaxel, the agent is structurally distinct. as noted in figure 2, hydroxyl groups present in docetaxel are replaced with methoxy groups in cabazitaxel. bissery reported preclinical data suggesting the in vitro activity of cabazitaxel.26 four cell lines were assessed, including p388 (lymphoblastic leukemia), hl60 (promyelocytic leukemia), kb (cervical adenocarcinoma), and calc18 (breast carcinoma). with a 4-day exposure to the drug, cytotoxicity was noted with relatively low cabazitaxel concentrations (ic50 = 329 ng / ml). in accompanying in vivo models, the agent was noted to have significant antitumor activity. in murine tumor xenografts (colon c38 and pancreas p03), cabazitaxel elicited complete tumor regressions. two schedules of the drug were assessed : 1) a day 1 and 5 schedule with a dose of 58 mg / kg and 2) thrice daily dosing on a day 1 to 5 schedule at 12 mg / kg. notably, in cell lines resistant to a variety of other cytotoxic agents (ie, anthracyclines, vinca alkaloids, and the older taxanes), cabazitaxel was noted to still induce tumor regression. the activity of cabazitaxel was subsequently documented in human tumor xenografts using a variety of intravenous schedules.27 in 3 human colorectal cell lines (hct-116, hct-8, and ht-29), high antitumor activity was observed. for instance, on a thrice daily schedule given every 3 days, cabazitaxel induced a 3.34 log cell kill (lck) at the total highest nontoxic dose (thntd), 36 mg / kg. in lung models, dosing at the thntd yielded 2.7 lck in the nci - h460 cell line, and 2.2 lck in the a549 cell line. as observed in murine tumor xenograft studies, multiple cases of complete regression were observed using human tumor xenografts. notably, long - term tumor - free survival (exceeding 133 days) and complete tumor regression were observed in pancreatic xenografts (mia paca-2), head and neck xenografts (sr475), and prostate xenografts (du145, a cell line that represents a hormone - resistant entity established from a prostate cancer brain metastasis).28 pharmacokinetic parameters associated with cabazitaxel were first documented in animal studies.29 using c - labeled cabazitaxel, doses of 15, 30, and 90 mg / m were delivered to mice as either 1-minute or 1-hour infusions. there was a correlation between dose and plasma exposure within the aforementioned dosing range, whereas brain exposure increased more than proportionally over the same range. the peak of brain concentrations occurred between 2 minutes and 1 hour post - infusion. parallel assessments performed in dogs using a dose of 15 mg / m over 80 minutes suggested lesser brain exposure as compared to mice. of note, brain concentrations of c - labeled cabazitaxel were detectable up to 168 hours after infusion in mice, and for up to 24 hours in dogs. the role of p - glycoprotein (p - gp) in the accumulation of cabazitaxel in the brain was assessed more extensively in a report by cisternino.30 again using c - labeled cabazitaxel, doses ranging between 15 and 90 mg / m were delivered to mice, and doses of either 15 or 60 mg / m were delivered to rats. it was noted that brain uptake of cabazitaxel was enhanced when concentrations exceeded 11 m. these saturable kinetics suggested the role of a critical transporter (ie, p - gp) in transporting cabazitaxel across the blood brain barrier (bbb) upon a certain threshold (saturation was found to be at 13 m). to further test this hypothesis, verapamil co - administration led to a 2.9-fold and 4.7-fold increase in brain uptake in mice and rats, respectively. harnessing these pharmacokinetic properties, 31 using sf-295 and u251 human glioblastoma cell lines, both orthotopic and subcutaneous murine xenografts were generated. furthermore, in orthotopic models, cabazitaxel led to complete tumor regression in 4 out of 10 u251 tumors. a phase i clinical trial of 3-weekly cabazitaxel enrolled patients with advanced solid malignancies refractory to conventional treatments.32 with respect to prior therapy, patients were limited to less than 2 prior chemotherapy regimens for metastatic disease and radiation affecting less than 25% of the available hematopoietic reserve. a starting dose of 10 mg / m was selected, representing one - tenth the severe toxic dose in mice (std10). given that the std10 in mice corresponded to a plasma level of 10.8 g / ml, pharmacokinetic monitoring was performed during the first course of therapy and dose - escalation was to be terminated for plasma levels beyond this value. in total, 25 patients were treated with 102 courses of 3-weekly cabazitaxel at 4 dose levels, ranging from 10 mg / m to 25 mg / m.32 a total of 22 patients had received prior chemotherapy (88%), and 8 patients had received prior taxane - based therapy (32%). although a diverse array of tumor types was enrolled, the largest subgroup comprised patients with prostate cancer (8 patients, 32%). a median of 4 cycles (range 19) was administered. a rapid initial phase was followed by a longer intermediate phase (t1/2 = 2.5 minutes and 1.3 hours, respectively). finally, a prolonged terminal phase (t1/2 = 77.3 hours) was observed. the dose - limiting toxicity (dlt) of cabazitaxel was neutropenia, with 1 case of febrile neutropenia and 2 cases of grade 4 neutropenia occurring at a dose of 25 mg / m. accordingly, the recommended phase ii dose emerging from this study was 20 mg / m.32 notably, support with granulocyte colony - stimulating factor (g - csf) or granulocyte macrophage colony - stimulating factor was not utilized in these studies, although it was ultimately administered in patients incurring grade 4 neutropenia. nonhematologic toxicities were generally mild in nature ; the most commonly encountered adverse events were diarrhea (52%), nausea (40%), and vomiting (15%). diarrhea in a patient dosed at 15 mg / m (resolving shortly after therapy with loperamide). in this initial clinical experience, an unconfirmed partial response was observed in a patient with bladder cancer, and minor responses were seen in 2 patients with osteosarcoma and prostate cancer, respectively. stable disease (sd) was recorded as a best response in 12 patients (48%). a phase ii study in breast cancer was originally designed as a randomized 3-arm study to explore 2 distinct dosing regimens of cabazitaxel and to further assess the activity of the novel taxane larotaxel. (the activity of larotaxel has been documented in phase i and ii studies in breast and lung cancer).3336 due to poor accrual, it was ultimately modified to be a single - arm study evaluating cabazitaxel alone in patients with taxane - resistant metastatic breast cancer. in the setting of patients who had received adjuvant or neoadjuvant taxane therapy, resistance was defined as metastatic progression within 12 months of systemic therapy. for patients with metastatic disease, the definition was more complex ; resistance was characterized as : 1) progressive disease (pd) representing the best response to treatment, 2) pd occurring within 4 months after first- or second - line therapy (after an initial clinical benefit), or 3) sd representing the best response if a taxane had been administered for 3 or more months. patients were treated initially with a dose of 20 mg / m, which was escalated to 25 mg / m in those patients who did not incur a significant adverse event during the first cycle of therapy. patients who were her2-positive were allowed to enroll if they had progressed on a trastuzumab - based regimen ; otherwise, the study was limited to her2-negative patients. the study was powered to assess objective response rate (orr) by response evaluation criteria in solid tumors (recist) guidelines, with secondary endpoints including duration of response, time to progression, and os.36 the study was stratified by the number of lines of previous taxane - based therapy. stratum 1 consisted of 47 patients who had progressed after either first - line systemic therapy for advanced disease or adjuvant / neoadjuvant taxanes ; stratum 2 consisted of 20 patients who had progressed on second - line therapy for advanced disease. the median age of enrolled patients was 53 years, with an expected distribution of hormone receptor - positive and her2-positive tumors (52% and 27%, respectively). the majority of patients had received prior chemotherapy for advanced disease, with only 1 patient having received adjuvant therapy. seven patients (10%) had received multiple forms of taxane therapy. among treated patients, the orr was 14% (95% confidence interval [ci ], 7%24%), with no differences between the two pre - defined strata (14% for stratum 1 and 12% for stratum 2).36 the median duration of response was 7.6 months (range 2.618.7 months). a significant proportion of patients also exhibited sd as a best response (38%). two patients were noted to have a complete response to cabazitaxel therapy. mirroring the phase i experience, the most common grade 3/4 toxicity incurred was neutropenia, present in 73% of the patients. two deaths were recorded within 30 days of on - study therapy ; both were secondary to nonhematologic toxicities. in the first patient, death occurred due to respiratory failure that was possibly related to study therapy, and in the second patient, the cause of death was unknown. the results for cabazitaxel in breast cancer have drawn multiple comparisons to the novel epothilone ixabepilone, which also impacts microtubule function and has been assessed in phase iii trials in this disease.37 the information garnered from phase i and ii studies, encompassing multiple malignancies, were used to inform the design of the phase iii tropic trial comparing cabazitaxel / prednisone with mitoxantrone / prednisone in patients with docetaxel - refractory prostate cancer.38 the study itself represented somewhat of a paradigm shift, given the absence of prior phase ii studies assessing cabazitaxel specifically in the setting of prostate cancer. however, no viable therapeutic options were available to the docetaxel - refractory patient at the time the study was initiated, generating a substantial area of need. furthermore, abundant preclinical data in docetaxel - refractory cell lines and an initial clinical demonstration of safety and efficacy in solid tumors supported this larger undertaking. in tropic, progression on docetaxel was defined by recist in patients with measurable disease, or by 2 consecutive prostate - specific antigen (psa) rises (at least 1 week apart) in patients with nonmeasurable disease.38 orchiectomy or prior pharmacologic androgen deprivation was mandated, and patients who were receiving lhrh agonists were instructed to continue taking them during protocol therapy. ultimately, 755 men were randomized (378 to cabazitaxel and 377 to mitoxantrone) in a total of 26 countries. the median age of the study population was 68 years, and the majority of patients were caucasian (84%).38 although enrollment was originally conducted irrespective of the amount of prior docetaxel therapy, the study was ultimately modified to exclude patients who had received a cumulative dose of less than 225 mg / m. this amendment was made in light of guidelines suggesting that castrate - resistant prostate cancer therapy be maintained for a period of at least 3 cycles prior to instituting any change. the mean docetaxel dose in the experimental arm was 576.6 mg / m, compared with 529.2 mg / m in the control arm. a substantial proportion of patients progressed on docetaxel therapy either during treatment (29%) or within 3 months of its completion (45%) ; the mean time from the last docetaxel dose to disease progression was 0.8 months in the experimental arm and 0.9 months in the control arm. although most patients had bony metastases (84%), a considerable proportion did have visceral metastases (25%). whereas the phase ii experience in breast cancer initiated 3-weekly dosing of cabazitaxel at 20 mg / m, in tropic, patients were initiated at 25 mg / m. patients randomized to receive mitoxantrone were started on a conventional dose of 12 mg / m every 3 weeks. both arms received prednisone 10 mg oral daily. in order to limit the risk of mitoxantrone - induced cardiac dysfunction, therapy on both arms while growth factor support was not allowed at the initiation of therapy, it was permitted to treat extended neutropenia (> 7 days), neutropenic infection, or neutropenic fever. the primary endpoint of the study was os, with a secondary endpoint of progression - free survival (pfs). pfs was defined by the occurrence of one of several clinical events, including psa progression, radiographic progression, progression of pain (measured by the mcgill - melzack present pain intensity scale, ppi) or death. the study met its primary endpoint, with an improvement in os of 2.4 months favoring cabazitaxel therapy (15.1 vs 12.7 months ; hazard ratio [hr ] 0.70, 95% ci 0.590.83, p < 0.001). the benefit of cabazitaxel for survival appeared to extend across the majority of subgroups assessed, including subgroups divided by performance status (ecog 0 - 1 or ecog 2), measureable disease (absent or present), number of previous chemotherapeutic agents (1 or 2), age (< 65 or 65), and pain (at baseline, absent or present). cumulative pfs (using the composite endpoint) was also improved with cabazitaxel therapy (2.8 vs 1.4 months, hr 0.74, 95% ci 0.640.86, p < 0.0001), although time to pain progression (as defined by the ppi inventory) did not significantly improve. psa response rate was 39.2% vs 17.8% (p = 0.002) and median time to psa progression was 6.1 vs 3.1 months (p = 0.001), both favoring cabazitaxel. mirroring the phase i and ii experiences, the most common toxicity associated with cabazitaxel therapy was neutropenia. grade 3 neutropenia occurred in 82% of cabazitaxel patients, with 8% of patients developing febrile neutropenia. common nonhematologic toxicities in patients receiving cabazitaxel included diarrhea, fatigue, and asthenias (all grades : 47%, 37%, and 20%, respectively). a total of 18 patients (5%) died within 30 days of the last cabazitaxel infusion, compared with 9 patients (2%) receiving mitoxantrone therapy within the same time frame. in the cabazitaxel arm, 7 patients (2%) died of complications related to neutropenia, while 5 patients (1%) died of cardiac causes., diarrhea appeared to be more prevalent in older patients (55.7% vs 44.5% in patients aged 75 or < 75, respectively ; p < 0.1) and in patients who had previously received radiotherapy (50.0% vs 41.4% in patients with and without prior exposure, respectively).39 the most prevalent toxicity, neutropenia, occurred at a frequency 6.6% higher in patients aged 65 compared with those < 65. furthermore, the incidence of neutropenia varied significantly by region, with rates of neutropenia in north america exceeding those in the europe. analyses are currently underway to determine the extent of growth factor use both in the study population at large and within these subgroups (notably, cycle 1 prophylaxis with growth factors was not allowed in the tropic protocol). until these data are available, the currently available fda label suggests the use of primary prophylaxis with g - csf in those patients who are considered high risk, as delineated in table 1. as yet, there are no head - to - head trials comparing docetaxel and cabazitaxel, making it challenging to juxtapose both the efficacy and toxicity of these agents. nonetheless, the rates of neuropathy with cabazitaxel were relatively low, only 1% of patients reporting a grade 3/4 event (14% for all grades).38 it should be noted that patients with grade 2 or higher peripheral neuropathy in association with docetaxel were excluded from tropic, confounding any comparisons with this agent. another important distinction between cabazitaxel and docetaxel is the premedication regimen proposed for each. in swog 9916 and tax 327, patients receiving 3-weekly docetaxel received 60 mg and 24 mg of oral dexamethasone divided over 3 doses, respectively.9,10 in contrast, patients receiving cabazitaxel in the tropic study received 8 mg of intravenous dexamethasone in conjunction with an antihistamine and h2-antagonist.38 in the setting of certain co - morbidities (ie, diabetes), the latter regimen may be preferable. therapy with cabazitaxel in docetaxel - refractory crpc has already been adopted as a category 1 recommendation in national comprehensive cancer network criteria.40 however, the challenge that lies ahead is multifold. given the efficacy of cabazitaxel in the heavily pretreated population in the tropic study, could cabazitaxel potentially be moved forward in the current therapeutic algorithm for prostate cancer (figure 1) ? furthermore, underway are numerous clinical studies assessing synergy of docetaxel with a range of agents. for instance, the phase iii cancer and leukemia group b (calgb) 90401 trial showed no os benefit with the addition of bevacizumab to docetaxel.41 nonetheless, several other phase iii efforts are underway, including studies pairing docetaxel with the endothelin antagonists zibotentan and atrasentan, and the antiangiogenic / immunomodulatory agent lenalidomide.4244 with its efficacy now demonstrated, the investigator may be inclined to assess cabazitaxel in the same combinations currently being investigated with docetaxel. the research community is cautioned to perform appropriate preclinical and clinical safety testing prior to embarking on larger efforts assessing such combinations. several ongoing clinical trials of cabazitaxel both alone and in combination with other cytotoxic agents are denoted in table 2. furthermore, cabazitaxel / prednisone (dosed at both 20 and 25 mg / m) will be compared to docetaxel / prednisone (at a standard dose of 75 mg / m) as first - line chemotherapy in metastatic crpc. the primary endpoint in this study is os. problematic in the trial design is the fact patients progressing on docetaxel (but not cabazitaxel) will have a known effective salvage therapy. the role of docetaxel in distinct settings of prostate cancer may similarly guide clinical implementation of cabazitaxel. for instance, calgb 90203 is a randomized, phase iii effort comparing 6 cycles of neoadjuvant docetaxel therapy preceding prostatectomy with prostatectomy alone in the setting of high - risk, localized disease.45 if the trial yields promising results, the application of cabazitaxel as neoadjuvant therapy could be explored. further, it remains to be seen whether cabazitaxel has specific activity in the context of aggressive prostatic cancer histologies, such as tumors bearing neuroendocrine features. available clinical data suggest limited efficacy of docetaxel and other standard cytotoxic agents in this setting.46 questions remain about the dosing regimen chosen in the tropic study ; ie, could toxicity have been mitigated by starting with a dose of 20 mg / m ? as previously noted, this represented the initial dose utilized in a phase ii study of cabazitaxel in breast cancer. in that study, allowance of dose escalation to 25 mg / m was contingent upon completion of the first cycle of therapy with no toxicity. the aforementioned phase iii first line trial in metastatic crpc will help to address this issue. finally, it is not known yet whether the activity of cabazitaxel in docetaxel - refractory crpc will translate to other tumor types. the previously noted phase ii study assessing the agent in taxane - refractory advanced breast cancer may stimulate further trials in this disease.36 furthermore, urothelial carcinoma, lung cancer, ovarian cancer, and countless other malignancies where taxanes have a described clinical benefit may represent new domains where cabazitaxel therapy could be examined. | docetaxel remains a cornerstone of therapy for the patient with metastatic castration - resistant prostate cancer (crpc). however, the landscape of crpc therapy is changing rapidly recently, data from the phase iii tropic study revealed a survival advantage with the novel taxane cabazitaxel / prednisone (compared with mitoxantrone / prednisone) in a cohort of 755 men with docetaxel - refractory metastatic crpc. interestingly, cabazitaxel bears substantial structural similiarity to docetaxel but appears to be mechanistically distinct. in preclinical studies, the agent has antitumor activity in a variety of docetaxel - refractory in vitro and in vivo models. subsequent to phase i testing in advanced solid tumors (where neutropenia was identified as a dose - limiting toxicity), the agent was assessed in a phase ii trial in advanced, taxane - refractory breast cancer and in the aforementioned phase iii tropic study. this review describes in detail the preclinical and clinical development of cabazitaxel. |
subcorneal pustular dermatosis (scpd) is a rare chronic - relapsing skin disorder that was first described by sneddon and wilkinson in 1956. the characteristic primary skin lesions are pustules, which can develop into flaccid blisters, with hypopyon - like accumulation of pus in their lower part and clear fluid in their upper part. typical localizations involve the trunk, intertriginous areas, and flexural sites of the extremities. the characteristic histological finding of scpd involves a superficial split below the stratum corneum, with an abundance of neutrophils in the blister but without spongiosis or acantholysis of the epidermis. the accumulation of neutrophils is nowadays recognized as an important mediator of scpd [4, 5 ], but its underlying causes remain insufficiently understood. however, a role for tnf has been implicated. elevated cytokine levels have been reported in serum and blister fluids, which might drive neutrophil activation and accumulation [7, 8 ]. in addition, several clinical reports have described effective off - label therapy using tnf blocking agents in cases that responded insufficiently to first - line therapy, such as with dapsone [7, 9 ]. a 29-year - old male it specialist presented with a 3-week history of severe, progressive pustular skin eruptions. the lesions had begun as a maculopapular rash on the patient 's axillae 1 day after he had visited a thermal bath, and had spread over the course of the next days. previous therapy with topical and high - dose oral corticosteroids, as well as co - amoxicillin, had been ineffective. upon initial evaluation at our department, the patient exhibited plaques and large flaccid blisters that covered most of the patient 's trunk (fig. some of the blisters showed hypopyon - like accumulation of pus in their lower part and clear fluid in their upper part. the patient suffered from pruritus ; however, fever and other symptoms of systemic disease were not present. due to the severity of the patient 's skin affection and the associated risk of septic complications, he was admitted to hospital for further evaluation and treatment. bacterial cultures obtained from intact blisters showed an abundance of -hemolytic streptococci and staphylococcus aureus species. thus, an (albeit very severe) bullous impetigo was suspected and treated with potassium permanganate containing baths and daily intravenously amoxicillin - clavulanate potassium (augmentin), in accordance with antibiotic susceptibility testing. however, bacterial cultures obtained aseptically from the bottom of newly punctured blisters were negative. at best, marginal improvement was found as a result of 2 courses of amoxicillin in total (oral, then intravenously) for 13 days (fig. direct immunofluorescence on skin specimen from primary lesions was negative, as was a serological screen for antibodies directed against skin antigens, including desmoglein 1 and 3. histopathology revealed subcorneal pustules, with only mild acantholysis and no spongiosis of the underlying epidermis (fig. additional tests were performed to screen the patient for systemic diseases that have previously been associated with scpd, namely iga or igg gammopathy and multiple myeloma, but these were inconclusive. a single dose of 350 mg infliximab was administered. consolidation was observed 1 day after treatment and regression of skin lesions occurred after a few days (fig. 1d). residual scarring and postlesional hyperpigmentation was seen at a 2-month follow - up appointment (fig. a daily maintenance therapy with dapsone was initiated, which led to a drop in hemoglobin and had to be stopped after 2 months. upon development of small, scaly lesions, a maintenance therapy with infliximab scpd is a rare skin disorder, which typically presents in women over the age of 40. some cases in children and adolescents have been reported, but several displayed atypical features. accompanying systemic symptoms or a history of other skin conditions might complicate the diagnosis. in particular, the differentiation between scpd and pustular psoriasis can be very challenging. furthermore, secondary infection with streptococci or s. aureus species is possible, which might complicate the initial distinction of scpd from bullous impetigo. it has further been debated whether scpd is an independent disease entity or rather represents a subentity of other diseases. an overlap with iga pemphigus has been described, which has been linked to the autoantigen desmocollin 1. although the course of scpd is generally considered to be benign, it can be associated with several systemic diseases. notably, iga or igg gammopathy and other lymphoproliferative disorders, especially multiple myeloma, have been observed [3, 14 ]. these findings might indicate a certain predisposition for immune dysregulation. although the subcorneal accumulation of neutrophils appears to be a hallmark of scpd, its exact pathomechanism is still not known. several chemotactic factors have been implicated in neutrophil recruitment and invasion, including the proinflammatory cytokine tnf. these findings correspond well with clinical reports of successful off - label use of tnf blocking agents in cases that were refractory to first - line therapy, mostly with dapsone [7, 9 ]. other reports describe initially good responses to infliximab, which were gradually lost with repeated infusions of infliximab or lost due to relapse. complete responses to etanercept were observed in 3 cases, 1 of which received a combination with low - dose acitretin [19, 20 ], and an additional report describes a near - to - complete response. in 1 case, a complete response to adalimumab could be observed. following the loss of efficacy of an anti - tnf agent, switching to a different agent has been successful. one case describes the stepwise loss of efficacy of infliximab, and then etanercept, but clearance under adalimumab. considering these cases reported in the literature to date and the severity of manifestation observed in this case, the drug interrupted the pustulation, demonstrating the power of targeted therapies in rare neutrophil - mediated diseases. | subcorneal pustular dermatosis (scpd, sneddon - wilkinson disease) is a rare chronic - relapsing skin disorder that typically manifests as flaccid sterile pustules without systemic symptoms. although the accumulation of neutrophils is acknowledged to be a hallmark of scpd, its exact pathomechanism is still not known. several chemotactic factors have been implicated in neutrophil recruitment and invasion, including the proinflammatory cytokine tnf-. these findings correspond well with clinical reports of successful off - label use of tnf blocking agents in cases that were refractory to first - line therapy, mostly with dapsone. we report the case of a 29-year - old male with atypical and severe manifestation of scpd that resolved after a single dose of infliximab. consolidation was observed 1 day after treatment and regression of skin lesions occurred after a few days. residual scarring and postlesional hyperpigmentation was seen at a 2-month follow - up appointment. the patient was initiated on a daily maintenance therapy with dapsone, which led to a drop in hemoglobin and had to be stopped. upon development of small, scaly lesions, a maintenance therapy with infliximab was started and the patient has had no recurrence to date. anti - tnf agents present a promising option for patients affected by severe scpd. we review the reports of similar cases in the literature to date. |
although fibrosis is considered the hallmark of cirrhosis, regeneration, necrosis and inflammation are important prognostic factors. the most relevant etiologic factors in cirrhosis are alcohol, viruses, nonalcoholic steatohepatitis and hemochromatosis. initial clinical symptoms are vague, while advanced disease is mainly associated to liver decompensation with ascites, esophageal variceal hemorrhage, jaundice and hepatic encephalopathy. the hepatocellular carcinoma (hcc) development rate is 8% at 5 years and patients with chronic hepatitis and cirrhosis are initially biopsied to establish a definitive diagnosis and stage the liver status. however, a biopsy is not necessary if the clinical, laboratory and radiological data suggest cirrhosis. furthermore, there is a small but significant risk to liver biopsy, and cirrhosis itself predisposes patients to complications due to liver biopsy. magnetic resonance (mr) imaging is mostly used in the diagnosis of tumor development because of its ability to reliably depict hcc. the currently available imaging tests (ultrasound, computed tomography and conventional mr imaging) are neither sensitive nor specific in the detection of early parenchymal changes. many signs of moderate and advanced cirrhosis can be detected as classical morphological and signal intensity changes. new functional mr imaging sequences can also depict fat and iron deposition, regenerative nodules, necroinflammatory infiltrate, fibrosis, varices, perfusion abnormalities and hepatocyte functionality [1, 2 ]. the evaluation of chronic hepatitis and cirrhosis with imaging modalities should be performed ideally on early stages of the disease. to be clinically useful, any method used to evaluate chronic hepatitis and cirrhosis must accurately identify regeneration, inflammation, necrosis, fibrosis, fat, iron and also neoplasia. fibrosis, necroinflammatory activity, fat and iron deposits are the most important parameters for antiviral treatment indication and follow - up. report and quantification of these parameters by the radiologist are fundamental in order to entail an impact on patient management. however, the different components of the parenchymal lesions may also need to be studied and quantitated. high - resolution expiratory breath hold dual - echo chemical shift spoiled gradient - echo (gre) sequencing is used for the acquisition of opposed - phase and in - phase t1-weighted (t1w) images. the dual - echo sequence can evaluate fat, although the use of t2 correction performs better for an accurate quantification [3, 4 ]. this t2 calculation can be used to measure iron accurately with a multiecho gre sequence. respiratory triggered short t1 inversion recovery turbo spin echo (stir tse) images should be optimized so that the signal intensity of the liver is close to that of the subcutaneous fat and paraspinal muscles (ti of 150160 ms at 1.5-t and 180190 at 3-t magnets), which serve as an internal tissue of reference for the necro - inflammatory activity. three - dimensional spoiled t1w gre contrast - enhanced dynamic examinations with fat suppression are mainly used to exclude tumor development and grade esophageal varices. controlling the bolus arrival interval time for the late arterial, portal and equilibrium phases maximum intensity projection (mip) vascular map images reconstructed from the arterial and portal phases show the extent of collateral vessels due to portal hypertension, as well as the arteries that perfuse abnormal regions and lesions. in order to calculate pharmacokinetic model parameters, the acquisition should have enough temporal resolution (less than 5 s for each image set, for at least 5 min) with a dual input double compartment model. a voxelwise statistical analysis is suggested. this dynamic acquisition can be acquired with a low dose of contrast media (a fifth of the regular dose) and also be used to calculate the bolus arrival time to properly initiate the late arterial phase of the high - resolution conventional dynamic sequence. the 3d high - resolution fat suppression t1w spoiled gre images obtained 30 to 60 min after the administration of hepatobiliary contrast media (hbcm), although not routinely used in the mr evaluation of cirrhosis and hcc development, may give information on hepatocyte functionality. the t2 gre images obtained after superparamagnetic iron oxide particle (spio) liver enhancement, with and without concomitant gadolinium - based contrast - enhanced dynamic images, can be used to rule out hcc in difficult cases and depict advanced fibrosis. specific contrast media with intracellular phases although large vessels can be observed with most mr images, the use of steady - state fully refocused transverse magnetization gre images (such as balanced, fiesta or true - fisp) facilitates the observation of abnormal parenchyma vessels. diffusion - weighted (dw) imaging is an mr technique that is based on intravoxel incoherent motion (ivim) and provides noninvasive quantification of water diffusion and microcapillary - blood perfusion. information provided with dw reflects tissue cellularity, integrity of cellular membranes and capillarity. in order to standardize dw acquisitions and apparent diffusion coefficient (adc) calculations, a biexponential signal modeling and respiratory - triggered precontrast acquisitions with at least 5 b - values (0, 50, chronic hepatitis does not modify the macroscopic architecture of the liver. on the other hand, advanced cirrhosis generates typical morphological changes in the liver parenchyma and surface contour because of regenerating nodules, necrosis and fibrous development, which alter the liver architecture. liver surface nodularity is usually fine and diffuse, being more prominent on the hypertrophied segments. although the finding has been claimed as characteristic, minor bulging of the liver surface can be found in normal cases, and also hepatic surface nodularity can be seen in patients with fulminant hepatic failure, usually reflecting a combination of alternating foci of confluent regenerative nodules and necrosis. regeneration and necrosis lead to liver global or segmental volumetric changes associated with regional variations in the portal venous blood supply. although any combination can be found, the caudate lobe and lateral segment of the left hepatic lobe usually develop hyperplasia, whereas the left medial segment and right lobe show atrophic changes (fig. transverse opposed - phase t1w images show caudate hypertrophy with nodular surface and prominent fat at the hepatic hilum (a). the right posterior hepatic notch sign is clearly defined in another patient (b) morphological changes of the liver. transverse opposed - phase t1w images show caudate hypertrophy with nodular surface and prominent fat at the hepatic hilum (a). the right posterior hepatic notch sign is clearly defined in another patient (b) one of the first imaging biomarkers for diagnosing cirrhosis was the evaluation of caudate lobe hyperplasia with the caudate - right lobe ratio. this index chose the bifurcation of the main portal vein as a reproducible landmark to divide these lobes. a ratio greater than 0.65 is associated with cirrhosis with an overall accuracy of 66%. the modified caudate - right lobe ratio uses the right portal vein bifurcation to set the lateral boundary, with an abnormal index being greater than 0.9. this index is more accurate (74%) for diagnosing cirrhosis and evaluating its clinical severity as significant differences were found among the three child - pugh classes (table 1) (p 50 (> 3)serum albumin (g / l)>3528352.20ascitesnonemildseverehepatic encephalopathynonegrade i ii (or suppressed with medication)grade iii iv (or refractory) the child - pugh score employs five clinical measures of liver disease. inr = international normalized ratio of prothrombine prolongation caudate hypertrophy is also responsible for the right posterior hepatic notch sign, defined as a sharp indentation on the medial posteroinferior liver surface between the caudate and right lobes (fig. another associated finding related with segmental parenchyma atrophy is the widening of the porta hepatis demonstrated as a prominent fatty space anterior to the main portal vein at the hepatic hilum. 2) bounded laterally by the right hepatic lobe and medially by the lateral segment of the left hepatic lobe is known as the expanded gallbladder fossa sign. although all these changes must be considered specific for relatively advanced cirrhosis, the enlargement of the hilar periportal space has been demonstrated in early cirrhosis. transverse in - phase t1w image shows an expanded gallbladder fossa sign morphological changes of the liver. transverse in - phase t1w image shows an expanded gallbladder fossa sign most regenerative nodules are small. macroregenerative nodules rarely exceed 2 cm in diameter, and therefore larger nodules should be carefully evaluated to exclude dysplasia and carcinogenesis. slightly hypovascularized large confluent areas of regenerative nodules may be seen mainly close to the interlobar and intersegmental territories. regenerative nodules are homogeneous, non - encapsulated, hypointense, rounded foci on t2w images, whereas they are usually isointense on t1w images. they are surrounded by fine reticular septa, slightly hyperintense on fat - suppressed t2w and stir images (fig. hypointense nodules on the in - phase second echo gre and t2w tse images are considered siderotic. some non - dysplastic and non - tumoral nodules may be hyperintense on the t1w gre images (fig. 4), but they do not contain fat (do not lose signal intensity on opposed - phase imaging) and are not arterialized (do not significantly enhance during the hepatic arterial dynamic phase). this high signal intensity in t1w images transverse stir image shows a reticular pattern of the fibrotic bands surrounding hypointense regenerative nodulesfig. transverse opposed (a) and in - phase (b) t1w images show hyperintense nodules without decreased signal intensity on opposed - phase imaging and hypointensity on stir image (c). note several gamna - gandy siderotic splenic bodies due to advanced portal hypertension fibrosis and regenerative nodules. transverse stir image shows a reticular pattern of the fibrotic bands surrounding hypointense regenerative nodules non - tumoral nodules. transverse opposed (a) and in - phase (b) t1w images show hyperintense nodules without decreased signal intensity on opposed - phase imaging and hypointensity on stir image (c). note several gamna - gandy siderotic splenic bodies due to advanced portal hypertension cirrhosis is not the only disease associated with morphological changes of the liver. regeneration and atrophy can also be found in disorders such as budd - chiari syndrome, postchemotherapy, nodular regenerative hyperplasia and portal cavernomatosis. standard t1w and t2w images are not sensitive to the inflammatory liver changes. on the contrary, respiratory triggered stir tse images depict an increase in liver brightness when there is an increase in the water content due to intracellular edema, inflammation or cell necrosis (fig. this increased signal can be qualitatively assessed if the inversion time t1 is properly adjusted so that the normal liver signal intensity is quite similar to the signal of the paraspinal muscles. in chronic hepatitis and cirrhosis, this increase in the liver signal intensity can be considered a surrogate marker of portal inflammation and periportal and lobulillar necrosis. the liver signal in stir images is not influenced by the presence of either fibrosis or steatosis. however, the presence of iron decreases the liver signal intensity and masks the increased signal of the necroinflammatory infiltrates. if iron is present, the necroinflammatory activity can not be properly estimated with tse - stir images. transverse stir image shows an increase in liver brightness due to intracellular edema, inflammation and necrosis inflammation and necrosis. transverse stir image shows an increase in liver brightness due to intracellular edema, inflammation and necrosis reactive lymph nodes at the hepatic hilus and gastrohepatic ligament are also well - known findings. superior diaphragmatic adenopathies are usually hyperplastic, even when an hcc is present (fig. a prominent cisterna chyli, with a diameter larger than 2 mm, is observed in uncompensated cirrhosis with a high positive predictive value of 96%. this phenomenon is due to the increased lymph production in these patients caused by disturbance in the drainage of vascular flow from the sinusoid to the central or terminal hepatic veins associated with lobular distortion - impaired lymphatic circulation in cirrhosis. note the increased signal intensity of the liver due to necroinflammatory activity reactive lymph node. note the increased signal intensity of the liver due to necroinflammatory activity late arterial phase dynamic contrast - enhanced mr images may demonstrate a heterogeneous pattern of patchy parenchymal enhancement with large geographical areas showing a slight hypovascularization. this frequent finding (50%) of perfusion heterogeneity relates to the presence of inflammatory macrophages, variable hepatocyte necrosis and increased steatosis. these areas may progress to areas of focal confluent fibrosis and collapse. in the cellular phase after hbcm, a decreased and heterogeneous enhancement relates to the presence of hepatocyte necrosis intermixed with fibrous bands. areas of regeneration demonstrate an increased enhancement related to the increase in the number of hepatocytes per voxel together with an impaired bile excretion. in cirrhosis, the severity of the hepatic injury relates to the down - regulation of the hbcm transporter expression, leading to a threshold response appearing in advanced stages (much lower enhancement), but not before (relative maintained enhancement). with this limitation in mind, the gd - eob - dtpa hepatic extraction fraction can be used as a direct, noninvasive technique for the quantitative evaluation of liver function. this extraction ratio is calculated from deconvolution analysis of aortic and hepatic parenchymal time - intensity curves obtained by dynamic mri and could be a promising alternative for the determination of noninvasive hepatic function in patients with liver disease. on the t2w gre images acquired with a long te (7 ms), the less hypointense areas are statistically related to reduction in their functional status. heterogeneous r2 shortening is also a reliable predictor of advanced fibrosis (fig. 7), with a positive predictive value of 93%. iron oxide particles will clearly depict the fibrotic bands surrounding the hypointense negatively enhanced regenerative nodules. liver heterogeneous decreased enhancement after spio is related to the presence of fibrous bands and subcapsular collapse areas fibrosis. liver heterogeneous decreased enhancement after spio is related to the presence of fibrous bands and subcapsular collapse areas in an early study in patients with liver biopsy, adc was not correlated with inflammation grades. however, more recent publications [22, 23 ] have shown a significant relationship between adc and inflammation scores, with adc being a predictor of inflammation grade 1 or greater. unfortunately, adc values are influenced by the choice of b - value ; they are multifactorial (steatosis, fibrosis, perfusion) and vary between different vendors, limiting the role of standard adc calculations. routine mr imaging can not observe early fibrosis, but these images are sensitive for detecting moderate and advance fibrosis by demonstrating the reticular pattern of the fibrotic bands surrounding regenerative nodules. this fine reticulation is hyperintense on t2w fat - suppressed images and on the equilibrium and delayed images after contrast administration (fig. the fine sieve appearance, occasionally associated with poorly defined subcapsular retractile stellate areas, are clear indicators of the presence of advanced fibrosis. the observation of this pattern is facilitated by decreasing the signal intensity of the nodules after spio administration while increasing the signal from the septa after gadolinium enhancement. although this method separates advanced fibrosis or cirrhosis (f3f4) from intermediate, early or no fibrosis (f2-f0), it does not allow to differentiate no fibrosis (stage f0) from minimal (stage f1) and intermediate (stage f2) fibrosis. transverse fat suppression t1w spoiled gre image after administration of contrast media shows reticular pattern of the fibrotic bands surrounding regenerative nodules fibrosis. transverse fat suppression t1w spoiled gre image after administration of contrast media shows reticular pattern of the fibrotic bands surrounding regenerative nodules areas of focal confluent fibrosis are usually found in long - standing cirrhosis, especially associated with alcohol abuse. they are frequently multiple, with the most classical locations being the interlobar and intersegmental fissures, as these areas have terminal territory perfusion. the collapsed area has a geometrical (often triangular or quadrilateral) capsular - based wedge shape pointing to the hepatic hilum, associating volume loss and capsular retraction with focal flattening or even concavity of the adjacent liver surface. the abnormality is moderately hyperintense in t2w images, isointense or slightly hypointense in t1w, with a progressive and delayed enhancement after contrast media administration (fig. trapped vessels and dilated biliary ducts can be seen within the abnormality. on the cellular phase images after hbcm and spio administration, the enhancement is usually decreased due to cell necrosis. the abnormality is moderately hyperintense in stir (a) and shows progressive and delayed enhancement after contrast media administration (b, arterial ; c, delayed phases) focal confluent fibrosis. the abnormality is moderately hyperintense in stir (a) and shows progressive and delayed enhancement after contrast media administration (b, arterial ; c, delayed phases) microscopic water diffusion is decreased in cirrhosis. the reduced liver diffusion can be qualitatively observed on the dw images (fig. some studies have analyzed the role of dw imaging, mainly throughout mean adc comparisons, in the evaluation of chronic diffuse liver diseases. although adc measurements vary in b - values and motion correction techniques, the adc values of cirrhotic livers are significantly lower. the shortest adc values in cirrhosis are mainly related to a decrease in the capillarity perfusion component and not to a true microscopic diffusion restriction associated with fibrosis and inflammation. transverse signal video inversion of the diffusion - weighted image shows decreased water diffusion in a patient with liver cirrhosis fibrosis. transverse signal video inversion of the diffusion - weighted image shows decreased water diffusion in a patient with liver cirrhosis taouli. evaluated dw technique as a predictor of the presence of moderate and advanced liver fibrosis. the liver adc value (breath hold, six b - values of 0, 50, 300, 500, 700 and 1,000 s / mm) in patients with chronic hepatitis versus healthy volunteers was a significant predictor of fibrosis stage f2 or greater and also stage f3 or greater. similar results were obtained by lewin., where dw (navigator - triggered, four b - values : 0, 200, 400, and 800 s / mm) was compared to other non - invasive methods to conclude that patients with moderate - to - severe fibrosis (f2-f3-f4) had hepatic adc values lower than those without or with mild fibrosis (f0-f1) and healthy volunteers. in discriminating patients staged f3-f4, the sensitivity, specificity, positive predictive value and negative predictive value were 87%, 87%, 72% and 94%, respectively, with an adc cutoff level of 1.21 10 - 3 mm / s. analyzed the influence of fibrosis in liver diffusion properties by ivim technique (respiratory triggered, ten b - values : 0, 10, 20, 30, 50, 80, 100, 200, 400, 800 s / mm). they observed a restricted diffusion in patients with cirrhosis mainly related to variations in the perfusion component, reflecting decreased perfusion, as well as alterations in pure molecular water diffusion in cirrhotic livers. confirming this observation, girometti. (breath hold acquisition ; six b - values : 0, 150, 250, 400, 600, 800 s / mm) also concluded that the perfusion component presents a higher accuracy at lower b - values for the assessment of liver fibrosis. the studies in rats with hepatic fibrosis, both in - vivo and immediately after death, also pointed in this direction. although ivim seems to be a promising technique in the diagnosis and staging of fibrosis, some bias must be clearly controlled to standardize this biomarker (fig. 11). the concomitant effect of mr machines, mr sequence parameters, fat, iron, inflammation and necrosis on the adc values should be evaluated. ivim parametric maps of pure diffusion (a, d), perfusion (b, d) and vascular components (c, f) of the diffusion - related signal attenuation fibrosis. ivim parametric maps of pure diffusion (a, d), perfusion (b, d) and vascular components (c, f) of the diffusion - related signal attenuation cirrhotic liver vascular perfusion changes are related to the disease activity and staging. although the arterial blood supply is increased due to the decreased portal flow, the buffer is not sufficient to maintain adequate liver perfusion because of the high level of extrahepatic portosystemic shunting. the overall reduction of the total liver perfusion can be quantified as a prolongation of the mean transit time and a decrease in mean peak liver enhancement. another phenomenon is observed as fibrosis development leads to progressive arterializations of the hepatic sinusoidal bed, with shunting and hyperdynamic circulation, and augmentation of the extracellular interstitial space with collagen deposition. these changes produce an overall increase of the liver enhancement during the equilibrium phase images of the dynamic series. some parametric pixel - by - pixel mapping, such as the mean and maximum enhancement ratios, show significantly higher values in cirrhosis than in normal livers. an increase in the liver enhancement can also be also quantified with the area under the curve and is statistically related to the degree of chronic hepatic insufficiency. a dual - input single compartment model demonstrates an increased distribution volume (related to the increased interstitial volume) and mean transit time (related to the collagen deposition in the extracellular spaces of disse). these perfusion modifications can be separated and objectively evaluated through the pharmacokinetic compartment model analyses. although the experience is limited, cirrhotic livers have an increased vascular permeability (k) and extracellular space (e) with a heterogeneous distribution. these parameters correlate with the degree of liver fibrosis and may be used as a hemodynamic biomarker in injured fibrotic livers. the shear mechanical compressional waves are transmitted through the liver, detected with phase - contrast type sequences, and analyzed as wave propagation and tissue deformation. the calculated elasticity maps show the shear elasticity modulus (kpa) at each point. quantitative stiffness parametric maps, also known as elastograms, become more heterogeneous with increasing fibrosis. while differences in stiffness between patients with early stages of fibrosis (f0 vs. f1 vs. f2) are small, with overlap between groups, the differences between higher stages (f2 vs. f3 vs. f4) are larger with less overlap. glutamine and glutamate complex (glx), phosphomonoesters (pme), glycogen and glucose complex (glyu), and lipids are clearly observed. chronic hepatitis and cirrhosis showed an increase in glx, pme and glyu levels relative to the lipid content. this increase is related to the severity of fibrosis, although data overlap is present between groups. early fibrosis associates deposition of collagen in the disse space with alteration of the sinusoidal architecture, resulting in a decreased portal venous flow, which is counteracted by an increase in hepatic arterial flow (buffer response). when the venous inflow blockade occurs and vascular resistance increases, the portal flow may be adequate for centrally located parenchyma areas, but not for the subcapsular regions. the arterial response may generate enhancement of these peripheral zones with relative hypointensity in the central perihilar areas. on the contrary, as previously mentioned, there may also be a heterogeneous pattern of slightly hypervascular behavior within geographical areas due to the presence of necroinflammatory infiltrates and steatosis. other common causes of perfusion abnormalities to be taken into consideration in cirrhotic livers are related to spontaneous arterio - portal shunts (fig. 12), shunts associated with vascular compression (hcc, inflammatory changes, biliary tree dilatation) and portal occlusion. fig. transverse fat suppression t1w spoiled gre image after administration of contrast media shows small and non - encapsulated areas with enhancement in the arterial phase (a), but without wash - out on the portal phase image (b) vascular changes : arterioportal shunts. transverse fat suppression t1w spoiled gre image after administration of contrast media shows small and non - encapsulated areas with enhancement in the arterial phase (a), but without wash - out on the portal phase image (b) portal hypertension frequently complicates liver cirrhosis. dilatation of the portal vein and its tributaries, and extrahepatic collateral circulation, splenomegaly and ascites are well - known findings. esophageal and gastric varices, paraumbilical, spleno - renal, retroperitoneal and puborectal shunts are well visualized with contrast - enhanced mr images and mip projections (fig. mr images after gd administration depict esophageal varices in most (81%) cases with a statistically significant relationship with the endoscopic grading of the severity. gallbladder wall thickening is associated with venous and lymphatic congestion in the presence of portal hypertension and drainage difficulty. mip vascular image reconstructed from the portal phase shows collaterals vessels and splenomegaly portal hypertension. mip vascular image reconstructed from the portal phase shows collaterals vessels and splenomegaly a relatively small main portal vein in patients with cirrhosis may indicate hepatofugal flow. early arterial phase enhancement of the portal vein has been also reported as a sign of hepatofugal flow, although this finding is misleading as the late arterial phase mixes with the early portal phase. in most cases, perfusion abnormalities are easy to interpret on the non - specific contrast media enhanced images as they have clearly defined and straight - line margins, corresponding to a vascular territory, and normal vessels coursing through the abnormality. when the portal flow is decreased or absent and the arterial flow volume is increased, they are often seen as hyperarterialization with fading or disappearance in the portal and equilibrium phases. cyst abnormalities occur in the peribiliary tissue adjacent to the large intrahepatic and extrahepatic ducts in association with severe liver disease, and are usually asymptomatic. these cysts have variable size and morphology and represent cystic dilatation of the extramural glands in the periductal connective tissue. peribiliary cysts show imaging findings of simple cysts, with low signal intensity on t1w and high signal on heavily t2w mr images (fig. mrcp image shows peribiliary cysts with variable size and morphology primary sclerosing cholangitis (psc) shows irregular intra- and/or extrahepatic bile duct dilatation and stenosis along with periportal t2w hyperintensity in the major portal tracts. cirrhosis from advanced psc develops marked atrophy of the posterior aspect of the right lobe and the lateral segment of the left lobe with hypertrophy of the caudate lobe. in primary biliary cirrhosis, on t2w images, a periportal hyperintensity, especially at earlier stages of disease, may be seen. cirrhotic livers may have an increase in fat content. also, steatosis and nonalcoholic steatohepatitis (nash) lead to fibrosis and cirrhosis. early detection and follow - up of steatosis would facilitate a better diagnosis and intervention before liver damage is irreversible. a large number of patients with nash and chronic hepatitis may also present increased iron content in the liver with all these factors affecting the imaging signal characteristics. for the quantitative evaluation, the fat content computed from in - phase and opposed - phase images may be erroneous unless a correction is made for the influence of t2 decay. the t2w tse with and without fat suppression method is easier and accurate enough in a clinical setting. however, single voxel proton mr spectroscopy, although limited by the volume sampling, will be needed in clinical trials and longitudinal studies to further increase accuracy. fig. transverse opposed - phase t1w image (a) shows decreased signal intensity of liver, while in the axial in - phase t1w image (b) the liver shows increased signal intensity liver steatosis. transverse opposed - phase t1w image (a) shows decreased signal intensity of liver, while in the axial in - phase t1w image (b) the liver shows increased signal intensity cirrhosis is frequently associated with an increased deposition of iron, mainly in alcoholic cirrhosis. conventional chemical shift dual echo gre t1w and tse - stir images will demonstrate the liver signal intensity drop, allowing the qualitative diagnosis of significant iron deposits (fig. transverse opposed - phase (a) and in - phase (b) t1w images show a decrease in the signal intensity of the liver in the in - phase image because of increased deposition of iron liver iron overload. transverse opposed - phase (a) and in - phase (b) t1w images show a decrease in the signal intensity of the liver in the in - phase image because of increased deposition of iron both the liver - to - muscle signal ratio and the liver r2 relaxation rate significantly correlate with the amount of iron content [3, 4 ]. although sequences and postprocessing of images for t2 and t2 relaxation rate calculations are more difficult to implement in a clinical environment, this approach must be preferred to the simple signal - to - ratio determination in clinical trials. the calibration between r2 and iron concentration is dependent on field strength, being different for 1.5- and 3-t magnets. the coupling of multiecho gre techniques with chemical shift imaging may simultaneously evaluate both iron and fat liver content. this is relevant as both entities may coexist in a significant number of cases with chronic liver disease. overt hcc is characterized by a mass lesion showing hyperarterialization with washout, becoming hypointense to the liver on the delayed phases after contrast administration (fig. dysplasia, atypia and early neoplasic degeneration are much more difficult to demonstrate. in this setting, any hyperintensity within a nodule in the t2w images and hyperintense nodules in the t1w images may be considered suspicious, and their perfusion characteristics should be carefully evaluated. dw images may help in depicting a malignant transformation if clear diffusion restriction is observed. transverse t1w images (a, opposed phase ; b, in - phase) show a slightly hypointense encapsulated lesion located in the right posteroinferior segment. the lesion is hypervascular (c) with washout (d) with a delayed enhancing outer rim capsule hepatocellular carcinoma. transverse t1w images (a, opposed phase ; b, in - phase) show a slightly hypointense encapsulated lesion located in the right posteroinferior segment. the lesion is hypervascular (c) with washout (d) with a delayed enhancing outer rim capsule some special situations in cirrhotic livers need further comment. nodular appearance of arterio - portal shunts is typically seen in cirrhosis as a small and non - encapsulated area with ill - defined margins. they are not seen on the plain non - contrast - enhanced images, delayed equilibrium and cellular phases after hbcm and reticuloendothelial system contrast media (recm), allowing the differentiation with small hcc. to differentiate a nodular arterio - portal shunt from a small hcc without washout on the equilibrium phase images, specific contrast media (either hbcm or spio) can be useful if no significantly different uptake is seen, a finding typical of arterio - portal shunt. this is also the situation with peribiliary transient hepatic arterialization caused by long - standing biliary obstruction. the typical histological features of chronic hepatitis and cirrhosis influence the liver s appearance and must be assessed separately by imaging biomarkers in order to be useful clinically. with advancements in mr technological quantification, several biomarkers have been developed to grade the liver status in cirrhosis. nowadays, research is focused on developing ways to improve detection of early and intermediate stages of fibrosis as well as hepatocyte dysfunction. the implementation of these biomarkers in a clinical environment is still waiting on an appropriate evaluation of their diagnostic accuracy. | objectivethe typical histological features of chronic hepatitis and cirrhosis are variable degrees of hepatocellular necrosis and inflammation (activity or grade of disease), fibrosis (stage of disease), and associated fat and iron deposition. these features influence the liver s appearance and must be assessed separately by imaging biomarkers in order to be clinically useful. hepatic morphologic alterations and features of portal hypertension identify most cases of established cirrhosis. nowadays, research is focused on developing ways to improve detection of early and intermediate stages of fibrosis as well as hepatocyte dysfunction. even more, most imaging - related measurements are subject to complex interactions and are influenced by different pathologic processes, such as fatty infiltration, edema, necrosis and iron accumulation.methods and resultsmr experience throughout the last 15 years at the dr peset university hospital is reviewed.conclusionnowadays, several biomarkers have been developed to grade the liver status in cirrhosis. this review will focus on these topics. |
patriarchal gender norms and unequal power relations between men and women may function as barriers to contraceptive use by supporting pronatal attitudes and control over women by men, limiting women 's decision - making power and inhibiting their access to resources, information and services. gender norms and unequal power relations play an important role in reproductive decision - making since men 's and women 's approval and demand for use of contraception may diverge, with women 's non - use of contraception, or sometimes their covert use, as potential consequences (blanc, 2001). numerous studies have found that women 's empowerment or autonomy is associated with lower fertility and/or greater contraceptive use (e.g. schuler & hashemi, 1994 ; gage, 1995 ; morgan & niraula, 1995 ; govindasamy & malhotra, 1996 ; schuler., 1997 ; do & kurimoto, 2012). in other research, perceived spousal disapproval was found to be enough to increase unmet need for family planning reported by wives (wolff., 2000), and lack of direct couple communication about reproductive intentions has been linked to unmet need or inaccurate reports about partners ' approval or disapproval of family planning (wolff., 2000 ; derose., 2004). while there is evidence that programmes that use a gender - transformative approach (that is, programmes that go beyond accommodation and seek to change inequitable gender norms) can lead to improved reproductive health outcomes (who, 2007 ; rottach., 2009 ; greene & levack, 2010), most of the programmes that have documented success in transforming gender norms have focused on hiv / aids or gender - based violence, including tuelimishane (tanzania) (maganja., 2007 ; mbwambo & maman, 2007), stepping stones (south africa) (jewkes., 2006, 2007, 2008) and programme h (brazil and india) (pulerwitz., 2006 ; verma., 2006). other programmes have worked with youth using multi - sectoral life skills approaches, including ishraq (egypt) (save the children, 2004 ; brady., 2007) and first - time parents (india) (population council, 2006 ; santhya & haberland, 2007 ; santhya., the links between gender attitudes and reproductive health outcomes may seem apparent to many, but the case for incorporating gender - transformative interventions into health and population programmes could be further strengthened by increasing the evidence base regarding the links between gender attitudes and contraceptive use. save the children 's male motivator project in malawi, which did directly measure gender attitudes, resulted in significant increases in contraceptive use, but increases in gender - equitable attitudes among husbands in the intervention group were not significantly greater than increases in the control group (shattuck., 2011). additionally, as the results were based on husbands ' reports of wives ' contraceptive use, there are questions about the reliability of the findings (becker & costenbader, 2001). however, later qualitative work with wives of men who participated in the intervention did reveal perceived improvements in spousal communication and an increase in shared decision - making, which women said directly contributed to their family planning use (hartmann., 2012). additional programmes that have successfully integrated a gender perspective into interventions to prevent unintentional pregnancies include together for a happy family (jordan) (jhuccp, 2003), prachar (india) (wilder., 2005 ; daniel., 2008), reward (nepal) (crehpa, 2002 ; cedpa, 2005), women 's empowerment model to train midwives and doctors (afghanistan) (family health alliance, 2007 ; salke, 2007) and procosi (bolivia) (palenque., 2004, 2007). however, many of these interventions either did not directly measure the impact on gender or showed only modest changes in gender attitudes (rottach., 2009). although there is an evolving field of research examining health implications of gender norms and attitudes, less attention has been made to the effects of gender attitudes and gender - related behaviours on contraceptive use specifically (rottach. the following analysis tests the hypothesis that gender attitude scales (which measure the degree of equity in gender attitudes) are associated with contraceptive use. the objectives were to : (1) determine which of four gender attitude scales, if any, predict contraceptive use ; and (2) determine whose gender attitudes, if anyone 's, predict contraceptive use husband 's and/or wife 's. an implicit assumption is that gender - equitable behaviour is reflected in stated attitudes and that a correlation between equitable attitudes and contraceptive use constitutes evidence (though not conclusive) that improvements in gender equity may lead to increases in contraceptive use. the data are from an intervention study implemented by the communication for change (c - change) project that aimed to test the effectiveness of radio programming addressing gender norms in support of a community - based contraceptive distribution programme implemented by the tanzania marketing and communication company (t - marc) and the private nurses and midwives association of tanzania (prinmat), a network of privately run clinics. the larger quasi - experiment tests the hypotheses that : (1) intensive radio campaign with gender - integrated family planning messaging affects gender equity attitudes ; (2) gender - integrated messages have greater impact on community - based contraceptive distribution programme method uptake than non - gendered messages ; and (3) gender equity is related to family planning use by couples. the overall findings of the odds ratio (or) study and details of the methodology will be published elsewhere. this analysis uses data from the baseline household survey on contraceptive use, conducted in 2009 in dodoma and mwanza regions in tanzania, which was carried out as part of the intervention study. the survey includes questions on attitudes about gender, as well as family planning knowledge, attitudes and behaviour. this paper focuses on the total sample of 400 respondents (200 male female couples, either married or co - habiting ; husbands and wives were interviewed separately), ranging in age from 15 to 49, including both users and non - users of family planning. institutional review board approval was obtained both in the united states and in tanzania. for this paper, the main outcome of interest is current contraceptive use as reported by wives. in using wives ' reports, the guidance of becker & costenbader (2001) is followed, which was adopted by the dhs in 2000. additionally, in tanzania, male method use was only 2.3% in 2010 (national bureau of statistics (nbs) [tanzania ] & icf macro, 2011), and there is some evidence of women using contraceptives without telling their husbands (becker & costenbader, 2001 ; schuler., 2011). the question asked in the survey was are you currently doing something or using any method to delay or avoid getting pregnant? the responses were coded into a dichotomous variable, whereby 0 indicates not using a method, and 1 indicates use of any contraceptive method, including both modern and traditional methods. as an additional point of interest, it was noted that the kappa statistic for women 's and men 's reported contraceptive use was 0.56, indicating a moderate level of agreement on this indicator. three scales were adapted from questions included in the standard dhs questionnaire, which are intended to measure the extent of women 's acceptance of norms that justify men 's control over women (upadhyay & karasek, 2010) and the fourth is the gender equitable men (gem) scale, which is a standardized measure of attitudes toward gender norms in intimate relationships (see pulerwitz & barker, 2008, for more details). the gem scale is a 24-item scale measuring equitability of attitudes in intimate relationships across four domains, including violence, sexual relationships, reproductive health and disease prevention, and domestic chores and daily life. respondents were asked about their agreement with a range of statements in each of these domains. for example, these include statements such as : there are times a woman deserves to be beaten, men need sex more than women do, a real man produces a male child, and a woman 's role is taking care of her home and family. responses were classified into one of the following three categories : agree, partially agree and disagree. for each gem scale item, a variable was constructed to capture the degree of equity in the attitude : the variable was coded as 1 to indicate the most equitable attitude, and 0 to indicate all other response options (i.e. agreement or partial agreement with the statement). a composite measure was constructed using a summated score for the gem scale ranging from 0 to 24, with 0 indicating the least gender - equitable attitudes and 24 indicating the highest level of gender - equitable attitudes. in addition, a categorical variable for the gem scale was created using a trichotomy of low equity (score of 0 to 7), medium equity (score of 8 to 15) and high equity (score of 16 to 24). this scale was derived from dhs questions on women 's role in household decision - making that are designed to measure attitudes towards women 's degree of control over their environment in five areas. the question posed was in a couple, who do you think should have the greater say in each of the following decisions ? making large household purchases ; making small daily household purchases ; deciding when to visit family, friends or relatives ; deciding what to do with money the woman earns from her work ; and deciding how many children to have and when to have them? responses were classified into one of the following categories : husband, wife, equal, it depends and do n't know. for each household decision, a variable was constructed to capture women 's decision - making, alone or jointly with her husband ; the variable was coded as 1 to indicate those who believe wives should clearly have a say (alone or jointly) in the household decision, and again, a summated score was used as a composite measure for the household decision - making scale ; scores ranged from 0 to 5, with 0 indicating the least gender - equitable attitudes and 5 indicating the highest level of gender - equitable attitudes related to household decision - making. a categorical variable for the household decision - making scale 1 to indicate if the wife has a say (either alone or jointly) in all five decisions, and 0 if the wife has a say (either alone or jointly) in fewer than five decisions or no say in any decisions. it measures acceptance of four hypothetical scenarios under which a wife may be considered justified in refusing to have sex with her husband. the question from the survey is husbands and wives / partners do n't always agree on everything. please tell me if you think a wife / partner is justified in refusing to have sex with her husband / partner when : she suspects her husband has a sexually transmitted disease ; she suspects her husband has sex with women other than his wife ; she has recently given birth within the last 6 weeks or has not fully recovered ; she is tired and not in the mood. responses were classified into yes (coded as 1) and no (coded as similar to the composite measure for household decision - making as described above, a summated score ranging from 0 to 4 was constructed, with 0 indicating the least and 4 the most equitable attitudes. again, a categorical variable was created for this scale such that the variable was coded as 1 if the respondent says the wife has the right to refuse sex for all four reasons given, and 0 if the respondent says the wife does not have the right to refuse sex for one or more of the reasons given. the third scale derived from the dhs standard questionnaire measures acceptance of five hypothetical scenarios under which a husband is justified in hitting or beating his wife. the question asked is sometimes, a husband is annoyed or angered by what his wife / partner does. in your opinion, is a husband / partner justified in hitting or beating his wife / partner in the following situations : if she goes out without telling him ; if she neglects the children ; if she argues with him ; if she refuses to have sex with him ; if she burns the food? responses were classified into one of the following categories : yes, no, it depends and do n't know. for each item, 1 for a no response, indicating that a husband / partner was not justified in hitting or beating his wife / partner in that situation, and the summated score ranged from 0 to 5, with 0 indicating the least gender - equitable attitudes and 5 indicating the most gender - equitable attitudes. a categorical variable for the attitudes toward wife beating scale was used as follows : one or more of the five reasons for wife beating are justified (coded as 0), and none of the reasons for wife beating was justified (coded as 1). in addition, the analyses included the following socio - demographic variables that served as controls : current age, education, number of living children and region of residence. the first stage of the analysis focused on describing the characteristics of the matched couple sample. means and prevalence rates on gender equity indicators, other related factors and socio - demographic characteristics were estimated. the levels of response to each item in the gender scales were examined separately for wives and husbands (results not shown), as well as the overall means on each scale. statistical tests (t - tests) were carried out to compare mean scores on each gender scale for wives and husbands to identify any significant differences. the next part of the analysis investigated associations between individual attributes, attitudes and contraceptive use using chi - squared tests to compare findings across husbands and wives. in the final stage of analysis, multivariate logistic regression techniques were employed to examine the associations between each gender scale and contraceptive use. models were run separately for male (husbands) and female (wives) respondents. table 1 shows the percentage distribution of selected socio - demographic and other background variables for husbands and wives in the baseline sample. the variable distributions indicate husbands had more education than their wives : 84% and 73.5%, respectively, who have completed primary school or higher. husbands in this sample also tended to be older than wives with a mean age of 33 years for husbands and 27 years for wives. similarly, husbands had higher levels of media exposure compared with wives : 58% of husbands and 41% of wives reported reading the newspaper, and 98% and 94.5%, respectively, listened to the radio. women reported relatively high current use of any method of family planning at 45.5%. table 1.characteristics of sample, dodoma and mwanza regions, tanzania, 2009variablewives(n=200)husbands(n=200)age1529 years66.0%31.5%3049 years34.0%68.5%mean27.4 years33.2 yearseducationno education12.5%3.0%some primary14.0%13.0%complete primary63.5%67.0%post - primary10.0%17.0%no. living childrennone7.5%6.5%121.5%21.5%226.5%24.5%3 or more44.5%47.5%religionchristian74.5%69.5%muslim25.0%29.5%traditional0.5%1.0%exposure to mediareads newspaper41.0%58.0%listens to radio94.5%98.0%current contraceptive useno54.5%yes45.5% characteristics of sample, dodoma and mwanza regions, tanzania, 2009 tables 2 and 3 describe the gender attitudes of respondents in order to assess how gender equitable husbands and wives were relative to each other. as shown in table 2, average scores on all four gender attitude scales were compared with t - tests. although scores overall tended to be at the lower end, suggesting support for many inequitable gender norms, husbands had higher average scores than wives on all four scales. husbands ' scores were significantly higher on the household decision - making scale (3.2 compared with 2.9, p<0.05) and attitudes toward wife beating scale (3.4 compared with 2.0, p<0.001). table 2.average gender scale scores for wives and husbands, dodoma and mwanza regions, tanzania, 2009gender attitude scalewiveshusbandsgender equitable men (gem) scale (0 to 24)9.09.4household decision - making scale (0 to 5)2.93.2attitudes toward wife refusing sex scale (0 to 4)3.53.6attitudes toward wife beating scale (0 to 5)2.03.4t - test : p<0.05 ; p<0.01 ; p<0.001. table 3.comparison of husbands ' and wives ' gender attitudes, dodoma and mwanza regions, tanzania, 2009variablewives(n=200)%husbands(n=200)%gem scale equity indexlow equity36.538.0moderate equity58.045.5high equity5.516.5household decision - making scalehas say in fewer than 5 decisions or no say in any decisions92.084.0has say (alone or jointly) in all 5 decisions8.016.0attitudes toward wife refusing sex scalewife does not have the right to refuse sex for one or more reasons25.527.5wife has the right to refuse sex for all 4 reasons given74.572.5attitudes toward wife beating scaleone or more of the 5 reasons for wife beating are justified94.569.5none of the reasons for wife beating is justified5.530.5pearson 's test : p<0.05 ; p<0.01 ; p<0.001. average gender scale scores for wives and husbands, dodoma and mwanza regions, tanzania, 2009 t - test : p<0.05 ; p<0.01 ; p<0.001. comparison of husbands ' and wives ' gender attitudes, dodoma and mwanza regions, tanzania, 2009 pearson 's test : p<0.05 ; p<0.01 ; p<0.001. as shown in table 3, the four gender attitude scales were treated as categorical variables to indicate different levels of gender equity. again, wives had less equitable gender attitudes compared with husbands on all gender scales. for example, for the gem scale, husbands were significantly more likely than wives to fall into the high - equity category (16.5% compared with 5.5%, p<0.01). similarly, among the three gender attitude scales derived from the dhs, there were significant differences between husbands and wives for the household decision - making scale, with 16% of husbands compared with 8% of wives reporting that a woman should have a say (alone or jointly) in all five household decisions (p<0.05), and the attitudes toward wife beating scale, with 30.5% of husbands compared with 5.5% of wives reporting that none of the reasons given for wife beating was justified (p<0.001). separate logistic regression models were fitted for each of the four gender attitude scales, and these analyses were run separately for husbands and wives. the models control for age, education, the number of living children and region of residence. tables 4 to 7 show the findings from the multivariable logistic regression analyses, including adjusted odds ratios and 95% confidence intervals. table 4.adjusted odds ratios (or) and 95% confidence intervals (95% ci) of reporting contraceptive use by the gem scale and socio - demographic factors, dodoma and mwanza regions, tanzania, 2009adjusted or (95% ci)variablehusbandswivesage1529 years (ref.)3049 years1.24 (0.60, 2.59)1.12 (0.53, 2.39)educationno education or some primary (ref.)complete primary or higher1.65 (0.65, 4.23)1.65 (0.78, 3.51)no. living children01 (ref.)22.76 (1.17, 6.53)4.37 (1.78, 10.76)3 or more2.94 (1.27, 6.80)5.74 (2.20, 14.97)regionmwanza (ref.)dodoma4.20 (2.10, 8.42)7.06 (3.24, 15.38)gem scale (continuous)1.01 (0.96, 1.07)1.11 (1.01, 1.21)p<0.05 ; p<0.01 ; p<0.001. table 5.adjusted odds ratios (or) and 95% confidence intervals (95% ci) of reporting contraceptive use by the household decision - making scale and socio - demographic factors, dodoma and mwanza regions, tanzania, 2009adjusted or (95% ci)variablehusbandswivesage1529 years (ref.)3049 years1.25 (0.60, 2.60)1.10 (0.52, 2.32)educationno education or some primary (ref.)complete primary or higher1.61 (0.62, 4.15)1.74 (0.83, 3.63)no. living children01 (ref.)22.71 (1.14, 6.43)4.09 (1.69, 9.93)3 or more2.85 (1.22, 6.68)5.36 (2.09, 13.77)regionmwanza (ref.)dodoma4.02 (2.10, 7.68)4.82 (2.38, 9.77)household decision - making scale (continuous)1.07 (0.84, 1.36)1.17 (0.90, 1.51)p<0.05 ; p<0.01 ; p<0.001. table 6.adjusted odds ratios (or) and 95% confidence intervals (95% ci) of reporting contraceptive use by the attitudes toward refusing sex scale and socio - demographic factors, dodoma and mwanza regions, tanzania, 2009adjusted or (95% ci)variablehusbandswivesage1529 years (ref.)3049 years1.22 (0.58, 2.54)1.14 (0.54, 2.42)educationno education or some primary (ref.)complete primary or higher1.61 (0.64, 4.05)1.82 (0.87, 3.84)no. living children01 (ref.)22.73 (1.15, 6.46)4.04 (1.66, 9.79)3 or more2.88 (1.24, 6.70)5.03 (1.96, 12.88)regionmwanza (ref.)dodoma3.81 (1.98, 7.35)5.48 (2.70, 11.16)attitudes toward refusing sex scale (continuous)1.22 (0.79, 1.86)0.78 (0.57, 1.08)p<0.05 ; p<0.01 ; p<0.001. table 7.adjusted odds ratios (or) and 95% confidence intervals (95% ci) of reporting contraceptive use by the attitudes toward wife beating scale and socio - demographic factors, dodoma and mwanza regions, tanzania, 2009adjusted or (95% ci)variablehusbandswivesage1529 years (ref.)3049 years1.26 (0.60, 2.62)1.07 (0.50, 2.28)educationno education or some primary (ref.)complete primary or higher1.77 (0.71, 4.46)1.54 (0.72, 3.27)no. living children01 (ref.)22.76 (1.17, 6.52)4.43 (1.80, 10.89)3 or more2.98 (1.28, 6.93)5.52 (2.13, 14.30)regionmwanza (ref.)dodoma4.12 (2.13, 7.97)6.43 (3.04, 13.62)attitudes toward wife beating scale (continuous)0.95 (0.77, 1.17)1.29 (1.02, 1.64) p<0.05 ; p<0.01 ; p<0.001. adjusted odds ratios (or) and 95% confidence intervals (95% ci) of reporting contraceptive use by the gem scale and socio - demographic factors, dodoma and mwanza regions, tanzania, 2009 p<0.05 ; p<0.01 ; p<0.001. adjusted odds ratios (or) and 95% confidence intervals (95% ci) of reporting contraceptive use by the household decision - making scale and socio - demographic factors, dodoma and mwanza regions, tanzania, 2009 p<0.05 ; p<0.01 ; p<0.001. adjusted odds ratios (or) and 95% confidence intervals (95% ci) of reporting contraceptive use by the attitudes toward refusing sex scale and socio - demographic factors, dodoma and mwanza regions, tanzania, 2009 p<0.05 ; p<0.01 ; p<0.001. adjusted odds ratios (or) and 95% confidence intervals (95% ci) of reporting contraceptive use by the attitudes toward wife beating scale and socio - demographic factors, dodoma and mwanza regions, tanzania, 2009 p<0.05 ; p<0.01 ; p<0.001. in all models, the odds of contraceptive use increased with the number of living children reported by the respondent, although the effects were greater for women than for men. region of residence was also associated with increased odds of contraceptive use in all models. neither age nor education was significant in any of the models. for women, two of the four gender attitude scales the gem scale and the attitudes toward wife beating scale were positively associated with contraceptive use. for men, none of the gender attitude scales was associated with contraceptive use, based on the wife 's report (tables 47). this study contributes to a growing body of couple studies on reproductive behaviour (see becker & costenbader, 2001 ; derose,., 2004 ; allendorf, 2007 ; kulczycki, 2008), and is one of the first couple studies using the gem scale to compare gender - equitable attitudes of spouses. the analysis reveals new evidence regarding the influence of gender attitudes on contraceptive use, and underscores the need for information from both members of a couple. in general, wives had less equitable gender attitudes as compared with their husbands and, for three of the four scales examined, the differences were statistically significant (the gem, household decision - making and attitudes toward wife beating scales). somewhat surprisingly, wives ' gender attitudes predicted contraceptive use (as reported by wives) but husbands ' gender attitudes did not. the more equitable a woman 's gender attitudes, the more likely she was to be using a method of contraception. age, parity and education are commonly accepted variables positively correlated with contraceptive use, but in this analysis, only parity was significantly correlated with contraceptive use, and highly so, showing that gender attitudes have predictive power even after controlling for known correlates of family planning. the study has several limitations, including its small sample size (200 couples), and the fact that the sample was drawn from only two areas of tanzania (the sites of an intervention study), thus limiting generalizability. sites were limited to districts in which t - marc was collaborating with prinmat clinics in a community - based contraceptive distribution initiative, and where there was a local radio station willing to promote the community - based distributors. additionally, there was unusually high contraceptive use among the sample, especially in one of the regions (30% of female respondents in mwanza, 61% in dodoma). this is taken into account by controlling for region. according to the 2010 dhs, current use of any method in these regions was reported as 15.2% in mwanza and 28.4% in dodoma (nbs & icf macro, 2011). although it can not be demonstrated with this dataset, it is possible that, in reality, men 's attitudes do not reflect a greater degree of gender equity than women 's and that the data collected from men suffers from a higher degree of in other words, men may be more likely to report what they assume to be the more if that is the case, the question of whether men 's gender attitudes are associated with contraceptive use in this context is still open. similar findings have been reported from research using matched couples in india where husbands had more equitable views of their wives ' autonomy than their wives did (jejeebhoy, 2002). acceptable responses in the survey than when they were questioned in depth. in this study, unfortunately, neither male nor female participants were interviewed in depth, so it was not possible to determine levels of socially desirable responses. similarly, a secondary analysis of survey data from monogamous couples in malawi that looked at couple agreement on a range of issues, including ownership of household items, livestock, number of children, communication about fertility, family planning and aids, found that for many questions, when spouses ' reports disagreed, husbands were more likely to answer yes was the answer that implied greater wealth, fertility or modernity (miller., 2001). while the primary arguments for pursuing gender transformative strategies in health and population programmes should be based on equity and human rights concerns, evidence that such strategies can also contribute to health and population objectives may help to strengthen and broaden commitment to them. the finding that contraceptive use is associated with gender - equitable attitudes among women suggests that one should not overlook women in the call to change male attitudes and support male involvement in reproductive health. women as well as men often support inequality in a social system, and in this setting, family planning programmes that support gender - equitable attitudes among women may help facilitate their use of contraception. | summarythis paper explores the hypothesis that gender attitude scales (which measure the degree of equity in gender attitudes) are associated with contraceptive use. four hundred male and female respondents (200 couples) were interviewed using a pre - tested, structured questionnaire. analyses included comparisons of means and prevalence rates on gender equity indicators, other related factors and socio - demographic characteristics ; t - tests to compare mean scores on each gender scale for wives and husbands to identify any significant differences ; chi - squared tests to compare associations between individual attributes, attitudes and contraceptive use ; and multivariate logistic regression to examine associations between each gender scale and contraceptive use. the findings revealed that, on average, wives endorsed more inequitable gender attitudes compared with husbands on all gender attitude scales. for wives, more equitable gender attitudes were positively associated with contraceptive use. for husbands, the role of gender attitudes had no significant association with wives ' reported contraceptive use. family planning programmes that aim to challenge inegalitarian gender norms should not overlook women in their efforts since both men and women often accept and support inequality in a social system and, in some cases, it may be women 's gender attitudes that most influence family planning decisions. |
mouse genome informatics (mgi) is the primary international database resource for the laboratory mouse, providing integrated genetic, genomic and biological data to facilitate the study of human health and disease. the mgi team curates the biomedical literature (11 000 publications per year) and normalizes and integrates sequence and functional data about mouse genetics and genomics from almost 50 other external database and informatics resources. mgi organizes curation teams around particular types of data including sequence data, phenotypes, embryonic expression data, comparative and functional information, mouse tumorigenesis and mouse models for human diseases. mgi utilizes multiple bio - ontologies and is the authority for mouse gene and strain nomenclature. the mouse genome database (1) includes data on gene characterization, nomenclature, mapping, gene homologies among mammals, sequence links, phenotypes, disease models, allelic variants and mutants and strain data. the gene expression database (2) integrates different types of gene expression information from the mouse and provides a searchable index of published experiments on endogenous gene expression during development. the mouse tumor biology (3) database provides data on the frequency, incidence, genetics and pathology of neoplastic disorders, emphasizing data on tumors that develop characteristically in different genetically defined strains of mice. the mgi group is a founding member of the gene ontology consortium (go, www.geneontology.org, (4)). mgi fully incorporates the go in the database and provides a go browser for access to mouse functional annotation. finally, the mousecyc database (5) focuses on mus musculus metabolism and includes cell level processes such as biosynthesis, degradation, energy production and detoxification. it is part of the biocyc collection of pathway databases created at sri international (6). here, we outline the workflow process for one component of the mgi data acquisition and integration process that associated with the gene ontology project at mgi (figure 1). mgi assigns functional annotations (go terms) to genes and protein products through semi - automated methods and manual curation. semi - automated annotation strategies include mapping and translating data from the enzyme commission, swiss - prot, interpro (see http://www.geneontology.org/go.indices.shtml), rat and human ortholog experimental data and others. curation of these data sets includes review and resolution of quality control reports generated through the process of data loading and comparisons to existing data in mgi. for the purpose of this paper, we will not discuss these semi - automated integration methods, but rather concentrate on the manual literature curation, which is a vital source of experimental mouse functional data. while we focus on the go component of mgi in this description, the literature curation process is very similar for other mgi components that curate literature. figure 1.go curation workflow : papers of interest are identified and entered into the database system (triage) and associated with genes (indexed). go curation workflow : papers of interest are identified and entered into the database system (triage) and associated with genes (indexed). the subcomponents of the literature curation process include the following : (a)literature triage : identifying and obtaining relevant scientific literatureeach curator is assigned a specific subset of journals from a set of 160 relevant journals out of 650 subscriptions carried by the jackson laboratory. journals are chosen for triage based on the numbers of articles that have been identified and curated for that journal over the previous years. others are selected on the basis of particular annotation processes such as full curation of the wnt family of proteins, or in another example, full curation of genes implicated in human diseases. on a yearly basis, the number of papers selected from all journals is tabulated, and the selection of which journals to regularly curate the next year is determined based on the relevancy of the journal publications during the previous year and the number of full time equivalents available for this task. typically, a few journals are dropped and a few are added each year to the formal triage process. the quosa application (http://www.quosa.com) is used as an aid to access and identify recent papers as represented in pubmed containing data about the mouse and determining which component of the database will be curated from the paper (go, expression, mutant alleles, phenotypes, mapping, tumor). quosa is used to retrieve full text pdfs for a journal issue or time frame of interest. because the experimental organism mouse is often not mentioned in the abstract (334% depending on the journal), a curator selects an issue of a journal and then searches the full text of papers containing the keywords mice. the application highlights these terms showing the context of the search keywords, which enables curators to quickly determine whether the experiments described are of a suitable nature to be used for go annotation (i.e. do the experiments aid in determining the normal function of the gene ?) (figure 2). the number of papers examined and the number selected are somewhat journal dependent. for example, out of 8090 papers per each weekly issue of journal of biological chemistry, roughly 60 contain one or more of the three keywords, and of those, the curator may select 1015 as being relevant for some area of the database. for an issue of nature, which may have 1520 research articles per issue, up to 5 may have the keywords and papers selected are uploaded to an in - house server for the next steps.(b)adding the publications to the mgi system through the editorial interfacepapers selected as containing data appropriate for go annotation are entered into a master bibliography module. each record is tagged for the area of use of the database for which it has information. information about journal, volume, pages and the abstract are automatically obtained nightly from pubmed using the pmid. used box would then have an x added automatically.(c)indexing the papers to determine the genes being studieda paper is then associated or indexed to the genes discussed within by adding the paper to each gene s detail module (figure 3b). prominer, a natural language processing (nlp) application, is used to assist in the gene indexing process (7). utilizing official nomenclatures and synonym lists for mouse / rat / human gene names and gene symbols, prominer marks up papers for review by a curator, who then associates genes - to - papers in the mgi editorial interface (ei) (figure 4). figure 2.quosa information manager showing part of a paper from an issue of jbc with mouse, murine or mice highlighted. curator quickly looks at context to select appropriate area of mgi that the paper best fits figure 3.(a) mgi master bib ei, showing a record for a paper that has been selected for alleles / phenotypes and go. the paper has not yet been curated for go (indicated by an x in the selected and x in not used). references to be associated with this gene are entered into the lower left hand panel. if a paper is associated with multiple genes, the same paper is entered for each gene. literature triage : identifying and obtaining relevant scientific literature each curator is assigned a specific subset of journals from a set of 160 relevant journals out of 650 subscriptions carried by the jackson laboratory. journals are chosen for triage based on the numbers of articles that have been identified and curated for that journal over the previous years. others are selected on the basis of particular annotation processes such as full curation of the wnt family of proteins, or in another example, full curation of genes implicated in human diseases. on a yearly basis, the number of papers selected from all journals is tabulated, and the selection of which journals to regularly curate the next year is determined based on the relevancy of the journal publications during the previous year and the number of full time equivalents available for this task. typically, a few journals are dropped and a few are added each year to the formal triage process. the quosa application (http://www.quosa.com) is used as an aid to access and identify recent papers as represented in pubmed containing data about the mouse and determining which component of the database will be curated from the paper (go, expression, mutant alleles, phenotypes, mapping, tumor). quosa is used to retrieve full text pdfs for a journal issue or time frame of interest. because the experimental organism mouse is often not mentioned in the abstract (334% depending on the journal), a curator selects an issue of a journal and then searches the full text of papers containing the keywords mice. the application highlights these terms showing the context of the search keywords, which enables curators to quickly determine whether the experiments described are of a suitable nature to be used for go annotation (i.e. do the experiments aid in determining the normal function of the gene ?) (figure 2). for example, out of 8090 papers per each weekly issue of journal of biological chemistry, roughly 60 contain one or more of the three keywords, and of those, the curator may select 1015 as being relevant for some area of the database. for an issue of nature, which may have 1520 research articles per issue, up to 5 may have the keywords and adding the publications to the mgi system through the editorial interface papers selected as containing data appropriate for go annotation are entered into a master bibliography module. each record is tagged for the area of use of the database for which it has information. information about journal, volume, pages and the abstract are automatically obtained nightly from pubmed using the pmid. for example, if this paper was used for a go annotation, the used box would then have an x added automatically. indexing the papers to determine the genes being studied a paper is then associated or indexed to the genes discussed within by adding the paper to each gene s detail module (figure 3b). prominer, a natural language processing (nlp) application, is used to assist in the gene indexing process (7). utilizing official nomenclatures and synonym lists for mouse / rat / human gene names and gene symbols, prominer marks up papers for review by a curator, who then associates genes - to - papers in the mgi editorial interface (ei) (figure 4). quosa information manager showing part of a paper from an issue of jbc with mouse, murine or mice highlighted. curator quickly looks at context to select appropriate area of mgi that the paper best fits. (a) mgi master bib ei, showing a record for a paper that has been selected for alleles / phenotypes and go. the paper has not yet been curated for go (indicated by an x in the selected and x in not used). references to be associated with this gene are entered into the lower left hand panel. if a paper is associated with multiple genes, the same paper is entered for each gene. output from prominer : prominer is used to assist in the gene indexing process. utilizing official nomenclatures and synonym lists for mouse / rat / human gene names and gene symbols, prominer marks up papers for review by a curator, who then associates genes - to - papers in the mgi ei. sometimes a paper discusses several genes, but not all of them may be objects for direct go annotation. for example, a paper describing the effects of a knock out of a particular gene may use analysis of other gene products to analyze the particular processes being affected, but the annotation to involvement in the process would only be made to the gene being knocked out. currently, the topical areas selected for each paper are not directly tied to the genes associated with the paper. thus, a paper selected for go for one of the genes will appear in the go ei interface of unused papers for each of the genes indexed to the paper. ideally, all papers would be immediately used for go annotation, but on average 300 new papers are added to the database each week, only less than half of these are curated each week due to resource limitations. therefore, various priority selection criteria are used to choose which genes and papers warrant immediate attention. reports of interest are generated, such as genes with no go annotation but have new indexed literature selected for go or genes with mutant alleles that have literature selected for go (figure 5). additionally, participation in various collaborative projects, such as the reference genome project (8), or the protein ontology (8) defines primary sets of genes to work based on community input. a curator at mgi uses the go ei module to enter annotations (figure 6). curators use the annotation guidelines set forth by the gene ontology consortium (http://www.geneontology.org/go.annotation.shtml). it is divided into two main data entry sections : the annotation area and the annotation properties area. a list of papers selected for go that are associated with this gene is shown in the lower right panel. individual protein isoforms or modified forms can be indicated using annotation properties where an i d for a specific isoform can be indicated. after reading a paper next, the reference number is added (2), as well as the evidence code (3). if required by the type of evidence code, additional information is added to the inferred from column (4). once the annotation is saved, information about the cell type that the experiment was done in, or the specific isoform, or tissue, is entered into the annotation properties section (5). additional ontologies such as the cell ontology (9), mouse adult (10) and embryonic anatomies (11), protein ontology (12), and psi - mod (13) are used in the properties fields. several of these are used to supply an extension to the annotation which are used in the gene association file (gaf) (5). curators can use the obo - edit tool (14) to load multiple ontologies to aid in searching for appropriate terms, as well as viewing the chosen term in the context of the rest of the ontology (figure 7). the go vocabulary is refreshed daily from the go site, and only current go terms can be used, otherwise data entry is prohibited. only reference identifiers previously entered into there are other mechanisms, such as data loads, that provide go annotation. in all cases, provenance is provided. the go ei also has a built - in report generator that highlights words matching go terms found in the abstracts of papers selected for go as an aid to suggesting the type of information and evidence present in a paper (figure 8). figure 6.mgi go data entry module : the interface is divided into three main sections : go annotation, annotation properties and search and reference tracking. drop - down menus display pick - lists of allowed entries in various fields (evidence property, go qualifier and evidence codes). numbered areas : 1, go i d entry ; 2, reference entry ; 3, evidence code entry ; 4, inferred_from entry required for certain evidence codes and 5, annotation properties entry. the far left panel shows the vocabularies that have been loaded for searching and viewing. the right panel displays the terms in all of the vocabularies that contain the word kidney. the go term kidney mesenchyme morphogenesis is selected and is visible as a tree view showing its children (middle panel), and as a graphical view showing its parents (lower left). figure 8.report generated using the abstracts of papers selected for go for the gene being annotated within the go ei. mgi go data entry module : the interface is divided into three main sections : go annotation, annotation properties and search and reference tracking. drop - down menus display pick - lists of allowed entries in various fields (evidence property, go qualifier and evidence codes). numbered areas : 1, go i d entry ; 2, reference entry ; 3, evidence code entry ; 4, inferred_from entry required for certain evidence codes and 5, annotation properties entry. the far left panel shows the vocabularies that have been loaded for searching and viewing. the right panel displays the terms in all of the vocabularies that contain the word kidney. the go term kidney mesenchyme morphogenesis is selected and is visible as a tree view showing its children (middle panel), and as a graphical view showing its parents (lower left). report generated using the abstracts of papers selected for go for the gene being annotated within the go ei. annotations are tracked based on a variety of criteria such as annotation source (mgi curation or data load) and evidence (experimental or predictive, such as through orthology or functional domain). scripts review changes to the go structure and provide qc reports for curators noting genes whose annotations may be affected by these changes (figure 9). additionally, we use the master bibliography tables and the go annotations to keep track of various areas that need focus, such as genes with no go annotation but have papers that are selected for go but not used. figure 9.go change log report showing changes to the go and genes with annotations using the term that may need to be looked at. go change log report showing changes to the go and genes with annotations using the term that may need to be looked at. all data assertions in mgi are supported by evidence and citation to the source of the information. for assertions that are associated with controlled vocabularies such as the go, links are provided to vocabulary browsers that provide the relationships between the assertion and other knowledge in that area of the ontology. using the table and associated information mgi also provides a graphical display of go annotations from the go detail page for each gene. figure 10.the go annotation details for drd2 displayed as summary text (a), table (b) or graph (c). the go annotation details for drd2 displayed as summary text (a), table (b) or graph (c). go annotations are also shared with the go consortium (goc) through a gaf. this is a tab - delimited file that contains most of the elements of a go annotation as outlined in the go ei section above. presently, only the cell type and gene product annotation properties are included in the gaf. this file is available on either the goc web site or along with other data sets, from the mgi ftp site (ftp://ftp.informatics.jax.org/pub/reports/index.html). the gaf and the go vocabulary file are used as input for many analytical tools such as go termfinder (15). instructions for construction of a go gaf file are found in go documentation at http://www.geneontology.org/go.format.annotation.shtml. although some groups have used nlp to expedite the curation of literature (16), this can be especially difficult to do when applied to mouse biology because of the integration of human and mouse studies within the same description of results. the concepts captured by the go can not be gleaned just from simple text matching of terms, but must also take into account inferences that reflect a given context. additionally, an understanding of the nature of an experimental assay is important to correctly use the information as evidence of a particular result. while we continue to work with nlp developers to design a system to automate identification and tagging of papers (17), it is clear that the complexity of understanding the information in a biomedical publication requires the intervention of an experienced biologist - curator in the process. funding for open access charge : mgi database resources are funded by grants from the national human genome research institute (hg00330, hg02273), national institutes of health / national institute of child health and human development (hd062499) and the national cancer institute (ca89713). | the mouse genome database, the gene expression database and the mouse tumor biology database are integrated components of the mouse genome informatics (mgi) resource (http://www.informatics.jax.org). the mgi system presents both a consensus view and an experimental view of the knowledge concerning the genetics and genomics of the laboratory mouse. from genotype to phenotype, this information resource integrates information about genes, sequences, maps, expression analyses, alleles, strains and mutant phenotypes. comparative mammalian data are also presented particularly in regards to the use of the mouse as a model for the investigation of molecular and genetic components of human diseases. these data are collected from literature curation as well as downloads of large datasets (swissprot, locuslink, etc.). mgi is one of the founding members of the gene ontology (go) and uses the go for functional annotation of genes. here, we discuss the workflow associated with manual go annotation at mgi, from literature collection to display of the annotations. peer - reviewed literature is collected mostly from a set of journals available electronically. selected articles are entered into a master bibliography and indexed to one of eight areas of interest such as go or homology or phenotype. each article is then either indexed to a gene already contained in the database or funneled through a separate nomenclature database to add genes. the master bibliography and associated indexing provide information for various curator - reports such as papers selected for go that refer to genes with no go annotation. once indexed, curators who have expertise in appropriate disciplines enter pertinent information. mgi makes use of several controlled vocabularies that ensure uniform data encoding, enable robust analysis and support the construction of complex queries. these vocabularies range from pick - lists to structured vocabularies such as the go. all data associations are supported with statements of evidence as well as access to source publications. |
emc was first described by enzinger and shiraki in 1972.1 emc accounts for approximately 2.5% of all soft tissue sarcomas.2 to our knowledge, bras. in 1985, we represent a case of emc arising from the orbit in a 34-year - old male. we report the presentation, diagnosis, treatment, histopathology, immunocytochemistry, and molecular genetic studies of this case. a 34-year - old male without any known medical illnesses was referred to our clinic with 3 years history of the left orbital mass. ophthalmic examination indicated a palpable firm inferotemporal orbital mass of the left globe with no skin changes. the patient 's visual acuity, pupil, anterior segment, posterior segment, and optic nerve examinations were all within normal limits. a brain and orbit computed tomography (ct) scan, both with and without contrast, demonstrated a well - circumscribed left inferotemporal orbital mass measuring 14 mm 9.6 mm 15.3 mm, with mild globe displacement but no involvement, bony invasion or remodeling and no attachment to the extraocular muscles and no optic nerve compression [figure 1 ]. coronal (a) and sagittal (b) computed tomography scan with contrast showing a left inferotemporal heterogeneous orbital mass measuring 14 mm 9.6 mm 15.3 mm. the bony orbit appeared to be intact a complete resection of the mass was undertaken for both diagnostic and therapeutic purposes. microscopically, the tumor comprised of spindle - shaped cells embedded within the myxoid matrix. the tumor cells were arranged in a reticular growth pattern, nests, and perivascular pseudo - rosette. there was no mitotic activity, there were small cells showing eosinophilic to vacuolated cytoplasm, oval to round nuclei with inconspicuous nucleoli, revealing perivascular arrangement in some regions, and nests and cords in a myxoid background [figure 2 ]. molecular genetic study utilizing fluorescence in situ hybridization was positive for ewing sarcoma breakpoint region 1 (ewsr1) chromosome. (a and b) histopathology study, (a) small cells showing eosinophilic to vacuolated cytoplasm, oval to round nuclei with inconspicuous nucleoli, revealing perivascular arrangement in some areas (h and e, 200). (b) nests and cords in myxoid background (h and e, 200). (c) immunocytochemistry study showing vimentin reveals strong and intense diffuse expression (avidin - biotin complex technique, 400) on the basis of immunohistopathology and molecular genetic studies, the patient was diagnosed with emc of the orbit. the patient was sent for additional systemic investigations to the oncology center, which reported no evidence of metastasis. throughout the 6 months follow - up after resection, there were no additional signs of local recurrence both clinically and with radiology studies. first described by enzinger and shiraki in 1972,1 emc accounts for approximately 2.5% of all soft tissue sarcomas.2 emc most commonly affects the lower extremities.3 differentiation of emc from other sarcomas is possible due to cytological and molecular genetic studies45 in conjunction with clinicopathological features. several cases of emc have been reported in different anatomical locations of the body,6 with only a single previously reported case of emc within the orbit.7 chondrosarcoma is a misnomer used to describe the emc at the histopathological level, and only minority of cases show evidence of well - formed hyaline cartilage. in addition, only a few cases of emc have been found to be s100 positive, whereas skeletal chondrosarcomas nearly always stain positive.8 gross examination of emc usually indicates neoplasms as soft tissue multinodular with smooth surface that is fairly circumscribed. microscopically, the tumors present mostly as multinodular comprising of uniform round to oval or spindle - shaped cells embedded within the myxoid matrix and round to oval hyperchromatic nuclei and eosinophilic cytoplasm. the tumor cells are arranged in a reticular growth pattern, nests, cords, or strands. immunohistochemistry9 indicates the tumor cells are usually intense diffuse positive staining for both vimentin and synaptophysin with a variable percentage of staining to s-100 protein, desmin, and epithelial membrane antigen (ema). poor prognosis and an aggressive tumor is based on size larger than 10 cm with high cellularity ; anaplasia and rhabdoid phenotype, with focal regions of ki-67 staining > 25%.10 localized emc is usually treated with a complete excision of the tumor with a wide surgical margin. currently, there is no evidence of the effectiveness of adjunctive therapy such as radiation or chemotherapy if there are no signs of metastasis. the only emc case previously reported in the orbit was treated with exenteration due to the aggressive tumor presentation cause very severe proptosis of the eye.7 in our case, the microscopic features were identical to previous reports with no mitotic activity or necrosis. cyclin d1 decorated more than 75% of the nuclei, and p53 and ki-67 were present in 15% and < 1% of nuclei, respectively. ema ; s100 ; glial fibrillary acidic protein ; pan - cytokeratin ; actin ms all failed to express any immune - positivity. the patient was treated with complete resection of the mass with a wide surgical margin without adjunctive therapy. to our knowledge | extraskeletal myxoid chondrosarcoma (emc) is a rare soft tissue tumor. numerous cases of emc have been reported in different anatomical locations. there is currently only a single case of emc of the orbit and that was reported in 1985. we report a second case of orbital emc in a 34-year - old healthy male. |
teeth show signs of the least amount of changes in morphology and they are easily accessible for examination. it is the hardest and chemically the most stable structure in the body. they are selectively preserved and fossilized, thereby providing by far the best proof for evolutionary alteration. their resilience in the case of fire and bacterial decomposition makes them important for identification in forensic science. sex determination with aid of skeletal remains pose a great dilemma to forensic experts particularly when part of the body are remained. to solve this difficulty, tooth size standards based on odontometric data can be used in age and sex determination. refers to those differences in size, stature and appearance between male and female that can be applied to dental identification because no two mouths (dentitions) are alike. studies on tooth morphology have in the past been conducted using either intraoral measurements or measurements on casts. (1999) have found the mandibular canines to exhibit the greatest sexual dimorphism among all teeth., various studies has been reported on south india, north india, western uttar pradesh population for sex determination using mandibular canine width dimorphism. the present study was undertaken to find out utility of maxillary and mandibular canine width as a tool for sex determination in central indian population.to find out the average size of canines in males and females of central indian population.to compare the findings with national and international studies. to find out utility of maxillary and mandibular canine width as a tool for sex determination in central indian population. to find out the average size of canines in males and females of central indian population. to compare the findings with national and international studies the casts were selected from vspm dental college and research centre, post graduate department in orthodontics. the age group of selected casts were 17 - 21 yrs, as attrition is minimal in this age group. casts were selected with all permanent canines erupted and with exclusion criteria of unerupted or partially erupted teeth and any prosthetic replacement. total 100 casts were selected out of which 50 were males and 50 were females. the mesiodistal diameter of maxillary and mandibular canines was measured using a digital vernier calliper. the measurements were recorded on excel spreadsheet and subjected to statistical analysis to assess sex difference using students all the mean values are in millimetres comparisons with wheeler comparison with wheeler comparison with other studies hashim and murshid (1993), conducted a study on saudi males and females in the age group of 13 - 20 years to determine the teeth in human dentition with the highest likelihood of dimorphism and found that only the canines in both the jaws exhibited a significant sexual difference while the other teeth did not. in a continuation of the same study, they also determined that there was no statistically significant difference between the left and right sides suggesting that measurements of teeth on one side could be truly representative when the corresponding measurements on other side was unobtainable. following this study, number of studies however, our study was limited to just canine widths of maxilla and mandible and comparison with other studies. a study by kaushal., found a statistically significant dimorphism in mandibular canines in 60 subjects in north indian population where the mandibular left canine was seen to exhibit greater sexual dimorphism. if the width of the canine is greater than 7 mm, the probability of the sex of the person under consideration being male was 100%. schield., observed sexual difference in tooth size among american black, european and mongoloid populations. the degree of sexual dimorphism of mandibular canine width was more in ohio caucasians and australian aborigines than in pima indians and tristanite population. only two studies were reported where maxillary canines were studied (mohd abdulla, gorea and sharma). latest study reported by sharma and gorea on north indian population (patiala) supported our findings that statistically significant sexual dimorphism is present in case of maxillary canines. abdulla reported the difference in saudi population but with a low degree of sexual dimorphism (not statistically significant)., in saudi arabian population and by a study of human fossil excavated at ra 's al- hamra, eastern arabian coast, which showed a general low degree of sexual dimorphism of mandibular canine teeth. acharya and mainalli found reverse dimorphism in the mesiodistal dimension of mandibular second premolar in nepalese population. the finding could be attributed to evolution resulting in a reduction in sexual dimorphism, causing an overlap of tooth dimension in modern males and females. similar finding was observed by karen boaz and chaavi gupta in a dimorphic study of maxillary and mandibular canines in 100 subjects in south indian population and revealed the lack of significant dimorphism in canines and also the finding of reverse dimorphism where the females exhibited larger canines than males. we derived the mean measurement of right and left maxillary canines for males and females and mean of these measurements were taken to arrive at a single value for maxillary canine. both the maxillary and mandibular canine measurements in the present study were found to be more than the wheeler 's. this finding is very important as it indicates that normative data based on one population can not be used for other population. comparing the mean canine measurement of our findings with other studies, the values of our study on central indian population one more interesting finding observed was that there is more variation in size of maxillary right and left canines of females which is reflected by more standard deviation [table 5 ]. the maxillary canines in males show more consistent mesiodistal width [difference in sd=0 ] as compared to female maxillary canines. in the literature, mandibular canines are found to be more reliable in sex determinations. in our study, measurements of maxillary canines are found to be more consistent and reliable for sex determination. the mandibular canines show more variations in mesiodistal width of right and left sides as compared to maxillary canines. the emerging field of forensic odontology in india relies a lot on inexpensive and easy means of identification of persons from fragmented jaws and dental remains. application of moire 's topography and fourier 's analysis requires sophisticated equipments and the use of complex mathematical equations, respectively, hence the present study measured by only linear dimensions because of the simplicity, reliability and in expensivity. the mandibular canine index may also be used as an adjunct to enhance accuracy. the mesiodistal width of canines of both the jaws is significantly greater in males than females. this finding can be used as a tool for sex determination in central indian population.the mean maxillary canine width in males and females is 8.02 mm. the mean mandibular canine width in males and females is 7.73 mm.the mean (male and female), maxillary and mandibular canine width is found to be more in central india population as compared to north indian and south indian population.the mean (male and female), maxillary and mandibular canine width is found to be more in central india population as compared to values given by wheelers and the saudi population. the mesiodistal width of canines of both the jaws is significantly greater in males than females. this finding can be used as a tool for sex determination in central indian population. the mean (male and female), maxillary and mandibular canine width is found to be more in central india population as compared to north indian and south indian population. the mean (male and female), maxillary and mandibular canine width is found to be more in central india population as compared to values given by wheelers and the saudi population. as tooth morphology is known to be influenced by cultural, environmental and racial factors, more studies on different populations will be of much use to make data base available on dental morphometric measurements with a view to determine variation among large population that may be beneficial for anthropological, genetic, legal and forensic applications. the mesiodistal width of canines of both the jaws is significantly greater in males than females. this finding can be used as a tool for sex determination in central indian population.the mean maxillary canine width in males and females is 8.02 mm. the mean mandibular canine width in males and females is 7.73 mm.the mean (male and female), maxillary and mandibular canine width is found to be more in central india population as compared to north indian and south indian population.the mean (male and female), maxillary and mandibular canine width is found to be more in central india population as compared to values given by wheelers and the saudi population. the mesiodistal width of canines of both the jaws is significantly greater in males than females. this finding can be used as a tool for sex determination in central indian population. the mean (male and female), maxillary and mandibular canine width is found to be more in central india population as compared to north indian and south indian population. the mean (male and female), maxillary and mandibular canine width is found to be more in central india population as compared to values given by wheelers and the saudi population. as tooth morphology is known to be influenced by cultural, environmental and racial factors, more studies on different populations will be of much use to make data base available on dental morphometric measurements with a view to determine variation among large population that may be beneficial for anthropological, genetic, legal and forensic applications. | background : teeth are an excellent material for genetic, odontological and forensic investigations and research purpose. from all the teeth, the mandibular canines are found to exhibit sexual dimorphism. however, very few studies have been published on maxillary canine 's measurements.aims:1. to find out utility of maxillary and mandibular canine width as a tool for sex determination in central indian population. 2. to find out the average size of canines in males and females of central indian population. 3. to compare the findings with national and international studiesmaterials and methods : the present study was conducted in 100 cases in the age group of 17 - 21 years. mesiodistal width of right and left mandibular and maxillary canines were measured on the casts with digital calliper and subjected to statistical analysis.statistical analysis : statistical analysis was done to assess sex difference using students t test (paired).results and conclusions : it was seen that a definite statistically significant sexual dimorphism exists when mandibular and maxillary canine measurements were compared. thus, it can be suggested that canine width measurements can be used as an adjunct for sex identification purpose in central indian population. |
pure titanium and titanium alloys are the most used biomaterials for fabrication of surgical implants due to their excellent mechanical properties, biocompatibility15 and resistance to corrosion16. they are considered ideal materials because they have shown better acceptability by human tissues than other metals under diverse circumstances16. the high biocompatibility of titanium derives partially from the stable and protective oxide layer, which apparently aids in connecting extracellular matrix to the implant surface16. the knowledge of the biomaterial - bone tissue interface is extremely important to define which material would promote a better tissue response and which kind of surface would be more adequate for the proliferation of bone cells21. after placement of an implant in the surgical cavity, ideally, these events should lead to wound healing by intimate apposition of the bone to the biomaterial, i.e., osseointegration6. osseointegration is a direct structural and functional connection between living bone and the surface of an implant5. regardless of their external shape, microscopically they can present smooth, porous or textured surfaces1,4,5,10,19,26. several studies have shown that the success or failure of surgical implants can be related to chemical3,12,19 and biological12 properties of their surfaces as well as to their micromorphology13,16,26. the differences in the microstructure of implant surfaces seem to influence stress distribution, bone retention, cellular response on its surface and consequently the osseointegration3,12,23. the porous surface has been considered as a good alternative to rough coating11. biomaterials with porous structure are aimed at optimizing the interfacial resistance between the material and the bone, leading to a more effective fixation of the implant. porous implants allow an interdigitation of the bone tissue to the implant, thus increasing the interfacial resistance18. additionally, a porous surface provides a more efficient fixation, shorter initial healing time19 and increased cellular adhesion potential. pilliar,.19 (1998) reviewed and compared several designs and surfaces of surgical implants and concluded that the surface with pores of 100 m in diameter would be ideal for the neoformation of a three - dimensional osseointegration net inside the implant. oliveira,.18 (2002) determined that, although the fabrication process parameters have been optimized, the ideal porous requirements for surgical implants have not yet been reached. these authors reported that some changes are necessary in order to increase porosity and advocated that an analysis of pore size distribution along the sample has been performed to indicate more efficiently which porous fraction would better meet implant requirements17. therefore, the purpose of this study was to analyze, by histological and histomorphometric methods, the bone repair around cylindrical grade-2 commercially pure titanium implants with rough and porous surface, fabricated using powder metallurgy technique, after their insertion in tibiae of rabbits. seven male new zealand albino rabbits aged 6 to 8 months and weighing 3.5 kg on average were used in this study. the animals were provided by the vivarium of so jose dos campos dental school and were kept in individual cages and fed a commercial pet chow (coelhil r ; socil, belo horizonte, mg, brazil) and water ad libitum. the commercially pure titanium implants with either rough or porous surface were fabricated at the department of materials of the air and space institute at cta (brazilian air and space technical center, so jos dos campos, sp, brazil) in an association with the national institute of technology of rio de janeiro (rio de janeiro, rj, brazil). the implants had 3.0 mm in diameter and pore diameters ranging from 250 and 350 m. the implants were cleaned, wrapped and sterilized in autoclave at 121c for 15 min. prior to surgery, the animals were weighed and injected intramuscularly with 13 mg / kg of aqueous solution of 2% hydrochloride of 2-(2,6-xylidine)-5,6-dihydro-4h-1,3-thiazin (rompum ; bayer, so paulo, sp, brazil), which is an analgesic, sedative and muscular relaxant substance. general anesthesia was obtained with administration of 33 mg / kg of ketamine (dopalen ; agribrands do brasil ltda, paulinia, sp, brazil).). a local anesthetic composed of 3% octapressin combined with prilocaine hydrochloride and felypressin (3%citanest dentsply) was also used. the implant was removed from the wrap, placed in the perforation and pressed into the surgical cavity until it was fixed to the cortical bone. in order to standardize the procedure, the right tibia received a rough surface implant (control) group, while the left tibia received a porous - surface implant. the muscular tissue was sutured with absorbable thread and the skin with mononylon 4 - 0 surgical thread. after that, all animals were given penicillin and were monitored until sacrifice 30 days after surgery. after euthanasia, the surgical segments with the implants were removed and the implants were tested for mobility using a clinical clamp. the segments were placed in 10% formalin solution for at least 48 h. next, the specimens were dehydrated in an increasing ethanol series (50%, 75%, 90% and 100%), embedded in polyester resin (orto cristal t 208 ; valglass com. so paulo, sp, brazil) and sectioned longitudinally in a sectioning machine for hard tissues (labcut 1010 ; extec technologies, inc., enfield, ct, usa) providing 80-m - thick serial sections on average. the sections were ground in a polishing machine (labpol 8 - 12 ; extec technologies, inc.) using sandpapers of decreasing abrasiveness (# 400, # 600 and # 1200) until reaching a thickness of 30 - 40 m. images were taken with a digital camera (dsc - s85, cyber - shot, sony) coupled to the optical microscope and evaluated on a television monitor (panasonic). three sections of each implant were evaluated for the percentage of bone neoformation at implant - bone interface. two areas of each section were digitized (x100), representing the medial and distal interfaces of the implant. bone neoformation rate and bone ingrowth into the pores were calculated using the image j software (nih, usa). the final data were submitted to statistical analysis using the minitab software version 12.3 (minitab inc, usa) and student 's t - test at 5% significance level. all animals presented satisfactory postoperative results, without any evidence of inflammation or infection of the surgical site. no adverse reaction was observed during the procedure. during the clinical evaluation, performed with a clinical clamp, none of the implants presented mobility. in both groups (rough- and porous - surface implants), there was bone neoformation around the implant leading to osseointegration (figures 1 and 2). this new bone formation was similar in both groups and was constituted by mature bone trabeculae that presented lamellar arrangement and by medullar spaces of different sizes. in several specimens, regardless of the analyzed group, bone neoformation above the implants and at their bottom surface was observed, sometimes contacting the internal cortical face on the opposite side. a clear delimitation between the new bone formation and the preexistent cortical bone was also noticed (figures 3, 4 and 5). the results showed that the types of implant used presented statistically significant difference to each other regarding the amount of new bone formation on the implant - bone interface, the porous - surface implants showing a larger amount neoformed bone. table 1 shows the means of bone neoformation (%) at bone - implant interface of rough- vs. porous - surface implants. statistically significant difference. in our study, cylindrical implants with porous surface were compared to cylindrical implants with rough surface regarding the quantity and quality of new bone formation on the implant - bone interface after implantation in rabbit tibiae. however, when the quantity of bone neoformation at implant - bone interface was evaluated, a larger formation of bone tissue was observed for the porous - surface implants, and this difference was statistically significant. the most important factors to dental implant osseointegration are related to the characteristics of its surfaces, which include topography and chemical and electric properties of the material19, since bone - implant interaction is mainly related to the most external layers of the implants13. important factors to a more successful osseointegration are : implant material, implant shape, surgical technique20, quantity of bone tissue20,25, load6 and implant resistance4. however, some other factors such as surface energy, sterilization techniques and chemical and topographic properties of the implant surface are extremely important for the final outcome of osseointegration2,8,12,16. in the present study, the results showed that all implants were well tolerated when placed in rabbit tibiae, thus corroborating the findings of previous studies that indicated titanium as the best biomaterial for surgical implants15,16. bone growth is also dependent on factors such as percentage of surface porosity, stability and degree of micromovement of the implant and the presence of gaps between the implant and the bone at the time of placement. implants placed under pressure inside the surgical cavity help minimizing the gaps and implant micromovement8. therefore, in order to obtain osseointegration, the surgical cavity must be prepared with the least injury possible15,20. in order to cause minimal damage to the surrounding bone tissues, in the present study, bone perforation was performed using burs of increasingly larger diameters, and under constant saline irrigation. after that, the implants were gently pressed into the surgical cavity, which diminished the gap between the implant and the bone and promoted efficient stability. additionally, all implants were evaluated with a clinical clamp at the moment of sacrifice, 30 days after surgery, and were stable. some previous studies used a 4-week healing period to evaluate the biocompatibility of metal materials7,14,17,22. deporter,.10 (1986) reported that healing periods (i.e., between implant placement and euthanasia of the animals) longer than 4 weeks added no benefits to increase the quantity of bone tissue ingrowth into porous - surface implants, and observed that only bone tissue maturation took place after this period. thus, in the present study, a 4-week period was used to evaluate the biocompatibility of porous - surface grade 2 commercially pure titanium implants fabricated by means of powder metallurgy technique. the purpose of studying and developing porous - surface implants is to promote a more stable and biocompatible fixation of titanium implants. the creation of a porous geometry surface aims not only at increasing contact area but also at allowing bone ingrowth into the pores, including those located more centrally. such ingrowth is due to pore intercommunication, which produces a three - dimensional net and allows a mechanical entanglement7,8,9,18. the porous - surface implant have an ideal diameter for the proliferation of bone cells2,12, and most studies report that pore diameter ranges from 100 to 400 m. in addition, the pores have been shown to be interconnected, so that bone interdigitation into the porous structure may occur, thus reaching maximum interfacial resistance18,24. however, nguyen,.17 (2004) reported that small pores measuring only 45 m in diameter also allow bone ingrowth. furthermore frosch,.14 (2002) found that 300 to 600 m pores showed a three - dimensional, reticular osteoblast - like cell development within 4 weeks, and that in 1000 m pores cell ingrowth was incomplete. in the present study, the diameter of the pores varied between 250 and 350 m, corroborating the findings of other studies9,19,23. svehla,.23 (2000) compared five different implant surfaces and verified that porous surfaces promoted an excellent substrate for the ingrowth of bone tissue and consequent implant fixation, whereas smooth surfaces resulted in the formation of a fibrous tissue and improper fixation. in the present study, when the cylindrical porous - surface implants were compared to the cylindrical rough - surfaces implants, osseointegration was observed in both groups. deporter,.11 (1990) concluded that a smaller segment of porous - surface implants allowed a more effective contact with the bone, due to a bigger contact area, determined by its topography. similar findings were observed in the present study in which a statistically significant difference was observed between the groups in relation to the quantity of bone neoformation at titanium - bone interface. therefore, smaller porous - surface implants could be developed to be used in complex clinical situations such as regions with low - quality bone9,19. the results of this study showed that because of the larger contact surface promoted by the presence of pores, there was more bone ingrowth on the implant - bone interface. such results are consistent with those of cook and rust - dawicki7 (1995), deporter,.9 (2002), deporter,.10 (1986), deporter,.11 (1990), frosch,.14 (2002), karabuda,.15 (1999), story,.22 (1998), svehla,.23 (2000), vidigal junior,.25 (1999), zinger,.26 (2005) who also observed more effectiveness of the porous - surface implants compared to other types of implants. based on the results of this study, it may be concluded that the cylindrical porous - surface implants yielded greater bone neoformation than the cylindrical rough - surface implants because of their larger area in contact with the bone tissue and the presence of an intercommunicating porous structure that allowed the formation of a three - dimensional osseointegration network. | the purpose of this study was to analyze the bone repair around commercially pure titanium implants with rough and porous surface, fabricated using powder metallurgy technique, after their insertion in tibiae of rabbits. seven male rabbits were used. each animal received 3 porous - surface implants in the left tibia and 3 rough - surface implants in the right tibia. the rabbits were sacrificed 4 weeks after surgery and fragments of the tibiae containing the implants were submitted to histological and histomorphometric analyses to evaluate new bone formation at the implant - bone interface. means (%) of bone neoformation obtained in the histomorphometric analysis were compared by student 's t - test for paired samples at 5% significance level.. the results of the histological analysis showed that osseointegration occurred for both types of implants with similar quality of bone tissue. the histomorphometric analysis revealed means of new bone formation at implant - bone interface of 79.69 1.00% and 65.05 1.23% for the porous- and rough - surface implants, respectively. statistically significant difference was observed between the two types of implants with respect to the amount new bone formation (p<0.05). in conclusion, the porous - surface implants contributed to the osseointegration because they provide a larger contact area at implant - bone interface. |
apoptosis (programmed cell death) is a physiological mechanism of cell death. during apoptosis, there is a rapid reduction in the cellular volume followed by chromatin condensation, associated with characteristic internucleosomal dna cleavage. this results in the production of nucleosomes of dna fragments complexes with core histones, which are distinct multiples of an 180200 bp subunit. world health organization statistics have estimated that cancer will cause 83.2 million deaths between 2005 and 2015 if the recommended measures are not respected. in 2007, cancer was the cause of 7.9 million deaths, which is 13% of world mortality. among males in the third world countries, damaged cells will undergo apoptosis, but in the case of cancer cells mutations may have occurred that prevent cells from undergoing apoptosis. understanding apoptosis regulation is a main concern in the development of chemotherapeutic anticancer drugs on malignant cells [3, 4 ]. traditionally, many extracts from roots, stems, and fruits have been used for maintaining health, enhancing overall immune status, and prevention and treatment of chronic diseases, and the modulation and treatment of different diseases. spinosa is known as apple punice from punicaceae family commonly widespread in the latitudes of 475 m above sea level in the north of iran. granatum extracts possess a plethora of biological activities including antibacterial, antiviral, antifungal, cytotoxic and immuno - potentiating activities. granatum tree (pomegranate), especially its fruit, possesses a vast ethno medical history and represents a phytochemical reservoir of heuristic medicinal value. the tree / fruit can be divided into several anatomical compartments : (1) seed, (2) juice, (3) peel, (4) leaf, (5) flower, (6) bark, and (7) roots, each of which has interesting pharmacologic activity. juice and peels, for example, possess potent antioxidant properties, while juice, peel, and oil are all weakly estrogenic and heuristically of interest for the treatment of menopausal symptoms. the use of juice, peel, and oil has also been shown to possess anticancer activities, including interference with tumor cell proliferation, cell cycle, invasion, and angiogenesis. accordingly, we have conducted our research on toxicity extracts of punica granatum l. var. the objective of this study was to examine the in vitro cytotoxic activities of a wildly ethanolic standardized punica granatum l. var. cell death elisa and tunel was employed to quantify the nucleosome production resulting from nuclear dna fragmentation during apoptosis. punica granatum l. var. spinosa (pgs) plants known as apple punice from punicaceae family were collected from the southeast of golestan province, iran (ramian). mazandarani from the medicinal plant research center of islamic azad university of gorgan, iran, identified the plant. the seeds and peels parts of the plant were separated, shade dried, and grinded into powder with mortar and pestle. extraction of ethanolic extract was carried out by macerating 100 g of powdered dry plant in 500 ml of 70% ethanol for 48 h at room temperature. then, the macerated plant material was extracted with 70% ethanol solvent by percolator apparatus (2-liter volume) at room temperature. the plant extract was removed from percolator, filtered through whatman filter paper (no. the ethanol extract was filtered and concentrated using a rotary evaporator and then evaporated to dryness. briefly, the concentrated plant extracts were dissolved in dimethyl sulphoxide (dmso) (sigma, usa) to get a stock solution of 10 mg / ml. the substock solution of 0.2 mg / ml was prepared by diluting 20 l of the stock solution into 980 l serum - free culture medium, rpmi 1640 (the percentage of dmso in the experiment should not exceed 0.5). the human prostate cancer cell line (pc3) and normal fibrosarcoma cell line (l929) were obtained from national cell bank of iran (ncbi, pasteur institute of iran). the cells were grown and maintained in a humidified incubator at 37c and in 5% co2 atmosphere. rpmi-1640 medium supplemented with 10% fetal bovine serum (fbs, invitrogen gibco), 100 units / ml penicillin, and 100 g / ml streptomycin (invitrogen gibco) was used for cell cultures of pc3. ten thousand cells from log phase cultures were seeded in 100 l of rpmi-164 medium supplemented with 10% fetal bovine serum per well of 96-well flat - bottom culture plates (nunc, denmark). proliferative response and cell death of the pgs extract - treated cells were determined using mtt assay and cell death elisa, respectively. a colorimetric assay using 3-(4, 5-dimethylthiazoyl)-2, briefly, cells were added onto flat - bottomed microculture plates in the presence or absence of various concentrations of the extracts (in triplicate) and incubated at 37c in a 5% humidified co2 incubator for 24 and 48 h. then, 10 ml of mtt (5 mg / ml, sigma) was added to each well and incubation was continued for a further 4 h at 37c. in each well, 100 l / well of solubilization solution, containing dmso and sorenson buffer, were added. after complete solubilization of the dye, plates were read at 570 nm on an elisa reader. the mean optical density (od) sd for each group of replicates was calculated. the inhibitory rate of cell growth was calculated using the formula : % growth inhibition = (1 od extract treated)/od negative control 100. cell death detection elisa (roche applied science, switzerland) was used to quantify histone - complexed dna fragments (nucleosomes) in cytoplasm of the apoptotic cells after induction of apoptosis, as described elsewhere. briefly, after incubation with the pgs extract (at concentrations determined by mtt assay) for 24 h, the pc3 cells were pelleted and lysed. the resulting color development, which was proportional to the amount of nucleosomes captured in the antibody sandwich, was measured at 405 nm (with reference wavelength at 490 nm) using a benchmark microtiter plate reader (bio - rad). results were expressed as the apoptotic and necrosis percentage, calculated from the ratio of absorbance of treated (apoptotic) sample to that of the untreated (control) sample. briefly, 1 10 cells were seeded into 96-well plates and treated with or without (as control) pgs extract at specified doses for 24 h. after the incubation period, the cultures were harvested and washed twice with pbs., 20 l of cell was mixed with equal volume of 0.4% trypan blue (sigma, usa merck) and was count with neubauer haemocytometer (weber, england) by clear field microscopy (nikon, japan). were assayed two times in triplicate. to assess cell death by apoptosis, an in situ cell death detection kit, pod (roche, germany) for dna chromatin morphologic features was used for quantification. cells grown on coverslips were washed twice with pbs, air dried, and fixed for 60 min in freshly prepared 4% paraformaldehyde / pbs (ph : 7.4) (sigma - germany), ph 7.4, at room temperature. then the cells were washed again twice with pbs (ph : 7.4) and incubated with 3% h2o2/methanol (merck - germany) for 10 min. following washing with pbs, cells were permeabilized in 0.2% triton x-100/pbs (ph : 7.4) (sigma - germany) for 2 min at 4c. samples were incubated in 50 l of tunel reaction mixture for 2 h at 37c in a humidified chamber and in the dark, covered with parafilm. omission of tdt provided the negative control for the assay, and preincubation of cells with 10 g / ml dnase i in 50 mm tris - hcl, ph 7.4, 1 mm mgcl2, and 1 mg / ml bsa for 10 min at room temperature to induce dna strand breaks artificially, served as positive control. min in a humidified chamber, at 37c with 50 l converter - pod (anti - fluorescein antibody, fab fragment from sheep, conjugated with horse - radish peroxidase). after rinsing in pbs, the samples were incubated for 10 min with 100 l dab (sigma - germany) substrate in the dark. at the end, the samples were mounted and analyzed under light microscope, where the apoptotic cells could be seen as condensed shrinked dark brown cells. the data are expressed as mean standard deviation (sd) for at least three independent determinations in triplicate for each experimental point. for all the measurements, tow - way anova followed by duncan 's new multiple range test (p 0.05) was used to assess the statistical significance of difference between control and pgs treated. peels of pgs extract at 10 to 600 g / ml exhibited significant dose - dependent inhibitory effects on the proliferation of pc3. growth inhibition of peel extract in 24 and 48 h was 61.2 2.3% and 67.1.75%, respectively (figure 1(a)), with more than 75% suppression. the concentrations producing 50% growth inhibition (ic50) of the pgs extract on pc3 were effectively suppressed with the ic50 value (250.21 g / ml) after incubation with the peel extract. however seed extract induced no significant suppression on the proliferation of pc3 cells (figure 1(b)) and the peel extract induced no significant suppression on the proliferation of normal l929 cells (figure 1(c)). pc3 cells were compared to elucidate the cytotoxicity of both peels of pgs extract and toxol (chemotherapeutic agent, control positive) with more than 75% in 600 g / ml and 90% in 20 g / ml growth suppression in 24 h (table 1). in 24 and 48 h dye exclusion assay evaluated viability of pc3 cells exposed to peel extract. as the result in figure 2, the viabilities of cells exposed to peel pgs extract at concentrations of 10 and 600 g / ml were 96.3 7.8% and 24.1 2.5%, respectively. as determined by mtt assay, peel extract at 50, 100, 200, and 300 g / ml was chosen for pc3 cell line in cell death detection elisa. the proportion of dead pc3 cells increased sharply (from 32 8.5%, to 55 1.9%) upon 24 h incubation with the peel extract at 50300 g / ml at 24 h. these results suggested that the apoptotic response of pc3 cell lines should be evaluated at different concentration points. to test whether or not peel extract that induced the decrease of cell viability and cytotoxicity contributes to apoptotic death in pc3 cell lines in vitro. cells were incubated with 250 g / ml of pgs for 24 h and then determined using tunel assay. it was found that the pc3 cells treated with peel extract (250 g / ml) for 24 h exhibited apoptotic body formation (figure 4). pc3 cells treated with peel extract displayed typical morphological features of apoptotic cells, with condensed and fragmented nuclei (figure 4(a)). however, homogenous nuclear chromatin was evident in control cells (figure 4(b)). tunel assay based on labeling of dna strand breaks generated during apoptosis revealed that peel extract induces apoptosis in pc3 cells. understanding apoptosis regulation is a main concern in the development of chemotherapeutic anticancer drugs on malignant cells. the present study has demonstrated that ethanolic peel extract of pgs in natural form could significantly suppress the proliferation of pc3 cells in vitro using the mtt assay. such antiproliferative activity of peel extract of pgs was characterized by the dose - dependent manner (figure 1(a)). however seed extract induced no significant suppression on the proliferation of pc3 cells (figure 1(b)) and the peel extract induced no significant suppression on the proliferation of normal l929 cells (figure 1(c)). toxol at an optimal in vitro concentration was found to selectively induce at least 90% growth suppression on pc3 cells but peel extract has more than 75.50% in 600 g / ml in comparison to 90% inhibition activity toxol in 20 g / ml growth suppression in 24 h (table 1). viability percentage was evaluated by dye exclusion assay at 24 h with peel extract at concentrations of 10 to 600 g / ml and viable cell decreases from 96.3 7.8% to 24.1 2.5% by increasing dose and time of treatment (figure 2). in order to determine whether the antiproliferative activity of peel extract is manifested by induction of apoptosis, cell death detection elisa was employed to quantify the nucleosome production during nuclear dna denaturation of apoptotic cells (figure 3), suggesting dose - and time - dependent induction of apoptosis. these results suggested that the extract exerts its cytotoxic effect on prostate cells possibly via an apoptosis - dependent pathway. it is known that dna strand breaks occur during the process of apoptosis, and the nicks in dna molecules can be detected qualitatively through tunel assay. in present study, typical apoptotic characteristic tunel staining was observed in treated cells (figure 4). the effects of pomegranate on prostate cancer have been investigated in the cell culture system, previously. each preparation suppressed prostate cancer cell growth and invasive potential pca lncap, pc-3, and du 145 cells, whereas normal prostate epithelial cells were significantly less affected. antiproliferative properties of pomegranate fruit extract (pfe) against human pca cells were demonstrated by the vidal. in the cell culture system and in a xenograft mouse model. human pca pc-3 cells treated with pfe (10100 g / ml) for 48 h resulted in a dose - dependent inhibition of cell growth and induction of apoptosis. taking together, the present study is the first to show toxicity of pgs in malignant cell lines in which apoptosis or programmed cell death play an important role. this study provides the evidence that in vitro cytotoxic activity of an ethanol standardized extract from wild punica granatum l. var. spinosa (pgs) from southeast of golestan province, iran (ramian), was found to dose dependently inhibit the proliferation of prostate cells possibly via an apoptosis - dependent pathway. | the punica granatum l. var. granatum (pomegranate) has been demonstrated to exert antitumor effects on various types of cancer cells. the present study aimed to evaluate the medicinal herbs punica granatum l. var. spinosa (apple punice) that are native to iran. this study was determined to test the possible cytotoxic activity and induction of apoptosis on human prostate cell lines. the effect of ethanol extracts of the herbs on the inhibition of cell proliferation was assessed by mtt colorimetric assay. pc3 cell lines treated with the extracts were analyzed for the induction of apoptosis by cell death detection (elisa) and tunel assay. dye exclusion analysis was performed for viability rate. our results demonstrated that the punica granatum l. var. spinosa extract dose dependently suppressed the proliferation of pc3 cells (ic50= 250.21 g / ml) when compared with a chemotherapeutic anticancer drug (toxol) (vesper pharmaceuticals) with increased nucleosome production from apoptotic cells. the punica granatum l. var. spinosa extract attenuated the human prostate cell proliferation in vitro possibly by inducing apoptosis. the punica granatum l. var. spinosa is likely to be valuable for the treatment of some forms of human prostate cell line. |
bees and wasps commonly sting because an intruder has neared the hive or nest, which has become quite common in india due to its large rural population and increasing deforestation. insect stings belonging to hymenoptera such as wasps, yellow jackets, bees, or hornets are generally associated with local reactions and occasionally anaphylaxis. systemic complications such as acute renal failure, liver dysfunction can rarely occur following multiple stings. a 55-year - old farmer attended our emergency department with history of multiple wasp stings on his right shoulder and trunk, while working in his fields. initially, he was treated at a nearby hospital with oral antihistamines, steroids, and local anesthetic application. at 3 days later, past medical history was unremarkable. on examination, patient was afebrile with pedal edema. his pulse rate was 94/min, respiratory rate 18/min and blood pressure of 126/82 mm of hg. 51 sting marks were found on the posterior aspect of neck, anterior aspect of right forearm, both anterior and posterior aspect of his right shoulder and chest wall [figure 1 ]. multiple wasp stings on front and back of right shoulder and chest investigations revealed hemoglobin 10.8 g / dl, total count 8320 cells / mm, blood urea 224 mg / dl, serum creatinine 6.2 mg / dl, serum sodium 136 meq / l, potassium 6.2 meq / l, serum bilirubin 2.2 mg / dl, aspartate aminotransferase 689 iu / l, alanine aminotransferase 864 iu / l, lactate dehydrogenase (ldh) 1640 iu / l, and creatinine kinase (cpk) 89252 his arterial blood gas analysis showed ph 7.31, pco2 24 mmhg, and hco312 meq / l. the patient had progressively worsening renal failure and remained oliguric in spite of adequate hydration. hence, the patient was initiated on intensive hemodialysis, and treated with antibiotics, antihistamines, and diuretics. he received a total of eight sessions of hemodialysis over a period of 2 weeks. iu / l, 876 iu / l, and 233 iu / l on 5, 7, and 14 day after admission, respectively [table 1 ]. he was discharged after 2 weeks of hospitalization with serum creatinine of 2.8 mg / dl and urine output > 1.5 l / day. bee and wasp stings are commonly observed in our country and are potential environmental hazard. their venom is a concentrated mixture of various biogenic amines, such as melittin, apamine, phospholipases, hyaluronidase, acid phosphatase, histamine, and kinin. phospholipase a and surface agents such as mellitin and apamine act on red cell membranes leading to hemolysis. though the exact mechanism of rhabdomyolysis is unknown, it is probably due to direct toxic effect of venom on muscular tissue. the systemic manifestations are seen only in patients with more than 50 stings and the potentially lethal number of stings has been estimated to be 500. common manifestations of hypersensitivity reactions to toxins of insects range from local swelling to angioedema and anaphylaxis. pain occurs immediately followed by erythematous papular lesions accompanied by urticaria and edema of varying degrees, which resolve in 4 - 6 h. wasp stings can result in multi system involvement ranging from hemolysis, rhabdomyolysis, acute renal failure, disseminated intravascular coagulation, myocardial dysfunction, hepatic dysfunction, and thrombocytopenia, which may occasionally become fatal. acute kidney injury can occur due to acute tubular necrosis secondary to shock, or pigment nephropathy resulting from rhabdomyolysis and intravascular hemolysis, interstitial nephritis from a hypersensitivity reaction to the wasp venom, or direct nephrotoxicity of venom. other systemic manifestations include myocardial necrosis and infarction, centrilobular necrosis of liver, and thrombocytopenia as a result of direct platelet toxicity. in a review of previously reported 24 cases of wasp sting, hemolysis and rhabdomyolysis were observed in 14 out of 21 evaluated cases and 11 out of 19 evaluated cases, respectively. the most common histological diagnosis was acute tubular necrosis, though acute interstitial nephritis, acute cortical necrosis, and thrombotic microangiopathy have also been reported. management of these patients includes early correction of hypotension, forced alkaline diuresis in case of rhabdomyolysis and hemolysis, and hemodialysis if required. in a large case series of 75 patients from china, 7 (9.3%) died, and 8 (10.7%) developed chronic kidney disease (ckd). further analysis showed no difference in the mortality rates between the modality of dialysis (continuous and intermittent with or without plasma exchange). wasp sting fatality rate has been found to be 0.02 and 0.2 per million population per year in an australian study and swedish registry, respectively. even though, wasp stings are quite common there are no such data available from india. since a kidney biopsy was not done, we could not identify the exact mechanism of acute renal failure in our patient. since, the muscle enzymes (cpk, and ldh) were highly elevated, and there was no evidence of hemolysis or no prior episode of hypotension, our patient developed acute kidney injury probably secondary to rhabdomyolysis causing pigment nephropathy and acute tubular necrosis. patients who have sustained multiple stings by bees or wasps should seek medical care as early as possible, even if they are apparently normal. primary as well as emergency care physicians must be well aware of these complications and investigate actively to exclude anaphylaxis, damage to the kidneys, blood, liver, and muscles. this case report highlights the importance of early recognition and treatment of acute kidney injury as delay in management may lead to ckd and mortality in a significant number of patients. | in most patients, wasp stings cause local reactions and rarely anaphylaxis. acute kidney injury and rhabdomyolysis are unusual complications of wasp stings. we report a case of acute kidney injury and rhabdomyolysis secondary to multiple wasp stings. a 55-year - old farmer developed multi organ dysfunction with acute kidney injury and rhabdomyolysis 3 days after he had sustained multiple wasp stings. the etiology of acute kidney injury is probably both rhabdomyolysis and acute tubular necrosis. he improved completely after hemodialysis and intensive care. |
cerebral palsy (cp) is a non - progressive developmental movement and posture disorder that occurs during fetal or infant development1. it is the most common physical developmental disability in childhood and encompasses a group of disorders of the development of movement and posture that cause activity limitations. these are attributed to non - progressive disturbances that occur in the developing infant brains2, 3. the impairment in cp, including secondary impairments such as spasticity, muscle contracture, bone deformity, muscle weakness, and coordination disorders, is multifactorial and primarily affects the lower extremities, such as deficits in walking ability4, 5. the varying levels of impairment affecting children with cp can be described according to the gross motor classification system (gmfcs)6. physical inactivity in children with cp increases the risks of secondary problems such as pain, depression, social isolation, fatigue, pressure sores, and mobility limitations. children with cp suffer from motor and cognitive disabilities, which usually require a long - term, multifaceted, and multidisciplinary approach7. children with cp also tend to have lower endurance, muscular strength, and cardiorespiratory fitness than the general population8. the consequences of chronic muscle imbalance and the resultant deformities can cause increasing disability with age9. one of the most significant problems faced by children with cp is defective postural control. maintaining postural control, which is required to perform activities of daily living, is often a major challenge for children with cp. active videogames such as those on the nintendo wii system may allow children with cp to perform many types of activities that are less affected by environmental barriers, which limit accessibility. such videogames allow children with cp to perform self - directed, on - demand, and affordable physical activity10. the growing popularity of interactive gaming in healthcare is partially due to the belief that the fun and competitive aspects of videogames can direct a patient s focus away from the repetitive and mundane nature of rehabilitation exercises11. some studies have evaluated interactive videogames, especially wii fit games, in different neurological diseases12. however, no trials have demonstrated the benefits of wii - based training for patients with cp13. nintendo wii interactive games provide children with cp many health - related benefits such as improved postural control, mobility, postural stability, functional independence, and self - esteem14. therefore, this study determined the effects of interactive play with nintendo wii fit games on motor performance in children with spastic cp. forty children diagnosed with cp spastic diplegia, their age ranged from 610 years old, with a matching functional capability of level 3 on the gmfcs, lower - limb muscle power no less than grade 4 according to the manual muscle test, and no fixed contractures in the lower limbs were recruited from the pediatric physical therapy department, king abdulaziz university hospital. children with severe hearing loss, marked visual impairment, epilepsy, or autism were excluded. the patients were randomly divided into two study groups : group a played the nintendo wii fit game for 20 minutes / day for 12 weeks, while group b did not (control group). this study was approved by the faculty of applied medical sciences scientific research ethics committee at king abdulaziz university hospital, and informed consent was obtained from the parents or guardians of all children prior to participation. each child in group a was provided a nintendo wii fit, which includes about 20 games, for use at home over a 12-week period. measures reported in the present study, interviews with the parents / guardians about the families measures rep with the intervention, and kinematic measurements of goal - directed arm movements were performed and will be presented in parallel papers. the movement assessment battery for children-2 (mabc-2) was used to assess motor performance, because it mainly involves motor tasks very similar to those involved in playing nintendo wii fit games, e.g., goal - directed arm movements, balancing, and jumping. this test is a new version of the mabc and was developed to be more appropriate for research. the mabc-2 targets children aged 316 years with coordination disorders and scores movement quality on the basis of 8 items included in 3 tests of manual dexterity, balance, and catching and aiming. the original mabc test was demonstrated to be both valid and reliable for assessing motor performance15. upper - limb coordination was assessed by subtest 5:6 (touching a swinging ball) from the bruininks - oseretsky test of motor proficiency (botmp). finally, the one - minute walk test, the validity of which has been established, was performed to assess the general motor function of children with cp16. the mean values of the investigated parameters were determined at the beginning and end of the study in both groups and compared by paired student s t - tests. the present study evaluated the motor performance in response to training with nintendo wii fit games in children with spastic cp. all children were able to practice the motion interactive games and were generally capable of handling the technology and setting up the gaming system. the motor performance scores of group a before and after the intervention are shown in table 1table 1.motor performance test variables before and after nintendo training of group(a) (unit : logs)testprepostmabc-2 total test score38.3 5.4244.1 5.21manual dexterity10.4 2.3217.3 1.25aiming & catching12.5 2.9115.9 3.18balance12.1 3.1216.1 3.10botmp 5:62.23 botmp 5:6 : bruininks - oseretsky test of motor proficiency subtest 5:6 (i.e., touching a swinging ball) ; mabc-2 : movement assessment battery for children-2. furthermore, subtest scores, included manual dexterity, aiming and catching, balance, one - minute walk test, and botmp 5:6 showed significant improvements. meanwhile, there were no significant changes in any parameter in group b (tables 2table 2.motor performance test variables in group b (control group) (unit : logs)testprepostmabc-2 total test score38.9 5.2739.1 5.16manual dexterity11.1 2.4411.3 2.42aiming & catching12.8 3.1513.1 3.11balance12.5 3.6212.7 3.74botmp 5:62.82 botmp 5:6 : bruininks - oseretsky test of motor proficiency subtest 5:6 (i.e., touching a swinging ball) ; mabc-2 : movement assessment battery for children-2 and 3table 3.motor performance parameters after the 12-week study period (unit : logs)testgroup agroup bmabc-2 total test score44.1 5.2139.1 5.16manual dexterity17.3 1.2511.3 2.42aiming & catching15.9 3.1813.1 3.11balance16.1 3.1012.7 3.74botmp 5:63.78 botmp 5:6 : bruininks - oseretsky test of motor proficiency subtest 5:6 (i.e., touching a swinging ball) ; mabc-2 : movement assessment battery for children-2). furthermore, the differences in parameters between groups at the end of the study were significant. botmp 5:6 : bruininks - oseretsky test of motor proficiency subtest 5:6 (i.e., touching a swinging ball) ; mabc-2 : movement assessment battery for children-2 data are mean sd. botmp 5:6 : bruininks - oseretsky test of motor proficiency subtest 5:6 (i.e., touching a swinging ball) ; mabc-2 : botmp 5:6 : bruininks - oseretsky test of motor proficiency subtest 5:6 (i.e., touching a swinging ball) ; mabc-2 : movement assessment battery for children-2 accordingly, children with cp, who already have additional challenges, should be urged to perform more physical activity18. playing motion interactive videogames however, the extent of improvement in daily physical activity motion among physically disabled children due to interactive videogames remains unclear owing to a lack of research ; furthermore, most existing studies are limited in size and do not provide strong evidence20. two controlled studies and a few uncontrolled reports show positive results of motion interactive videogames on movement control21. a few small studies also demonstrate motion interactive videogames can be useful for enhancing children be useful disposition, motivation to practice, and self - esteem22. in the present study, the standing domains of gross motor function increased significantly after the intervention. this can be attributed to the regular physical activities performed by the children during the 12-week period, which is concordant with a study indicating virtual reality games such as the nintendo wii system improve arm and hand movements in children with cp23. the impairment in cp, including secondary impairments such as spasticity, muscle contracture, bone deformity, muscle weakness, and coordination disorders, is multifactorial and primarily affects the lower extremities, such as deficits in walking ability24. moreover, one study demonstrates children with cp who play interactive videogames for 3 weeks show improvements in balance and motor performance25. children with cp may have impaired postural balance, which contributes to gait abnormalities26, 27. furthermore, loaded sit - to - stand exercises improve basic motor abilities, functional muscle strength, and walking efficiency28,29,30. in conclusion, nintendo wii fit games improve the motor performance of children with spastic cp, warranting further study. | [purpose ] motor control and muscle strength impairments are the prime reasons for motor behavior disorders in children with spastic cerebral palsy. these impairments lead to histological changes in muscle growth and the learning of motor skills. therefore, such children experience reduced muscle force generation and decreased muscle flexibility. we investigated the effect of training with nintendo wii fit games on motor performance in children with spastic cerebral palsy. [subjects and methods ] forty children with cerebral palsy spastic diplegia aged 610 years diagnosed with level-3 functional capabilities according to the gross motor classification system (gmfcs) were enrolled. participants were divided randomly into equal groups : group (a) that practiced with the nintendo wii fit game for at least 20 minutes / day for 12 weeks and group (b) that underwent no training (control group). the movement assessment battery for children-2 (mabc-2) was used to assess motor performance, because it mainly involves motor tasks very similar to those involved in playing nintendo wii fit games, e.g., goal - directed arm movements, balancing, and jumping. [results ] there were significant improvements in the subscales of the motor performance test of those who practiced with the nintendo wii, while the control group showed no significant changes. [conclusion ] using motion interactive games in home rehabilitation is feasible for children with cerebral palsy. |
parkinson 's disease (pd) is a complex neurodegenerative disorder that affects different regions of the nervous system, although it is clinically recognized by typical motor manifestations. even though much of the etiology of pd remains unknown, early - onset pd (eopd) (onset before 50 years old), which accounts for approximately 5%10% of all pd cases, can be explained by monogenic causes. mutations in coding regions of park2 gene are often implicated as the most common cause of eopd, followed by pink1 gene variants. these two genes are associated with the autosomal recessive forms of parkinsonism and may act in the same pathway, controlling mitochondrial homeostasis [24 ]. parkin is an ubiquitin e3 ligase and, therefore, participates in the ubiquitin - mediated proteasomal degradation pathway, whose activity seems to be compromised by pathogenic mutations. since the first description of park2 mutations in japanese patients with juvenile eopd, more than 170 different mutations have been described throughout its sequence, including large deletions or amplifications, small deletions / insertions as well as missense mutations. as a common molecular consequence, such mutations are known to cause loss of parkin function, leading to impaired mitochondrial integrity. pink1 (park6) encodes a mitochondrial serine / threonine kinase, which is expressed ubiquitously in the human brain. recent evidence suggests that the physiological role of pink1 comprises the phosphorylation of mitochondrial proteins in response to cellular stress and the protection of mitochondria against various stressors [8, 9 ]. up to date, homozygous and compound heterozygous losses of function mutations affecting the kinase domain of pink1 gene were observed, all of them reducing its enzymatic activity [10, 11 ]. the frequency of these mutations varies according to the geographic region from 0 to 15% all over the world [7, 12 ]. parkin and pink1 are thought to participate in the same pathway concerning controlling mitochondrial integrity and function, with pink1 functioning upstream from parkin [13, 14 ]. in the present study, we investigated the presence of park2 and pink1 sequence mutations in patients with eopd from the southeast and midwest regions of brazil. we analyzed 136 unrelated brazilian patients (86 men and 50 women ; mean age 49.8 13.3 years ; mean age at onset 39.5 10.3 years) with idiopathic pd manifesting before 51 years old and 200 healthy brazilian controls. all patients and healthy volunteers were from the same geographic area, both with similar age and socioeconomic status. amongst the patients included in this study, 31 cases had familial history of pd in relatives of 1st and 2nd degrees and the ethics committee of state university of rio de janeiro approved this study and a written informed consent was obtained from all subjects. the presence of exon rearrangements in park2 and pink1 genes was formerly screened in 102 probands of our sample through the mlpa analysis. the sequencing reactions were prepared according to the manufacturer, using the big dye terminator v3.1 kit (applied biosystems). all reactions were processed on an automated sequencer abi prism 3130 (applied biosystems), and the sequence analysis was performed using the softwares chromas lite 2.0 (technelysium) and bioedit sequence alignment editor version 6.0.6 (isis pharmaceuticals, inc.). dna samples which showed sequence variations underwent pcr - rflp analysis or taqman snp genotyping to confirm the alteration found. the prediction analysis of the effects of changes which cause amino acid substitutions in proteins was performed using electronic tools polyphen and pmut. to exclude the possible effect of silent mutations on splicing, we have used spliceview and nnsplice softwares. we identified twelve sequence variants in park2 gene : three silent variants and nine missense mutations (table 1). between the alterations found, five are nonpathogenic polymorphisms : c.500 g > a (p.s167n), c.1138 g > c (p.v380l), c.783a > g (p.l261l), c.1180 g > a (p.d394n), and c.111 g > a (p.p37p). the already - known c.245c > a (p.a82e) and c.719c > t (p.t240 m) variants were found in one patient each, both in heterozygous state. the p.a82e substitution had also been found in control subjects worldwide [17, 18 ]. we also identified the heterozygous c.1310c > t (p.p437l) variant in two probands and one healthy individual. this well - known alteration had already been reported in pd patients and controls in some populations [18, 19 ]. the patient found with the heterozygous c.434 g > a (p.s145n) substitution also harbors the p.s167n polymorphism in park2 exon 4. this variant has an uncertain pathogenic nature, and it has never been identified in controls worldwide. three changes found in this study have never been described : c.659a > g (p.k220r), c.1016c > t (p.a339v), and c.1021c > t (p.l341l) (table 1). the new variant recognized in exon 9 of park2 gene, c.1021c > t, was identified in two patients and resulted in a silent mutation (p.l341l). among the 200 healthy controls, the c.1310c > t (p.p437l) substitution in park2 gene was found just in one individual in heterozygous state. besides, we investigated the presence of c.1016c > t (p.a339v) and c.659a > g (p.k220r) variants in the same population, and we were unable to detect these mutations in any of the control individuals analyzed. in pink1 gene we found only known benign polymorphisms, including the exon 5 variant c.1018 g > a (p.a340 t) in seven patients, the silent variant c.1173 t > c (p.d391d) in one patient (exon 6), the exon 7 variant c.1426 g > a (p.e476k) in two patients, and exon 8 variant c.1562a > c (p.n521 t) that was found in half of our patients (66/132) (table 2). the quantitative analysis, previously realized by our group, identified 4 patients with dosage mutations : one proband with exon 1 heterozygous deletion of pink1 gene ; an index case with heterozygous deletion of park2 exon 4 ; one patient with heterozygous duplication of park2 exon 4, and a compound heterozygous patient that harbors two park2 mutations, a deletion of exons 5 - 6, and a duplication of exon 3. among pd patients with heterozygous pathogenic or probably pathogenic park2 variants, so far, park2 and pink1 are the genes most frequently associated with autosomal recessive eopd, and both coding products participate in the same metabolic pathway centered on maintenance of the morphological integrity of mitochondria. studies of park2 and pink1 genes in latin american populations are very scarce. in our study, we found twelve substitutions in park2 gene, from which nine are nonpathogenic variants and three putative pathogenic mutations. among the heterozygous variants identified, three are silent mutations, including one that is novel (p.l341l). the exon 3 c.245c > a (p.a82e) variant was identified in one proband and was previously described by other authors in pd patients and controls [17, 18 ], suggesting that the p.a82e variant is probably nonpathogenic. besides, the in silico analysis through the polyphen and pmut softwares showed that this alteration is benign. we also identified the c.719c > t (p.t240 m) substitution in one patient. this change had already been described, and it had been previously classified as a known pathogenic variant [18, 20 ]. our in silico analysis corroborated these data and considered that this alteration affects the parkin function, being probably pathogenic. another mutation, c.1310c > t (p.p437l), was found in two patients and one healthy subject. other groups have reported this variant in a similar frequency between cases and controls in north american and european populations [18, 19 ]. although our in silico predictions have classified this variant as probably pathogenic, this mutation was present in a brazilian control individual (59 years old), which leads us to believe that it is a nonpathogenic polymorphism. we have identified this mutation in three asymptomatic daughters (35, 33, and 31 years old) and a granddaughter (12 years old) of a carrier patient. functional analyses should help to clarify if the p.p437l substitution is a risk factor for pd. the c.434 g > a (p.s145n) variant was found in a patient that also harbors the polymorphism c.500 g > a (p.s167n) in the same exon of park2 gene. the in silico analysis had contradictory results, and this mutation has its pathogenic nature unclear in the literature. although the pathogenicity is not yet confirmed, we believe that functional analyses are important to establish if the p.s145n variant is related with a high risk of developing pd. three changes identified in this study have never been described : c.659a > g (p.k220r), c.1016c > t (p.a339v), and c.1021c > t (p.l341l). the first one (p.k220r) was found in only one patient, in heterozygous state. in addition, this mutation does not encode any part of the catalytic domain of the protein. we believe that it might be a rare polymorphism, but functional studies and segregation analysis would be valuable tools to determine the pathogenicity of this substitution. two novel mutations were found in exon 9 of park2 : p.a339v in one patient with familial history of pd and p.l341l in two sporadic cases. we did not observe the p.a339v variant in 400 control chromosomes, reinforcing the rarity of this variant or its potential association with the patient 's phenotype. on the other hand, the in silico analysis showed contradictory results, which did not help to clarify the pathogenicity of this mutation. another known missense mutation in the same amino acid residue had already been identified and does not cause phenotypic changes. we can not classify the pathogenicity of this variant until functional studies are performed. the second new alteration found in park2 exon 9 (c.1021c > t) results in a silent mutation, p.l341l. the in silico analysis using spliceview and nnsplice programs showed that this variant does not affect the recognition of donor and receptor sites of splicing, and, because of its silent nature, this change is considered nonpathogenic. as this substitution has already been found in some healthy control subjects by other studies [1, 25 ], it was predicted to be benign by bioinformatic programs and is poorly conserved even within mammals, and we suggest it is a nonpathogenic variant. all putative pathogenic point mutations identified in this study in park2 gene were in heterozygous state (table 1). until now, the role of homozygous and compound heterozygous variants was already established as a cause of autosomal recessively inherited eopd, but the pathogenic significance of heterozygous mutations is still uncertain, particularly for missense substitutions in the context of recessive inheritance. different mechanisms have been suggested to explain the effects of single heterozygous variants like loss - of - function mutations by lowering the biological activity of the encoded protein (haploinsufficiency), dominant - negative property, or gain - of - function dominant mutations. so, increasing evidence indicates that heterozygous variants are noncausative mutations but rather genetic susceptibility factors which may contribute to the risk of developing pd. our findings showed that park2 point mutations are more frequent than pink1 pathogenic variants in our sample of brazilian eopd patients. the absence of pathogenic mutations in pink1 gene in our population is consistent with other studies [19, 23, 27 ], supporting the hypothesis that mutations in pink1 may not be a relevant cause of eopd among brazilian sporadic and familial patients. our results of park2 gene are in agreement with studies worldwide that have found similar frequencies of park2 point mutations in different populations [1820, 23, 24 ] (table 3), although they differ from the frequency identified by other brazilian groups (5.5% and 11.1%) [21, 22 ]. one possible explanation for these different results would be the reduced sample sizes tested by them (72 and 45 patients, resp.) we strengthen that the functional analyses of the missense variants found by us are still missing and would help us to clarify the real pathogenic value of these mutations in our population. besides, whether heterozygous mutations in recessive genes act as susceptibility factors or as causal agents in the pd process remains to be determined. | parkinson 's disease is the second most frequent neurodegenerative disorder in the world, affecting 1 - 2% of individuals over the age of 65. the etiology of parkinson 's disease is complex, with the involvement of gene - environment interactions. although it is considered a disease of late manifestation, early - onset forms of parkinsonism contribute to 510% of all cases. in the present study, we screened mutations in coding regions of park2 and pink1 genes in 136 unrelated brazilian patients with early - onset parkinson 's disease through automatic sequencing. we identified six missense variants in park2 gene : one known pathogenic mutation, two variants of uncertain role, and three nonpathogenic changes. no pathogenic mutation was identified in pink1 gene, only benign polymorphisms. all putative pathogenic variants found in this study were in heterozygous state. our data show that park2 point mutations are more common in brazilian early - onset parkinson 's disease patients (2.9%) than pink1 missense variants (0%), corroborating other studies worldwide. |
obesity is a worldwide epidemic with a major burden on the healthcare systems and is now recognized as a disease (http://www.ama-assn.org/). obesity is an important determinant of cardiovascular diseases (cvd) as it promotes a cluster of cvd risk factors including dyslipidemia, type-2 diabetes (t2d), and hypertension. the arab population of the gulf cooperation council (gcc) countries has experienced rapid socioeconomic growth and changes over the last two decades, resulting in a tremendous increase in obesity as well as its associated comorbidities. indeed, gcc countries including kuwait are ranked among top 10 countries with the highest prevalence of t2d worldwide. kuwait has also the highest rate of obesity in the region with more than 45% of adults being obese : 53% in females as compared to 39% in males. chronic inflammation and excessive oxidative stress are key pathophysiological hallmarks of obesity and their contribution to downstream complications is well established [5, 6 ]. sedentary lifestyle, physical inactivity, and overconsumption of a calorie - rich diet are among the modifiable factors that lead to this chronic condition. white adipose tissue has been identified as the predominant site that produces various bioactive molecules including cytokines, adipokines, crp, and free fatty acids which lead to aberrant regulation of inflammatory and stress kinases and ultimately a loss of cellular homeostasis. although chronic inflammatory response is an established fact in obesity, the molecular determinants that trigger this response and maintain it in a sustained state are still poorly understood. reactive oxygen species (ros) and free fatty acids have however been proposed as potential contributors to this process. indeed, conditions that lead to increased oxidative stress are also known for their ability to lead to inflammation, in large part through the activation of nf-b. in turn, activated inflammatory cells release high levels of ros that potentiate the inflammatory response. thus, the relationship between oxidative stress and inflammation is more complex than it was originally thought, and it is clear that inflammation and oxidative stress are mutually inclusive and most likely they operate by creating a cycle which exacerbates them. it is well established that oxidative stress is caused by an imbalance between increased production of ros and reduced antioxidant defense. for instance, paraoxonase-1 (pon1) is an antioxidative enzyme preventing the formation of oxidized lipoproteins. pon1 is synthesized primarily in the liver and is secreted into the bloodstream, where it protects phospholipids by associating with high - density lipoproteins (hdl). the status of oxidative stress and inflammatory response with respect to gender effect on cvd risk factors has been investigated in different ethnic populations and clear gender - related effects were reported [1315 ]. these dissimilarities in plasma levels are often attributed to physical and physiological differences between males and females, such as adiposity distribution, lean muscle mass, and differences in hormone levels [1315 ]. therefore, expanding these studies by further stratifying various ethnic groups according to gender will provide insights regarding the gender effect on the relationship between inflammatory and stress markers and their association with cvd risk factors. as studies that investigated these two responses in the arab population are scarce, we designed the present study as a cross - sectional analysis on 378 arab adults living in kuwait in order to assess gender differences in oxidative stress and inflammatory circulating markers and their association with cardiometabolites. this study is of emerging interest in cvd research, since the use of circulating biomarkers might help in the stratification of cvd risk factors. this population - based cross - sectional survey was carried out between june 2011 and august 2012 and was undertaken on adult male and female arab emigrants in kuwait. according to the 2011 kuwait consensus, 67.7% of the population in kuwait were expatriates hailing mostly from arab countries such as egypt, syria, lebanon, palestine, jordan, and arab gulf countries. subjects were selected randomly from the computerized register of the public authority of civil information. the study conformed to the principles outlined in the declaration of helsinki and was approved by the institute 's scientific advisory board and ethical review committee at dasman diabetes institute (ddi). an informed written consent was obtained from all the participants before their enrolment in the study. physical and anthropometric measurements included body weight, height, waist circumference (wc), and blood pressure (bp). bp was measured with omron hem-907xl digital sphygmomanometer and an average of 3 readings, with 510-minute rest between each, was noted. height and weight were measured with participants wearing light indoor clothing and bare - footed using calibrated portable electronic weighing scales and portable inflexible height measuring bars. wc was measured using constant tension tape at the end of a normal expiration, with arms relaxed at the sides, at the highest point of the iliac crest at the midaxillary line. body mass index (bmi) was calculated, using standard metric bmi formula (kg / m). obesity was assessed using bmi cut - off standard criteria ; bmi between 18.5 and 24.9 was considered normal, 25 to 29.9 was considered overweight, and equal to or higher than 30 was considered obese. according to international diabetes federation (idf), abdominal obesity (central obesity) was defined as wc 94 cm in males and 80 cm in females. blood samples were obtained after an overnight fast of at least 10 hours and analyzed for fasting blood glucose (fbg), hba1c, fasting insulin, and a lipid profile that included triglycerides (tg), total cholesterol (tc), low - density lipoprotein (ldl), and high - density lipoprotein (hdl). all laboratory tests were performed by certified technicians at the clinical laboratories of ddi using the ministry of health approved methods and quality standards. insulin resistance was calculated using homeostasis model assessment (homa - ir) formula : fasting insulin (mu / l) fbg (mmol / l)/22.5. to measure inflammatory and metabolic biomarkers, plasma was obtained after centrifugation at 1700 g for 15 min at 4c, aliquoted, and then stored at 80c until assayed. plasma levels of inflammatory markers were assessed with the multiplexing immunobead array using the bio - plex pro human cytokine 27-plex immunoassay kit that included il-6, il-8, and tnf- (bio - rad, hercules, ca). data was analyzed with bio - plex manager software version 6 (bio - rad, hercules, ca). lipid peroxidation was assessed by measuring plasma levels of malonaldehyde, using tbars assay kit (cayman chemical company, ann arbor, mi). serum levels of ros were determined using the oxiselect ros assay kit (cell biolabs inc., san diego, ca). paraoxonase-1 activity was measured using enzchek paraoxonase assay kit (life technologies, grand island, ny). c - reactive protein (crp) levels were measured using high sensitivity crp hscrp all analyses were performed using sas (version 9.4 ; sas institute, cary, nc). comparisons of subjects characteristics between genders were made by student 's t - test or wilcoxon test for nonparametric analyses in variables with nonnormal distribution. spearman correlation coefficients were estimated to determine associations between levels of the various inflammatory and oxidative stress markers investigated and anthropometric and clinical measurements. age, bmi, and wc / gender adjusted geometric mean values of various inflammatory and oxidative stress markers concentrations in men and women were calculated using analysis of covariance (ancova). to examine independent correlates of various inflammatory and oxidative stress markers, multivariate linear regression models were constructed with log - transformed biomarkers as the dependent variable. all data were reported as mean standard deviation (sd) and range, unless otherwise stated. research electronic data capture (redcap) was used for data collections and data management. all statistical assessments were two - sided and considered significant at p value < 0.05. a total of 378 participants consisting of 185 males and 193 females matched for age were enrolled in this study with a mean age of 44.4 11.7 years. females had higher bmi but lower wc than males (p < 0.0001 and p = 0.0153, resp.). by contrast, the sbp and dbp were significantly higher in males compared to females (p < 0.0001 and p = 0.0252, resp.). likewise, the glycemic markers were higher in males than in females as monitored by fbg (p = 0.0424) and hba1c (p < 0.0001). there was also an alteration of lipid profile between the two groups as females had increased levels of both cholesterol (p = 0.0353) and hdl (p < 0.0001) but decreased levels of tg (p = 0.0045). in our study population, 206 subjects showed high blood pressure, including 105 subjects previously diagnosed, of which only 77 were taking medication. regarding diabetes, 109 were diabetic ; among them 98 subjects were previously diagnosed and 92 receiving treatment. 27 subjects (7.2%) already had a history of cvd ; however, for our study, we only collected data related to diabetes treatment. according to our survey, females were more physically active and smoked less than males (p = 0.0026 and p < 0.0001, resp.). gender differences in circulating makers of inflammation (crp, il-6, il-8, and tnf-) and oxidative stress (ros, tbars, and pon1) were measured and the medians are displayed in table 2. accordingly, the levels of crp, ros, and tbars were all significantly high in females compared to males (p < 0.0001). by contrast, males had higher levels of il-8 (p = 0.0032) and the levels of il-6, tnf-, and pon1 were comparable between the two genders (table 2). after adjustment for age, bmi, and wc, similar patterns between males and females were observed for crp (p = 0.0022), il-8 (p = 0.0092), tbars (p < 0.0001), and ros (p while there was still no significant difference between genders in the activity of pon1, the concentration levels of il-6 and tnf- were significantly higher in males than females (p < 0.05). these observations prompted us to investigate the possible associations between these markers and various anthropometric and clinical parameters of our study population. to this end, we performed an initial spearman correlation on all subjects and the data are displayed in table 3. as shown, crp correlated positively with most of the parameters but the highest significant correlations were found with bmi, wc, and homa - ir (p < 0.0001). other positive correlations were also found for tc and ldl (p < 0.001), tg (p < 0.01), and dbp and hba1c (p < 0.05). similar to crp, il-6 correlated positively with age and bmi (p < 0.01), wc (p < 0.001), and fbg, hba1c, and homa - ir (p < 0.05) but negatively with hdl (p < 0.05). in the case of il-8, a positive correlation was found for age, hba1c, and tg (p < 0.001) as well as wc, homa - ir, and tc (p < 0.05) but a negative correlation with hdl (p < 0.05) (table 3). by contrast to the inflammatory markers, we observed less correlations with the oxidative stress markers but, as shown in table 3, a negative correlation was observed between tbars and bmi (p < 0.05) and a positive correlation between ros and hba1c (p < 0.0001). to investigate the specific gender effect of each of these markers with the anthropometric and clinical parameters, we performed a gender - based spearman correlation and the results are displayed in table 4. under our conditions, crp levels in females were associated positively with tc (p < 0.001). in males, however, crp correlated positively with sbp and negatively with hdl (p < 0.001). il-6 correlated in females positively with fbg, homa - ir, tc, and tg (p < 0.05). il-8 correlated also in females positively with homa - ir, tc, and tg (p < 0.05) but negatively with hdl (p < 0.05) (table 4). for the markers of oxidative stress, tbars levels correlated positively in females with hdl (p < 0.05), whereas pon1 levels correlated negatively with fbg, hba1c, and tg (p < 0.05) and positively with hdl (p < 0.05). in males, pon1 levels correlated positively with tc (p < 0.01) and ldl (p < 0.05) (table 4). we next investigated the possible association of central obesity with the inflammatory and oxidative stress markers after adjusting for age, gender, and bmi. to this end, we choose two groups within our study population with either normal or higher central obesity as measured by wc and the data are illustrated in figure 2. as shown, our data indicated that, out of the seven markers investigated, central obesity was associated with increased levels of tnf- (p < 0.05), il-6 (p < 0.01), and ros (p < 0.05). the increase in the incidence and prevalence of obesity and its related comorbidities in the countries of the gcc are of extreme health and socioeconomic concerns. nonnational arabs living in the gcc region are also vulnerable to these chronic disorders, highlighting the pivotal role of environmental factors, namely, excessive food energy intake, sedentary lifestyle, and physical inactivity, as major contributors to these chronic conditions. in this study, we investigated the status of the inflammatory and oxidative stress responses in nonnational arabs of kuwait with and without central obesity and evaluated whether there was a gender difference in these two responses as well as their association with classical cvd risk factors. after adjustment for bmi and wc, the main findings of the study are as follows : (1) higher levels of tnf-, il-6, and il-8 were observed in males, whereas females had higher levels of crp, ros, and tbars ; (2) the levels of tnf-, il-6, and ros were associated more with wc rather than the bmi ; (3) despite their apparent healthier status, arab females might be at risk of cvd complication due to increased oxidative stress. these results support the hypothesis that central adiposity dysfunction may play a critical role in the cross talk between biomarkers of inflammation and oxidative stress and probably other metabolites to constitute major cvd risk factors that may indirectly contribute to cvd. to the best of our knowledge, this was the first cross - sectional study that investigated the status of the inflammatory and oxidative stress responses in nonnational arabs of kuwait with relationship to central obesity and the risk of cvd. although direct causality can not be inferred from these correlation studies, our data provide an additional support that warrants further investigations to understand the cross talk between inflammation and oxidative stress with respect to gender and their pathophysiological impacts on metabolic diseases. the status of the inflammatory and oxidative stress responses with respect to obesity and its association with cvd risk factors has been investigated intensively in several cross - sectional studies involving many ethnic groups in both males and females [1825 ]. these studies were pivotal in pointing to the existence of gender differences in the inflammatory and stress responses. more importantly, they revealed the existence of a myriad of factors including the proportion of fat tissue and its distribution, the level of sex hormones, genetic factors, lifestyle habits that could contribute to these differences, and ultimately the gender - specific risk of metabolic diseases. consistent with these studies, the levels of circulating tnf-, il-6, and ros in our study population were associated with central obesity after adjusting for age, gender, and bmi (figure 2). the observed profile appeared to be specific to wc as there was no significant association between these markers and the bmi of our study population when adjusted for age, gender, and wc (data not shown). our findings corroborate and further extend those obtained from the framingham heart study in which indicators of central adiposity and not global obesity were strongly associated with inflammation and oxidative stress markers. in agreement with these findings, a more recent study carried out on 100 emirati obese - diabetic subjects consisting of 41 males and 59 females reported also that wc indicated adiposity better than bmi and that increased wc associated with increased inflammation and oxidative stress. they also reported that females had higher crp and decreased antioxidant status (vitamin c) and hdl and that these levels were significantly associated with adiposity as measured by wc. likewise, a more closely related study that investigated the gender differences in fat distribution and inflammation in arabs was carried out on 58 healthy qatari consisting of 29 males and 29 females. in their study, while there was no significant difference in the levels of crp and il-6 between genders, they found a strong correlation of crp and il-6 with wc and sbp in males. the above findings did not fully agree with another investigation that was carried out recently on 1005 relatively lean chinese females in which both wc and bmi were associated with various markers of inflammation, namely, crp, tnf-, soluble tnf - receptor 1 (stnf - r1), and il-6, and the marker of oxidative stress f2-isop - m. other studies have also shown that female gender and bmi are independent risk factors of metabolic syndrome in turkish and gaza strip populations [26, 27 ]. relationships between anthropometric adiposity indicators and cvd risk factors have been already thoroughly explored in many ethnic groups. our spearman correlation analysis showed that inflammatory markers (il-6, il-8, and crp) correlated positively with adiposity (wc and bmi) and with the classical cardiometabolites (blood pressure, sugar index, and lipids index) in whole population (table 3). however, after adjusting our data for wc, correlation with bmi became statistically insignificant (data not shown). indeed, the majority of studies suggest that wc is a better indicator of cvd risk than bmi and has been proven to be an appropriate clinical measure of the pathogenic potential of adipose tissue amongst populations [2830 ]. in our current investigation, we adjusted for age, bmi, and wc and found that crp, ros, and tbars levels were significantly higher in females whereas males had increased levels of tnf-, il-6, and il-8 (figure 1). reported also increased levels of crp in premenopausal females and increased levels of il-6 and tnf- in males. other independent studies have also demonstrated higher levels of oxidative stress markers in females as compared to males [31, 32 ] and it was suggested that oxidative stress may be a key contributor to increased cvd risk in females, presumably through crp [30, 31 ]. crp was initially described as an inflammatory marker of cvd ; however, recent evidence indicated its link to oxidative stress [34, 35 ]. moreover, cartier. concluded from their study that the observed gender difference in crp concentrations can not be explained by the visceral adipose tissue depots. instead, they suggest that the higher crp concentrations found in women are linked to higher subcutaneous fat in women. in agreement with these hypotheses, our results showed that females display higher crp levels after correction for adiposity markers as compared to males and when adjusted for gender and bmi, crp did not associate with wc (figure 2). the high bmi and wc as well as elevated tc and ldl in females enrolled in our study may explain this increase in oxidative stress response as compared to males. it is generally accepted that the prevalence of cvd is higher in males and postmenopausal females [36, 37 ]. however, epidemiological data carried out on arab population indicated that cvd associated risks are elevated even in younger females compared to other ethnic groups [38, 39 ]. the current study sheds light on those risks in premenopausal females and may suggest that impairment of the oxidative stress response may be a key cvd risk factor rather than classical risk factors. in support of this, lower levels of glucose levels, dyslipidemia, and hypertension were observed in females as compared to males (table 1). by contrast, males displayed lower oxidative stress despite their high bmi, wc, fbg, tg, and ldl (table 2). classifying the study subjects into different categories of obesity might reflect better the link of adiposity distribution on inflammation and oxidative stress. indeed, obesity is described as being a state of low - grade chronic inflammation in which increased il-6, il-8, and tnf- levels have been described as obesity - induced risk factor of cvd [40, 41 ]. adipose tissue is a potent source of proinflammatory and redox mediators, suggesting that sex differences in total and regional adiposity could influence risk of cvd. evidence showed that lipid ratio, in particular tg / hdl, performs better than individual lipids in predicting cvd risk. our data showed that this ratio is higher in males than in females (data not shown). moreover, lipid ratios displayed a strong correlation with inflammation (il-6, il-8, and tnf-), particularly the tg / hdl and its logarithmic function (aip), which also associated negatively with the antioxidant enzyme pon1 in males (data not shown). the latter associations could reflect a direct effect of systemic inflammatory stress on lipoproteins or indirectly through metabolic alterations. accordingly, recent study reported that dysfunctional hdl might be considered as novel target in cvd. although the interaction between inflammatory mediators and lipoproteins is not clear, there is increasing evidence that the systemic inflammatory state potentiates cvd such as atherosclerosis. it should also be noted that not all obese individuals are at a similar risk of these complications, since a significant proportion of obese individuals are metabolically healthy. prevalence of this healthy obese phenotype was reported to be 2 to 4 times higher in females as compared to males. interestingly, a recent study showed a positive association between metabolically healthy obese phenotypes in females having a moderate to high level of physical activity. they further suggested that metabolically healthy obese individuals may have a reduced inflammatory status. in agreement with this observation, our data indicated that females displayed healthier metabolic profile and lifestyle as compared to males. this suggests that factors other than simple energy imbalance, such as oxidative stress, might determine the obesity risk outcomes. hence, the increased oxidative stress observed in the current investigation in apparently healthy females supports the concept that oxidative stress could represent a reliable predictor of obesity and cvd in females. thus, arab females are probably more prone to the detrimental effects of oxidative stress at lower blood glucose, lipid thresholds, and inflammation than males. our study has, however, several limitations that deserve consideration for future follow - up studies. for instance, this is a cross - sectional study from which it is difficult to extract meaningful conclusions regarding direct causality of cvd. second, given the complex cross talk between the inflammatory and stress responses, it is still difficult to identify the triggers that initiate such responses and to establish the hierarchical sequence of events that lead to a perturbation of normal homeostasis. furthermore, our study used an indirect measurement for generalized adiposity and central adiposity indicators, which probably have less accuracy, compared to direct measurement methods. furthermore, other confounding factors such as diet, genetic determinants, and sociocultural conditions may affect our findings. additionally, we used international standards for cut - off points for bmi and wc and this might not be fully accurate for arabs as was reported previously. our findings might be improved once appropriate cut - off points for arabs are established. despite these limitations, our study is the first that investigated the association of classical cvd risk factors with oxidative stress and inflammation in the arab population living in kuwait. having a relatively healthier metabolic profile compared with males, females showed an association with oxidative stress markers which may be considered as a risk factor of cvd regardless of their lower inflammatory status. males, however, displayed a metabolically unhealthy profile with increased inflammation levels probably reflecting their poor lifestyle including lower physical exercise and higher smoking rates. a further study is currently underway to prospectively follow up this cohort to determine directionality of the observed associations and trends. | background. the impact of gender difference on the association between metabolic stress and cardiovascular disease (cvd) remains unclear. we have investigated, for the first time, the gender effect on the oxidative and inflammatory stress responses and assessed their correlation with classical cardiometabolites in arab population. methods. a total of 378 adult arab participants (193 females) were enrolled in this cross - sectional study. plasma levels of crp, il-6, il-8, tnf-, ros, tbars, and pon1 were measured and correlated with anthropometric and cardiometabolite parameters of the study population. results. compared to females, males had significantly higher fbg, hba1c, tg, and blood pressure but lower bmi, tc, and hdl (p < 0.05). after adjustment for bmi and wc, females had higher levels of ros, tbars, and crp (p < 0.001) whereas males had increased levels of il-8, il-6, and tnf- (p < 0.05). moreover, after adjustment for age, bmi, and gender, the levels of tnf-, il-6, and ros were associated with central obesity but not general obesity. conclusion. inflammation and oxidative stress contribution to cvd risk in arab population linked to gender and this risk is better reflected by central obesity. arab females might be at risk of cvd complications due to increased oxidative stress. |
over 100 years ago kraepelin conceptualized dementia praecox and manic - depressive psychosis as two distinct diseases. however, many similarities between these two classes have been noted and the validity and heuristic value of this dichotomy is in question, especially when recent genetic findings are considered (lake, 2007 ; craddock & owen, 2005 ; moller, 2003). with the diagnostic and statistical manual of mental disorders (dsm) set to undergo some significant changes in its fifth edition, we defend the position of separate disorders and point out two major problems in joining the diagnoses. we explain why overlapping features, including psychosis, are not decisive in the debate. we also posit that each disorder is a clinical syndrome rather than a specific disease entity. as such, a domains of pathology approach is a more heuristic paradigm for etiologic, pathophysiologic, and therapeutic discovery, and may clarify points of similarity and decisive differences between the two syndromes. the problem with using psychosis, especially reality distortion symptoms such as delusions and hallucinations to define a disease entity, is simple. psychosis is a common manifestation of many diseases that are distinguished at the level of etiology. causes of psychotic experience range from sensory isolation to temporal lobe epilepsy and a number of psychotic conditions have known causes (e.g., pcp psychosis or huntington s disease). psychosis is on a continuum in human experience and is not uncommon in population surveys (rossler., 2007 ; van os., 2000 ; psychosis also occurs in circumstances not considered pathological (e.g. religious ecstasy). defining psychosis as an illness has required secondary criteria relating to disability and/or distress. but for schizophrenia and bipolar illnesses, disability and discomfort are more robustly associated with cognitive impairments, negative symptoms (in schizophrenia), and mood pathology (in bipolar). indeed, the brain generates hallucinations and delusions in so many conditions that it is difficult to understand how these symptoms have maintained primacy in the diagnosis of any specific disease. psychotic experience is to the diagnosis of mental illness as fever is to the diagnosis of infection important, but non - decisive in differential diagnosis. for example, mcintosh (2008) probe language dysfunction and observe decreased brain activation in both disorders, but an anatomic distinction with anterior insula in bipolar and dorsal prefrontal cortex in schizophrenia. similar forms of symptoms may be phenomenologically distinguished (e.g, pressured thought in mania and dissociative thought in schizophrenia ; mood congruent delusions in bipolar and more bizarre delusions in schizophrenia [solovay., 1987 ; shenton., 1987 ]), and causal mechanisms may be different even if the neural substrate is similar. with important similarities and differences, the paramount issue is whether the two diagnostic classes comprise one heterogeneous disorder with an artificial boundary or two disorders with overlapping features. the challenge to the kraepelinian dichotomy may not have become relevant had the core pathologic features, which originally distinguished dementia praecox and manic - depressive psychosis, survived in dsm - iii and iv. kraepelin described the two general maladies of dementia praecox : dissociative pathology, i.e. disorganization of thought and/or behavior, and a weakening of the well - springs of volition, bleuler also considered these pathologies fundamental (bleuler, 1950/1911) and considered reality distortion symptoms as secondary. the fundamental features of dissociation and avolition were critical in distinguishing schizophrenia from manic - depressive psychosis. debate as to whether the kraepelinian dichotomy is valid presumes that the current concept and criteria for schizophrenia relate closely with the original construct. this is not the case. for these pioneers it was the closely linked dissociative and avolitional pathology that described schizophrenia, and their co - occurance in individuals that defined caseness. the presence of hallucinations and delusions, or just delusions if bizarre, satisfy criteria a for schizophrenia even in the absence of dissociative and avolitional pathologies. avolition was not even included as a criterion in dsm - iii. in an effort to improve diagnostic reliability, schneider proposed that certain reality distortion symptoms (referred to as first rank symptoms) were highly discriminating of schizophrenia (1959). in europe, coupled with langfeldt s distinction between true and pseudoschizophrenia (1969), these concepts gelled in the concept of nuclear schizophrenia. during this time in the united states, scientists from washington university were organizing diagnoses with explicit criteria and validation based on onset, manifest pathology, course, treatment, and associated biologic features. this approach, modified and put forward as the research diagnostic criteria (rdc) by spitzer and colleagues (1979), joined the european nuclear schizophrenia tradition as the foundation for the dsm - iii approach to psychoses. however, in the transition from kraepelin / bleuler to dsm - iii, the concept of schizophrenia has been remarkably altered. that the original conceptualization of schizophrenia differs from current diagnostic criteria however, studies conducted during the late 1960s and early 1970s tested several versions of the nuclear schizophrenia concept and suggested just such a conclusion. first rank symptoms were observed in psychotic classes other than schizophrenia (carpenter & strauss, 1974 ; strauss., 1974 ; carpenter., 1973a) ; none of the definitions of nuclear schizophrenia based on supposedly pathognomonic reality distortion symptoms predicted course and outcome (hawk., 1975 ; strauss & carpenter 1974a ; strauss & carpenter 1974b ; carpenter. 1973b) ; and functional outcomes were mainly related to other aspects of the syndrome (hawk., 1975 ; strauss & carpenter 1974a ; strauss & carpenter 1974b ; carpenter. the most robust symptomatic distinction between schizophrenia and non - schizophrenia psychotic diagnostic classes were restricted affect, poor rapport, and poor insight (carpenter., 1973b). for dsm - iii, presumptions of validity of the nuclear schizophrenia concept trumped this empirical data falsifying the key hypotheses relating to the schneiderian and langfeldt systems. this shift in concept has profound implications and moves bipolar illness and schizophrenia closer together by emphasizing common features while de - emphasizing the pathological attributes that originally distinguished the conditions. kraepelin also distinguished manic depression from dementia praecox based on course, the former being cyclical and the latter chronic. does this difference carry any nosological information in an era of chronic mania (malhi., 2001) and remission / recovery in schizophrenia (fischer & carpenter, 2008) ? two current issues complicate testing of kraepelin s original observation : the first is the concept alteration in our modern diagnostic scheme. recent longitudinal studies in schizophrenia use diagnostic criteria that emphasize psychosis and do not require avolitional pathology. restricted affect was the most robust predictor of poor 5 -year outcome in the washington center of the international pilot study of schizophrenia (carpenter., 1978), and prominent negative symptoms are associated with a more chronic course (strauss. onward, treatment has been robustly influenced by movement away from chronic institutionalization and by pharmacotherapy. nonetheless, longitudinal data from pre- and post- antipsychotic drug eras reveal heterogeneity of course for schizophrenia (marengo, 1994). since most studies follow cohorts of already chronic cases, a bias towards chronic morbidity is present and good outcome cases may be excluded. a typical course for schizophrenia is difficult to define, use as a validating criteria for classification. the course pattern early in bipolar disorder is primarily mood pathology and episodic, but long - term follow - up also report eventual chronic course sometimes including apparent negative symptoms. the debate over whether schizophrenia and bipolar disorder are one disease or two is based on the assumption that each diagnosis is homogeneous enough to consider the combination. but there has been no documentation of a unifying etiopathophysiology within either schizophrenia or bipolar disorder. if this were the case, the debate would be settled by determining whether the specific pathological causal pathway is the same for both diagnostic classes. the hypothesis that deficit schizophrenia represents a separate disease within the syndrome of schizophrenia illustrates this concept (kirkpatrick., 2001 ; carpenter., 1988). realization that schizophrenia and bipolar disorders have syndrome status shifts the question to whether there is a disease entity within one of the syndromes that is a better fit in the other syndrome. and consider asking whether dementia and delirium are one disease or two. sharing some prominent cognitive impairment the heterogeneity within each syndrome would undermine any investigation designed to answer the one disease or two question. then remove multi - infarct dementia and the syndrome is more narrowly defined, but still a heterogeneous condition comprising pernicious anemia dementia, dementia associated with traumatic brain injury, parkinson s disease, and other discrete conditions. most investigations of schizophrenia and bipolar disorders are conducted as though a disease entity has been defined. unlike dementia and delirium, the tools to reduce heterogeneity of these two mental illness syndromes are not yet clearly established. nonetheless, study designs that treat a syndrome as though it were a specific disease entity provide a weak methodology for decisive hypothesis testing (carpenter., 1993). studies within schizophrenia that contrast subjects with and without primary negative symptoms (deficit versus non - deficit schizophrenia) reveal differences in some clinical features (e.g. vulnerability to depression and substance abuse) while being similar in psychotic symptoms (kirkpatrick., 1996 ; fenton & mcglashan, 1994 ; kirkpatrick. importantly, risk factors (e.g. an excess of summer births in the deficit syndrome as opposed to late winter - early spring births in schizophrenia in general [messias., 2004 ; kirkpatrick., 2002a ; kirkpatrick., 2002b ; tek., 2001 ; messias & kirkpatrick, 2001 ; kirkpatrick., 2000 ; kirkpatrick., 1998 ]) and neuropathology [kirkpatrick., 2003 ; kirkpatrick., 1999 ]) seem to distinguish the two forms of schizophrenia. results of comparing bipolar subjects to schizophrenia subjects would be quite different on key variables if the schizophrenia cohort is exclusively deficit or exclusively non - deficit. in principle the question of one disease or two is meaningless if each construct subsumes two or more entities that are importantly distinct from each other. this seems to be the case in schizophrenia (tandon and maj, 2008) and is likely the case in bipolar disorder as well (potash., 2003 ; potash. there are many features commonly observed in cases within both schizophrenia and mood disorders such as anxiety, depression, reality distortion, insomnia, and cognitive impairment. family pedigree studies generally support the dichotomy, but linkage and association studies suggest overlap or shared genetic vulnerability (berrettini, 2000). endophenotypes have been identified in both syndromes, often overlapping, sometimes not (ivleva., 2008 ; hill., 2008 ; thaker, 2008). consider this thought experiment : risk alleles for genes x, y and z have been identified for hallucinatory experience in the general populationthese risk alleles distinguish bipolar from non - ill controlsthese risk alleles distinguish schizophrenia from non - ill controlsthe association is stronger in schizophrenia compared to bipolar this pattern of finding seems plausible, but would not suggest that the two illness syndromes are the same disease. rather, it would suggest that hallucinatory behavior is associated with risk genes across cohorts that differ in the proportion of hallucinating subjects. testable hypotheses include : a. risk alleles for genes x, y, and z will distinguish bipolar subjects with hallucinations from bipolar subjects without a history of hallucinations ; and, b. overlap between schizophrenia and bipolar associations to these risk alleles will increase if all subjects in each class are required to have a history of hallucinations. how, then, should the field proceed in order to advance knowledge on the relationships among the diseases contained in the two syndromes ? risk alleles for genes x, y and z have been identified for hallucinatory experience in the general population these risk alleles distinguish bipolar from non - ill controls these risk alleles distinguish schizophrenia from non - ill controls the association is stronger in schizophrenia compared to bipolar one approach is illustrated by owen, craddock and jablensky (2007) in their genetic deconstruction of psychosis. they propose that overlapping genes such as disc1 and nrg1 contribute to psychotic and mood pathology and that other genes (e.g., daoa and bdnf) lead to the mood disorders prototype. genes such as dysbindin would move the picture towards typical schizophrenia. this concept places the psychotic disorders on a continuum with differential etiopathophysiological factors defining the two extremes. if mood and psychotic features are central, this is compatible with one disease with different co - morbid pathologies at the two extremes. if the pathologies at the two extremes are considered central to diagnostic class, this model would imply that mood disorder and schizophrenia are separate diseases with shared psychotic and mood pathology. a more explicit approach to this second alternative domains of pathology paradigm (strauss, 1974 ; carpenter and buchanan, 1989). breaking down each diagnostic class into domains of pathology gives more specificity to developing treatments and elucidating etiopathophysiology. the unit of study moves from diagnostic classes with porous boundaries to specific psychopathologies which are important to class but not unique. rather than porous boundaries confounded study designs, the pathological domains become the focus even though observed in more than one class [and not necessarily in all subjects within a class ]. a focus on certain pathological domains to make samples more homogeneous may lead to more consistent findings and, therefore, better understanding of the genetic etiology of diseases. this appears to be the case in preliminary studies where a different pattern of morbid risk was observed between deficit schizophrenia probands and non - deficit schizophrenia probands (kirkpatrick., 2001). indeed, an analysis of genetic data using latent class analysis to identify subgroups of psychosis revealed genes associated with the deficit subgroup that were not observed when data was analyzed by diagnostic class (fanous., endophenotypes (gottesman and gould, 2003) may be even more decisive in this regard (braff., 2007 ; turetsky., 2007 ; aukes., 2008 ; javitt., 2007 ; thaker, 2007 ; freedman., 1999) and were critical in identifying an alpha 7 nicotinic receptor gene on chromosome 15 as a candidate for involvement in schizophrenia pathology (freedman., 1997). a recent article by thaker (2008) describes the current status and challenges involved in application of endophenotypes across bipolar and schizophrenia. genes that confer risk for both bipolar disorder and schizophrenia may best be understood at the level of a specific psychopathological dimension. the general hypothesis is that shared risk factors and pathophysiology will be associated with domains of pathology that overlap between classes. non - shared risk factors and pathophysiology will be associated with non - overlapping pathology. the importance of addressing these issues is made clear by lichtenstein (2009) in a study of over 76,000 schizophrenia and bipolar probands and their families. in the best estimate to date, about 60% of the variance in each group is attributed to genetic factors, about equally divided between shared and unique genetic effects. do the unique factors simply add to liability for diagnostic class, or are they more specifically related to domains such as avolition in schizophrenia and episodic affect disruption in bipolar ? it remains to be determined which pathologies are critical for diagnostic class or, for that matter, how classification will be revised based on new data related to this paradigm shift. another crucial issue comes from the observation that both people with schizophrenia and people with bipolar disorder demonstrate cognitive impairments. in general, people with schizophrenia have worse cognitive impairment than people with bipolar disorder (keefe & fenton, 2007 ; krabbendam., 2005). cognitive impairments in schizophrenia are observed during pre - psychotic development and are remarkably constant in individuals over the course of their illness whereas cognitive impairments in mood disorders have shown variability depending on phase of illness (keefe & fenton, 2007 ; hill., 2008). however, evidence is accumulating that, among mood disorders, the presence of psychosis indicates worse cognitive impairment and people with psychotic bipolar disorder demonstrate cognitive impairment similar to that observed in schizophrenia (seidman., 2002 ; glahn.. impaired cognition associated with bipolar disorder has also recently been observed during non - medicated and euthymic states and in first - degree relatives suggesting at least a subtle trait impairment (malhi. 2008). is impaired cognition in the two disorders based on the shared genetic effects with greater load for schizophrenia, or does similarity of impairment represent a common final pathway based on unique genetic or environmental effects ? similarly, vulnerability genes for depressed mood may be shared among depressed cases in both syndromes. kempf, hussain and potash, (2007) conclude that dimensions are the critical unit of analysis when comparing mood disordered schizophrenia subjects to major mood disorder subjects experiencing psychosis. another interesting example is reported by mcdonald., (2007) who reduced syndrome heterogeneity by relating genetic risk for schizophrenia or for bipolar disorder with brain structural endophenotypes. anatomical variations in white matter overlapped between the two disorders while each disorder was associated with a distinctive pattern of variation in gray matter. the examples above help clarify differences in scientific methodology used to address observations of porous boundaries between current diagnostic classes. we propose that it is data at the level of pathologic domains rather than syndrome class that will provide the information critical to re - conceptualizing nosology and advancing knowledge on biomarkers and therapeutic targets. this approach presupposes that unique effects are associated with pathological processes that occur in several diagnostic classes, but in substantially different proportions [e.g, depression is ubiquitous in mood disorders, less frequent in schizophrenia ]. an alternative supposition is that unique factors combine with shared factors to create distinctive diagnostic classes and that porous boundaries are caused by the shared factors rather than pathological domains in different proportions. the question as to whether schizophrenia and bipolar disorder are one disease or two is relevant only if, in fact, these are two diseases at most. if either or both are a syndrome comprising several disease entities, the question is two or more, not two or less. if these classes are syndromes, then their combination creates a broader and more heterogeneous syndrome. this will decrease robustness of study designs, lead to type ii error, and be an unnecessary impediment to hypothesis testing. to ask the question in the dichotomous, kraepelinian context ignores the remarkable alteration in diagnosis associated with modern criteria. furthermore, psychosis is a final common pathway produced by the brain in response to many different insults. similarity across diagnostic classes based on psychotic symptoms may not be more definitive than similarity in anxiety across different classes. we propose that the current separation of schizophrenia and bipolar disorder into two syndromes captures some important distinctions. however, much more information is needed to determine the critical areas of similarity and difference. for this purpose, the failure to make use of strong inference in psychiatry has contributed to the slow pace of advancement in the field (carpenter., 1993). platt proposed that scientific fields with the most rapid progress design studies where the data can force theory modification and move the field to the next branch point (1964). a century of research on psychoses has been rich in hypothesis generation and slender on theory falsification. data have not yet been produced to decisively falsify the kraepelinian dichotomy or even to identify specific disease entities within the major syndromes. it seems very likely that dsm - v will take the approach of maintaining current classes of disorders with refined criteria while bringing a new emphasis to the domains of pathology paradigm (regier, 2007 ; van os & tamminga, 2007 ; allardyce., 2007 ; dutta., 2007 ; keller., 2007 ; research in this context will then clarify similarities and differences at the domain or dimension level. only then will the field be sufficiently informed to radically re - conceptualize classification of what we identify today as the psychotic and mood disorders. | kraepelin proposed dementia praecox and manic - depressive illness as the two major psychotic disorders. this paradigm is still prevalent, but observations of overlapping boundaries between bipolar disorder and schizophrenia challenge this dichotomy. however, the concept of schizophrenia has been radically altered from the original kraepelinian proposal. we defend the two psychoses position, but suggest two flaws in the heuristic application : 1) overlapping features such as psychotic symptoms are not decisive in differential diagnosis ; and 2) each disorder is a syndrome, not a disease entity. an alternative paradigm based on domains of pathology is more powerful for studies of etiology, pathophysiology, and therapeutic discovery. |
non - fatal injury posts a substantial burden on the nation 's health and safety. the us national center for health statistics (nchs) estimated that in 2012 over 30 million non - fatal injuries required medical attention;1 the lifetime medical and work - loss costs associated with non - fatal injuries totalled over $ 457 billion in 2013.2 there is no daily routine or activity that is genuinely free from injury risk, but if certain activities are known to have higher injury risk, appropriate task - based intervention strategies could be developed and tracked, which may prevent more injuries from occurring. in order to calculate injury rates by activity, both injury cases, known to occur while performing specific activities, and exposure time of the same activities need to be collected. in reality, few national surveys have the capacity to collect both numerator and denominator statistics. for example, the national health interview survey (nhis) asks respondents to report the occurrence of injuries and also inquires about activities performed at the time of injury (working, driving, etc.). however, the survey does not record the exposure time corresponding to these activities except for usual weekly work hours. as such, with data from the nhis alone it is not possible to estimate injury rates in terms of exposure hours. in the absence of information about exposure hours in the estimation of risk, researchers typically report the frequency of injuries by activity grouping and often report the injury rate per population, that is, the number of injuries that occur in a population at risk in a given time period, to describe injury risk at the population level.1 36 as a basic and important epidemiological measure, this expression of injury risk is widely used. in the case of the nhis since no exposure time in each activity is gathered, the population (estimated directly from the nhis)4 is used as the denominator, and the rate per population by activity (or by cause) basically reflects the relative distribution of injury cases by activities (or by cause).1 36 however, the injury rate per population calculated this way does not take into account exposure hours in the calculation and may conceal the true picture of injury risk per unit time exposed.7 8 moreover, because the population at risk is often treated as a constant,36 this expression of risk implicitly assumes that the exposure for the entire population is identical for all different types of activities, which may overstate the exposure time of activities where people typically spend less time (eg, sports and exercising) relative to the activities that people spend more time (eg, working). thus, rate per population should not be used to compare injury risk between different activities or demographic groups where time exposed to specific activities is known to vary. a further refinement of the expression of injury risk is warranted given the availability of adequate denominator statistics. to estimate injury rates by activity that take into account exposure time, we combine statistics from two national complex surveys for the numerator and denominator as inputs for the numerator and denominator of rate calculation. we adopt a method to estimate the variance of the injury rates that takes into account the complex survey designs. using different national surveys for the numerator and denominator of rate calculation is an approach first employed to assess the association between smoking and lung cancer,9 and has been applied to other topics, particularly cancer mortality.1015 some studies have examined the injury rate of certain activities in the usa such as transportation fatalities16 or fatal workplace injuries17 similarly using different data sources for the numerator and denominator. however, these studies are concerned about specific injuries or specific subpopulations ; no previous research has attempted to examine a wide spectrum of activities in which people are engaged on a daily basis. we add to the literature by estimating injury rates of all possible activities (known as person - time incidence rate or incidence density rate) for which no national estimates have been produced. we also demonstrate the ability to approximate the variance of the rates that integrates the complex survey designs of both numerator and denominator data. the numerator data, or injury outcomes, came from the 2010 nhis18 ; the denominator data, or exposure time data, came from the 2010 american time use survey (atus).19 the nhis is a cross - sectional household interview survey that has been monitoring the us population 's health since 1957, where the target universe of the nhis is the civilian non - institutionalised population.18 the nhis uses multistage sampling that involves stratification, clustering and oversampling of specific population subgroups. the survey is administered by the us census bureau under a contractual agreement with the nchs. we used nhis data for 2010, extracted from the integrated health interview series database,20 and restricted analyses to those aged 18 +. there were 27 157 adult observations in our analysis, and the response rate in 2010 was 79.5%.18 for the main portion of the interview, family core, all members of the household 18 years of age and over who are at home at the time of the interview are invited to participate and to respond for themselves.18 for adults not available for interview, information is provided by another adult family member in the household. the family core includes an injury and poisoning section, in which the respondents report each injury or poisoning episodes that was severe enough to seek medical treatment 3 months prior to the interview. we excluded the poisoning episodes, and only used injury episodes that occurred within a 6-week recall period (as opposed to 3 months) because studies4 21 showed that recall bias increases with the time elapsed between injury and interview, especially after 6 weeks. the atus measures the amount of time people spend performing various activities ; the survey started in 2003 and has been collected annually. the target universe is composed of the civilian, non - institutionalised population that are at least 15 years of age residing in occupied households in the usa.19 the atus is a stratified, three - stage sample where an eligible person is randomly selected from the household to conduct the interview and to participate in a 24-h time - use diary that documents the time spent in specific activities that are then coded using a standardised lexicon.22 the atus is conducted by the us census bureau for the us bureau of labor statistics. we used atus data for 2010, extracted from the atus - x database,23 and restricted analyses for persons 18 +. there were 16 679 adult observations in our analysis and the response rate of the atus in 2010 was 56.9%.19 the nhis inquires about activities that were being performed at the time of injury after the respondent describes the circumstances leading to the incident ; there are 11 categorical options.24 we estimated the rate of injury for the following five categories (as worded in the nhis questionnaire):24 (1) working at a paid job, (2) sports and exercise, (3) sleep, resting, eating, drinking, (4) working around the house / yard and (5) driving / riding in a motor vehicle. these activities were selected because the atus has recorded time use in a 24 h period in a much more detailed manner, which could be appropriately collapsed to allow matching of hours exposed corresponding to each of those five categories in the nhis. we let be the number of injury episodes of us adults in 2010 and related to a specific activity, and let be the same population 's total hours of exposure to the specific activity in 2010 that is also related to the same activity as the injury episodes. the rate per hour exposed is simply the ratio of to, which we estimated using the ratio of the corresponding sample means and, 25 assuming both surveys generalise to the same population:1 the sample means and were calculated individually, incorporating the survey - appropriate sampling weights. both and were annualised, because in the calculation of a rate the numerator and denominator need to have the same calendar period time, which in our study was the year of 2010. since we have restricted the recall period of injury episodes to 6 weeks (42 days), the annualised estimate was calculated by multiplying the weighted 6-week averages by 365/42,4 with the assumption that the injury pattern over a 6-week period represents that of a year.26 the time - diary data of atus were collected for a single day and weekend days were over - represented (50% of the sample data). the weighting variables were constructed to reduce the impact of over - representation of weekend days so that the weighted estimates represent a typical day in a calendar quarter.19 the annualised estimates were obtained by multiplying the weighted daily averages by 365,19 assuming that the typical day in the calendar quarter (after weighting) represents the time - use pattern throughout the year. the variance of,, was approximated using the delta method,27 28 which is essentially a first - order taylor series expansion. the delta method has been used extensively in statistics25 and can be applied to different sampling designs.25 29 (statacorp, lp. this method is the default approach for variance estimation with complex surveys of sas, sudaan30 and stata (statacorp, lp. stata survey data reference manual, release 13. college station, texas : stata press publication 2013.) and has been employed in a wide array of studies including those using complex surveys as their data.31 32 chapter 9 of sampling : design and analysis (lohr)25 describes the method in detail. the delta method28 shows that the variance can be approximated by a combination of,, and (online supplementary material provides the details of the derivation):2 if the data source for the numerator or denominator is a complex survey, the calculation of,, and needs to include the survey designs so that the design information is appropriately reflected in. operationally, we did this by using the stata command svy : mean(stata corporation lp, 2013) to calculate,,) and individually, and substituted them into equation (2) to obtain. stata v.13 was used for all analyses. to illustrate the difference between different expressions of injury risk, we estimated injury rate per population and contrasted the results with rate per hour exposed. similar to the method used to calculate rate per hour exposed, the numerator was the number of injury episodes. converse to the rate per hour approach was that the adult population (estimated directly from the nhis)1 4 was used as the denominator. the nchs has constantly provided annualised injury risk estimates,1 4 we followed this convention. the numerator data, or injury outcomes, came from the 2010 nhis18 ; the denominator data, or exposure time data, came from the 2010 american time use survey (atus).19 the nhis is a cross - sectional household interview survey that has been monitoring the us population 's health since 1957, where the target universe of the nhis is the civilian non - institutionalised population.18 the nhis uses multistage sampling that involves stratification, clustering and oversampling of specific population subgroups. the survey is administered by the us census bureau under a contractual agreement with the nchs. we used nhis data for 2010, extracted from the integrated health interview series database,20 and restricted analyses to those aged 18 +. there were 27 157 adult observations in our analysis, and the response rate in 2010 was 79.5%.18 for the main portion of the interview, family core, all members of the household 18 years of age and over who are at home at the time of the interview are invited to participate and to respond for themselves.18 for adults not available for interview, information is provided by another adult family member in the household. the family core includes an injury and poisoning section, in which the respondents report each injury or poisoning episodes that was severe enough to seek medical treatment 3 months prior to the interview. we excluded the poisoning episodes, and only used injury episodes that occurred within a 6-week recall period (as opposed to 3 months) because studies4 21 showed that recall bias increases with the time elapsed between injury and interview, especially after 6 weeks. the atus measures the amount of time people spend performing various activities ; the survey started in 2003 and has been collected annually. the target universe is composed of the civilian, non - institutionalised population that are at least 15 years of age residing in occupied households in the usa.19 the atus is a stratified, three - stage sample where an eligible person is randomly selected from the household to conduct the interview and to participate in a 24-h time - use diary that documents the time spent in specific activities that are then coded using a standardised lexicon.22 the atus is conducted by the us census bureau for the us bureau of labor statistics. we used atus data for 2010, extracted from the atus - x database,23 and restricted analyses for persons 18 +. there were 16 679 adult observations in our analysis and the response rate of the atus in 2010 was 56.9%.19 the nhis inquires about activities that were being performed at the time of injury after the respondent describes the circumstances leading to the incident ; there are 11 categorical options.24 we estimated the rate of injury for the following five categories (as worded in the nhis questionnaire):24 (1) working at a paid job, (2) sports and exercise, (3) sleep, resting, eating, drinking, (4) working around the house / yard and (5) driving / riding in a motor vehicle. these activities were selected because the atus has recorded time use in a 24 h period in a much more detailed manner, which could be appropriately collapsed to allow matching of hours exposed corresponding to each of those five categories in the nhis. we let be the number of injury episodes of us adults in 2010 and related to a specific activity, and let be the same population 's total hours of exposure to the specific activity in 2010 that is also related to the same activity as the injury episodes. the rate per hour exposed is simply the ratio of to, which we estimated using the ratio of the corresponding sample means and, 25 assuming both surveys generalise to the same population:1 the sample means and were calculated individually, incorporating the survey - appropriate sampling weights. both and were annualised, because in the calculation of a rate the numerator and denominator need to have the same calendar period time, which in our study was the year of 2010. since we have restricted the recall period of injury episodes to 6 weeks (42 days), the annualised estimate was calculated by multiplying the weighted 6-week averages by 365/42,4 with the assumption that the injury pattern over a 6-week period represents that of a year.26 the time - diary data of atus were collected for a single day and weekend days were over - represented (50% of the sample data). the weighting variables were constructed to reduce the impact of over - representation of weekend days so that the weighted estimates represent a typical day in a calendar quarter.19 the annualised estimates were obtained by multiplying the weighted daily averages by 365,19 assuming that the typical day in the calendar quarter (after weighting) represents the time - use pattern throughout the year. the variance of,, was approximated using the delta method,27 28 which is essentially a first - order taylor series expansion. the delta method has been used extensively in statistics25 and can be applied to different sampling designs.25 29 (statacorp, lp. this method is the default approach for variance estimation with complex surveys of sas, sudaan30 and stata (statacorp, lp. college station, texas : stata press publication 2013.) and has been employed in a wide array of studies including those using complex surveys as their data.31 32 chapter 9 of sampling : design and analysis (lohr)25 describes the method in detail. the delta method28 shows that the variance can be approximated by a combination of,, and (online supplementary material provides the details of the derivation):2 if the data source for the numerator or denominator is a complex survey, the calculation of,, and needs to include the survey designs so that the design information is appropriately reflected in. operationally, we did this by using the stata command svy : mean(stata corporation lp, 2013) to calculate,,) and individually, and substituted them into equation (2) to obtain. to illustrate the difference between different expressions of injury risk, we estimated injury rate per population and contrasted the results with rate per hour exposed. similar to the method used to calculate rate per hour exposed, the numerator was the number of injury episodes. converse to the rate per hour approach was that the adult population (estimated directly from the nhis)1 4 was used as the denominator. the nchs has constantly provided annualised injury risk estimates,1 4 we followed this convention. there were 12 679 adult observations in the 2010 atus, and 27 157 in the 2010 nhis. the mean age was 46.2% and 46.3%, and the proportion of females versus males was 51.7% and 51.6% for the nhis and atus, respectively. the left panel of table 2 summarises the average numbers of injuries an adult would sustain over a year (ie, the in equation 1) as well as the ses (ie, the square root of in equation 2). the right panel summarises the average activity exposure time over a year (ie, the in equation 1). nhis average number of injuries and atus average exposure hours by activity atus, american time use survey ; nhis, national health interview survey. table 3 reports the estimated injury rates per hour of activity exposed. to compare the injury risk associated with different activities, the rates were normalised to number of injury episodes per 100 000 h ' for each type of activity. injury rate per 100 000 h (number of injuries per 100 000 exposure hours) of us adults by activities performed at the time of injury, using 2010 nhis (numerator) and atus (denominator) atus, american time use survey ; nhis, national health interview survey. us adults sustained 1.45 injuries per 100 000 work hours (table 3). the comparable figure was 12.64, 0.23, 6.14 and 2.98 injuries per 100 000 h for the other four categories, respectively. table 4 describes injury rates per 1000 persons (population) for the same five activity types. the injury rate - per - hour approach depicts a ranking of injury risk quite different from the rate - per - population approach. for example, the injury rate per hour exposed in a sport activity was 4.24 times as high as the injury rate per hour exposed to driving (table 3). by contrast, the rate per population in a sport activity was only 1.08 times as high as rate per population exposed to driving (table 4). annualised injury rate per 1000 population (number of injury episodes per 1000 persons) of us adults by activities performed at the time of injury, using 2010 nhis for the numerator and denominator nhis, national health interview survey. we combined statistics from two different complex national surveys for the numerator and denominator to estimate injury rates per hour exposed by activity, and approximated the variance using the delta method, a commonly used method for variance estimation with complex surveys.25 31 32 researchers in other fields have combined statistics from two surveys to generate ratio estimates,915 although this approach has not been accomplished to estimate injury rates by activity groupings. by combining these two complex surveys and incorporating the time spent in specific activities, we identified a different rank order of injury risk compared with using a per person risk metric. we compared our estimates for work - related injuries with those using the 2010 nhis data alone ; the results were close. usual work hours per week (but not time use for the other five activities) which can serve as the denominator. the estimate was 1.40 injuries per 100 000 work hours (95% ci (1.02 to 1.78)), slightly lower but comparable with the current study (1.45 injuries per 100 000 work hours, table 3) which used the atus for the denominator. usual hours worked compared with time - diary measures.33 as such, the average exposure hours of the nhis may be greater than the atus, rendering a rate estimate (1.40 injuries per 100 000 h) slightly lower than the current study (1.45 injuries per 100 000 h). the major advantage of our approach (ie, injury rate per hour) is that we could control for the amount of time a person was exposed to a specific activity by explicitly using exposure hours as the denominator, which may more reliably indicate the underlying injury risk than the rate - per - population approach. rate per hour is also more suitable for comparing injury risks across different activities or demographic groups than rate per population.7 8 34 furthermore, by allowing the numerator and denominator to use different data sources, it becomes possible to measure the injury risk when data availability is constrained, in particular when no data source has documented both the injury and exposure time for a subject of interest. however, the rate - per - population approach is useful to understand the magnitude of injury burden across different populations or demographic groups ; estimates based on this approach are also more readily accessible.1 36 both approaches have their unique strengths and limitations, and should be interpreted based on the research context. the major limitation of our approach is that for a given domain of interest (eg, leisure), the information collected by the different surveys may not be perfectly comparable. sleeping, resting, eating, drinking, the atus does not record time specifically spent resting, but only sleeping, eating or drinking. thus, the exposure time in our study may be underestimated and the rate overestimated. while the atus and nhis do collect information on industry and occupation, there are not sufficient numbers of observations for cases and exposure hours to generate reliable estimates for individual industries or occupations on an annual basis. moreover, both the atus and nhis do not capture specific activities within any occupations, such as mining or logging. working as a miner is conceivably more dangerous than working at a computer, yet for the work - related injuries and activities as collected by the nhis and atus all these different types of work (both of high and low risk) are combined into a single work category. finally, an activity that has lower - ranked injury rates may still be a major public health problem whose significance should not be downplayed. for example, the injury rate per hour of work is much lower than playing sports. however, people on average spend much more time working than playing sports ; because of a greater amount of exposure time, work - related injuries still outnumber sports injuries by a factor of two (table 4). beyond injury rates, we note that activities vary in the extent to which they provide health or other life benefits. however, as our focus in this manuscript was methodological, we did not assess these potential trade - offs. an additional limitation is that both the atus and nhis are self - reported data ; incorrect recall may vary between different types of injuries which may bias the estimate for injury risk of one category relative to others. we assumed that the two surveys, both conducted in 2010, generalised to the same population (people age 18 +). indeed both surveys have been poststratified to make the population estimates by major demographic variables match the known population distributions as much as possible. however, nhis and atus used different poststratification procedures, and the estimated population totals of one survey may be different from the other. some categories have a small number of injury cases and the estimates and the cis may not be reliable. this issue could be potentially addressed by pooling multiple years of atus and nhis data, but it is unclear how to adjust for the correlation across years when combining data from two complex surveys for ratio estimates. non - fatal injury posts a substantial burden on the nation 's health and safety, and it is important to quantify and compare the injury risk of different activities that people are engaged in on a daily basis for accurate estimation of injury risk. we adopted a procedure that allows the use of different national complex surveys for the numerator and denominator to derive estimates of the rate of injury by activity as well as variance estimates. our results depicted a ranking of injury rates using hours spent engaged in an activity that are different from estimates using population as the denominator.3 6 this procedure produced estimates that may more accurately reflect the underlying injury risk and may be used to compare across different types of injuries or demographic groups. this procedure also overcomes data availability constraints frequently encountered by injury researchers, making it possible to estimate injury risk and cis even when no single survey has collected information on both injury cases and activity exposure time. few surveys collect both injury cases and exposure time, making it difficult to generate exposure time - based estimates for injury risk of common day - to - day activities. in the absence of exposure time data, researchers often report the number of injuries that occur in a population at risk in a given time period, or injury rate per population. the rate - per - population approach reflects the magnitude of injury burden of a population, but may conceal the true picture of injury risk per unit time exposed. to estimate injury rates by activity that account for exposure time, statistics from two national complex surveys were separately used as inputs for the numerator and denominator in the rate calculation. to calculate the variance of the rates, we use the delta method. our results demonstrate a different ranking of injury rate by activity as compared to ranked estimates using population as the denominator. our approach enables one to estimate injury risk and variance by combining injury cases and activity exposure time from separate surveys. few surveys collect both injury cases and exposure time, making it difficult to generate exposure time - based estimates for injury risk of common day - to - day activities. in the absence of exposure time data, researchers often report the number of injuries that occur in a population at risk in a given time period, or injury rate per population. the rate - per - population approach reflects the magnitude of injury burden of a population, but may conceal the true picture of injury risk per unit time exposed. to estimate injury rates by activity that account for exposure time, statistics from two national complex surveys were separately used as inputs for the numerator and denominator in the rate calculation. to calculate the variance of the rates, we use the delta method. our results demonstrate a different ranking of injury rate by activity as compared to ranked estimates using population as the denominator. our approach enables one to estimate injury risk and variance by combining injury cases and activity exposure time from separate surveys. | backgrounda common issue in descriptive injury epidemiology is that in order to calculate injury rates that account for the time spent in an activity, both injury cases and exposure time of specific activities need to be collected. in reality, few national surveys have this capacity. to address this issue, we combined statistics from two different national complex surveys as inputs for the numerator and denominator to estimate injury rate, accounting for the time spent in specific activities and included a procedure to estimate variance using the combined surveys.methodsthe 2010 national health interview survey (nhis) was used to quantify injuries, and the 2010 american time use survey (atus) was used to quantify time of exposure to specific activities. the injury rate was estimated by dividing the average number of injuries (from nhis) by average exposure hours (from atus), both measured for specific activities. the variance was calculated using the delta method, a general method for variance estimation with complex surveys.resultsamong the five types of injuries examined, sport and exercise had the highest rate (12.64 injuries per 100 000 h), followed by working around house / yard (6.14), driving / riding a motor vehicle (2.98), working (1.45) and sleeping / resting / eating / drinking (0.23). the results show a ranking of injury rate by activity quite different from estimates using population as the denominator.conclusionsour approach produces an estimate of injury risk which includes activity exposure time and may more reliably reflect the underlying injury risks, offering an alternative method for injury surveillance and research. |
open globe injuries are a major cause of vision loss worldwide,14 with an estimated 200,000 open globe injuries occurring worldwide each year.5 because of the heterogeneity of traumatic injuries, standardized terminology to describe traumatic eye injuries has been developed,6 and these terms were summarized as the birmingham eye trauma terminology.7 multiple classification schemes have also been developed, including by the ocular trauma classification group, who classified traumatic globe injuries based on four variables that had predictive value in estimating final visual outcomes : mechanism of injury, grade of injury as defined by presenting visual acuity, presence or absence of an afferent pupillary defect (apd), and zone of injury based on its most posterior extent.8,9 several scoring systems have been devised to isolate factors at presentation predictive of poor prognosis. one widely used scoring system is the ocular trauma score (ots), which was developed following analysis of > 2,500 open globe injuries and identified presenting visual acuity, presence of apd, mechanism of injury, retinal detachment, or endophthalmitis as key variables.10 the ots assigns numeric values to each of the variables mentioned earlier to determine a scoring category that is predictive of final visual acuity. subsequent studies have demonstrated the ots validity in predicting final visual acuity,1114 though the scoring system may be less reliable in pediatric populations.15 while numerous published reports have examined the prognostic factors for traumatic globe injuries, fewer studies have evaluated the types and number of surgeries required for treatment of traumatic globe injuries. andreoli and andreoli recently described long - term surgical rehabilitation of open globe injuries and found that patients on average required 1.7 surgeries and that more severe ots predicted the need for follow - up surgery.16 to our knowledge, no studies have investigated whether outcomes differ based on the subspecialty training of the surgeon at time of the initial open globe repair. for example, a patient with a zone iii injury (defined by the ocular trauma classification group as extending > 5 mm posterior to the limbus) may be referred to a vitreoretinal (vr) surgeon for primary repair, but data are limited as to whether visual acuity outcomes are better with a trained vr surgeon compared to a non - retina specialist, or whether having a subspecialist perform the initial repair will decrease the need for further surgeries after the initial repair. in this study, we describe the characteristics of open globe injuries presenting to an urban, tertiary trauma center in comparison to prior studies from our institution, and evaluate outcomes based on previously studied prognostic indicators including ots and zone of injury. we further evaluate whether outcomes such as visual acuity, number and type of post - repair procedures, and need for enucleation differ based on the subspecialty training of the surgeon performing initial repair. this is a single - center, retrospective chart review of consecutive adult patients (18 years old) who presented to the wilmer eye institute, the johns hopkins hospital, from july 1, 2007 to july 1, 2012 with open globe injuries as defined as a full - thickness wound of the eye wall. patients at the wilmer eye institute at johns hopkins hospital are treated for open globe injuries with a standardized protocol including complete history, ocular examination, and consent for surgical repair. all patients are evaluated by ophthalmology house staff and examined by a fellowship - trained attending ophthalmologist. a non - contrast computed tomography scan with thin cuts through the orbits initial treatment includes a single dose of intravenous antibiotics (usually moxifloxacin, a fourth - generation fluoroquinolone) and an updated tetanus shot. surgical repair is performed as soon as possible, generally within 24 hours unless prohibited by active medical issues. the initial globe repair is performed by the attending on call, in general regardless of zone of injury, unless the initial injury necessitates a subspecialist (eg, metallic intraocular foreign body [iofb ] in the posterior segment requiring a vr specialist at time of initial repair). intravitreal antibiotics (typically vancomycin and ceftazidime) are administered intraoperatively. patients are typically discharged to home if medically stable or admitted to address outstanding medical issues. patient charts were reviewed to determine patient demographics including age and sex as well as previous ocular history. data from examination findings at presentation included mechanism of injury, visual acuity, presence of a relative apd, descriptive extent of injury, hyphema, uveal prolapse, vitreous hemorrhage, retinal detachment, iofb, orbital fracture, or adnexal laceration. data recorded at follow - up visits included visual acuity, intraocular pressure, and details regarding any follow - up procedures. for each patient, otss were calculated when possible based on available data,10 and each injury was also classified by mechanism, grade, and zone of injury based on the ocular trauma classification group categories.8 data collected regarding initial surgical repair included the need for additional procedures, including anterior chamber (ac) washout, anterior vitrectomy, extraocular muscle involvement (including need to disinsert and reinsert muscles to determine extent of injury), lensectomy, pars plana vitrectomy (ppv), scleral buckle (sb), iofb removal, and enucleation. for follow - up surgeries, defined as procedures performed at a separate visit following the initial repair of the globe injury, the surgical data recorded included any of the procedures listed earlier, as well as need for wound revision, intraocular lens placement, penetrating keratoplasty, glaucoma filter or tube placement, membrane peel, use of intraocular gas or oil, retinectomy, or choroidal drainage. the subspecialty training of the attending surgeon was noted for both the initial repair and any follow - up surgeries. for the purposes of primary enucleation, which were all performed by attendings with oculoplastic subspecialty training, the sub - specialty training of the attending who either attempted initial repair or referred primarily for enucleation was noted. primary outcome measures included visual acuity at 6 months and 12 months post - repair, number of follow - up surgeries, and time until first follow - up surgery or enucleation. eyes were excluded from specific analysis if measures being analyzed were not included or if follow - up data were unavailable for past 6 months. visual acuity was categorized into five categories based on the ots : 1) no light perception (nlp), 2) light perception or hand motion, 3) 1/20019/200, 4) 20/20020/50, and 5) 20/40. eyes that were enucleated were considered to be of nlp category for the purpose of analyzing visual acuity outcomes. statistical analysis was performed with a p - value of < 0.05 considered statistically significant. multiple logistic regression analysis was performed using statacorp lp 2013 (statacorp lp, college station, tx, usa). there were a total of 283 open globe injuries from 282 adult patients seen from july 1, 2007 to july 1, 2012. seventy - five percent (212/282) of the patients were male, and 25% (71/282) were female. two hundred and fifty - two patients (89%) were not documented as wearing glasses, goggles, or any other form of eye protection at time of injury. out of the 282 patients, one patient had insufficient clinical information to characterize the type of injury or zone of injury per chart review. the majority of injuries were considered ruptures (130/282 ; 46%), most commonly extending into zone ii (121/282 ; 43%) per the open globe injury classification scheme (table 1).8 out of the 283 total injuries reviewed, 193 eyes from 192 patients reached 6 months of follow - up for inclusion for further analysis. the demographics of this patient population were similar to the overall patient population in our study, with 143 patients (75%) being male and with a mean age of 46 years. the distribution of type of injury was also similar, with ruptures (91/192 ; 47%) involving zone ii (75/192 ; 39%) being most common (table 1). the most common additional procedures performed in addition to globe closure at the time of initial globe repair are shown in table 2. approximately 20% of patients required ac washout or manipulation of an extraocular muscle. when comparing rates of additional procedures performed between vr surgeons and non - vr surgeons, ac washout, sbs, and ppv a total of 86 out of 193 eyes (45%) required follow - up surgery within the first year after initial injury, with average time until first follow - up surgery of 7.1 weeks. the most common follow - up procedures were ppv (70/193 ; 36%), ac washout (52/193 ; 27%), and lensectomy (48/191 ; 25%). thirty - nine out of the 193 eyes (20%) were enucleated within 12 months of initial repair, with average time to enucleation of 3.2 weeks. twelve eyes (7%) underwent primary enucleation at time of initial examination and/or attempted repair (table 3). there were a total of 36 different surgeons who performed open globe repairs in this study : twenty - one vr, six cornea, three uveitis, five oculoplastic, and one glaucoma. one hundred and thirty - one out of the 193 eyes were repaired by a vr surgeon, and 62 repaired by other surgeons (28 eyes cornea, 24 uveitis, nine oculoplastic, one glaucoma). patients treated by the 15 non - vr surgeons were compared against those treated by the 21 vr surgeons. there was no statistically significant difference in initial ots raw score, ots category, or zone of injury between these two subsets of patients (table 4). the rate and distribution of follow - up surgeries performed were similar regardless of the subspecialty training of the initial surgeon, with notable exceptions of ac washout (p=0.02) and sb (p=0.03). there were more primary enucleations performed in the non - vr group (p=0.01), though the rates of secondary enucleation were similar (table 3). at 6 months, visual acuity outcomes for all patients at 6 months and 12 months are shown in table 5. visual outcomes were compared for those initially treated by a vr surgeon and a non - vr surgeon. eyes initially treated by a vr surgeon were 2.3 times more likely to improve by one ots visual acuity category at 6 months (p=0.003) compared to eyes treated by non - vr surgeons, and this difference remained statistically significant at 12 months (p=0.019). when controlling for initial zone of injury, patients with more anterior injuries treated by a vr surgeon were more likely to improve by one ots visual acuity category compared to those treated by non - vr surgeons (p=0.004 and 0.016 for zones there was no statistically significant difference in visual acuity outcomes for eyes with posterior injuries (p=0.52 for zone iii). eyes initially treated by vr surgeons were also 1.9 times more likely to have at least one more follow - up surgery at 6 months compared to non - vr surgeons (p=0.027), and this difference was no longer statistically significant at 12 months (p=0.07). when controlling for zone of injury, eyes with zone ii injuries received more surgical rehabilitation when initially treated by a vr surgeon versus non - vr surgeon (p=0.048), while rates of follow - up surgery for zone i and iii patients were similar (table 6). the average time to the first follow - up surgery for the vr group was 6.7 weeks compared to 7.6 weeks for the non - vr group, and this difference was not statistically significant (p=0.52). the data presented in this study represent our experience of 5-year period treating adult patients with traumatic open globe injuries at an urban, tertiary referral center. numerous previous studies performed at our institution have characterized mechanisms of injuries, identified key prognostic factors, and described visual outcomes for patients with traumatic globe injuries.1720 when compared to a similar series of 476 eyes from patients who presented to our institution from 1970 to 1981,18 and 290 eyes from patients who presented from 1985 to 1993,20 our patients actually had a lower rate of final visual acuity 20/40 (21% in our series from 2007 to 2012 compared to 39% from 1970 to 1981 and 36% from 1985 to 1993). this may be reflection of demographics, as our patient population, which included only adults, tended to be older (mean age 46 years compared to 31 years for 19851993). in addition, injuries in our study tended to be more posterior (only 28% of eyes with isolated corneal injuries compared to 41% from 1985 to 1993). furthermore, there was a much higher proportion of globe ruptures in our study (47% compared to 30% from 1985 to 1993), which also predispose toward a poorer outcome per previous prognostic studies. while many previous studies have focused on identifying factors important for prognosticating outcomes following open globe injuries, fewer studies have evaluated the surgical rehabilitation required for patients who suffer traumatic globe injuries. in our study, 45% of eyes (86/193) required follow - up surgeries within the first year. this rate is the same as that reported in a recent study by andreoli and andreoli.16 as in their study, lensectomy and ppv were the two most common follow - up procedures performed after initial globe repair. recognizing that nearly half of patients with traumatic globe injuries require follow - up surgeries, our study further addresses the issue of whether the subspecialty training of the surgeon performing the initial globe repair affects final visual outcomes or number of follow - up surgeries required. the data suggest that eyes initially treated by a vr surgeon were 2.3 times more likely to improve by one ots visual acuity category by 6 months (p=0.003) compared to eyes treated by non - vr surgeons, and this difference remained statistically significant at 12 months. the differences in visual acuity outcomes in our study population may be explained by several different factors. for one, patients treated initially by a vr surgeon have more procedures performed at time of initial repair. these included retina - specific procedures such as ppv or sb, as might be expected, given the vr background of these surgeons. while a non - vr surgeon may opt simply to close the open globe at time of initial repair, surgeons with vr training may pursue additional steps in the initial surgery to secure the globe injury and start surgical rehabilitation. this may indicate not only that the subspecialty training of the attending surgeon influences outcome but also that vr - specific procedures conducted at the time of open globe injury may be important for improved patient outcome. even when a vr surgeon performs the initial globe closure, numerous factors may contribute to staging surgical rehabilitation, including the stability of the eye at time of primary closure for further surgery, the presence of adequate view for posterior segment surgery, and restoration of ocular anatomy more representative of normal once primary closure has been performed. in our study, despite undergoing more additional procedures on initial repair, patients treated by a vr surgeon were also 1.9 times more likely to have at least one follow - up surgery within the first year. as no significant differences exist between the type and zone of injury between the two groups of patients to explain the relatively greater number of follow - up surgeries, we believe that this may reflect a referral bias, perhaps due to recognition of the need for further posterior segment surgery at the time of initial repair, as the most common follow - up procedure was ppv. this pattern may also reflect a more aggressive mindset for surgical rehabilitation if initially treated by a vr surgeon, or suggest that the ability to perform vr - specific procedures necessary for surgical rehabilitation may influence a vr surgeon s willingness to employ these procedures in his / her approach to rehabilitation after globe trauma. while one may hypothesize that patients with more posterior injuries have better visual acuity outcomes when initially treated by a vr surgeon, the data in our study do not support this hypothesis. when controlling for initial zone of injury, there was no statistically significant difference in visual acuity outcomes for eyes with posterior injuries (p=0.52 for zone iii). these data reflect the overall poor prognosis for eyes with zone iii injury due to irreparable damage to structures such as the retina or optic nerve, regardless of the subspecialty training of the initial treating surgeon. by contrast, patients with more anterior (zones i and ii) injuries treated by a vr surgeon were more likely to improve by one ots visual acuity category compared to those treated by non - vr surgeons (p=0.004 and 0.016 for zones eyes with zone ii injuries received more surgical rehabilitation when initially treated by a vr surgeon versus non - vr surgeon (p=0.048), while rates of follow - up surgery for zone i and iii patients were similar. patients with anterior (zones i and ii) injuries, thus, likely represent the group with the best chance for improvement with aggressive surgical rehabilitation. for example, injuries in zones i and ii may result in hyphema, vitreous hemorrhage, or lens damage causing poor presenting visual acuity. however, in the absence of damage to posterior segment structures, significant improvements in visual acuity may be possible with ac washout, vitrectomy, lensectomy, and intraocular lens implantation as part of surgical rehabilitation. when comparing the rates of follow - up surgeries in patients initially treated by a vr versus non - vr surgeon, only the rates of ac washouts and sbs were different to a statistically significant degree. there was no statistically significant difference in the rate of secondary enucleations in the vr versus non - vr groups. when comparing the rate of primary enucleations between the vr and non - vr groups, however, the difference was significant (p=0.01). out of the 191 eyes that had at least 6 months of follow - up, nine of these cases were initially staffed by a non - vr surgeon (15% of the patients in the non - vr group vs 6% in the vr group). this in combination with the greater number of follow - up procedures within the vr group may suggest that vr surgeons, with knowledge and ability to perform vr - specific procedures at time of initial repair or on follow - up, may opt for initial attempt at surgical repair and subsequent rehabilitation despite poor presenting visual acuity. while our study provides evidence that procedures performed by vr surgeons may improve patient outcome following traumatic globe injuries, the results may not be generalized to younger patients with globe trauma, as only adult patients were included in the study due to difficulty in assessing visual acuity for calculation of otss in a pediatric population. approximately a third of the patients in this study did not have follow - up data at our institution past 6 months, in most cases because the patients were referred at time of trauma for initial repair and had follow - up care performed locally. despite the lack of follow - up data, the presenting characteristics of eyes lost to follow - up our study was not powered to compare outcomes across each individual subspecialty, but only compares outcomes between surgeons with vr training versus those without. the study was also not sufficiently powered to analyze the influence of surgeon experience on outcomes. overall, our data suggest that nearly half of patients who suffer traumatic globe injuries undergo follow - up surgical rehabilitation within the first year. eyes that were initially repaired by a vr surgeon had better visual outcomes than those treated by a non - vr surgeon. however, these eyes also underwent more surgical rehabilitation within the first year, and the difference was only apparent in anterior (zones i and ii) injuries. notably, while there was a trend toward improved visual outcomes for zone iii injuries, there was no statistically significant difference between vr and non - vr groups, confirming the generally poor prognosis of these eyes and suggesting that posterior injuries may not mandate involvement of a vr surgeon versus a non - vr surgeon at time of initial repair. eyes initially treated by a non - vr surgeon were more likely to undergo primary enucleation, and this may contribute to the difference in final visual acuity outcomes between the two groups. taken as a whole, these findings suggest that patients with anterior (zones i and ii) but not necessarily posterior (zone iii) traumatic globe injuries may have superior visual outcomes if initially treated by a vr surgeon, perhaps related to the use of vr - specific procedures at the time of initial repair and on follow - up surgeries for optimal rehabilitation in anterior injuries where visual prognosis is better. future studies are needed to isolate factors that indicate when initial repair by a vr surgeon may be most beneficial. in circumstances where a vr surgeon is not immediately available for initial repair, early referrals to a vr surgeon | purposethe purpose of this study was to evaluate whether subspecialty training of the initial treating surgeon affects visual acuity and surgical outcomes in patients with open globe injuries.designthis study is a single - institution, retrospective case series.methodsthe charts of adult patients with open globe injuries requiring surgical repair at the wilmer eye institute between july 1, 2007 and july 1, 2012 were retrospectively reviewed. clinical findings at presentation were recorded, and details of initial repair and follow - up surgeries were analyzed. differences in visual acuity and surgical outcomes were compared based on subspecialty training of the initial surgeon.resultsthe charts of 282 adult patients were analyzed, and 193 eyes had at least 6 months of follow - up for analysis. eighty - six eyes (44.6%) required follow - up surgery within the first year, and 39 eyes (20.2%) were enucleated. eyes initially treated by a vitreoretinal (vr) surgeon were 2.3 times (p=0.003) more likely to improve by one ocular trauma score (ots) visual acuity category and 1.9 times (p=0.027) more likely to have at least one more follow - up surgery at 6 months compared to eyes treated by non - vr surgeons. patients with more anterior injuries treated by a vr surgeon were more likely to improve by one ots visual acuity category compared to those treated by non - vr surgeons (p=0.004 and 0.016 for zones i and ii, respectively). there was no difference in visual acuity outcomes for eyes with posterior injuries (p=0.515 for zone iii).conclusioneyes initially treated by a vr surgeon are more likely to improve by one ots visual acuity category than those initially treated by a non - vr surgeon. however, patients initially treated by a vr surgeon also undergo more follow - up surgical rehabilitation, and improvement in visual acuity is more likely for anterior (zone i and ii injuries) than posterior (zone iii) injuries. |
the term darwinian medicine has been applied to the study of the interplay between inherited genetic characteristics and the very rapid societal changes, notably those in the industrialised nations, that have occurred over the last few centuries (rook 2009). studies in this field have, in particular, focused on the increasing isolation of the human population from many micro - organisms that millions of years of evolution have led the immune system to the considerable increase in the industrialised nations of a number of conditions, including allergies, a wide range of autoimmune disorders and certain cancers has been attributed to inadequate or inappropriate development of the immune system and its regulatory pathways. these conditions are all characterised by chronic inflammation (rook 2007), which, in contrast to acute inflammation, is a manifestation of dysregulated, and potentially inappropriate immune reactivity (bottasso. as well as initiating certain cancers, the key relevance of chronic inflammation to the subsequent development and maintenance of most if not all cancers has been highlighted in recent years ; indeed such inflammation has been termed the other half of the tumour (mantovani. initially, the hygiene - related factor responsible for the changing pattern of disease was thought to be a reduced exposure to a range of previously common infectious diseases during childhood, but it now seems likely that the relevant micro - organisms from which we are increasingly isolated also include commensals and saprophytes, such as actinomycetes, that are acquired by the oral route. intestinal helminth infestations, once universal and usually harmless, may have protected against allergic disorders and, possibly, some of the other conditions referred to above. the term another consequence of improving standards of hygiene is that certain infections once almost universally acquired in infancy are now acquired at a later age and the immune response to such infections in older people may be quite different than in infants, sometimes with deleterious consequences. the outcome of the nature and sequence of infections and other antigenic challenges during the lifetime of a person has been linked with the biography of the immune system (krone. 2009). to date, darwinian medicine has focused on external factors, notably the acquisition, or lack of acquisition, of commensal and pathogenic micro - organisms from the surrounding environment, but there is another key emerging theme in this important field of human medicine. the complete sequencing of the human genome revealed many surprises, not least of which is that almost half of the genome is composed of, or derived from, virus - like elements. as our planet, with its abundant supply of water and the eventual emergence of life on its surface, evolved as the result of many catastrophic collisions with asteroids and comets, so that which was ultimately to become the human genome appears to have enlarged and evolved as a result of numerous collisions with viral dna (kazazian 2004). the term virolution has been applied by frank ryan to this key aspect of our overall evolution (ryan 2009a), and the subject has been summarised in a series of excellent reviews by the same author (ryan 2009b). the components of the viral content of the human chromosome that may well have had the most significant impact on evolution are the human endogenous retroviruses (hervs), which comprise around 8 percent of the chromosome and represent around 400,000 genetic loci. these hervs have been acquired over millions of years and many different families and sub - families have been described (bannert and kurth 2004 ; dolei and perron 2009). the earlier the thus hervs of the w series are the oldest yet found, having been acquired by our primate ancestors around 40 million years ago. by contrast, those of the k series were acquired between 3 and 6 million years ago, at the time of the human - chimpanzee split, and may indeed have played a key role in mediating this split (sverdlov 2000). probably due to their relatively recent origin, those of the k series are the most complete and biologically active of the hervs and are able to induce the formation of replicating virus particles (ryan 2009a), though infectivity has not been demonstrated (boller. these whole virus particles can, however, co - package rna from hervs that are unable to directly induce such particles (ruprecht. 2008). whether also exogenous xenotropic retroviruses could become involved is far from clear (lo. hervs contain a small group of functional genes (env, gag, and pol, similar to those in other retroviruses including hiv) flanked by regulatory genetic units termed long terminal repeats (ltrs). thus, for example, the expression of the env regions of three hervs erv-3, herv - w and herv - frd is essential for the formation of the placental syncytiotrophoblast and the herv - frd component appears to be involved in the suppression of immune reactivity that might otherwise reject the foetus (rote. once established in the host dna, hervs produce multiple copies of themselves, in some cases up to 1,000, widely distributed throughout the genome and the ltrs are able to regulate functional genes of the host as well as those of the retrovirus. such ltrs play a key role in the intra - uterine development of the foetus, shepherding it through the complex recapitulation of evolution. accordingly, hervs have played a central role in the evolution of the human species and still have an indispensable function in our maturation (ryan 2009a ; gogvadze. herv antigens are not normally expressed, or are only expressed at low levels, by the host but in circumstances of inappropriate control and regulation, expression of herv genes may initiate or maintain pathological processes (larsson and andersson 1998). such gene products can be complete or incomplete viral env or gag proteins or products of recombinational processes (laufer. 2009). in this context, there is growing evidence that, by affecting immune regulation, the hygiene - related factors mentioned above may facilitate the abnormal expression of herv - encoded genetic information. the exact mechanisms underlying abnormal herv gene expression are poorly understood but in some cases it is associated with hypomethylation of the relevant viral genes (gimenez. there is, however, an achilles heel in the herv - mediated induction of pathological changes in cells ; namely, the immune presentation of herv - encoded epitopes on the surface of affected cells, thereby serving as targets for immune processes that repair or destroy the compromised cell. in principle, genetic expression of hervs can be controlled by self - specific cd8 t - cells although there may well be a need for the relevant sub - populations of t - cells to be expanded and maintained by contacts with environmental micro - organisms, particularly those encountered early in life, which by bearing epitopes homologous to the relevant herv epitopes induce cross - reactive immune responses. this raises the possibility that hygiene - related factors may modify such cross - reactive immunity with, in some cases, deleterious consequences, but it also raises the prospect of vaccination strategies to prevent, or immunotherapy to treat, herv - related disease processes. an example of a disease in which abnormal herv gene expression appears to play a primary role in the pathological processes is melanoma, which has undergone a considerable increase in incidence in the industrialised nations where, with the exception of lung cancer in women, its incidence is rising more rapidly than any other cancer (garbe and leiter 2009). indeed, in the light of current understanding, it is a disease that affords a very good model for elucidating the complex interactions between hervs, exogenous infections and the hygiene hypothesis. in view of occasional reports of spontaneous regressions of melanoma, suggesting the occurrence of effective immune responses, the febrile infections and melanoma (febim) working group within the epidemiological section of the melanoma cooperative group of the european organization for research and treatment of cancer (eortc) was established to conduct epidemiological studies on the impact of prior infectious diseases and vaccinations on the risk of melanoma in several european countries and israel. the febim group established that a history of serious but uncommon infections with fever > 38.5c, including pneumonia, sepsis, pulmonary tuberculosis and staphylococcus aureus infection, was associated with protection against melanoma, with the level of protection being directly related to the number of such infections (krone. 2005). in addition to naturally occurring infection, it was demonstrated that vaccination early in life against smallpox and/or tuberculosis (vaccinia and bcg vaccines) confers an enduring degree of protection against melanoma. adjusted odds ratios (95% confidence intervals) of risk were 0.40 (0.180.85) for bcg alone, 0.60 (0.360.99) for vaccinia alone and 0.41 (0.250.67) for both vaccines. in addition to this conferred protection, patients developing melanoma have a significantly better prognosis if they have received one or both vaccinations (klmel. there is evidence that expression of a human endogenous retrovirus of the herv - k family is involved in several stages of carcinogenesis in melanoma, from initiation of malignant change to development of the clinically evident tumour (muster., an unexpected finding was made : a healthy subject recruited for a trial of a melanoma vaccine had an expanded population of cytotoxic t - cells recognizing an epitope coded for by a herv - k (schiavetti. 2002), suggest a mechanism by which the certain vaccinations and infections might protect against melanoma. as the herv - encoded peptide, herv - k - mel, is presented by hla - molecules on the majority of human melanomas, the relevant infections and vaccinations might elicit protection inducing populations of cd8 t - cells cross - reacting with this target epitope. amino - acid sequences with homologies to the herv - k - mel peptide were therefore sought among pathogens and vaccines and were found in those that conferred protection but not in those that did not (krone. this finding raised the possibility that other vaccines in routine use might be able to confer protection and could be identified by the presence of epitopes with structurally similar amino acid sequences. such a sequence was found in the 17d yellow fever vaccine and its postulated protective effect was confirmed in a pilot study that also showed that there was a period of around 10 years between vaccination and observed protection (mastrangelo. this time lag suggests that the induced immune response exerts its protective effect at the time of tumour initiation which precedes the clinical manifestation of the disease by several years or a decade. immunotherapeutic strategies have been investigated in a mouse model based on a melanoma and a colorectal cancer, both of which express tumour associated mouse endogenous retrovirus antigens (mervs) (kershaw. it was postulated that immune responses targeting the envelope protein of the mervs might confer protection. accordingly mice were vaccinated either with a merv envelope peptide pulsed dendritic cell vaccine or a recombinant vaccinia vaccine and challenged with tumour cells injected into the tail vein. three weeks later, lung metastases were counted and showed that vaccination conferred a high degree of protection by generating immune responses specifically targeting the merv envelope. this vaccination strategy was, however, not effective against tumours that were already established. it is highly likely that such protection is mediated by self - specific cd8 t - cells, the developmental biology and function of which has been studied in recent years, although not all their modes of action have been determined (jordan. they belong to a distinct lineage and have developmental requirements distinct from those of conventional cd8 t - cells, being independent of tec kinases and of ras grp1 for their development but instead being dependent on il-15 and strong interaction with self - antigen. in vitro, they undergo marked proliferation when stimulated with either il-15 or il-12 in the absence of tcr stimulation. upon activation by il-15, they produce significant amounts of ifn- and can, for example, protect against infection with listeria via recognition of a self antigen. while exposure to foreign antigen may not be essential for the initial induction of these self - specific cd8 memory t - cells, contact with homologous antigens in an appropriate co - stimulatory context can increase and maintain relevant sub - populations of them. during an immune response they can activate other immune cells to produce a wide range of cytokines and, in addition, they provide an innate source of ifn- during an immune response such as against infection. as discussed above, epidemiological data indicate that certain infectious diseases and vaccinations against infectious diseases including vaccinia, bcg and yellow fever vaccinations confer protection against tumour development at the time of tumour initiation, years or decades before a clinical manifestation of the malignant disease, either by cell killing or repair. a candidate immune mechanism for cell repair is based on gangliosides of the neo - lacto series, in particular lm1 (ritter. 1986 ; nojiri. 1988 ; schaade. 1999, 2000), which are transmitted from leucocytes to the target cell by direct cell - to - cell contact. lm1 has effects on cell growth and differentiation and can normalise a malignant phenotype in various pre - malignant cell - lines by causing cell cycle arrest within the g0/g1 phase of the cell cycle (nojiri. 1999). a differential display analysis of gene expression (krone. 2005 ; schaade. 1999) shows that lm1 mediates suppression of retroviral rna ; moreover, it also induces a kelch 1-like protein, one of a family of proteins known to be involved in transcriptional repression (wang. 2008), and it induces s - adenosyl - homocysteine - hydrolase, an enzyme that is essential to the generation of active methyl groups. these methyl groups are needed for methylation of dna which is the most important mechanism by which cells silence the genetic expression of endogenous retroviral genes. further studies on this mechanism would be of great interest and relevance to carcinogenesis and its suppression. the immunoregulatory networks at any given time are the result of a complex and convoluted history of repeated encounters with the microbial environment, resulting in either long term or transient commensalism or acute, chronic or latent infection. this history, termed the biography of the immune system (krone. 2009), plays a key role in health and disease and is crucially affected by hygiene - related factors. even very small antigenic contacts, so small that they may not induce measurable immune responses, are able to imprint the immune system with patterns of subsequent reactivity (menon and bretscher 1996). in addition, changes in the timing of sequential infections, due in some cases to hygiene - related factors, may have major impacts on the outcome of infection, a phenomenon termed original antigenic sin (editorial 1958). thus the immune response to a pathogen often acquired very early in life, such as the epstein - barr virus, may be quite different if the infection occurs later in life and after infection by pathogens bearing cross - reactive epitopes that generate expanded populations of specific memory cells (krone. depending on the biography of the immune system, a wide range of effector and regulatory t - cells, and corresponding regulatory networks, with quite different relevance to protection and pathogenesis, will be generated. thus various populations of effector and regulatory cells may be generated, suppressed or eclipsed and inappropriate cells that promote pathological processes may be induced (krone. in general, in respect to melanoma, the label of good or bad, it seems that several infecting micro - organisms contribute to the development of immunoregulatory pathways that protect against melanoma and the absence of one or more may be compensated by others and, collectively, these may be regarded as old friends (rook 2007). compared with exogenous viruses, the situation with hervs is quite different as they are an integral part of inherited genetic material, yet their expression and effect may be just as hygiene - related as encounters with microorganisms in the external environment. while on the basis of their central role in human evolution they are certainly old friends, they can act as old foes under some circumstances which are becoming progressively less avoidable as human populations increasingly distance themselves from the environmental micro - organisms encountered by their ancestors. in addition to the example of melanoma discussed above, hervs could induce abnormal immune reactions in two ways. firstly, abnormally expressed herv encoded peptides could be recognised as foreign and, by inducing cross - reactive immune responses including antibody, could initiate a range of autoimmune diseases. secondly, a translocating herv long terminal repeat could promote the expression of a gene which is usually inactive, yielding a product which could likewise be recognised as although, as mentioned above, caution must be observed in determining whether abnormal herv expression is the cause or consequence of a disease process, such expression has been reported in a number of autoimmune disorders including rheumatoid arthritis, lupus erythematosus and sjgren s syndrome, other connective tissue diseases, various inflammatory neurological diseases (balada. thus, for example, significantly higher levels of mrna for a gag protein of herv k10 were found in patients with rheumatoid arthritis than in those with osteoarthritis and in healthy controls (ejtehadi. the expression of members of three herv families, herv - e, -k and -w, has been detected in 29 of 43 of psoriatic skin lesions but in only three of 14 lesions of atopic dermatitis and in two of 21 normal skin samples, but the relevance to pathogenesis has not been determined (mols. 2005). in schizophrenia, genes with mrna sequences homologous to the herv - w env gene were detected in plasma from 42 of 118 patients with recent - onset schizophrenia but in none of 106 healthy subjects, and this retroviral protein was found to upregulate several schizophrenia - associated genes (huang. in addition, herv expression has been described in several cancers in addition to melanoma, including breast, teratoma, seminoma, renal and haematological malignancies (schiavetti. the expressed hervs are of the k series, but gene products of herv - e are expressed on renal cell carcinomas and may serve as targets for donor cytotoxic t - cells (takahashi. high levels of herv - k rna was detected in sera from patients with either lymphoma or breast cancer, with whole viral particles being visualised in sera by electron microscopy, but the levels dropped markedly on treatment (contreras - galindo. 2008). despite these intriguing findings, the diverse roles of herv expression in cancers, and their causal significance, a similar degree of caution is required in the interpretation of findings of herv expression in disease processes as in the determination of the pathogenic role of exogenously acquired micro - organisms., herv expression may represent a normal physiological process that could well mediate a protective response to disease. this is particularly the case in the brain in which constitutive and genetically linked expression of several hervs has been detected in brain tissue from subjects with and without neuroinflammatory conditions (frank. thus, all human cells have genetic information encoding hervs and expression of herv genes is not detected in all patients with the putatively related disease, since such expression may have initiated the disease process years or decades before the clinical onset. moreover, there may be a low level of expression of the relevant hervs in persons without the disease. there are numerous copies of the herv genes within the human genome and, as a result of somatic mutations leading to genetic diversity, certain herv genes could be expressed in some healthy individuals. a critical point in the regulation of herv activity is immune control by self - specific cd8 t - cells, and those with specificities for herv targets are already present early in life. immune surveillance may depend on recognition of a single, or perhaps a very few, target peptides and may vary from person to person, depending on the profile of antigen - presenting hla - molecules. the efficiency of immune surveillance may well depend on expansion of the relevant sub - populations of self - specific cd8 t - cells by contact with homologous epitopes of exogenous micro - organisms. thus a lack of such contact due to hygiene - related factors enhances the risk of disease. alternatively, according to the principle of original antigenic sin, beneficial immune responses that are dominant if a micro - organism is encountered early in life may, if it is encountered later in life, be eclipsed by responses induced by prior contacts with antigenically related micro - organisms. the infectious and immunological background of herv - related disease is therefore far more complex than that of the classical infectious diseases so that none of koch s postulates can be used to confirm a causal relationship. instead, collectively, the following factors provide strong indications that a disease is caused by herv activity : the human genome codes for the relevant herv genes.at least one particular antigen of a specific herv is detected more frequently in tissues affected by the disease under consideration as compared with material from healthy control persons. in some cases two or three such antigens such as the env, gag and a peptide from a related open reading frame would be detected.the incidence of the disease has been significantly increasing in recent decades in countries with improved standards of hygiene.epidemiological studies reveal that a history of certain infections and vaccinations confer protection against the given disease and the use of genetic data banks reveal that protective micro - organisms express epitopes that could cross - react with the relevant herv epitopes but that these are not present in micro - organisms that are not associated with protection.vaccines with appropriate cross - reacting epitopes confer protection against the disease if administered at the time that the initial pathological changes leading to the disease occur. in the case of melanoma, this would be around ten years before clinical presentation.a product of a herv - encoded gene is shown to be directly involved in pathogenesis of a disease, such as altered melanin in melanoma (krone. 2005), or that it upregulates genes associated with expression of the disease, as in schizophrenia (huang. at least one particular antigen of a specific herv is detected more frequently in tissues affected by the disease under consideration as compared with material from healthy control persons. in some cases two or three such antigens such as the env, gag and a peptide from a related open reading frame would be detected. the incidence of the disease has been significantly increasing in recent decades in countries with improved standards of hygiene. epidemiological studies reveal that a history of certain infections and vaccinations confer protection against the given disease and the use of genetic data banks reveal that protective micro - organisms express epitopes that could cross - react with the relevant herv epitopes but that these are not present in micro - organisms that are not associated with protection. vaccines with appropriate cross - reacting epitopes confer protection against the disease if administered at the time that the initial pathological changes leading to the disease occur. in the case of melanoma, this would be around ten years before clinical presentation. a product of a herv - encoded gene is shown to be directly involved in pathogenesis of a disease, such as altered melanin in melanoma (krone. 2005), or that it upregulates genes associated with expression of the disease, as in schizophrenia (huang. herv postulates all apply to melanoma, as described above, and may find a more general application in the future. although one of koch s postulates, establishing the disease in an experimental animal, is inapplicable, proof of principle can be obtained by use of analogous animal models. a key issue in the emerging discipline of darwinian medicine, as recently reviewed in detail (rook 2009), is the impact of a rapidly changing environment on the acquisition of a range of micro - organisms that millions of years of evolution have led the developing immune system to expect. the other key aspect of this discipline, which can equally be considered darwinian, is the impact on health and disease, and indeed on evolution itself, of the endogenous relics of numerous successive retroviral infections over huge expanses of time. we postulate that these two aspects of darwinian medicine are very closely related, with fine balances between exogenous and endogenous immune challenges which, if disturbed, can result in disease. the study of herv - induced disease, in contrast to exogenous viral infections, is in its infancy. likewise, and for this reason, no specific vaccines against human herv - induced disease have been introduced, although vaccines eliciting tumour protective immune responses against endogenous retroviral epitopes in the mouse have been developed (kershaw. an alternative to developing novel vaccines is to screen currently available ones for their ability to generate cross - reacting protective immune responses to expressed herv epitopes involved in, or indicative of, disease processes. thus, as reviewed above, epidemiological studies demonstrated that vaccination with bcg and vaccinia early in life or with yellow fever in adult life induced significant protection against melanoma. although further studies are required, in particular to confirm the impact of yellow fever vaccination, immunization strategies of the future may have a two - fold aim to compensate for the loss of natural encounters with | introductiona diverse range of human diseases, including allergy, asthma, autoimmune disease, cancer and chronic neurologic diseases, notably multiple sclerosis and endogenous depression, is becoming more prevalent in industrialized countries. it has been postulated that environmental factors associated with improved standards of hygiene play a leading role in this process since the immune system seems to need extrinsic challenges for its proper maturation.the inner worldan added dimension has now emerged the impact on disease of the inner world, principally the numerous endogenous retroviruses (hervs) within the human genome. taking melanoma as an example, we propose a framework for understanding how a complex infectious and immunological background can induce or inhibit expression of a herv - related disease process. the central role of a failure to induce or to maintain certain populations of self - specific cd8 + t - cells mediating immune surveillance, the expression of herv - encoded peptides on affected cells and pathological mechanisms directly attributable to herv proteins are discussed.conclusionsthe presented concepts explain events preceding the clinical manifestation of diseases by several years and provide a rationale for the use of currently available vaccines to protect against certain herv - induced diseases, especially melanoma. criteria for establishing the causal role of hervs in a given disease are proposed. |
natural orifice surgery has evolved from a preclinical setting into a common occurrence at the university of california san diego (ucsd). with close to 40 transvaginal cases, the laparoscopic repair of recurrent umbilical defects, or defects greater than 3 cm in size, has been shown to decrease recurrence rates. we have chosen to further investigate the benefits of laparoscopy by applying a natural orifice approach. one of the perceived advantages in natural orifice surgery is the potential reduction in the incidence of hernia formation. patients with abdominal wall hernias may be at increased risk of forming additional hernias at incision sites. in addition, patients with recurrent incisional hernias may, likewise, be at increased risk. we believe that reducing or eliminating abdominal wall incisions may be of benefit in the repair of abdominal wall hernias. to this end, a preclinical research protocol in transvaginal incisional herniorrhaphy was undertaken in a porcine model. after obtaining comfort with the technical procedure, here, we describe what we believe to be the first natural orifice transluminal endoscopic surgical (notes) approach to the repair of an abdominal wall hernia. the patient is a 38-year - old female with a painful recurrent umbilical hernia who was repaired 8 years prior with a polypropylene - based mesh. the patient has had three children since the time of her repair and had a recurrence of her umbilical hernia, which was approximately 2 2 cm in size. the patient denied any obstructive signs or symptoms, but rated her daily pain at the site as 5/10. her past medical and surgical history is only pertinent for a cesarean section and the aforementioned hernia repair. the risks, benefits, and alternatives to a transvaginal incisional hernia repair she was consented under an approved ucsd transvaginal incisional hernia repair institutional review board (irb) protocol. importantly, all current ucsd notes protocols stipulate that an abdominal port be utilized for visualization of the transvaginal access. the patient was brought to the operating room, sequential compression devices applied, 1 g of cefazolin was given, and general anesthesia induced without complication. the patient was placed in the lithotomy position and prepped and draped in standard sterile fashion. a lower left quadrant 5-mm trocar was placed below the pubic hairline using optical assistance. a well incorporated mesh was noted in the umbilical region with a small herniation of falciform material through a 2-cm defect superiorly. given that the mesh was well incorporated and peritonealized, it was left in place. a uterine retractor was then placed and a 15-mm dual - lumen trocar (applied medical) was placed through the retrocervical cul - de - sac. the dual - lumen trocar allows for placement of the scope, as well as another 5-mm rigid or flexible instrument. the herniated falciform was then dissected free of the fascial edges using a combination of endoscopic and flexible instrumentation delivered through the vaginal port. next, the peritoneum was cleared superiorly approximately 4 cm using the same method. having dissected the facial edges free, the defect was then re - approximated primarily in the manner of franklin.. this was done transcutaneously using a suture passer through the center of the skin overlying the defect, and sequentially passing an 0-ethibond suture approximately 1 cm lateral to each fascial edge. next, a 7 7-cm flex hd acellular human dermis mesh (johnson & johnson) was inserted through the vaginal trocar once four anchoring ethibond sutures were tied to the corners. a needle passer was passed in four quadrants, tacking the mesh in a transfascial manner. lastly, a protack (autosuture) device was used to anchor the remainder of the material to the patient s anterior abdominal wall at approximately 1-cm intervals circumferentially (fig. 2final view of the hernia repair entrance of the vaginal trocar final view of the hernia repair the abdomen was inspected and had good hemostasis ; the 5-mm abdominal port was removed. the gynecology team then closed the patient s vaginal access site with a 2 - 0 vicryl suture in a running fashion. the 5-mm trocar was removed and the skin closed with a 4 - 0 monocryl suture. the patient was admitted to the hospital for observation as per the irb and was discharged on postoperative day 2 in good health. at 6 weeks she was then seen at 3 and 5 months and is still completely satisfied with her repair. the repair of primary and incisional hernias of the ventral abdominal wall via a transvaginal approach is technically feasible, and the result of our initial case was exceptional. however, there are still significant obstacles which must be addressed before this approach can be widely utilized. some have suggested that blind or primary access of the vagina for natural orifice surgery is safe [3, 4 ]. we do not agree. at this time, we still believe that visualization of the trocar insertion through the vagina is necessary. though all of the key steps of the described operation were performed utilizing the vaginal access port, we still believe that blind access to the abdomen with a trocar through the vagina may put the patient at risk for visceral or vascular injury. an additional concern in the application of natural orifice surgery to the repair of ventral hernias is the selection of the proper material for the hernia repair. several authors have described the preclinical technical aspects of notes hernia repair in a cadaver or animal model, and most have questioned whether a prosthetic material can be used in regard to potential mesh infection [5, 6 ]. we believe this to be a very valid point. additionally, many recurrent ventral hernias have also had prior implantation of prosthetic materials, as did this patient. her mesh was firmly incorporated and peritonealized. given this, we decided to progress with the operation. had the initial mesh been exposed or not incorporated, we would have abandoned the notes approach in favor of a more traditional method. we do not believe that there is sufficient preclinical data from our own laboratory, or others, to advocate the placement of a prosthetic device in the face of transvaginal or transgastric abdominal access. therefore, we used a biologic material for the repair. the use of biologic materials for abdominal wall reconstruction in contaminated or infected fields is well established. however, there is increasing concern that the re - herniation rate will be unacceptably high if the material is used to bridge the defect, rather than to buttress an appropriately re - approximated fascia. fascial re - approximation was fairly simple with this small defect, but this severely limits the patient population which may benefit from a natural orifice approach to ventral hernias. for the procedure to have widespread utilization, prosthetic devices will have to be used. one of the main benefits that we have seen in our transvaginal cholecystectomy population has been the reduction of postoperative pain. while our patient was fairly comfortable postoperatively, she still experienced some pain (5/10) at the transfacial fixation sutures on postoperative day 1. whether or not these sutures are still necessary when using a material that is ultimately absorbed or incorporated is questionable. there are no large case series from which to draw observations in regard to the laparoscopic placement of biologic materials for the repair of ventral hernias. given the minimal penetration of the abdominal tacks into the posterior fascia once passed through biologic material, we still believe them to be necessary. the initial application of natural orifice techniques to the repair of an abdominal wall defect was technically feasible and clinically successful. the patient presented here did very well postoperatively, with minimal complaints of pain or any signs of infection. the outcome from this operation was encouraging and we look toward further investigation to answer the aforementioned questions noted in this paper. | introductionnatural orifice surgery has evolved from a preclinical setting into a common occurrence at the university of california san diego (ucsd). with close to 40 transvaginal cases, we have become comfortable with this technique and are exploring other indications. one of the perceived advantages in natural orifice surgery is the potential reduction in the incidence of hernia formation. patients with abdominal wall hernias may be at increased risk of forming additional hernias at incision sites. in addition, patients with recurrent incisional hernias may, likewise, be at increased risk. we believe that reducing or eliminating abdominal wall incisions may be of benefit in the repair of abdominal wall hernias. here, we describe what we believe to be the first natural orifice transluminal endoscopic surgical (notes) approach to the repair of an abdominal wall hernia.methodsthe patient is a 38-year - old female with a painful recurrent umbilical hernia, previously repaired 8 years prior with a polypropylene - based mesh. the patient underwent a transvaginal recurrent umbilical hernia repair with one other 5-mm port in the abdomen for safety.resultsthe patient had no intraoperative or postoperative complications. at 5 months follow up, the patient had no complaints, no evidence of hernia recurrence, and was very pleased with her result.conclusionsthe repair of primary and incisional hernias of the ventral abdominal wall via a transvaginal approach is technically feasible, and the result of our initial case was exceptional. however, there are still significant obstacles which must be addressed before this approach can be widely utilized. these obstacles include safe entrance into the abdominal cavity via a transvaginal approach, the proper mesh to be placed during the repair, and the risk of infection. |
the study was carried out in the obafemi awolowo university teaching hospitals complex (oauthc), ile patients who were recruited into this study were diagnosed with depressive episode using the criteria of the international classification of diseases, 10th edition (icd 10) (20). those who had psychotic symptoms or who had co - morbid physical disorders were excluded from the study. the patients included were those who presented for the first time in their life and had not been started on medication. seventy - eight consecutive patients who met the criteria and gave consent to participate were recruited into the study. severity of depression was rated using the english language version of the 17-item hamilton depression rating scale (hdrs). max hamilton originally published the hdrs in 1960 and reviewed and evaluated it subsequently (2125). the scale is clinician - rated and is currently one of the most commonly used scales for rating depression in medical research and practice. nine of the 17 items are scored on a five - point scale, while the remaining eight items are scored on a three - point scale. a score of 1013 is regarded as mild depression, 1417 as mild to moderate depression, and > 17 as moderate to severe depression (appendix 1). eds was evaluated in the patients using the english language version of the epworth sleepiness scale (ess). this is a self - administered, eight - item, well - validated, and widely used subjective sleepiness scale. the patients were asked to score the likelihood of falling asleep in eight different situations. scores on the ess range from 0 to 24, with higher scores indicate greater likelihood of sleepiness. a score of 10 but less than 18 is considered sleepy, while a score of 18 or more is considered very sleepy (appendix 2). some previous studies variously defined eds as an ess score>10 (26), > 11 (19), or 10 (27). in this study, eds was defined as a score equal to or above the whole number immediately higher than the mean ess score. the data were analyzed using the statistical package for the social sciences, version 16 (spss 16.0). regression analysis was done with ess score and hdrs score as dependent and independent variables, respectively. the data from 67 of the 78 patients (85.9%) were included in the analysis. of the 67 patients included, 42 were females (62.7%) and 25 were males (37.3%). the age range of the patients was 3155 years with a mean of 41.4 years (sd=7). socio - demographic characteristics of the patients the mean ess score was 9.2 (sd=2.8), and so the cut - off point for eds was taken as an ess score 10. thirty of the 67 patients (44.8%) had eds defined as ess score of equal to or greater than 10. the mean score on the hdrs was 14.8 (sd=3.6). the correlation (r) between the ess score and the score on forty - eight percent (r = 0.48) of the total variance in the ess score is accounted for by its linear relationship with the score on the hdrs. results of regression analysis of ess score and hdrs score as dependent and independent variables, respectively note : ess, epworth sleepiness scale ; hdrs, hamilton depression rating scale. eds is one component of hypersomnia (the other component being prolonged nocturnal sleep). eds refers to sleepiness that occurs when the individual is reasonably expected to be awake and alert. many of the patients (44.8%) had eds defined as ess score of 10. in a similar study, 57.2% of depressed patients had eds and their ess scores correlated highly (r=0.85) with ratings on the beck depression inventory (27). some other studies also suggested a significant association between daytime sleepiness and severity of mood symptoms (15, 28). in the present study, it has been suggested that eds and depression have some genes in common and might both be inherited (8). eds has also been found to be strongly associated with suicidal ideation in depressed patients (27). it is thus clear that any clinical evaluation of depressive illness that does not include the evaluation of eds is incomplete. eds in a setting of depressive illness may respond to energizing antidepressants such as fluoxetine or bupropion. the clinician should be familiar with features of obstructive sleep apnea, narcolepsy, and idiopathic hypersomnia, all of which are common causes of hypersomnia and any of which may also coexist with depression. obstructive sleep apnea is characterized by snoring and periods of apneic attacks during sleep with daytime symptoms of fatigue and sleepiness. narcolepsy is characterized by a tetrad of eds (many sleep episodes and drowsiness), cataplexy (sudden decrease or loss of muscle tone often precipitated by intense emotion such as laughter or anger), sleep paralysis (which occurs on falling asleep or on waking up, with the individual unable to move or speak), and hypnagogic (sleep onset) hallucinations. all narcolepsy patients experience excessive sleepiness, most have cataplexy and some have three or four features of the narcoleptic tetrad. nighttime sleep may also be disrupted with repeated awakenings. the features of idiopathic hypersomnia include chronic sleepiness without cataplexy or other features of narcolepsy. in view of the established association between eds and depression, and given the harmful consequences of both conditions (operating separately or jointly), there is a need for proper clinical evaluation of eds in every case of depressive illness. it is therefore suggested that an effort be made by appropriate national and state authorities to establish facilities for dealing with sleep complaints. in terms of human resources, there are not enough personnel in the field of sleep medicine in the country. it is hoped that with adequate and proper facilities in place, the number of personnel in this field will gradually increase. in the meantime, clinicians should be aware of the magnitude of the problem of eds, particularly among depressed patients, and do their best to assist the patients. the author has not received any funding or benefits from industry to conduct this study. | backgroundexcessive daytime sleepiness (eds) has been reported among depressed patients in many populations. many depressed patients seek medical attention partly to deal with eds, but this sleep disorder is often overlooked in clinical practice.objectivesthe objectives of this study were to determine the prevalence of eds among depressed patients and determine its relationship with the severity of depression.methodssixty-seven patients diagnosed with depressive episode took part in the study. the severity of depression was rated using the 17-item hamilton depression rating scale (hdrs). eds was evaluated using the epworth sleepiness scale (ess).resultsthe mean ess score was 9.2 (sd=2.8). eds, defined as an ess score10, was present in 44.8% of the depressed patients. the mean score on the hdrs was 14.8 (sd=3.6), representing the mild moderate depression range. ess scores correlated highly and positively (r=0.69, p=0.000) with scores on the hdrs.conclusionin the light of the high prevalence of eds among depressed patients and its undesirable consequences, it is suggested that daytime sleepiness be evaluated in depressed patients. |
as presented in part 1 of this epidemiologic study, a bladder cancer cluster occurred within a cohort of white male criminal investigators working with the bureau of alcohol, tobacco, firearms and explosives (atf), united states department of justice, between 1994 and 2005. the cluster was identified through the self - reporting of employees participating in a medical surveillance program which was initially set up to monitor the health of employees dedicated to the investigation of fires and explosions. six of the seven were pathology report verified as urinary bladder cancer, five as low grade papillary transitional cell carcinoma, and one as transitional cell carcinoma in situ. in part 1, analysis of bladder cancer incidence in the study population determined that white male criminal investigators, without regard to work history, were at statistically significant increased risk for bladder cancer. because six of the seven cases reported holding special assignments associated with post - fire / post - blast scene investigation while employed with atf, the bladder cancer cluster appeared to be associated with the work on such scenes. most scenes investigated by atf are post - fire rather than post - blast and involve municipal structures. although atf employees who investigate post - fire scenes typically wait until the fire is out to enter the scene to search for origin and cause, the work still puts them at risk for exposure to a mix of hazardous chemicals and products of incomplete combustion which potentially includes known and suspect bladder carcinogens such as aromatic amines and polycyclic aromatic hydrocarbons (pahs) [220 ]. guidotti and clough expressed particular concern for exposures during the clean - up phase related to the smoldering of synthetic materials and the release of trapped gasses from porous materials, and this concern applies to fire investigation activities. several authors offer detailed reviews of the general toxicologic aspects of fire smoke and summaries of findings of fire scene exposure monitoring projects [2123 ]. most recently, the international agency for research on cancer published a monograph on firefighting which focuses on the results of studies measuring exposures to carcinogens found in smoke at fires. studies attempting to characterize exposures during firefighting activities have in general not monitored for aromatic amines and relatively few have monitored for pahs [2227 ]. as critiqued by golka and weistenhfer, the studies on characterization of smoke at fire scenes do not support the premise that firefighters are at increased risk for bladder cancer. independently, atf sponsored two efforts to characterize exposures occurring during fire scene investigation by atf employees, (1) a health hazard evaluation performed by niosh in 1997 and (2) a comprehensive industrial hygiene study carried out by a certified industrial hygienist between 2005 and 2009 (f. fitzpatrick, cih, unpublished data provided in report to atf entitled exposures to chemicals and other hazardous substances during atf post - fire / blast scene investigations, may 2010). both studies monitored for exposure to pahs, and the more recent study also monitored for aromatic amines. the first study monitored five different fire scenes, while the second study monitored 13 different fire or blast scenes. low levels of pahs were obtained at fires scenes in both studies, and no detectable levels of aromatic amines were obtained in the second study. in both studies all detectable levels of pahs were well below established occupational exposure limits, and none of the detectable pahs in fitzpatrick 's study were classified as a1 or a2 carcinogens by the american conference of governmental industrial hygienists. as no two fire scenes are alike and specific exposure risks vary from one scene to another, these industrial hygiene findings may not typify investigator exposures at all present - day fire scenes and may not characterize exposures occurring at fire scenes 10 to 20 years ago. investigator variables such as time spent on scene, use of personal protective equipment, and adherence to cleanup procedures will also influence exposure risks. potential for internal exposure to pahs during firefighting activities has been demonstrated in a study on firefighters exposed to burning diesel fuel during training exercises. this study found slight but statistically significant increases in pah urine levels over several days of fire suppression training, reductions in levels with use of respirators, and higher levels among smokers, and may have application to the job of post - fire investigation. since regular and recurring work as a fire investigator poses increased risk for exposure to variable and mixed products of combustion, such work might also be associated with increased risk for bladder cancer. since firefighters and fire investigators share a potential for similar exposures, and firefighters may participate in fire investigation, a review of the epidemiologic studies on bladder cancer in firefighters is appropriate. since the mid-1980s numerous epidemiologic studies [3044 ] and review articles [6, 23, 24, 45 ] have explored the relationship between firefighting and bladder cancer risk. at least 10 studies have looked at cancer mortality [3032, 3436, 38, 40, 41, 44 ], and six studies have looked at cancer incidence [32, 33, 37, 39, 42, 43 ]. in addition, other epidemiologic studies, evaluating the association of bladder cancer incidence and occupations in general, have also addressed the cancer risk in firefighters [4650 ]. there have been inconsistencies in results among these studies, and in those studies showing an increased risk of bladder cancer incidence or mortality among firefighters, few achieved statistical significance. these included one mortality cohort study with a standardized mortality ratio (smr) of 2.86 (95%ci 1.305.40), one incidence case - control study with an odds ratio (or) of 1.59 (95%ci 1.022.50), and one incidence cohort study with a standardized incidence ratio (sir) of 1.29 (95%ci 1.101.62). three recently published meta - analyses [5153 ] and one unpublished meta - analysis (x. tao, md, phd, unpublished data provided in report to atf entitled evaluation of a bladder cancer cluster among a cohort of criminal investigators with the bureau of alcohol, tobacco, firearms and explosives, november 2007) of the combined findings of epidemiologic studies addressing firefighters and bladder cancer risk found a 1.17-fold to 1.39-fold increase in cancer incidence risk and a 1.14-fold to 1.29-fold increase in cancer mortality risk, but these increases were of no or only marginal statistical significance. in the most recently published meta - analysis of bladder cancer incidence in firefighters, which included nine studies, the summary relative risk (srr) was 1.17 (95%ci 0.921.49). part 2 of this study uses data from the atf medical surveillance program to evaluate the association between post - fire / post - blast investigation and bladder cancer risk within the study 's white male cohort through an internal nested case - control analysis which controlled for both age and tobacco use history. part 1 previously detailed the atf medical surveillance program, which was established on a voluntary basis in 1995 and became mandatory in 2002, and the full roster cohort of 3768 employees, predominantly criminal investigators, used in the bladder cancer incidence study. as 2003 was the first year of the program in which participating atf employees completed detailed work history questionnaires, this analysis focused on program participants between 2003 and 2007. all seven bladder cancer cases identified in the atf cohort study of part 1 were self - reported by employees participating in the medical surveillance program. verification of six of the seven cancers by pathology report was addressed in part 1. as all reported bladder cancers occurred in white males, this study was limited to white males. the cases for this analysis, by definition, included all individuals with self - reported bladder cancers who were employed by atf at the time of diagnosis and who had at least one medical surveillance exam during the period 20032007 with complete data on all study parameters. the selected control group, by definition, included all white males in the atf cohort of part 1 who had at least one medical surveillance exam during the period 20032007 with complete data on all study parameters and no self - reported history of any type of cancer. dates of birth for cases and controls were self - reported through the medical surveillance program and then verified through cross - referencing with dates of birth obtained from annual atf staffing rosters. year of diagnosis of bladder cancer for cases was self - reported through the medical surveillance program and then verified through cross - referencing with a pathology report, as available. for study purposes, case age was the age the case turned in the year of diagnosis and control age was the age the control turned in the year of the most recent complete examination in the database. as cigarette smoking is a significant risk factor for bladder cancer, this study controlled for tobacco use history. tobacco use history for cases and controls was self - reported through the medical surveillance program and included the following : (1) yes or no if tobacco products were ever used, (2) whether use was current or past, (3) if past, the year quit, (4) type of product used (cigarettes, pipe / cigar, snuff / chew), (5) number of years used, and (6) amount per day. for study purposes, tobacco use data for the cases reflected tobacco use status in the year of diagnosis and tobacco use data for the controls reflected tobacco use status in the year of the most recent complete examination in the database. for cases diagnosed prior to 2003, historical medical surveillance data was accessible to verify tobacco use status in the year of diagnosis. tobacco use status of cases after year of diagnosis was excluded from study as it was not relevant. the selected work history parameters for this study included (1) number of years reported working on team assignments and with special designations relevant to post - fire / post - blast investigation (special assignment years), (2) number of years reported working post - fire / post - blast scenes (fire scene exposure years), and (3) number of days reported working post - fire / post - blast scenes (fire scene exposure days). as atf investigations are predominately post - fire, these work history parameters serve as surrogate measures or variables of exposure to products of combustion associated with fire scenes. these parameters applied only to work with atf and did not include work with prior employers. the team assignment and special designation categories included (1) national response team (nrt), (2) division response team (drt), (3) arson task force, (4) certified fire investigator (cfi), and (5) certified explosives specialist (ces). the terms nrt, cfi, and arson task force were clarified in part 1 of this study. the drt is similar in concept to the nrt but responds to requests for assistance on a regional level rather than a national level. cess are criminal investigators who have gone through a special training to receive certification as explosives experts. work history information was collected through the medical surveillance program in the same way for both cases and controls. for cases, work history data reflected the reported number of years worked or number of days worked up to the year of diagnosis. a projection was used to calculate the number of days worked on fire and explosives scenes up to the year of diagnosis, if this information was first reported after the diagnosis year. this projection was based on the assumption that the days worked were evenly distributed over the years worked on fire scenes. for controls, work history data reflected the reported number of years worked or number of days worked up to the year of the most recent complete examination available in the database. evaluation of the association between post - fire / post - blast scene investigation and bladder cancer incorporated a nested case - control study design to compare the work histories of the bladder cancer cases and the work histories of the controls with no history of any cancer. as stated earlier, since all identified bladder cancers in the study population occurred in white males, the nested case - control study focused only on white males. see section 2.1 for previously provided definitions of the cases and the controls used in this analysis. odds ratios (ors), based on logistic regression models, were computed to estimate the relative risk of bladder cancer associated with each fire scene exposure study parameter, while controlling for the confounding factors tobacco use and age. analyses of the special designation and team assignment categories included both years worked in each category and years worked in any one or more of the five categories combined. controls were not matched with cases in terms of the exposure parameters under study in order to avoid overmatching bias. to control for age, cases and controls were grouped into 10-year age increments : less than 30 years, 3039 years, 4049 years, 5059 years, and 60 or more years. to control for tobacco use, cases and controls were grouped into the following categories : non - user, user less than 10 years, and user 10 or more years. the small sample size limited more sophisticated covariate matched analysis involving age or further stratified analysis of tobacco use history. during the period 20032007, 2549 members (68%) of the full roster cohort of 3768 individuals (previously detailed in part 1) completed at least one examination and one work history questionnaire. these 2549 individuals represented 87% of the 2928 cohort members who were employed by atf for one or more years during 20032007. table 1 shows the distribution of individuals by race and sex for these 2549 individuals, as well as the distribution of self - reported bladder cancers and all other cancers, by race and sex. all seven of the self - reported white male bladder cancers of the cohort study met the definition as a case for this study and had complete study parameter data. of the 1771 white males in the population of table 1, 1525 individuals met the definition as a white male control and had complete study parameter data. excluded from the control group were 79 white males with a reported history of some other cancer and 160 white males with incomplete study parameter data. thus, the study population for the internal comparison analysis comprised seven reported bladder cancer cases and 1525 controls with no history of cancer. all cases and 97% of controls the remaining controls were primarily explosives enforcement specialists, forensic chemists, and fire protection engineers, typical members of the nrt. table 2 shows the distributions of cases and controls among the age increments and tobacco use parameters selected for analysis. about the same percentage of cases and controls was also tobacco nonusers, but the percentage of cases with less than 10 years of tobacco use was about half the percentage of controls with less than 10 years of tobacco use, and the percentage of cases with 10 or more years of tobacco use was about twice the percentage of controls with 10 or more years of tobacco use. by controlling for any form of tobacco use rather than cigarette use alone, greater weight was given to the cancer risk attributed to tobacco use, and lesser weight was given to the cancer risk associated with fire investigation work. among the seven cases, six reported work histories associated with investigation of fire scenes while employed with atf, as mentioned in the introduction. these six cases also comprised the six cancer cases verified by pathology report. at the time of diagnosis, three cases were both cfis and members of the nrt, one was a cfi but not a nrt member, one was a member of the nrt but not a cfi, and one was a member of the division response team (drt). three cases were also members of the arson task force and two of the cfis with membership on the nrt were also cess. none of the seven reported work histories associated with fire scene investigation prior to employment with atf. table 3 shows the distributions of cases and controls for each analyzed work parameter. for fire scene exposure days, a considerably lower percentage of cases reported 1 to 199 days of exposure compared to their control counterparts, and a considerably higher percentage of cases reported 200 or more days of exposure compared to their control counterparts. for fire scene exposure years, the distributions of cases and controls among the parameter 's incremental categories were fairly similar. for the study parameters of years spent on special assignment, when applicable, the percentages of cases with one to four years and with five or more years on special assignment exceeded the respective percentages of controls for any one or more of the special assignments combined and for each individual assignment. table 4 presents the odds ratios (ors) for each exposure variable, while controlling for tobacco use and age. analysis of fire scene exposure days did not show a statistically significant increase in bladder cancer risk with either one to 199 days or 200 or more days on fire scenes compared to zero days on fire scenes and actually suggested a protective effect with one to 199 days of exposure compared to zero days (or 0.05 (95%ci 0.000.82)). likewise, analysis of fire scene exposure years did not show a statistically significant increase in bladder cancer risk with either one to nine years or 10 or more years on fire scenes compared to zero years on fire scenes. analyses of years spent on any one or more of the special assignments combined and on each individual special assignment did, however, identify significant associations between special assignment work and increased risk for bladder cancer. for participation on any one or more of the special assignments, the or was 19.01 (95%ci 1.94186.39) for one to four years on any special assignment compared to zero years and 12.56 (95%ci 1.14138.58) for five or more years compared to zero years. for individual team assignments, nrt work with both one to four years and five or more years, drt work with five or more years, and arson task force work with one to four years exposure were all associated with statistically significant increase in bladder cancer risk. the cfi designation was associated with the highest ors for both one to four years and five or more years compared to zero years. the ces designation was the only individual special assignment which was not associated with increased risk of bladder cancer. as previously detailed in part 1 of this epidemiologic study, a bladder cancer cluster occurred within a cohort of white male criminal investigators working with atf between 1994 and 2005. the cluster consisted of seven self - reported cases, of which six were verified by pathology report. all six of the verified cases also had work histories of participation in fire scene investigations. this observation raised concern that fire scene investigation might be linked to increased risk for bladder cancer in this population. the nested case - control study of part 2 shows a statistically significant association between work on special assignments involving post - fire scene investigation and increased risk for bladder cancer and complements the findings of part 1, which show a significant increase in bladder cancer incidence in the criminal investigator employee population. no previous study reported in the literature has addressed the association of bladder cancer risk and fire investigation. specifically, the nested case - control analysis showed participation on any one or more of the special assignments combined to be associated with a greater than 12-fold increase in bladder cancer risk for both one to four years and five or more years of exposure, when compared to zero years of exposure, as detailed in section 3.4. with individual special assignments, greater than ninefold increases in cancer risk were seen for nrt members and for cfis with both one to four years and five or more years exposure, for drt members with five or more years exposure, and for arson task force members with one to four years exposure, when compared to zero years exposure. only the ces assignment, the one typically least involved with fire scene investigation, was not associated with statistically significant increase in risk of bladder cancer. the greatest increase in cancer risk was associated with the cfi special assignment, which was held by four of the seven bladder cancer cases, with ors of 43.84 (95%ci 6.70287.02) for one to four years and 22.76 (95%ci 2.52205.91) for five or more years. this finding is likely explained by the focused and intense role cfis play in fire investigation. also noteworthy, since individuals serving in any one of these special assignments frequently participate in one or more of the other special assignments, the individual assignment categories are not independent of one another. consequently, the ors for participation on any one or more of the special assignments combined may best characterize general estimations of cancer risk associated with fire scene investigation. another interesting point is the finding that the ors for one to four years of exposure were greater than the ors for five or more years of exposure for the nrt, cfi, and any one or more of the special assignments combined analyses. this observation suggests a very short latency period for some of the cases and may pertain to the requirement for cfi candidates to participate in 100 fire scene investigations during the cfi two - year certification process. it should also be mentioned that, aside from the statistical significance of the ors found with the special assignment analyses, the observed wide confidence intervals limit any perceived importance of differences in the magnitude of the ors between assignment categories or between one and four years of exposure and five or more years of exposure. as addressed in the introduction, known and suspect bladder carcinogens are potentially present in postcombustion products present at fire scenes, and the threat for exposure to postcombustion products from smoldering and off - gassing materials exists during investigation of those scenes. primary routes of exposure to these products include inhalation and skin absorption. exposure risk is dependent on a variety of factors including scene parameters (e.g., presence of smoldering hot pockets, amount of ventilation) and work practices (e.g., use of personal protective equipment, eating and drinking on site, maintaining adequate hydration, containment and decontamination of soiled clothing and equipment, timeliness of personal cleanup upon leaving the scene). while the use of self - contained breathing apparatus (scba) and firefighter turnout gear is an established practice during fire suppression, the use of respirators and other personal protective equipment during post - fire overhaul and investigation activities is less routine and, exposure to hazardous chemicals may in fact be greater during post - fire overhaul and investigation than during fire suppression. some fire investigators with atf have in the past described clearing their nose of black mucus for several days following a three to five day post - fire investigation or experiencing their vehicles reeking of smoke from soiled clothing and equipment. this anecdotal information appears to support the findings of this nested case - control study by illustrating that fire investigators could potentially be exposed to bladder carcinogens through both inhalation and skin absorption. even though the air - monitoring industrial hygiene studies reviewed in the introduction did not find bladder carcinogen concentrations of concern, the studied fire scenes may not represent all fire scenes and may not adequately define investigator risk for exposure to bladder carcinogens, especially historical risk occurring during the years prior to bladder cancer diagnosis. with each fire scene being unique, investigator use of respirators and other personal protective equipment, adherence to housekeeping measures related to personal hygiene and cleanliness, and decontamination of clothing and gear can be expected to moderate potential internal exposure to combustion products and any associated cancer risk. after concern was first raised that fire investigation appeared to be associated with increased risk for bladder cancer, atf elected to take precautionary actions to raise awareness among investigators, formalize a respiratory protection program, and improve work practices associated with fire investigation to reduce potential for exposure to hazardous chemicals while at and upon leaving fire scenes. it has now been over six years since the most recently reported case of bladder cancer. this is the first known epidemiologic study to evaluate the association of bladder cancer risk and fire scene investigation. the odds ratios generated in this study are relatively high when contrasted with findings of individual epidemiologic studies of bladder cancer risk in other occupations and industries. in these other studies, statistically significant increases in bladder cancer risk are typically found in the 1.1-fold to fivefold range but also occur in the sixfold to tenfold range, as addressed in the discussion section to part 1. for example, one study of occupational factors and bladder cancer incidence in canada showed statistically significant ors for jobs in the chemicals industry (2.37), with tars or asphalt exposure (3.11), in dye manufacturing (3.62), and in the dyeing of cloth (4.63). another case - referent study on occupational risk factors for bladder cancer in southern israel found statistically significant ors of 4.67 and 6.25 for occupational exposures to dusts and to multiple chemicals, respectively. in one study on chimney sweeps, the standardized morbidity ratio for bladder cancer was elevated and statistically significant at 2.36. in the recent meta - analysis by reulen., however, statistically significant elevations in summary relative risks for bladder cancer, which were found for several occupations, were relatively low, in the 1.1-fold to 1.3-fold range, as explained in part 1. the elevated ors of the current study even exceed the twofold to sixfold increase in risk for bladder cancer typically found in cigarette smokers compared to nonsmokers [5659 ]. additional epidemiologic studies are warranted to corroborate the findings of this study and in particular the magnitude of the elevated risk found with fire scene investigation. in the interim, the findings of both the incidence study and the nested case - control study support atf 's preliminary initiatives to educate the employee population regarding the potential cancer risks associated with post - fire investigation, monitor the health of the employee population through the medical surveillance program, perform bladder cancer screening as part of the medical surveillance program, provide appropriate personal protective equipment to those investigating fire scenes, and establish guidance for appropriate cleanup and decontamination measures following fire scene work. continued monitoring of current employees through the medical surveillance program for another five to 10 years is warranted to track the future pattern of bladder cancer occurrence in the population. in addition, with cancers being typically associated with latency periods, inclusion of retirees in some form of health - monitoring program should be a consideration. one strength of this nested case - control study is that work history data were available on 87% of the 2928 members of the full roster cohort who were employed with atf for one or more years during the period 20032007, when the work history questionnaire was part of the medical surveillance program. with this level of participation in the program, another strength of the study is that the atf employee population under study was fairly stable during the time frame of both the cancer incidence study of part 1 and the case - control study of part 2. although the seven individuals with reported bladder cancer were diagnosed over a 12-year period, 19942005, while employed with atf, all were still employed with atf during the period 20032007 and completed at least one work history questionnaire. in addition, of all white males in the full roster cohort of the incidence study, 65% were still employed with atf during 20032007 and completed work history questionnaires. the actual control group for the nested case - control study comprised 56% of all white males in the full roster cohort of the incidence study. the small number of cases is a limitation of this study and can be expected to contribute to statistical instability and wide confidence intervals of the ors, but the large size of the control group counters the effect of the small case size and restores some statistical stability to the ors or the confidence intervals would be even wider. another limitation of this nested case - control study concerns employee self - reporting of bladder cancer diagnoses. as presented in a prospective cohort study by bergmann. on the accuracy of self - reported cancer diagnoses when compared to state cancer registries, the sensitivity of self - reporting bladder cancer was 0.67 and the positive predictive value was 0.72. although the sensitivity in the bergmann study was only 0.67, the seven reported cases of the atf cohort were sufficient in number to achieve statistically significant elevations in the sirs computed in part 1 and in the ors of this study associated with fire scene special assignments. even though six of the seven identified cancer cases reported work on fire scene special assignments, any selective underreporting of bladder cancer among those with no fire scene special assignments would clearly affect the or outcomes. from a positive predictive value perspective, six of the seven (86%) reported cases in this study were pathology report verified. interestingly, the one unverified case was the same case with no reported work on fire scenes ; even with inclusion of this unverified case, the ors for the majority of fire scene special assignment analyses were significant. employee self - reporting of all exposure parameters, with potential for recall bias, also presents a study limitation. in this study, several work history parameters were selected for evaluation as surrogate measures of exposure to products of combustion associated with fire scenes. the most reliably reported work parameter is conceivably the number of years spent working on special assignments associated with fire scene investigation, where assignment membership is formally established. this work parameter was the only one in the study found to be associated with statistically significant increased bladder cancer risk and appears to qualify as a surrogate measure of fire scene exposure. the reported number of years spent working on fire scenes might also be a reliably reported work parameter, but unlike the special assignment parameter, it may not have functioned as a good indicator of actual fire scene exposure, for there was not a significant association between fire scene years and increased cancer risk. the number of fire scene days is likely the most unreliably reported work parameter due to recall bias and employee retrospective estimation of days spent on fire scenes, especially for work predating the institution of the work history questionnaire in 2003. in addition, for the or analysis of bladder cancer risk associated with reported fire scene days, as six of the seven cases were diagnosed prior to initiation of the work history form, the number of accrued days on fire scenes at the time of diagnosis was retrospectively calculated for these six cases through a systematic approach which applied the assumption that total accrued days were evenly distributed over the years worked on fire scenes with atf. inaccuracies in reported number of fire scene days and calculation assumptions could account for the lack of association between this work parameter and increased risk of bladder cancer and for the apparent protective effect of working 1199 days on fire scenes versus zero days, as noted in section 3.4, or this exposure variable may just not have been an appropriate surrogate of actual fire scene exposures. to conclude, in this nested case - control study on the atf medical surveillance population, white males with work histories of holding special assignments associated with post - fire / post - blast investigation had statistically significant elevated risk of bladder cancer compared to white males with no work histories of holding these special assignments. the other work parameters, days spent and years spent working fire scenes, were not associated with statistically significant increased risk for bladder cancer compared to no days spent and no years spent working fire scenes, respectively. | this study evaluated the association of bladder cancer risk and fire scene investigation within a cohort of white male criminal investigators with the united states bureau of alcohol, tobacco, firearms and explosives that was found to be at increased risk for bladder cancer. medical surveillance data were used in a nested case - control study to determine odds ratios (ors) estimating the relative risk of the cancer associated with post - fire investigation. the study comprised seven bladder cancer cases and 1525 controls. six of the cases reported holding assignments associated with post - fire investigation. the or for bladder cancer was 19.01 (95% confidence interval = 1.94186.39) for those holding any one or more of these assignments for one to four years versus zero years and 12.56 (1.14138.58) for those holding any one or more of these assignments for five or more years versus zero years. the risk for bladder cancer is significantly elevated for those holding post - fire investigation assignments compared to those not holding these assignments. |
myocardial infarction due to coronary atherosclerotic rupture is one of the main causes of mortality in young adults (1). despite the recent advancements in various imaging modalities, identification of high - risk coronary plaque is still difficult. anatomical imaging to assess luminal patency, such as computed tomography (ct) coronary angiography, have failed to detect high - risk plaque, because the vulnerable vessel normally has non - flowing limiting plaque, which is a high - risk plaque called " vulnerable plaque. " to overcome the limitations of anatomical imaging, several molecular imaging probes are currently being investigated to target biomarkers in vulnerable plaque. molecular markers for atheromatous plaque can provide clinically crucial information regarding the vulnerability and the degree of progression. in addition, molecular imaging has a potential to permit the development of a novel therapeutic agents and non - invasive imaging tool to monitor the therapy response (23). in this study, we reviewed the pathophysiology of calcification in atherosclerotic plaque and f-18 fluoride positron emission tomography (pet) as an imaging probe for vulnerable plaque. the pathogenesis of atheromatous plaque formation involves cholesterol deposition, macrophage accumulation, inflammation, and smooth muscle proliferation. the characteristics of vulnerable plaque include a large necrotic core, thin fibrous cap, inflammation, macrophage aggregation, hypoxia, and microcalcification (456). early plaque calcification was observed in the thin fibrous cap overlying the necrotic core of atherosclerotic plaque. studies have shown that microcalcifications in the thin fibrous cap increased the risk of plaque rupture, and subsequent stress - related microfractures result in acute thrombosis (78). similar to the calcification observed in tuberculosis patients, calcification occurs in patients with atheroma during the healing process after inflammation in the necrotic core (9). the early phase is not visible on conventional imaging, but is associated with plaque instability. the latter phase of macroscopic calcification can be observed on a radiograph or ct (10). normally, the diameter of microcalcification is small (1000) did not show significant f-18 fluoride uptake. this finding suggested that f-18 fluoride pet provides different information relating to metabolically active calcific plaque and developing microcalcification. coronary fdg pet images were not adequate in 49% of the cases, mainly due to high myocardial fdg uptake, small size, and partial volume effect. moreover, f-18 fluoride pet was related to symptomatic status, prior major adverse cardiac events, and cardiovascular risk scores, which showed the clinical significance. the diagnostic performance of f-18 fluoride pet for identification of ruptured and high - risk atherosclerotic plaque was assessed by a prospective clinical trial (24). the patients with myocardial infarction (n = 40) and stable angina (n = 40) underwent fdg pet, f-18 fluoride pet, and invasive coronary angiography. 93% of the patients with myocardial infarction showed high f-18 fluoride uptake at the culprit vessel, while only 33% of the patients showed high fdg uptake. in addition, coronary fdg could not be distinguished from background activity in 52% of the vessel territories. by comparison with histologic examination, microcalcification, macrophage infiltration, apoptosis, and necrosis they clearly showed that high f-18 fluoride uptake localized to recent plaque rupture through prospective clinical trial. another important finding by this study was the direct comparison between f-18 fluoride and fdg. however, more studies will be needed to determine that vascular f-18 fluoride is superior to vascular fdg images. vascular microcalcification is regarded as an early marker for atheromatous plaque formation, and has a potential as a predictor for future cardiovascular events. the correlation between vascular microcalcification and its vulnerability is yet to be clarified, due to lack of a non - invasive imaging method. recently, f-18 fluoride pet for vascular imaging can provide useful in vivo information on vascular microcalcification, and holds possibility for identifying high - risk and ruptured coronary atherosclerotic plaques. f-18 fluoride pet represents early stage, active microcalcification, while conventional imaging methods targeting macrocalcification is about late stage of atherosclerotic plaque. non - invasiveness, easy accessibility, and high reproducibility of f-18 fluoride pet in vascular microcalcification warrant further clinical investigation. prospective clinical trials to assess the prognostic value of f-18 fluoride uptake will determine whether f-18 fluoride uptake is generally accepted as a novel biomarker for plaque vulnerability. | a large number of major cardiovascular events occur in patients due to minimal or some lumen narrowing of the coronary artery. recent biological studies have shown that the biological composition or vulnerability of the plaque is more critical for plaque rupture compared to the degree of stenosis. to overcome the limitations of anatomical images, molecular imaging techniques have been suggested as promising imaging tools in various fields. f-18 fluorodeoxyglucose (fdg), which is widely used in the field of oncology, is an example of molecular probes used in atherosclerotic plaque evaluation. fdg is a marker of plaque macrophage glucose utilization and inflammation, which is a prominent characteristic of vulnerable plaque. recently, f-18 fluoride has been used to visualize vulnerable plaque in clinical studies. f-18 fluoride accumulates in regions of active microcalcification, which is normally observed during the early stages of plaque formation. more studies are warranted on the accumulation of f-18 fluoride and plaque formation / vulnerability ; however, due to high specific accumulation, low background activity, and easy accessibility, f-18 fluoride is emerging as a promising non - invasive imaging probe to detect vulnerable plaque. |
major depressive disorder (mdd) is a mental disorder characterized by low mood, low self - esteem, and loss of interest in normally enjoyable activities, the changes lasting for a minimum period of two weeks and causing impairment in social, occupational, sleeping and eating habits, general health, or other important areas of functioning. depression is characterized by a typically chronic course with associated anxiety symptoms as co - morbidity. associated anxiety symptoms may result in greater symptom severity, higher suicidal risk, and poor treatment response than either depression or anxiety alone. a significant overlap exists in the pathophysiologic components of depression and anxiety involving serotonergic, noradrenergic, and gabaergic systems in brain and their treatment. the selective serotonin reuptake inhibitors (ssri) and serotonin - norepinephrine reuptake inhibitors (snri) are reported to be effective in treating anxiety disorders associated with mdd. desvenlafaxine is a snri that received united states food and drug administration (us fda) approval in february 2008 for the treatment of mdd, generalized anxiety disorder (gad), panic disorder, and social anxiety disorders ; in india, central drugs standard control organisation (cdsco) approved desvenlafaxine on july 2009 for mdd. desvenlafaxine 's novelty lies in the fact that it inhibits both norepinephrine and serotonin uptake. escitalopram, a ssri is approved for mdd and gad in adults and children over 12 years of age. head - to - head comparison of the two drugs in general population is lacking except one such trial conducted on post - menopausal patients. therefore, this study was undertaken to compare the clinical effectiveness and safety of desvenlafaxine versus the standard antidepressant, escitalopram in patients with mdd associated with symptoms of anxiety. the study was a randomized, controlled, parallel group, open label, phase 4 trial. eligible subjects were adults 18 years and 60 years and of either sex who attended the psychiatry outdoor clinic of a teaching hospital in eastern india with a clinical diagnosis of mdd as per diagnostic and statistical manual of mental disorders, fourth edition, text revision (dsm - iv - tsr). subjects with a baseline hamilton depression rating scale (ham - d) score of 7 - 18 (mild and moderate depression cases) and a baseline hamilton anxiety rating scale (ham - a) score 12 (vide infra) were included. exclusion criteria included pregnant, lactating women, suicidal tendencies, catatonic features, patients on antidepressants for last one month, concurrent medical illnesses (hypertension, uncontrolled diabetes, ischemic heart disease, chronic renal failure, cirrhosis, malignancy), subjects with other coexistent psychiatric disorders (e.g. associated psychotic and maniac features, dementia, low i.q, obsessive compulsive disorders) or subjects on drugs (quetiapine, duloxetine, buproprion, cough preparations, aspirin, fluoxetine, sertraline, monoamine oxidase inhibitors, tricyclic antidepressants), which are known to interact with the study medications. number iec/1055) and written informed consent was obtained from all subjects or their legally acceptable relative (lar) as applicable. successive eligible patients were randomized (unstratified) using a computer generated random number table with 1:1 allocation ratio to receive either desvenlafaxine (50 mg) or escitalopram (10 mg) once daily for a period of 8 weeks. responder rate was defined as 50% of baseline ham - d score improvement at the 4 and 8 week visit. the study medications were purchased by the department for trial purpose and the entire consignment (both study drug and comparator) were of the same manufacturer. the manufacturing company had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the effectiveness outcome measures were changes from baseline to study end of ham - d17 and ham - a. the ham - d scale is a 17 - 21 items observer scale to assess the presence and severity of depressive state in patients, amongst the first 17 items, 9 items are scored 0 - 4 (0 = absent, 1 = doubtful or slight, 2 = mild, 3 = moderate, 4 = severe) whereas the next 8 items are scored 0 - 2 (0 = absent, 1 = doubtful or slight, 2 = clearly present). the ham - a scale consists of 14 items, each defined by a series of symptoms, and measures both psychic anxiety (mental agitation and psychological distress) and somatic anxiety (physical complaints related to anxiety). response to treatment was assessed by 50% decrease in ham - d and ham - a score. safety assessment included changes in vital signs and treatment emergent adverse effects - those reported by the subjects and those elicited by the clinician at every visit. laboratory investigations viz., hematogram (hemoglobin, total leucocyte count, differential leucocyte count, and erythrocyte sedimentation rate), liver function test, and serum urea and creatinine were done at baseline and at study end visit as a part of safety check. detailed clinical history, drug history, and baseline ham - d and ham - a scores were recorded in a pre - designed case report form. three post - baseline visits were scheduled at 2 weeks, 4 weeks, and 8 weeks after randomization. at each follow - up visit, they were evaluated clinically using ham - d and ham - a rating scores. evaluation of the effectiveness and safety parameters were done in all subsequent follow - up visits as described above. sample size calculation was based on changes in the primary outcome variable (ham - d score). with a 80% power, = 0.05, standard deviation of ham - d score of 3, effect size of 2 with equal allocation ratio, the number of evaluable subjects required per group was 36. however, assuming a 20% dropout rate, we recruited 43 subjects per group in the study. effectiveness analysis was done by modified intention- to - treat basis whereby subjects who had baseline and at least one post - baseline data of ham - d and ham - a were evaluable. missing visit data were substituted by the last observation carried forward strategy. for safety analysis, all randomized subjects who had received at least one dose of the trial medication were considered evaluable. continuous variables (normal distribution) were compared between groups by independent sample t - test and within group by paired t - test. for between group comparisons of non - parametric variables, whitney u test and for within group comparison friedman 's anova followed by post - hoc dunn test were used. categorical data were compared using chi - square or fisher 's exact test as appropriate. detailed clinical history, drug history, and baseline ham - d and ham - a scores were recorded in a pre - designed case report form. three post - baseline visits were scheduled at 2 weeks, 4 weeks, and 8 weeks after randomization. at each follow - up visit, they were evaluated clinically using ham - d and ham - a rating scores. evaluation of the effectiveness and safety parameters were done in all subsequent follow - up visits as described above. sample size calculation was based on changes in the primary outcome variable (ham - d score). with a 80% power, = 0.05, standard deviation of ham - d score of 3, effect size of 2 with equal allocation ratio, the number of evaluable subjects required per group was 36. however, assuming a 20% dropout rate, we recruited 43 subjects per group in the study. effectiveness analysis was done by modified intention- to - treat basis whereby subjects who had baseline and at least one post - baseline data of ham - d and ham - a were evaluable. missing visit data were substituted by the last observation carried forward strategy. for safety analysis, all randomized subjects who had received at least one dose of the trial medication were considered evaluable. continuous variables (normal distribution) were compared between groups by independent sample t - test and within group by paired t - test. for between group comparisons of non - parametric variables, mann whitney u test and for within group comparison friedman 's anova followed by post - hoc dunn test were used. categorical data were compared using chi - square or fisher 's exact test as appropriate. detailed clinical history, drug history, and baseline ham - d and ham - a scores were recorded in a pre - designed case report form. three post - baseline visits were scheduled at 2 weeks, 4 weeks, and 8 weeks after randomization. at each follow - up visit, they were evaluated clinically using ham - d and ham - a rating scores. evaluation of the effectiveness and safety parameters were done in all subsequent follow - up visits as described above. sample size calculation was based on changes in the primary outcome variable (ham - d score). with a 80% power, = 0.05, standard deviation of ham - d score of 3, effect size of 2 with equal allocation ratio, the number of evaluable subjects required per group was 36. however, assuming a 20% dropout rate, we recruited 43 subjects per group in the study. effectiveness analysis was done by modified intention- to - treat basis whereby subjects who had baseline and at least one post - baseline data of ham - d and ham - a were evaluable. missing visit data were substituted by the last observation carried forward strategy. for safety analysis, all randomized subjects who had received at least one dose of the trial medication were considered evaluable. continuous variables (normal distribution) were compared between groups by independent sample t - test and within group by paired t - test. for between group comparisons of non - parametric variables, whitney u test and for within group comparison friedman 's anova followed by post - hoc dunn test were used. categorical data were compared using chi - square or fisher 's exact test as appropriate. the flowchart of study participants is depicted in figure 1. of the 86 randomized subjects, 77 (escitalopram group = 39, desvenlafaxine group = 38) were evaluable as per the modified intention - to - treat analysis, since 9 subjects were lost to follow up. the mean age in the escitalopram group was 40 10.6 years and in the desvenlafaxine group was 41.9 8.5 years ; the difference was not statistically significant (p = 0.27). a total 64.1% were males in the escitalopram group and 60.5% in the desvenlafaxine group. the disease duration at screening were also comparable (p = 0.19) between groups. no concomitant psychiatric medications were used, as elicited during drug history ; however, 5 subjects (3 in test and 2 in control) were on anti - hypertensives and were well - controlled throughout their study period. consort flowchart of study participants baseline ham - d (p = 0.63) were comparable in the two treatment arms. the responder rates defined as 50% improvement in ham - d score from baseline at 8 weeks were 79.48% in the escitalopram group versus 73.68% in the desvenlafaxine group ; the difference was not statistically significant (p = 0.59). responder rates for ham - a score at 8 weeks were 76.92% and 71.05% in the escitalopram and desvenlafaxine groups, respectively. the difference was not statistically significant (p = 0.61) [table 1 ]. the responder rates assessed at 8 weeks visit for both ham d and ham a were higher in the escitalopram group compared to the desvenlafaxine group, though the difference was not statistically significant. number of patients responding to 50% decrease in ham - d and ham - a scores ham - d scores showed comparable results at all visits. however, escitalopram faired significantly better in ham - a scores than desvenlafaxine at all visits. comparison of ham - d and ham - a scores are listed in table 2, 3, and 4. between group comparison of ham - d scores between group comparison ham - a scores between group comparison of changes in ham - a score from baseline within group changes of both scores, from baseline to subsequent visits in both treatment arms, were statistically significant (p < 0.01). between groups comparison of the median ham - d scores showed no significant changes, but there was a statistically significant difference in ham - a scores between the treatment arms both at baseline, subsequent follow - up visits, and at the end of the study. safety data analysis revealed that 10 out of 39 subjects (25.64%) in the escitalopram group and 14 out of 38 (36.84%) in the desvenlafaxine group reported at least one adverse event. the common adverse events were nausea, vomiting, discomfort, insomnia, somnolence, and fatigue. all adverse events were non - serious and mild in severity and did not require treatment interruption or study drug withdrawal. the study results indicate that both escitalopram and desvenlafaxine, which are widely used antidepressants, were also beneficial in reducing anxiety symptoms. the effectiveness of both drugs in controlling anxiety symptoms in mdd subjects appear to be comparable as there was no statistically significant difference in the responder rates. this difference was, however, present since baseline ; therefore, this baseline difference could attribute to the differences in subsequent visits. reduction of median ham - a score was more in the escitalopram group (baseline 24 - 5 at the end of study) compared to the desvenlafaxine group (baseline 26 - 14 at the end of study). in clinical practice, a 50% decrease in the scores holds greater relevance than an absolute score decrease. since the 50% responder rate were similar in both the groups in the follow - up visits, escitalopram and desvenlafaxine have comparable effectiveness in controlling symptoms of anxiety with depression. our result findings were in concurrence with a randomized, double blind placebo - controlled, multicenteric, flexible dose trial in adult subjects of generalized anxiety disorder (gad) by bose a., where escitalopram was compared with venlafaxine extended release ; the overall efficacy analysis suggested that escitalopram and venlafaxine are both effective treatments for gad, but escitalopram was better tolerated. compared the efficacy and safety of desvenlafaxine and escitalopram in mdd with symptoms of anxiety in postmenopausal women, and the study demonstrated that there was no significant difference in their efficacy as evaluated on ham - d17 scores. desvenlafaxine treatment achieved 50% response in 56 - 58% (ham - d score) and 41 - 48% our study achieved a higher response rate, 73.68% in ham - d score and 73.7% in ham - a score. also, a review conducted by ali and lam showed that escitalopram demonstrates better efficacy than other ssris and similar efficacy to snris which is consistent with the results of our study however, a systematic review on the comparative effectiveness of various ssris and snris on mdd accompanied by anxiety or insomnia or pain in 19 head - to - head trials suggested that ssris do not differ in effectiveness, though the evidence was moderate in nature. the current study is consistent with previously reported individual results and adverse events for the study medications. in terms of tolerability, escitalopram was better tolerated than desvenlafaxine, as escitalopram indicated lesser incidence of adverse effects. the study limitations were that we could not undertake a double blind study due to logistic reasons and a longer active duration of treatment would be more informative. over the last two decades, there has been much progress in our understanding of epidemiology, neurology, and treatment of depression and anxiety disorders. whilst ssris are regarded as first - line treatment in most depression and anxiety disorders, snris are receiving increasing considerations and desvenlafaxine received regulatory approval. in conclusion, this study indicates that desvenlafaxine is comparable to escitalopram in patients of depression with symptoms of anxiety, and escitalopram is better tolerated. | aim : selective serotonin reuptake inhibitors (ssri) and serotonin - norepinephrine reuptake inhibitors (snri) are effective in treating anxiety disorders associated with major depressive disorder (mdd). this randomized, controlled, parallel - group, open - label, phase 4 trial (ctri/2012/08/002895) was undertaken to compare the effectiveness and safety of desvenlafaxine versus escitalopram, a standard antidepressant.materials and methods : effectiveness was assessed using the hamilton depression rating scale (ham - d17) and hamilton anxiety rating scale (ham - a). response to treatment was assessed by 50% decrease of baseline scores (responder rate). safety and tolerability was evaluated by changes in routine laboratory parameters, vital signs, and adverse events reported by the subject and/or observed by the clinician.results:responder rates for both ham - a and ham - d scores at 8 weeks were better in the escitalopram group compared to the desvenlafaxine group (ham - a 76.92% vs. 71.05% ; ham - d 79.48% vs 73.68%) but the differences were not statistically significant (p = 0.59 and p = 0.61). within group changes of both scores, from baseline to subsequent visits in both treatment arms were statistically significant (p < 0.01).conclusion : the effectiveness of desvenlafaxine was comparable to escitalopram, but escitalopram was better tolerated. |
cutaneous melanoma is a malignant tumour that originates from melanocytes, the pigment producing cells of the skin. melanoma development is a multistep process driven by the acquisition of genetic and epigenetic abnormalities. although a subset of melanomas develop in normal skin de novo, in many instances premalignant naevoid pigmented lesions can be discerned in melanoma formation. approximately a third of melanomas develop from a precursor naevus that is most commonly dysplastic. dysplastic naevi can progress into radial and subsequently vertical growth phase melanomas [3, 4 ]. the complex multistage development process of melanoma is characterized by various morphological, cellular, and biochemical alterations. histopathologically dysplastic naevi show characteristic morphological alterations, including proliferation and variable atypia of epidermal melanocytes, formation of irregular cell nests in the epidermis and basement membrane zone, and the interconnection of these nests and layers (bridging). melanocytes in dysplastic naevi (dnmc) furthermore exhibit morphological alterations in melanosomes and mitochondria, similar to those observed in melanoma cells. we have previously shown that dnmc in comparison with normal melanocytes show higher pheomelanin, iron, and calcium levels resulting in elevated reactive oxygen species (ros) levels [6, 7 ]. the diminished levels of antioxidant enzymes in dnmc further highten ros levels and reinforce chronic oxidative stress in these cells [8, 9 ]. in the present study we compared the gene expression and protein expression patterns of melanocytes from dysplastic naevus and from normal skin of 18 individuals. from a surgically removed dysplastic naevi and adjacent normal skin, melanocytes were isolated and briefly cultured for gene expression analysis using whole genome expression arrays and proteome analysis by peptide mass fingerprinting. gene ontology - based gene expression analysis and protein expression analysis revealed differentially expressed pathways involved in cellular metabolism, detoxification, and cell shape organization. these findings appear to signify deregulated processes that all together may contribute to an increase in the levels of reactive oxygen species and oxidative stress, as is often observed in dnmc. the resultant oxidative dna damage is an endogenous mutagenic force that might contribute to melanoma development. after approval by the medical review board of leiden university medical center and patient consent, 18 clinically atypical naevi were excised from 18 different patients. all 18 samples were confirmed after pathologist review as dysplastic naevus. the elliptical incision consisted of the dysplastic naevus in the central part and normal skin at the tips. to ensure separate isolation of melanocytes from the dysplastic naevus and from adjacent normal skin, the central part and tip of each surgical specimen were first divided before further processing. for isolation of the melanocytes from the dysplastic naevus and normal skin from the epidermal compartment, briefly, after removal of subcutaneous fat, epidermis and dermis were separated by overnight incubation with dispase grade ii (boehringer mannheim inc, indianapolis, in). the cells from this single cell suspension were plated in ham 's f10 melanocyte medium supplemented with penicillin (100 u / ml), streptomycin (100 u / ml), l - glutamine (invitrogen, breda, the netherlands), 1% ultroser g (biosepra, fremont, ca), endothelin-1 (5 ng / ml), basic - fgf (5 ng / ml), cholera toxin (30 g / ml), ibmx (33 m), and tpa (8 nm, sigma - aldrich, zwijndrecht, the netherlands). this melanocyte medium contains ingredients that are toxic to keratinocytes present in the epidermal cell suspension. under these conditions purity of these melanocyte cultures was confirmed by microscopic examination of cell morphology by clockwise assessment of the culture plate surface ; after 10 days all cells invariably had the dendritic, star - shaped or spindle - like morphology of melanocytes and no cells with epithelioid morphology were present in the culture dish. additional stainings of representative melanocyte cell cultures with melanocytic cell markers hmb45 and melan - a confirmed the purity of these melanocyte cultures. melanocytes isolated from dysplastic naevus (dnmc) and from normal skin (mc) were cultured for a maximum of 6 passages to ensure logarithmic growth and keep the time in culture as short as possible. to minimize the variation introduced by cell culture on gene and protein expression, all mc and dnmc patient samples underwent identical treatment in culture and rna was isolated from melanocytes with the rneasy mini kit (qiagen, venlo, netherlands). antisense amplified rna (arna) was generated with the messageamp ii arna kit (ambion, austin, tx) for hybridization to the human genome u133 plus 2.0 array (affymetrix, santa clara, ca) following manufacturer 's protocol. rna concentrations were determined by nanodrop measurements and rna quality control was performed prior to hybridization by lab - on - a - chip on agilent 's bioanalyzer. rna quality was further confirmed by posthybridization quality control measurements performed as part of the affymetrix microarray analysis, including normalization scaling factor across the microarrays and crna transcript integrity as assessed by 3 to 5 probe set signal intensity ratios, as routinely performed at the leiden genome technology center. protein was isolated from melanocyte cell pellets using trizol reagent (invitrogen) ; protein concentrations were determined by the pierce bca protein assay kit. protein samples (10 g) were labelled with 1000 pmol fluorescence amine - reactive cyanine dyes for two - dimensional difference gel electrophoresis (2d dige) following manufacturer 's protocol (amersham - pharmacia biotech). probe set signal intensities derived from microarray scans were subjected to the robust multiarray average (rma) algorithm as previously described. this algorithm results in background - corrected, quantile normalized and log(base2)-transformed final probe intensities that serve as measures of gene expression ; this log scale measure of expression will be further referred to as (gene) expression value. to identify genes that were differentially expressed in dnmc when compared to mc, a statistical analysis method (random variance model (rvm)-based t - test) suited for detection of gene expression differences in microarray experiments comprising relatively smaller sample sizes was used, while controlling the number and proportion of false discoveries with a multivariate permutation test as described previously. additional hierarchical clustering based on euclidean distance was performed ; clustering reproducibility was assessed using calculated r (reproducibility) and d (discrepancy) indices. all gene ontology annotations were obtained through http://www.geneontology.org/. gene ontology (go) classes with fewer than 5 and more than 100 genes represented in the array data were not considered in order to exclude very small and very large classes. the dnmc sample of one individual patient was compared to the paired mc sample, in order to assess whether differential expression of go classes existed across all microarrays between the paired melanocyte sample set. functional class scoring, based on semisupervised gene expression data analysis, was performed according to the statistical method as previously described. this method does not require prior gene selection and p values are assigned based on interrogated genes in a go class. gels were scanned in low - fluorescent glass plates with a typhoon 9400 scanner (ge health care uk ltd., tiff images of the gels were analysed with delta2d v3.4 software (dodecon, greifswald, germany). the matched spot volumes were subsequently normalised across the whole set of gels and normalised spot volumes were compared between images in the set. statistical significant differences between normalised spot volumes were determined using the welch - modified student 's t - test and step - down westfall and young method as correction for the false discovery rate. protein spots of interest were excised from the gel for analysis on a maldi - tof / tof instrument (ultraflex, bruker, germany). all mass fingerprint and ms / ms data were searched against the human protein database with the mascot program (matrix science, boston, ma). cdna was generated from dnase - treated rna (1 g) with the iscript cdna synthesis kit (biorad, hercules, ca). qrt - pcr was performed with iq sybr green supermix on a myiq real - time pcr detection system (biorad). previously it was demonstrated that melanocytes isolated from dysplastic naevus (dnmc) show differences in morphology, pigmentation, and growth abilities when compared to melanocytes isolated from normal skin (mc) in culture. for this study paired dnmc and mc were obtained from dysplastic naevi and normal skin of 18 patients. similarly we observed that the cultures of dysplastic naevus cells differed in morphology from that of normal melanocytes ; figure 1(a) displays a representative picture from melanocyte cultures derived from either normal skin (left, mc) or from dysplastic naevus (right, dnmc), showing that the mc have more equally - shaped dendrites as compared to the dnmc that consistently show a distinct stretched and thinner dendrite morphology. microarray gene expression profiling was performed on these 18 paired melanocytic cell sets to gain insight into differences between these melanocyte sample groups on molecular level. analysis of affymetrix u133 plus 2.0 microarray data revealed that melanocytes from dysplastic naevi and from normal skin expressed 45% of the interrogated genes at a measurable level. figure 1(b) depicts a scatter plot where the expression ratio 's of a representative sample set of dnmc and mc of one patient is plotted, indicating that gene expression differences between the dnmc and mc of the same patient were generally modest. except for two samples, hierarchical clustering of the microarray data showed that the dnmc samples resembled their autologous normal counterparts more closely than they resemble the dnmc of another individual (figure 1(c)). of note, samples 0310b and 0223b clustered separately from the remaining samples ; this distinction was not reflected in a different morphology of these cell samples, since cell morphology across the 18 dnmc samples was homogeneous without notable differences between the samples. in addition, all dysplastic naevi included in this study were histopathologically very similar and also in this respect samples 0310 and 0223 did not significantly differ from other patient samples. we considered the possibility that in our melanocyte sample sets, smaller but parallel effects among groups or sets of genes may exist that act together within pathways. in this way we would not so much identify individual marker genes differentially expressed by dnmc, but rather obtain an impression of dysregulated biological processes in dnmc. we aimed to uncover genes that were highly relevant when placed in a larger network of genes that interact with it, even though showing small differential expression individually. we proceeded to analyze for each pair of dnmc and mc from a single patient, which functional groups of genes were differentially expressed. the top go classes identified by functional class scoring as differentially expressed in dnmcs when compared to mcs, are listed in table 1(a). among the top represented go classes were organellar ribosome, mitochondrial ribosome, hydrogen ion transporter activity, prefoldin complex, and camp - dependent protein kinase regulator activity. table 1(b) presents the details of the top 5 go classes that are differentially expressed between 18 sets of dnmcs and mcs. additional comparative statistical analysis of the gene expression data between the entire group of 18 dnmcs and 18 normal mcs did not yield significantly differentially expressed genes (data not shown). not all genes belonging to a certain go class will necessarily exhibit differential expression between the dnmc and mc samples. to subsequently validate and assess whether individual genes from the identified go classes are differentially expressed in dnmc as compared to mc, we selected 14 genes that were among the top 4 go classes and analyzed expression levels by qpcr in the sample groups. table 2 provides an overview of the 14 selected genes for validation and the corresponding qpcr primer sequences. figure 2 shows the qpcr results for 5 of these validated genes (mrpl42, psmd10, hspd1, mrpl47, and mrsp22) with expression depicted as the ratio of gene expression in the dnmc to the corresponding expression in mc (vertical axis) plotted per sample for all 18 samples (horizontal axis). differential gene expression seemed to vary per patient, as gene expression was either increased or decreased in dnmc as compared to mc for all interrogated paired melanocyte sample sets ; this pattern was observed for all five genes. some specific patient samples showed a higher increase in expression than other sample sets that also showed increased expression (sample 8018 ; expression psmd10 and mrpl47), but a consistent differential expression pattern similar for all five genes was not observed for individual samples. this heterogeneous differential expression pattern in dnmc when compared to mc across the 18 samples seems to be in line with the observations from the microarray expression analysis. the 18 matched melanocyte sample sets, derived from either dysplastic naevus or normal skin, were additionally subjected to mass spectrometry analysis to assess whether protein expression differences existed between the melanocyte sample groups. figure 3 shows a representative example of proteins expressed in mc (visualized as green signal - cy3) and the corresponding protein expression in dnmc (visualized as red signal - cy5). dye swap experiments to control for dye labelling efficiency performed on various samples indicated that no large differences in efficiency were present between both dyes (data not shown). each of the 2d dige gels contained on average 2205 spots, 90% of these spots were reproducible in at least 15 gel sets. protein spots (n = 99) that were differentially expressed between normal and atypical melanocytes in at least 15 gel sets determined by statistical analysis were excised and subsequent maldi tof / tof analysis was performed. database mining resulted in the annotation of 70 proteins (see table 1 available as supplementary material online at doi:10.1155/2012/981308). additional statistical analysis yielded 16 most differentially expressed proteins (p t transversions reflecting the relevance of oxidative dna damage as a mutagenic force in melanoma development. via a proteomic approach, we found several proteins differentially expressed in melanocytes from dysplastic naevus when compared to melanocytes from normal skin. notably, proteins involved in the mitochondrial respiratory electron transport chain and overall oxidative phosphorylation process showed a decrease in expression in dnmc. a decrease in expression of proteins involved in oxidation reduction, cytoskeletal organization, and protein folding some of the proteins that showed significant higher level of expression in the dnmc were involved in protein metabolism, lipid oxidation, positive regulation of inflammatory responses, and protein refolding. the pattern of differential protein expression that our comparative analysis has yielded, may indicate that dnmc are indeed exposed to elevated levels of oxidative stress due to their own deregulated cellular metabolic processes, particularly mitochondrial oxidative phosphorylation, additionally suffering from faulty protein folding, and cytoskeletal organization. which (epi)genetic alterations are causing oxidative stress to occur in melanocytes within a dysplastic naevus and whether these sets of alterations can serve to identify the dysplastic naevus cells are important issues that remain to be elucidated. genome - wide expression profiling of melanocytes derived from dysplastic naevus (dnmc) and melanocytes from normal skin (mc) resulted in significantly differentially expressed organellar ribosome, mitochondrial ribosome, hydrogen ion transporter activity, and prefoldin complex gene ontology classes, with overall gene expression differences between both melanocyte groups being relatively small. mrpl42, psmd10, hspd1, mrpl47, and mrsp22 were among the top go classes ; validation of these genes showed a heterogeneous differential expression pattern across the dnmc and mc samples. proteomic analysis revealed differentially expressed proteins in dnmc compared to mc with a role in cellular metabolism, detoxification, and cytoskeletal organization processes. together these results indicate possible deregulated processes such as metabolism and protein synthesis in the melanocytes from dysplastic naevus ; this is in line with the observed elevated oxidative stress levels that can potentially result in oxidative dna damage in these cells. furthermore, investigations into which set of (epi)genetic alterations underlie the deregulated cellular processes that lead to increased oxidiatve stress, and whether these aberrations can specifically identify dysplastic naevus cells are warranted. | cutaneous melanoma, a type of skin tumor originating from melanocytes, often develops from premalignant naevoid lesions via a gradual transformation process driven by an accumulation of (epi)genetic lesions. these dysplastic naevi display altered morphology and often proliferation of melanocytes. additionally, melanocytes in dysplastic naevi show structural mitochondrial and melanosomal alterations and have elevated reactive oxygen species (ros) levels. for this study we performed genome - wide expression and proteomic analysis of melanocytes from dysplastic naevus (dnmc) and adjacent normal skin (mc) from 18 patients. whole genome expression profiles of the dnmc and mc of each individual patient subjected to go - based comparative statistical analysis yielded significantly differentially expressed go classes including organellar ribosome, mitochondrial ribosome, hydrogen ion transporter activity, and prefoldin complex. validation of 5 genes from these top go classes revealed a heterogeneous differential expression pattern. proteomic analysis demonstrated differentially expressed proteins in dnmc that are involved in cellular metabolism, detoxification, and cytoskeletal organization processes, such as gtp - binding rho - like protein cdc42, glutathione - s - transferase omega-1 and prolyl 4-hydroxylase. collectively these results point to deregulation of cellular processes, such as metabolism and protein synthesis, consistent with the observed elevated oxidative stress levels in dnmc potentially resulting in oxidative dna damage in these cells. |
iron deficiency anemia in adolescents can negatively impact on growth, increase susceptibility to infection, and also impair mental development and learning.(1) during this time 20% of final adult height and 50% of adult weight are attained. the prevalence of anemia in girls (hb < 12 gm%) and in boys (hb < 13 gm%) is high as per the reports of nfhs-3 and the national nutrition monitoring bureau survey.(23) an estimated 56% girls and 30% boys in the age group 10 - 19 years in india are anemic according to nfhs-3 data.(4) in india, the highest prevalence of anemia is reported between the ages 12 - 13 years, which also coincides with the average age of menarche.(3) to combat anemia in adolescents, government of india has decided to implement the weekly iron and folic acid (ifa) supplementation (wifs) programme under nrhm in 2013 in class vi to xii enrolled in government / municipal schools through the platform of schools and out of school through anganwadi centers across all states in india(5) this method of weekly ifa supplementation through school teachers at school level proved effective as a strategy.(6) but the coverage of this scheme is low hens we tried its delivery through peers. the present study on anemia was conducted to find out the burden of anemia on adolescent age group in the tribal area of jhagadia and to assess the change in the hemoglobin through the weekly iron and folic acid (dots) supplementation by peer educators at community level among adolescent boys and girls. a community based cross - sectional study was conducted before and after introducing intervention in the months of april, may and june 2013 under sharda mahila vikas society (smvs) in five tribal villages of jhagadia block, gujarat, india. smvs is a voluntary organization has been working since 2002 towards the development of women, youth and children. universal sample size was considered for this study as all adolescent girls (117) and boys (127) of the age 10 - 19 years, registered under the adolescent awareness programme were enrolled in this study. hemoglobin level was determined using hemocue method (standardized by who) and sickling estimation done using dtt and electrophoresis. administration of weekly iron and folic acid ifa (dots) (100 mg elemental iron and 500 g folic acid) tablets with the distribution of albendazole tablets for de - worming bi - annually under the supervision of trained peer educators along with iec materials. data on the hemoglobin level was collected by trained health supervisors, while data on number of iron tablets consumed was collected ongoing by the peer educators and thereby all the data was entered in excel and statistically analyzed by applying paired t - test in spss 16.0. informed verbal consent was taken from the adolescent girls and boys and their mother before hemoglobin collection. administration of weekly iron and folic acid ifa (dots) (100 mg elemental iron and 500 g folic acid) tablets with the distribution of albendazole tablets for de - worming bi - annually under the supervision of trained peer educators along with iec materials. data on the hemoglobin level was collected by trained health supervisors, while data on number of iron tablets consumed was collected ongoing by the peer educators and thereby all the data was entered in excel and statistically analyzed by applying paired t - test in spss 16.0. informed verbal consent was taken from the adolescent girls and boys and their mother before hemoglobin collection. adolescents were 100% tribal, 76% living in kutcha house and below poverty line, only 5% households had toilets, 67% were school going and 23% were dropouts. baseline hemoglobin estimation was performed on adolescent 141 girls and 143 boys but statistical analysis was done on adolescents who were available, girls 117 and boys 127 for both the pre and post test. mean rise of hemoglobin seen among adolescent boys was 1.5 gm / dl and for adolescent girls was 1.3 gm / dl in the tribal area of south gujarat. there was significant reduction in anemia of all severity among adolescent girls and boys at the end of intervention compared to before. the result of this study shows that 79.5% girls and 64% boys were anemic during baseline survey [figure 1 ]. the prevalence of anemia among adolescent boys was 64% before intervention and it reduced to 25% after intervention making the prevalence to be 39%. [table 1 ] 2 gm increase was seen among 24.8% girls and 19.7% boys. it was also seen that the adolescent girls who did not consume iron tablets are 0.73 times more likely to develop anemia than those who consumed iron tablets and adolescent boys who did not consume iron tablets are 0.60 times more likely to develop anemia than those who consumed iron tablets thus the relationship between consuming iron and folic acid tablet in preventing anemia was statistically significant (p < 0.05). reduction in anemia on consumption of iron and folic acid (dots) tablet among adolescents prevalence of anemia according to severity before and after intervention among adolescent girls (n = 117) and boys (n = 127) compliance with weekly ifa among adolescents by gender the body mass index estimation was done for adolescent girls and boys and it was found that before intervention 88% adolescent girls and 92% boys were mal - nourished. baseline hemoglobin estimation was performed on adolescent 141 girls and 143 boys but statistical analysis was done on adolescents who were available, girls 117 and boys 127 for both the pre and post test. mean rise of hemoglobin seen among adolescent boys was 1.5 gm / dl and for adolescent girls was 1.3 gm / dl in the tribal area of south gujarat. there was significant reduction in anemia of all severity among adolescent girls and boys at the end of intervention compared to before. the result of this study shows that 79.5% girls and 64% boys were anemic during baseline survey [figure 1 ]. the prevalence of anemia among adolescent boys was 64% before intervention and it reduced to 25% after intervention making the prevalence to be 39%. [table 1 ] 2 gm increase was seen among 24.8% girls and 19.7% boys. it was also seen that the adolescent girls who did not consume iron tablets are 0.73 times more likely to develop anemia than those who consumed iron tablets and adolescent boys who did not consume iron tablets are 0.60 times more likely to develop anemia than those who consumed iron tablets thus the relationship between consuming iron and folic acid tablet in preventing anemia was statistically significant (p < 0.05). reduction in anemia on consumption of iron and folic acid (dots) tablet among adolescents prevalence of anemia according to severity before and after intervention among adolescent girls (n = 117) and boys (n = 127) compliance with weekly ifa among adolescents by gender the body mass index estimation was done for adolescent girls and boys and it was found that before intervention 88% adolescent girls and 92% boys were mal - nourished. in the present study, the prevalence of anemia among adolescent girls was found to be 79.5% before intervention studies conducted in wardha, hyderabad, india reported a similar prevalence of 59.8%, 60% and 63% respectively.(789) other studies conducted in various part of the country showed the prevalence of anemia of 44.8%, 34.5%, 25.9%, 23.9% in tamil nadu, meerut, varanasi and chandigarh among adolescent girls respectively.(6101112) who global data base on anemia in karnataka showed a prevalence of anemia among adolescent girls to be around 50.7%.(13) interestingly very few studies have been conducted on anemia among adolescent boys, where one of the studies in nepal reported a prevalence of 47.7% among boys and 52.3% among girls.(14) while study in gujarat reported 75% anemia among girls and 16% reduction in the prevalence of anemia in the tribal area among school going adolescent girls where weekly iron and folic acid supplementation was done by school teachers(15) while in our study the reduction was 21.5% among girls and 25% among boys in the tribal area. it may be due to better compliance of ifa (dots) tablets provided through peer educators. world health report 2002, identified anemia as one among the top 10 risks for infant mortality, maternal mortality and preterm birth. who / unicef has suggested that the problem of anemia is of very high magnitude in a community when prevalence rate exceeds 40%.(16) there were various reasons for decrease in hemoglobin level due to non - compliance of iron and folic acid tablets of its taste or due to migration of families, abortions or possibility of sickle cell anemia etc. a significant association was found in change in hemoglobin before and after intervention (p = 0.000) among adolescents. this study shows that anemia among these rural tribal adolescents is high like other parts of the country. the hemoglobin level increased among adolescents suggesting that anemia can be corrected by weekly prophylaxis if right approach is made. the study gives an insight that anemia is not only common among girls but also among boys. this study indicates that that weekly ifa under the supervision of trained peer educators may reduce the severity of anemia. in - depth study should be conducted to know the serum ferritin and serum trans - ferritin level. schemes like mid - day meal and supplementary food at anganwadi center should be regularly monitored in terms of quality and quantity of food and its supply. no randomization was done, universal sample was taken.there had been few dropouts during the post intervention test. shobha shah, pankaj shah, shrey desai, dhiren modi, gaytri desai, honey arora declare that they have no conflict of interest. no randomization was done, universal sample was taken.there had been few dropouts during the post intervention test. shobha shah, pankaj shah, shrey desai, dhiren modi, gaytri desai, honey arora declare that they have no conflict of interest. | background : anemia during adolescence affects growth and development of girls and boys increasing their vulnerability to dropping out - of - school. hence investing in preventing anemia during adolescence is critical for their survival, growth and development.objective:to find out the burden of anemia on adolescent age group in the tribal area of jhagadia block and to assess the change in the hemoglobin level through the weekly iron and folic acid ifa (dots) directly observed treatment supplementation under supervision by peer educators at community level among adolescents.methods:community based intervention study conducted with adolescents (117 girls and 127 boys) aged 10 - 19 years, through supplementation of ifa (dots) by trained peer educators for 52 weeks in 5 tribal villages of jhagadia. hemoglobin level was determined by hemocue method before and after intervention and sickle cell anemia by electrophoresis method. primary data on hemoglobin and number of tablets consumed was collected and statistically analyzed in spss 16.0 software by applying paired t-test.results:the overall findings suggest that the prevalence of anemia reduced from 79.5% to 58% among adolescent girls and from 64% to 39% among boys. mean rise of hemoglobin seen was 1.5 g / dl among adolescent boys and 1.3 g / dl among girls. a significant association was found in change in hemoglobin before and after intervention (p = 0.000)conclusion : prevalence of anemia among girls and boys can be reduced in their adolescent phase of life, through weekly supplementation of iron folic acid tablets under direct supervision and nutrition education by peer educator at community level. |
breast cancers are potentially life - threatening malignancies that develop in one or both breasts. now there is an ever - expanding array of therapeutic management options for invasive breast cancer in the modalities of surgery, radiotherapy, and systemic therapy.1 distant metastases in breast cancer may spread to almost any region of the body ; about 50% to 75% of patients relapse first in a single organ. typically, metastases to regional lymph nodes are observed in nearly one - third of patients with cancer in the breast, colon, uterus, neck oral cavity, and pharynx.2 metastases in the oral regions, including soft tissues and jaw bones, account for only 1% to 8% of all oral malignancies,3 and are derived from primary tumors located elsewhere in the body.4 although most patients are previously diagnosed with primary neoplasms and treated, in one - third of the metastases the oral region presents the first clinical sign of the malignancy.4,5 bone metastases, including those in the jaw bones, are the second most frequent malignant bone tumors after osteosarcomas.6 nearly all types of malignancy may metastasize to the mouth.4 the mandible is the most common site of metastasis of the oral and maxillofacial region.4 mandibular lesions are the early sign of generalized metastatic disease in 67% of cases.6 the formation of secondary foci of tumor colonization occurs by hematogenous dissemination of tumor emboli, mainly accumulating in regions with larger amounts of bone marrow and low circulatory velocity.6 although no particular malignancies seem to favor spread in the oral cavity, some primary tumors are found to occur more often than others. the most common primary tumors are from lung cancer in men and breast cancer in women.4 a 36-year - old female was referred to the department of oral and maxillofacial surgery of ataturk university, the faculty of dentistry, with complaint of pain over the left half of the face, trismus, extraoral swelling, hard on palpation on the anterior body of the mandible, and numbness in the left half region of the mandible. intra - oral examination showed a hard swelling over the posterior corpus of the mandible. the trabecular pattern and bone density of the left mandibular body and ramus were distinctly different on panoramic radiograph (figure 1). diffuse osteolytic defect sites in the mandible, maxilla, and orbit floor were observed on ct (figure 2). patient s medical history revealed that 2 years ago, a mastectomy for the treatment of invasive ductal carcinoma was performed and chemotherapy was followed because of recurrence. under local anesthesia, sections from the biopsied material, stained with haematoxylin and eosin, showed the presence of cells with hyperchromatic nuclei and large eosinophilic cytoplasm. the cells were arranged in nests and glandular - like structures and were located between segments of bone and into bone marrow spaces (figure 3). these histological results supported evidence of metastatic invasive ductal carcinoma. subsequently, a static scintigraphic image of the whole body was obtained. bone scintigraphy showed isotope (technetium tc 99 m) accumulation in the left half of the mandibular body, floor of the orbit, maxilla, left parietal bone, iliac bone, and cervical and thoracal vertebras (figure 4). the patient was sent to consult with the department of medical oncology and radiooncology for chemotherapy and radiotherapy. metastasis to the bone occurs more frequently than to the soft tissues.3 metastases to the soft tissues account for 0.1% of all oral lesions. of all metastatic malignancies in the mouth and jaw bones, approximately 61% occur in the mandible, 24% in the maxilla, and 15% in soft tissues. the most frequent primary sites of malignancy with the potential to metastize in the mandible are, in decreasing order, the breast (31%), lung (18%), kidney (15%), thyroid, prostate and colon (6%), stomach and skin (5%), testicle (3%), bladder, liver, uterus, and ovary (1%).6 a study for a 30-year period reported 1,537 cases of oral malignant tumors, of which 24 cases were metastatic.4 fourteen out of 24 cases were located in the mandible, 4 in the maxilla, and only 1 in the maxilla and mandible. women were affected in 12 cases and 4 cases were metastatic tumors from the breast into the mandible. the present case was a metastatic breast carcinoma in the left half of the mandible, maxilla, floor of the orbit, parietal bone, and some cervical and thoracal vertebras in a 36-year - old female. metastatic lesions occur more often in the posterior region of the mandible, which is an area that retains its normal hematopoietic function.6,7 this possibly explains why the maxilla, although rich in spongy bone tissue, is a less common site for metastasis.6 in our case, the metastatic lesion was located in the posterior corpus of the mandible and the whole region of the ramus. the clinical symptoms of our case may simulate a benign proliferative process, osteomyelitis, and primary bone tumor. the metastatic lesions of the jaws do not cause specific signs or symptoms.6 in our case, symptoms were pain, trismus, swelling, and paresthesia of the left half of the mandible. if histologically distant metastasis in the oral and maxillofacial region or elsewhere in the body of breast cancer is diagnosed, a bone scintigram or pet - scan has to be made to identify other tumor locations and to evaluate therapeutic options. | female breast cancer is one of the major causes of death among women. metastatic tumors to the maxillo - facial bones are rare. we present diagnosis and treatment of multiple metastatic invasive ductal carcinoma involving massive and early stage the left half of the mandibular body, the floor of the orbit, maxilla, left parietal bone, the iliac bone and cervical and thoracal vertebras in a 36 years old female one and half years after operated. |
acute poisoning is one of the main childhood accidents and is as an important public health problem. among accidents, poisonings are responsible for around 7% of all cases in children under 5 years of age, and are implicated in approximately 2% of deaths of children in developed countries, corresponding to 3000 deaths per year in european countries. the american association of poison control centers has recorded an annual increase in accidents involving poisons in children. in 2012, there were 2 275 141 cases of exposure to poison notified in humans, with 48.3% of these occurring in children under the age of 6 years, mainly unintentionally. when adolescents from 13 to 19 years of age were considered, approximately 24% were intentional exposures. in children under 6 years of age, mortality was 2.4% ; however, it increased to 6.1% when the entire pediatric age - group was considered. in brazil, in 2011, only 23 of the 35 regional poison centers provided the national system of toxic - pharmacological system (sistema nacional de informaes txico - farmacolgicas [sinitox ]) with data, with 98 765 cases of human poisoning being registered. approximately 36% of the notifications were made concerning children under 14 years of age, resulting in 10.4% deaths in this age - group. the most frequent occurring toxic agents were medications, in 36% of the cases, followed by household cleaning products at 18.4% and venomous animals at 12.7%. acute poisonings plays a relevant role in the present context of childhood accidents because of its frequency of attendances in emergency care units, the possibility of sequelae, and the corresponding health care costs. over the past few years, due to rapid industrialization these products are sold without adequate control, exposing children to greater risk of contact with these agents. in spite of this, there is a scarcity of studies with the aim of identifying the epidemiological characteristics of these accidents, and which may subsidize prevention strategies to reduce the morbidity and mortality in childhood. the present study seeks to contribute to filling this gap in researches that characterize the profile of acute poisoning in children. for this reason, the aim of this study was to describe the epidemiological characteristics and clinical development of acute poising in children attended to at a public hospital in bahia. a descriptive study was conducted of acute poisoning in children attended to at the hospital geral roberto santos (hgrs) and by the poison control center (centro antiveneno da bahia [ciave ]) of bahia. hgrs is the largest tertiary public hospital in bahia, and ciave is the only reference center in toxicology of the state, both being located in the capital (salvador, bahia). all the children who are victims of poisoning attended to at hgrs are notified and followed - up simultaneously by ciave up to the time of discharge or death. the study population consisted of children from 0 to 14 years of age, victims of acute poisoning, attended to at the emergency unit of hgrs, and notified by ciave, in the period from january 1, 2008, up to december 31, 2012. data collection was performed retrospectively in the period from june 2013 to march 2014, from the record charts of hgrs and standardized forms of ciave. the option was taken to use the ciave form, because it is filled out in accordance with the manual for filling out the notification and attendance form, ministry of health, oswaldo cruz foundation / sinitox. as inclusion criteria, the following were used : report of contact with toxic agent associated with clinical manifestation. poisoning was considered the exposure to a certain type of product and/or chemical substance, with the appearance of biochemical reaction, functional alterations, and/or clinical signs compatible with the condition of poisoning. the age of inclusion was based on the statute of the child and adolescent of brazil, which considers a person up to the age of 12 incomplete years to be a child, and an adolescent as one between the ages of 12 and 18 years. the age - group adopted in this study was, therefore, due to the fact that attendance at the pediatric service of hgrs is restricted to patients up to 14 years of age. the variables studied were the following : age, sex, origin, time elapsed between accident and attendance, type of occurrence, circumstance, zone of occurrence, place of occurrence, pathway and type of exposure, site of the body affected, toxic agent, main clinical manifestations, severity of poisoning, indication of observation in the emergency unit or hospitalization, time of stay in hospital, and clinical development. after data collection from the ciave notification form, the patient record charts of attendance at the emergency units and hospitalization were evaluated, and complementary and clinical evolution data were recorded. data were collected by a previously trained team and supervised by the authors, who included undergraduate medical students and resident doctors of pediatrics at hgrs. after filling out the collection instrument, all the variables were transferred to the database of the software program statistical package for the social sciences (spss), version 21.0. from then on, the results were presented by means of descriptive statistics, using tables of distribution by frequency and percentages for categorical variables, and as mean and standard deviation, or median and interquartile interval (iq), for quantitative variables. this research was approved by the ethics committees of the hospital geral roberto santos and bahian school of medicine and public health, report no. 05/2013. access to the registered data was authorized by the boards of directors of hgrs and ciave. initially, 735 notification forms were selected ; however, 64 cases corresponded to exposure and 14 to adverse reaction of patients. these cases were not included because they did not meet the criteria of the manual for filling out the notification and attendance form / sinitox with reference to the terms poisoning, exposure, and adverse reaction. therefore, for analysis of the study, 657 cases of acute poisoning were registered. the median age was 4 years (iq = 2.0 - 10)minimum age of 1 month and maximum of 14 years. the mean number of cases per annum was 132, and in the year 2011, an increase of 20.8% occurred, totaling 166 cases. in table 1, one observes that 349 cases (53.1%) of poisoning occurred in children under the age of 4 years. the majority of cases originated from salvador and little variation occurred for the male sex. the most frequently found group of toxic agents in all the periods was medications, followed by venomous animals, particularly in the last 2 years of the study. in the male sex category, there was greater frequency of accidents involving household cleaning products (54.2%) and venomous animals (63%), while in the female sex category, there was predominance of accidents involving medications (53.5%) and raticides (54.4%). in the distribution of the groups of toxic agents according to age - group (table 2), medications were observed to predominate in children of up to 4 years of age, accounting for 66.6% of the occurrences. between 5 and 14 years, approximately 87% of poisoning due to household cleaning materials occurred between the ages of 1 and 4 years. the poisoning involving raticides predominated in 2 age - groups : 47.4% between 1 and 4 years of age and 35% of the cases from 10 to 14 years of age. the occurrences were accidental in 92% of the cases, and all poisoning due to suicide attempts occurred in the age - group between 10 and 14 years (5.8%). error of administration occurred mainly in the age - group of under 1 year, accounting for 40% of the cases. acts of violence occurred in all age - groups and predominated between 1 and 4 years of age (42.9%). the large majority of accidents in children under the age of 5 years occurred in the residence (94.2%), and in this age - group, around 41% occurred in an outside environment. the main exposure pathway was oral, followed by bite / sting (table 3)., one observes that the majority of accidents were slight, mainly in children under 4 years of age. cure was confirmed in 98.2% of the cases and lethality was low, with 3 deaths occurring (0.5%). the median of time elapsed between the accident and hospital attendance was 3.0 hours (iq = 1.5 - 11.0). time of observation in emergency unit presented a median of 4.0 hours (iq = 2.0 - 10.0). hospitalization was necessary in 117 cases (17.8%), with a median of 3.0 days (iq : 2.0 - 6.0), with 91.4% of cases being in the infirmary and 8.6% in intensive care units. the main groups of agents that led to hospitalization were venomous animals (38.5%), followed by medications (29.9%) and raticides (13.7%). in the present study, cases of intoxication in children under 4 years of age were the most frequent, and in this age - group, 94.2% of the accidents occurred in the home. the most prevalent toxic agents were medications, particularly neuroleptic agents (20.9%) and benzodiazepines (17.1%). in the age - group between 4 and 14 years, we observed predominance of poisonings by venomous animals, due to accidents with scorpions (41.7%) followed by poisonous snakes (37.8%) and spiders (20.5%). the largest portion of poisonings were classified as light (73.5%), and perhaps, for this reason, the rate of deaths was relatively low (0.5%). the prevalence of poisonings in children aged 4 years reported in other studies has been higher than it was in the findings of our study (53.1%). in brazil, the agency sinitox registered a prevalence of 67.3% in this age - group. the highest frequency of these accidents the age - group up to 4 years of age perhaps results from the peculiarities of growth and development. in the majority of instances, children in this age - group are victims of unintentional poisoning due to immaturity, making them vulnerable and defenseless to protect themselves from accidental and intentional risks. outstanding too is their curiosity and motivation to perform tasks, inability to foresee and avoid situations of danger, greater tendency to imitate and repeat behaviors, in addition to their motor coordination not yet being fully established. the slightly higher frequency in the male gender ratifies the greater exposure of boys to toxic agents. kouta, in france, observed 54.4% of the cases in the male gender, and tavares, in brazil, also obtained a similar prevalence (52.2%). there are cultural aspects that may be involved in these findings, such as less family vigilance of boys, resulting in greater freedom to perform activities with less adult supervision. this permissiveness related to male upbringing has been found in the higher number of accidents and deaths due to external causes registered in this gender. the domestic environment is the place where the child remains the greater part of the time, and is also the place with the highest number of accidents involving poisoning in the first 5 years of life. bentur, in israel, related that, in 89.3% of cases, the residence was the place of occurrence ; however, these authors found differences with regard age - group : 92% in the group from 0 to 5 years and 79% in the group from 5 to 18 years. the toxic agents found in the home, without adequate storage, may be transformed into a risk of accidents, as observed in a study in spain, in which 75% of the accidents occurred in the residence and the family members mentioned storing products for the home in nonoriginal receptacles in 26% and medications in unsafe places in 15.6% of the cases. in brazil, loureno observed that 81% of the accidents occurred in the residence, with 65.4% involving 5-year - old children, and the parents or grandparents were present in 69.2% of the occurrences. the frequency of accidents with toxic agents depends on the socioeconomic and cultural conditions, the enforcement of legislation for the control of these products, climate, and the predominance of local industrial or agricultural activity. in developed countries, poisoning by medications, cosmetics, household cleaning products, and alcohol are more common. in sweden, where the majority of the population resides in urban areas, 29 848 cases of poisoning in children under the age of 10 years were registered in 2013, with 42% involving chemical products, mainly cleaning products (44%), followed by medications (28%), plants (11%), cosmetics (8%), and only 1% by snakes / insects. in developing countries, where the economy is based on agriculture, in addition to poisoning by medications, cases involving other agents such as insecticides and venomous animals are frequent. in a study conducted in india, chowdhury verified that pesticides, mainly organophosphates, were the most frequent agents involved (53.3%), followed by chemical household products (33.7%), mainly kerosene (24%). poisoning in children is ranked the 12th most common cause of admission to hospitals, and the authors warned about the growing use of agricultural chemical substances. a study conducted in mexico by olguin et in addition to the higher frequency, different from the findings of our study, the most prevalent group was analgesics (42.3%), followed by anticonvulsant (22.9%) and anxiolytic (17.9%) drugs. it is important to point out that the sale of the most frequent groups of medications in our study, neuroleptic agents and benzodiazepines, is subject to restrictions, and these drugs present potential risk of serious adverse effects. other multiple studies also ratified that medications are the most frequent agents involved in acute poisoning in children. in the majority of cases, medications are used by family members, stored in an inadequate manner, and easily accessible to children, as was verified in a case - control study in brazil with child victims of acute poisoning. in this study, it was observed that storage at a height below 150 cm presented 16 times more chances of leading to poisoning ; another variable associated with a risk factor was distraction of caregivers at the time of the accident. household cleaning products were also prevalent agents in poisonings in our findings, with predominance of bleaching / whitening products (55%). among the raticides, the (pb) lead - containing product marketed illegally in brazil was mentioned as the causal agent in 77.2% of the cases. another study conducted in brazil by werneck and hasselmann, with children under the age of 6 years, observed predominance of chemical products for domestic use (39%), followed by medications (35%) and raticides (15%), with lead also being involved in over half of the cases. in the same way as medications, these products are stored in the kitchen and in cupboards located very low down, in packages, at times not the original ones, and easily accessible to children. another important factor associated with poisoning by medications and chemical products is lack of standardization in special child - resistant and poison prevention packaging, which has been regulated in several developed countries, with a view to making it difficult to open and handle the contents by the majority of children under the age of 5 years. after standardization of special child - resistant packaging in england, there was a reduction in deaths due to poisoning in children under 10 years of age, from 151 per 100 000 in 1968, to 23 per 100 000 in 2000. in the united states, special child - resistant packaging was regulated in 1970, and since then, a significant reduction has been observed in the unintentional ingestion of toxic products listed in this standardization. in brazil, the draft bill of law 4841/1994 instituting special child - resistant packaging was presented in 1994 and is still pending in national congress. in the age - group of 5 to 14 years, predominance of poisoning by venomous animals was observed in this study. the high frequency of these accidents in comparison with other studies may be related to the extensive rural zone of bahia, and the fact that ciave, coordinator of the national program of control of accident involving venomous animals in the state, performs the distribution and replacement of antivenom sera according to the notifications received. in general one of the variables related to the prognosis is the time elapsed between the accident and attendance, which in the present study was a median of 3 hours. after classification of the severity, a higher frequency of light conditions was observed, which may justify a short time of hospital observation. hospitalization was necessary in 117 cases (17.8%), with a median of 3 days, of which 91.4% were in the infirmary and 8.6% in intensive care units. in 98.3% of the cases, cure was confirmed and there was low lethality, with 3 deaths occurring (0.53%) by medications and pesticides for agricultural use. the low number of deaths may be attributed to the low level of severity of the majority of cases, early attendance, and the characteristics of the hgrs as a reference hospital in toxicology. when the variables time of stay in hospital and development were evaluated, we observed that the majority of articles analyzed make no correlation with the severity of cases. a study conducted in iran demonstrated a mean time between the accident and attendance of 6.9 3.1 hours. approximately 31% of the children were discharged within the first 6 hours, 51% remained under observation for a period lasting from 6 to 24 hours, and 19% remained in hospital for over 24 hours, with 5 deaths occurring (1.5%). another study conducted in turkey, with patients who needed intensive care, observed that 57% of the children were attended in up to 2 hours and the mortality rate was 1.9%. therefore, it was observed that there was high morbidity of poisoning in childhood ; however, it presents low lethality when early initial attendance occurs, even in more severe accidents. one of the limitations of this study results from the fact that it was retrospective and depended on the quality of the records. another aspect to consider was that it was conducted in a public hospital and reference center, and this may not be representative of other health units, wherein patients may be attended to and not be duly notified. nevertheless, the profile of acute poisonings obtained in this study may contribute to a description of these accidents, which is broader in scope, and due to the small number of publications concerning children, this study may contribute to increasing our understanding and power of stimulating preventive measures with a view to reducing morbidity in childhood. cases of acute poisoning are frequent accidents in childhood, which result in a high level of morbidity, particularly in children under 4 years of age. knowing the profile of these occurrences, including the frequency of toxic agents and the area under study, may subsidize the adoption of educational and other measures with a view to guaranteeing the safety of children, for example, the mandatory use of child - resistant packaging of medications and chemical products for domestic use, greater surveillance of the production and sale of illegal toxic agents, and special care with regard to the exposure of children to venomous animals. | acute poisoning is a frequent accident in childhood, particularly in children under 4 years of age. this was a descriptive study with data collected from standardized forms of the poison control center and patient record charts. all the cases of acute poisoning in children aged 0 to 14 years during the period 2008 to 2012 were selected. the variables studied comprised characteristics of the events and toxic agents, clinical development, and outcome. a total of 657 cases of acute poisoning, with higher frequency in the age - group from 1 to 4 years (48.7%) and male sex (53.4%), were recorded. the occurrences were accidental in 92% of the cases, and 5.8% were due to suicide attempts. among the toxic agents, medications (28.5%), venomous animals (19.3%), nonvenomous animals (10%), household cleaning products (9.0%), and raticide agents (8.7%) predominated. the majority of cases were characterized as light (73.5%) and around 18% required hospitalization, and there was low lethality (0.5%). |
category iii chronic prostatitis / chronic pelvic pain syndrome (cp / cpps) is the category with the highest incidence, accounting for 60% to 90% of those with prostatitis. bacterial pathogens have not been found in prostatic tissue, urine, or prostatic fluid by conventional culture ; however, prostatic inflammation and inflammatory markers are often identified. this suggests the existence of yet unknown infectious pathogens and the importance of identifying these pathogens for the diagnosis and treatment of type iii prostatitis. the female genital tract represents a highly dynamic environment, with a resident microflora consisting of a variety of different species. the coexistence of different sexually transmitted microorganisms is very common and is due to several factors, including a common route of transmission, the sexual behavior of the host, and the resident flora. infectious vaginitis accounts for 90% of all cases in women of reproductive age and is represented by the triad of candidiasis, bacterial vaginosis, and trichomoniasis. other less common infections are caused by gonorrhea, chlamydia, mycoplasma, herpes, campylobacter, and some parasites. vaginal infections are often (varies between 20% and 40% of vaginal infections) a mix of various etiologies, which presents challenges for treatment. indeed, when only one cause is treated, the other pathogens can gain resistance and induce relapses and recurrences. therefore, the key factor is to obtain a precise diagnosis and to provide treatment with a broad - spectrum anti - infective. nanobacteria (nb) are newly discovered infectious agents of 100 - 500 nm in size with a 16s ribosomal rna (rrna) gene sequence and slow growth and a doubling time of about 3 days. they are fastidious and difficult to culture but can be detected with standard microbiological methods [5 - 7 ]. in vivo, nb are found to be voided mainly through the urinary system, and they have been isolated within the genitourinary tract, including in polycystic disease, renal calculi, and chronic prostatitis. furthermore, nanobacterial infection of malignant ovarian tissue contributes to mechanisms leading to the formation of calcified deposits known as psammoma bodies. in this study, we aimed to investigate the detection of nb from expressed prostatic secretions (eps) in patients with cp / cpps and from vaginal swabs in patients with vaginitis by reverse transcriptase polymerase chain reaction (rt - pcr) and to evaluate the association between nb and neisseria gonorrhea (n. gonorrhea), chlamydia trachomatis (c. trachomatis), ureaplasma urealyticum (u. urealyticum), mycoplasma hominis (m. hominis), trichomonas vaginalis (t. vaginalis), and mycoplasma genitalium (m. genitalium). a group of 11 men attending a specialized cp / cpps clinic of the urology department of the hospital (mean age, 43.5 years) were enrolled in the study. prostatic fluid samples were collected from outpatients with refractory type iiib prostatitis by aseptic technique by use of the mearses - stamey four glass urine collection method. all patients must have abstained from sexual activity for at least 4 days before sample collection. symptoms were quantified by the national institutes of health chronic prostatitis symptom index (nih - cpsi). all patients had a complete history, physical examination, wet mount examination, urine culture, and eps. because the routine culture results of the first voided bladder specimen, second midstream bladder specimen, eps, and urine sample after prostatic massage were negative, we could exclude cystitis and urethritis. the control group included 5 healthy men (mean age, 40.9 years) without symptoms. a group of 157 women of reproductive age attending the obstetrics and gynecology department of the same hospital (mean age, 38 years) were enrolled in this study. all women reported symptoms of lower genital tract infection (vaginal discharge or vulvar or vaginal complaints). twenty - nine healthy women (mean age, 39.7 years) without symptoms of lower genital tract infection who visited the hospital health center were selected as a control group. a qiaamp rna / dna mini kit (qiagen, hilden, germany) was used according to the manufacturer 's instructions. vaginal swabs and eps specimens were swirled in lysis buffer containing 1% triton x-100, 0.5% tween 20, and 1 mmol edta. after mixing the samples with 200 l buffer al (qiagen) and 20 l proteinase k, the samples were incubated for 30 min at 56 followed by 15 min at 95. for synthesis of cdna, reverse transcription was carried out by using the reverse aidtm first strand cdna synthesis kit (fermentas, burlington, canada). ten microliters of each bacterial rna was denatured at 80 for 3 min ; mixed with a master mix consisting of 4 l of 5x rt - buffer, 2 l of dntps, 1 l of rnase inhibitor, 1 l of reverse random primer, 1 l dithiothreitol (dtt), and 1 l of reverse transcriptase ; and incubated for 90 min at 37. after inactivation of reverse transcriptase by incubation at 94 for 2 min, cdnas were processed immediately for amplification. cdnas (3 l) were mixed with a pcr premix consisting of 10x pcr buffer, 1 l of forward primer (5'-acaatggtggtgacagtggg-3 '), and 1 l of reverse primer (5'-cgggtaaaaccaactcccat-3 ') (table 1). forty cycles were carried out at 94 for 30 s and 60 and 72, each for 90 s. then the pcr mix (10 l) was subjected to 4% agarose gel electrophoresis at 100 v for 30 min and nucleic acid bands were visualized by ethidium bromide staining. a multiplex pcr has been designed for simultaneous detection of n. gonorrhea, c. trachomatis, u. urealyticum, m. hominis, t. vaginalis, and m. genitalium., seoul, korea) uses two separate primer mixes and can detect the dna for 6 types of sexually transmitted pathogens (table 1). differences in proportions were assessed by 2-tailed fisher 's exact tests. any p - value. 14.0 (spss inc., chicago, il, usa) was used. a group of 11 men attending a specialized cp / cpps clinic of the urology department of the hospital (mean age, 43.5 years) were enrolled in the study. prostatic fluid samples were collected from outpatients with refractory type iiib prostatitis by aseptic technique by use of the mearses - stamey four glass urine collection method. all patients must have abstained from sexual activity for at least 4 days before sample collection. symptoms were quantified by the national institutes of health chronic prostatitis symptom index (nih - cpsi). all patients had a complete history, physical examination, wet mount examination, urine culture, and eps. because the routine culture results of the first voided bladder specimen, second midstream bladder specimen, eps, and urine sample after prostatic massage were negative, we could exclude cystitis and urethritis. the control group included 5 healthy men (mean age, 40.9 years) without symptoms. a group of 157 women of reproductive age attending the obstetrics and gynecology department of the same hospital (mean age, 38 years) were enrolled in this study. all women reported symptoms of lower genital tract infection (vaginal discharge or vulvar or vaginal complaints). twenty - nine healthy women (mean age, 39.7 years) without symptoms of lower genital tract infection who visited the hospital health center were selected as a control group. for rna / dna isolation, a qiaamp rna / dna mini kit (qiagen, hilden, germany) was used according to the manufacturer 's instructions. vaginal swabs and eps specimens were swirled in lysis buffer containing 1% triton x-100, 0.5% tween 20, and 1 mmol edta. after mixing the samples with 200 l buffer al (qiagen) and 20 l proteinase k, the samples were incubated for 30 min at 56 followed by 15 min at 95. for synthesis of cdna, reverse transcription was carried out by using the reverse aidtm first strand cdna synthesis kit (fermentas, burlington, canada). ten microliters of each bacterial rna was denatured at 80 for 3 min ; mixed with a master mix consisting of 4 l of 5x rt - buffer, 2 l of dntps, 1 l of rnase inhibitor, 1 l of reverse random primer, 1 l dithiothreitol (dtt), and 1 l of reverse transcriptase ; and incubated for 90 min at 37. after inactivation of reverse transcriptase by incubation at 94 for 2 min, cdnas were processed immediately for amplification. cdnas (3 l) were mixed with a pcr premix consisting of 10x pcr buffer, 1 l of forward primer (5'-acaatggtggtgacagtggg-3 '), and 1 l of reverse primer (5'-cgggtaaaaccaactcccat-3 ') (table 1). forty cycles were carried out at 94 for 30 s and 60 and 72, each for 90 s. then the pcr mix (10 l) was subjected to 4% agarose gel electrophoresis at 100 v for 30 min and nucleic acid bands were visualized by ethidium bromide staining. ten microliters of each bacterial rna was denatured at 80 for 3 min ; mixed with a master mix consisting of 4 l of 5x rt - buffer, 2 l of dntps, 1 l of rnase inhibitor, 1 l of reverse random primer, 1 l dithiothreitol (dtt), and 1 l of reverse transcriptase ; and incubated for 90 min at 37. after inactivation of reverse transcriptase by incubation at 94 for 2 min, cdnas were processed immediately for amplification. cdnas (3 l) were mixed with a pcr premix consisting of 10x pcr buffer, 1 l of forward primer (5'-acaatggtggtgacagtggg-3 '), and 1 l of reverse primer (5'-cgggtaaaaccaactcccat-3 ') (table 1). forty cycles were carried out at 94 for 30 s and 60 and 72, each for 90 s. then the pcr mix (10 l) was subjected to 4% agarose gel electrophoresis at 100 v for 30 min and nucleic acid bands were visualized by ethidium bromide staining. a multiplex pcr has been designed for simultaneous detection of n. gonorrhea, c. trachomatis, u. urealyticum, m. hominis, t. vaginalis, and m. genitalium., seoul, korea) uses two separate primer mixes and can detect the dna for 6 types of sexually transmitted pathogens (table 1). differences in proportions were assessed by 2-tailed fisher 's exact tests. any p - value less than 0.05 was considered statistically significant. in order to detect nanobacterial rna in eps and vaginal swabs, rt - pcr was performed with primers specifically designed for direct detection of nanobacterial genomic elements. 1 shows the results of agarose gel electrophoresis of rt - pcr products (band at 208 bp). in eps samples, the detection rate of nb in patients with cp / cpps was 9.1%, and all of the 5 healthy volunteers were negative. there was no significant difference in the detection rate of nb by rt - pcr between the two groups (p=0.48). nine (5.7%) of 157 vaginitis patients who showed positive results on rt - pcr for nb in vaginal swabs and all of the 29 healthy volunteers were negative. there was no significant difference in the detection rate of nb by rt - pcr between the two groups (p=0.19). the associations observed among the 7 microorganisms in the group of symptomatic men analyzed are summarized in table 2. the associations observed among the 7 microorganisms in the group of symptomatic women analyzed are summarized in table 3. nineteen patients were co - infected with two organisms (25.3%), 5 patients were co - infected with three organisms (6.7%), and 2 patients were co - infected with 4 organisms (2.7%). our data demonstrate the association in vivo between monoinfection of nanobacteria and vaginitis (6.7%). nb were co - infected with c. trachomatis, u. urealyticum, and m. hominis. in order to detect nanobacterial rna in eps and vaginal swabs, rt - pcr was performed with primers specifically designed for direct detection of nanobacterial genomic elements. 1 shows the results of agarose gel electrophoresis of rt - pcr products (band at 208 bp). in eps samples, the detection rate of nb in patients with cp / cpps was 9.1%, and all of the 5 healthy volunteers were negative. there was no significant difference in the detection rate of nb by rt - pcr between the two groups (p=0.48). nine (5.7%) of 157 vaginitis patients who showed positive results on rt - pcr for nb in vaginal swabs and all of the 29 healthy volunteers were negative. there was no significant difference in the detection rate of nb by rt - pcr between the two groups (p=0.19). the associations observed among the 7 microorganisms in the group of symptomatic men analyzed are summarized in table 2. the associations observed among the 7 microorganisms in the group of symptomatic women analyzed are summarized in table 3. nineteen patients were co - infected with two organisms (25.3%), 5 patients were co - infected with three organisms (6.7%), and 2 patients were co - infected with 4 organisms (2.7%). our data demonstrate the association in vivo between monoinfection of nanobacteria and vaginitis (6.7%). nb were co - infected with c. trachomatis, u. urealyticum, and m. hominis. nb have recently been described as novel microorganisms characterized by a small size (0.2 to 0.5) with a 16s rrna gene sequence and slow growth and a doubling time of about 3 days. they are gram - negative, have a unique structure and apparent nucleic acid, and their growth in vitro is best inhibited by tetracycline. raoult and young thought that nb were mineral - fetuin complexes or pleomorphic mineralo - protein complexes ; nevertheless, they could not exclude the possibility that nb are pathogenic microorganisms. in the clinical situation, nb may initiate kidney stone formation. they have been found in periodontal disease, calcified human valves, and in urine and bladder tissue samples of patients with interstitial cystitis / painful bladder syndrome and have been shown to participate in the clinical pathological process of those diseases. in addition to the culture method, several other diagnostic tools have been developed for identification of nb. one of the most powerful methods is transmission electron microscopy (tem) ; however, this method requires very expensive equipment. conventional nb culture and antigenic detection do not require expensive equipment ; however, these methods are often time consuming with cumbersome procedures. for dna sequencing, genomic rna is isolated from nb cultures, which is similar to the above methods. therefore, we developed a primer for conventional rt - pcr, which makes it possible to find nb from uncultured direct specimens. this method was superior to tem, conventional nb culture, and dna sequencing of isolated nb cultures. our method was rapid, simple, low in cost, and easily available because of the use of uncultured specimens and conventional rt - pcr. zhou found a 62.5% and 12.5% nb - positive rate in cultured eps and urine samples, respectively, after prostatic massage in men with type iii prostatitis. another study found indirect evidence of nb on antigen and antibody by using enzyme - linked immunosorbent assay (elisa) in 40% of urine samples and 0% of eps in patients with cp / cpps. shen postulated that the pathogenesis of prostatic calculi involves a certain mechanism : 1) nb form calcifications and mineral deposition cores ; 2) the prostatic epithelial membrane is damaged by nanobacterial infection, causing exposure of tissue components that may form crystal cores ; 3) nb mix with prostatic secretions ; 4) with urine backflow, high metabolite concentrations result. shen concluded that nb might be an important etiological factor for type iii prostatitis. in our 11 direct eps samples, the detection rate of nb in patients with cp / cpps was 9.1% and nb were co - infected with c. trachomatis. c. trachomatis was most commonly detected with cp / cpps and the frequency of co - infection with nb was higher than that for other infectious organisms. in our study, the nb - positive rate in direct eps was lower than 62.5% with cultured eps and higher than 0% with the elisa method. in our opinion, because we used only the direct eps samples, and not cultured eps samples, the nb - positive rate was low. the culture method showed a high positive rate but required a minimum of 5 weeks and had opportunity for contamination. the elisa method for detection of antibody showed a positive rate that was too low. rt - pcr for nb has the advantages of being rapid, simple, low in cost, and easily available. the sequences obtained were confirmed as nb by comparison with those in the genbank (national center for biotechnology information) database by using the basic local alignment search tool (blast). however, when applying molecular assays as a routine diagnostic test, one should be aware of false - positives resulting from the amplified method. also, clinical diagnosis by pcr only may be inaccurate, because vaginitis and prostatitis caused by nb can not be distinguished from that caused by the normal flora and contamination, and the positive results of pcr are not always the cause of the disease. according to current opinion, type iii prostatitis is probably related to nanobacterial infection, mainly because nb have been shown to cause multiple organic infections, especially urologic infections. also, after anti - nb treatment, the nb - positive rate decreased significantly, and the patients ' symptoms resolved. it is important that we precisely identify the cause of infection and provide the correct treatment. therefore, we attempted to develop a rapid, simple, low - cost, and easily available method for use with uncultured specimens. vaginal infection encompasses a broad range of symptoms, ranging from a state of severe inflammation and irritation with a frothy malodorous discharge, pain, and dyspareunia to an asymptomatic carrier state, which is estimated to constitute up to 50% of cases. infections by u. urealyticum, m. hominis, and t. vaginalis during pregnancy can lead to premature rupture of the placental membranes, premature labor, and low - birth - weight infants. in this study, 9 (5.7%) of 157 vaginitis patients showed positive results in rt - pcr for nb in vaginal swabs and all of the 29 healthy volunteers were negative. there was no significant difference in the detection rate for nb by rt - pcr between the two groups (p=0.19). however, we found five patients who were not positive for n. gonorrhea, c. trachomatis, u. urealyticum, m. hominis, t. vaginalis, or m. genitalium who were only infected with nb. the prevalence of u. urealyticum among the four patients with nb co - infection was 75% ; the presence of u. urealyticum might raise suspicion for nanobacterial infection. a symbiotic association between nb and u. urealyticum was implied ; however, the number of subjects co - infected with nb was too small, which was a limitation of the study. unfortunately, physicians could not determine whether nanobacteria were the cause of infection because it would take too much time to detect the nanobacteria and it would not result in appropriate treatment. although the controversies about whether nb are living organisms are continuing, the results of our study suggest that conventional rt - pcr for nb is rapid, simple, low in cost, and easily available for the detection of nb and that nb may be an etiological factor for vaginitis and prostatitis. however, there were several limitations to our study. first, there were a significant number of patients, both with cp / cpps and vaginitis, for whom the positive results of nb testing were not available. second, we could not compare our results with the results of the culture method and tem. finally, we did not attempt to correlate the clinical presentation of our patients with their test results. we found that conventional rt - pcr for nb was rapid, simple, low in cost, and easily available for the detection of nb and that nb may be a possible etiological factor for vaginitis and cp / cpps. the prevalence of u. urealyticum among the four patients with nb co - infection was 75% ; the presence of u. urealyticum may therefore raise suspicion for nanobacterial infection. physicians may want to consider nb as the cause of infection and attempt to provide treatment with an appropriate drug. | purposewe aimed to investigate the detection of nanobacteria (nb) from expressed prostatic secretions (eps) in patients with category iii chronic prostatitis / chronic pelvic pain syndrome (cp / cpps) and from vaginal swabs in patients with vaginitis by reverse transcriptase polymerase chain reaction (rt - pcr) and to evaluate the association between nb and neisseria gonorrhea, chlamydia trachomatis, ureaplasma urealyticum (u. urealyticum), mycoplasma hominis, trichomonas vaginalis, and mycoplasma genitalium.materials and methodsa group of 11 men attending a specialized cp / cpps clinic and a group of 157 women who reported symptoms of lower genital tract infection were enrolled in this study. nb were detected by rt - pcr. a seeplex sexually transmitted disease detection assay (seegene inc., seoul, korea) was used that could detect dna for 6 types of sexually transmitted pathogens.resultsin eps samples, the detection rate of nb in patients with cp / cpps was 9.1%, and 9 (5.7%) of 157 vaginitis patients showed positive results in rt - pcr for nb in vaginal swabs. associations observed among the 7 microorganisms included 6 (54.5%) patients who tested positive on eps and 75 (47.8%) patients who tested positive on vaginal swabs. five patients with vaginitis were found to have monoinfection of nb (6.7%).conclusionswe found that conventional rt - pcr for nb was rapid, simple, low in cost, and easily available for the detection of nb, and that nb may be a possible etiological factor for vaginitis and cp / cpps. the prevalence of u. urealyticum among the four patients with nb coinfection was 75% ; the presence of u. urealyticum might therefore raise suspicion for nanobacterial infection. |
it is in this small subset of patients, where mortality is high, ranging from 28% to 60%. deaths occur within 24 h from exsanguination, and after 24 h from multi - organ failure. the likely cause of this late mortality is a direct result of the bloody vicious cycle associated with a delay to definitive hemorrhage control and associated massive blood product transfusion. therefore, therapeutic interventions for patients exsanguinating from a pelvic fracture have been aimed at prompt hemorrhage control. anatomically, bleeding in the pelvis originates from three potential sources including the fractured cancellous bone surface, posterior pelvic venous plexus, and named branches of the internal iliac artery(ies). irrespective of the source, pelvic bleeding associated with fractures, usually manifests as a retroperitoneal hematoma. hemostatic techniques have included any combination of skeletal stabilization (both noninvasive and invasive), angioembolization (angio) or packing. although it is recognized that a combination of techniques is required to arrest significant pelvic bleeding, controversy exists over the correct sequence of interventions. most contemporary algorithms for exsanguinating pelvic fracture management include early angio, as this intervention been associated with improved survival. this is compounded by the apparent lack of resources out - of - hours, even in a level i trauma center. extraperitoneal pelvic packing (epp) is a technique that may be a valuable tool in the management of exsanguinating pelvic fractures. the main potential advantage with epp is earlier hemorrhage control, by overcoming the delay in accessing angio. it is likely to be a component of damage control for these severely injured patients. at present, there is no prospective comparison between epp and angio as the primary intervention for patients with exsanguinating pelvic fracture. therefore, the aim of this present study was to determine the utility of epp in controlling hemorrhage from exsanguinating pelvic fractures. westmead hospital is the adult major trauma centre for western sydney and the western part of the state of new south wales, australia, and services a population of approximately 1.5 million people. approximately, 1500 patients are admitted to the trauma service per year, of which 450 have an injury severity score (iss) of > 12. all adult (age > 15 years) patients between september 2011 and may 2014 presenting with an exsanguinating pelvic fracture were included in this prospective cohort study. exsanguinating pelvic fracture was defined as the presence of a pelvic fracture (as per young and burgess classification, i.e., lateral compression, anterior - posterior compression, or vertical shear) on pelvic x - ray and hemodynamic instability (sustained systolic blood pressure [sbp ] 5). exclusion criteria included death before arrival, or death in resuscitation room before determination of primary intervention for the pelvic fracture. included patients were treated with a standard protocol and allocation to the epp group or angio group determined by the on - call trauma surgeon 's proficiency with the epp technique [figure 1 for protocol and allocation method ]. this pragmatic experimental design was required due to state restrictions on delayed consent, therefore impeding the ability to perform a randomized control trial in this severely injured cohort. pelvic fracture management protocol and allocation method. fast : focused assessment with sonography in trauma, dpa : diagnostic peritoneal aspirate epp was performed as described by pohlemann. for patients requiring concomitant laparotomy, the peritoneum was preserved below the umbilicus to facilitate packing into the extraperitoneal pelvic space. evidence of bleeding was defined as the presence of contrast extravasation, pseudoaneurysm, or acute vessel truncation. embolization method (gelfoam or coils) was determined by the interventional radiologist performing the procedure. all relevant variables were imported and/or entered into microsoft office excel 2011 (microsoft corp., selected clinical variables included emergency department (ed) lowest sbp, highest pulse rate (pr), glasgow coma score. outcome variables included time to primary intervention, pre - angio packed red blood cell (prbc) requirement, time to definitive orthopedic fixation, postoperative complications, intensive care unit (icu) length of stay (los), total hospital los and mortality. continuous data are presented as means and standard deviation or medians and interquartile range (iqr, range from the 25 to the 75 percentile). chi - square test or fisher 's exact tests were used to compare proportions and to test for trends. the student 's t - test and mann whitney u - test was used to compare unpaired groups of continuous data. for all analyses, actual p values were reported and where possible, 95% confidence intervals presented. westmead hospital is the adult major trauma centre for western sydney and the western part of the state of new south wales, australia, and services a population of approximately 1.5 million people. approximately, 1500 patients are admitted to the trauma service per year, of which 450 have an injury severity score (iss) of > 12. all adult (age > 15 years) patients between september 2011 and may 2014 presenting with an exsanguinating pelvic fracture were included in this prospective cohort study. exsanguinating pelvic fracture was defined as the presence of a pelvic fracture (as per young and burgess classification, i.e., lateral compression, anterior - posterior compression, or vertical shear) on pelvic x - ray and hemodynamic instability (sustained systolic blood pressure [sbp ] 5). exclusion criteria included death before arrival, or death in resuscitation room before determination of primary intervention for the pelvic fracture. included patients were treated with a standard protocol and allocation to the epp group or angio group determined by the on - call trauma surgeon 's proficiency with the epp technique [figure 1 for protocol and allocation method ]. this pragmatic experimental design was required due to state restrictions on delayed consent, therefore impeding the ability to perform a randomized control trial in this severely injured cohort. pelvic fracture management protocol and allocation method. fast : focused assessment with sonography in trauma, dpa : diagnostic peritoneal aspirate epp was performed as described by pohlemann. for patients requiring concomitant laparotomy, the peritoneum was preserved below the umbilicus to facilitate packing into the extraperitoneal pelvic space. evidence of bleeding was defined as the presence of contrast extravasation, pseudoaneurysm, or acute vessel truncation. embolization method (gelfoam or coils) was determined by the interventional radiologist performing the procedure. all relevant variables were imported and/or entered into microsoft office excel 2011 (microsoft corp., selected clinical variables included emergency department (ed) lowest sbp, highest pulse rate (pr), glasgow coma score. outcome variables included time to primary intervention, pre - angio packed red blood cell (prbc) requirement, time to definitive orthopedic fixation, postoperative complications, intensive care unit (icu) length of stay (los), total hospital los and mortality. continuous data are presented as means and standard deviation or medians and interquartile range (iqr, range from the 25 to the 75 percentile). chi - square test or fisher 's exact tests were used to compare proportions and to test for trends. the student 's t - test and mann whitney u - test was used to compare unpaired groups of continuous data. for all analyses, actual p values were reported and where possible, 95% confidence intervals presented. the epp group was younger, more severely injured, and physiologically deranged than the angio group, although this was not significant [table 2 ]. the angio group received significantly more ed rbc transfusion (6.6 3.4 vs. 3.7 3.2, p = 0.04). time to primary procedure was significantly shorter in the epp group, as was the total time to angio [table 3 ]. the pre - angio transfusion rate was also significantly less in the epp group [table 3 ]. although mortality was decreased, both icu los and total hospital los were significantly increased in the epp group [table 3 ]. exsanguinating pelvic fracture cohort clinical features between extraperitoneal pelvic packing and angioembolization outcomes from extraperitoneal pelvic packing and angioembolization in the epp group, a mean of 8 4 packs was used. all patients undergoing epp subsequently underwent angiography, with 8 out of the 14 patients requiring embolization of arterial bleeding. sbp was significantly increased after epp (74 mmhg vs. 83 mmhg, p = 0.03), with a corresponding decrease in pr (120 bpm vs. 111 bpm, p = 0.026). there were 3 wound complications associated with epp (21%), but otherwise no significant difference in overall complications between epp and angio groups. the single death in the epp group occurred in a 59-year - old male pedestrian versus train. he had evidence of multicavitory bleeding and required a thoracotomy, laparotomy, and epp ; however, hemorrhage control was unable to be obtained, and he died in the operating room, without receiving angio. two deaths in the angio group occurred in the interventional radiology suite. the time to angio for these two cases was 80 min. the 2 death in the interventional radiology suite was due to intraabdominal hemorrhage, i.e., pelvic hemorrhage was thought to be the main source of bleeding in the resuscitation room due to a reported negative fast. the additional death in the angio group was an 80-year - old male pedestrian versus truck. exsanguinating pelvic fractures are an uncommon problem but are associated with a significant risk of death from hemorrhage. angio is the current choice of hemorrhage control in most pelvic fracture guidelines / algorithms. although effective, delay to embolization has been shown to increase mortality. in this study, the mortalities in the angio group can be directly attributed to pelvic hemorrhage or incorrect choice at the critical decision node of abdominal versus pelvic hemorrhage. it is important to recognize that operative management of the retroperitoneal pelvic hematoma has been controversial. historically, opening the retroperitoneal hematoma in an attempt to control bleeding from the internal iliac arteries has resulted in catastrophic failure. packing from within the abdominal cavity was so ineffective, as to result in recommendations for massive transfusion as being the optimal management of exsanguinating pelvic fractures 27. a recent report from a level i trauma center, revisiting bilateral internal iliac ligation for bleeding pelvic fractures, demonstrated a mortality rate of 64.3%. therefore, the longstanding surgical dogma of never opening a pelvic / retroperitoneal hematoma seems to hold true. epp does not violate the retroperitoneum and applies hemostasis through direct pressure onto the sacral plexus of veins, as well as onto the iliac vessels. time to operative intervention was significantly faster in this study and is consistent with reports by tai. and osborn. although there is no difference in 24-h prbc transfusion requirements between the groups, the transfusion rate was lower in the epp group before angio, suggesting that bleeding had slowed after packing. 57% of the epp group demonstrated an arterial injury requiring embolization, supporting the statement that epp is not a replacement for angio. when considering the faster time to the operating room, epp seems well placed as a damage control procedure, while angiographic resources are being mobilized. the findings in this study are consistent with those reported by ttterman. and tai., where time to the operating room was significantly shorter, and arterial injury found in > 50% of case undergoing epp in addition, the overall mortality in this study is lower than previously reported (16.7% compared with > 40%). the current study did not reach the target accrual to detect a 30% difference in mortality between groups. the incidence of severe pelvic injury was low (ten patients per year), and therefore an additional 3 years of enrollment would be required. institutional change in clinical management occurred after this analysis when it was concluded that there was sufficient evidence to perform epp primarily as a bridge to angio. although enrolled prospectively, potential bias exists with the treating surgeon. it is not known if the trauma surgeons were more comfortable with using angio for those patients perceived as less severely injured. furthermore, the time of day may have influenced the therapeutic decision, as consultant surgeons are off - site after hours. the final limitation of this study is that epp has not been proven to be more effective than angio at controlling hemorrhage. the procedure could be interpreted as a method of avoiding a system issue, namely, the inefficient access to angio services at all hours. epp has been demonstrated to be a valid technique with improvement in time to definitive hemorrhage control. mortality might be improved in the epp group, despite the epp group appearing to be more significantly injured than the angio group. therefore, epp should be considered a part of all general surgical / orthopedic surgeons skillset in the management of exsanguinating pelvic fracture, but as damage control component. this may have relevance to the rural / regional setting, where efficient access to angio services is not readily available. although this study was unable to reveal a statistically significant difference in mortality when comparing epp to angio, it forms the basis for further investigation in a multicenter, prospective randomized trial. | introduction : exsanguinating pelvic fractures are still associated with a significant mortality rate of 28 - 60%. extraperitoneal pelvic packing (epp) has been proposed as an optimal method of early haemorrhage control. the aim of this study was to determine the effect of epp compared with angioembolization as a primary intervention for patients with exsanguinating pelvic fracture.method:a prospective observational trial was performed at westmead hospital between september 2011 and may 2014. adult patients with exsanguinating pelvic fracture were allocated into one of two treatment groups determined by the primary / initial haemorrhage control technique : 1. epp followed by angioembolization or 2. angioembolization alone. the intervention was determined by the on - call surgeon 's proficiency with epp. demographic, clinical and laboratory data were collected. univariate analysis of the two groups was performed with student 's t - test, mann - whitney - u test and fisher 's exact test.results:24 exsanguinating pelvic fracture cases were included. 14 underwent epp while 10 underwent angioembolization as the primary intervention. although not statistically significant, the epp group was more severely injured (injury severity score 32 vs. 23), more acidotic (base deficit 7.9 vs. 6.2), and more hypotensive (systolic blood pressure 74.2 vs. 84.3). despite these differences, mortality was reduced (7.1% vs. 30%, not significant). time to epp compared with angioembolization was reduced (67.6 vs. 130.2 minutes, p = 0.017). pre - angioembolization transfusion requirement was also reduced with epp (0.032 vs. 0.052 units / min, p = 0.04). arterial injury was found in 51% of the epp group. there were no significant differences in complication rates between the groups.conclusion:epp appears to be a safe and efficient technique for primary haemorrhage control in exsanguinating pelvic fractures. given the high rate of associated arterial injury, epp should be considered as the first part of a damage control approach for exsanguinating pelvic fractures. |
medicine is repeatedly transformed by the discovery of biological processes and ultimately disease indicators that inform and refine clinical care. a century ago, exposure and contraction of tuberculosis were common with little ability to prevent, predict, or treat this condition. the simple and now widely available bacillus calmette - gurin (bcg) vaccine, coupled with selective screening by x - ray chest examination, has transformed the disease profile of tuberculosis and almost eliminated tuberculosis in the developed world. more recently, cancer biomarkers are offering the very real capacity for early detection and for selective and targeted therapeutic strategies based on molecular markers. in contrast, the quest for cures for schizophrenia seems to be limited while our definition of the disease remains a largely opaque scientific venture where clinical diagnosis is cast and dependent upon the quicksand of symptomatology. any biological feature of living humans has the potential to be an informative indicator of schizophrenia risk, occurrence, or progression. in a narrow sense, a biomarker refers to a molecular change in body tissues and fluids that can be used as a clinical indicator. those measures that prove to be reliable (consistent) and valid (true) predictors through research efforts can be used as clinical biomarkers. thus, a biomarker has to have clinical utility and biomarkers will take time, money, and coordinated research to develop. to prove clinical utility in schizophrenia will take large scale and coordinated efforts of biologists, doctors, and patients and is by nature translational. in considering the effort required to identify, replicate, and validate biomarkers for clinical use, it may not be surprising that no biomarker has been accepted for use in schizophrenia, although there are some leads. the goal of finding biomarkers for schizophrenia is not new. a pubmed search with the keywords biomarker and schizophrenia reveals a nature paper from 1965 entitled phenolic and indoleamines in the urine of schizophrenics and demonstrates biomarkers have been sought after by our field for decades and a review by professor sabine bahn points out that studying the blood of the insane was taking place as early as the mid-1800s by w. lauder lindsey in scotland. in more modern times, the published literature in biomarkers and schizophrenia grew slowly over the 15 years following 1965 reaching double - digit figures / year by 1980 and then undergoing a substantial increase more recently with over 100 papers / year in 20052012 and ~2,000 papers in total including almost 400 review articles. this increase in biomarkers research articles in schizophrenia may be due to more sensitive and sophisticated assessment tools that can provide thousands of measurements in parallel and the development of modelling approaches and computing power to store and analyse large amounts of data ; however, despite the increase in research papers, there has not been a corresponding increase in clinical use of biomarkers in schizophrenia. since the use of multiple molecular measures is a fairly recent development, and the standards against which we assess new markers are likely imperfect and evolving, the power of any proposed biological marker to predict disease or treatment response, even in some people with schizophrenia, in a real - world setting has not had adequate time to be developed, tested, modified, and implemented. when considering a nascent field with almost 20% reviews, we were challenged to consider what another review article could provide when most biomarkers for schizophrenia are still in the realm of speculation ; or, said another way : are there any schizophrenia biomarkers with unassailable data and widespread agreement for use available for review ? from our perspective, this ambitious goal has not yet been realized. in this paper, rather than review the controversial evidence for or against any one biomarker in particular, we will raise major concepts and questions regarding how biomarkers can be chosen, prioritized, and evaluated in schizophrenia. generally, a biomarker can be developed for three main purposes (1) diagnostic (to classify as having a disease), (2) prognostic (to make predictions on who will develop a disease), or (3) theranostic (to predict an individual response to a particular therapy). it is important to consider that the biomarkers useful for one purpose (i.e., diagnosis) do not necessarily have to be useful for the others (i.e., response to treatment). also worth emphasizing is that when dsm5 was being developed, biomarkers were considered to be forthcoming as external validations that may help to define and group diagnoses and inform reclassifications. however, this thinking seems to have been premature, as biomarkers do not feature in dsm5. in this paper, we will discuss some of the concepts around developing biomarkers in an effort to understand why the use of biomarkers for schizophrenia has not been realized. one argument for a diagnostic biomarker for schizophrenia is that while diagnosis can be made based on clinical interviews and careful observations by trained medical staff, the diagnostic process is by nature more subjective and variable than in some other areas of medicine and thus would benefit from a more objective test. psychiatrists working at the turn of the last century recognized that schizophrenia was not a single entity, but that heterogeneity was present in the illness. the fact that schizophrenia has multiple causes each with distinct biological mechanisms means that attempting to find a single biomarker or group of biomarkers that would coincide with all cases of dsm - defined schizophrenia is unlikely. perhaps it would be beneficial for the biomarker field to look for biomarkers in subsets of people with schizophrenia and, in this way, biomarkers may be developed for the most common biological underpinnings of schizophrenia, but we suggest schizophrenia researchers developing biomarkers may not want to attempt to capture all people with this diagnostic label. a diagnostic biomarker test (even if for a subgroup) may be of clinical benefit if a positive prediction can be made, as even though psychiatrists are highly trained to provide reliable diagnoses, some clinicians may have difficulty discriminating schizoaffective disorder from bipolar i disorder. furthermore, those without extensive experience in psychiatry, such as general practitioners, may have limited experience in discriminating one major mental illness from another ; for example, symptoms of major depression (particularly psychotic depression), schizophrenia, schizoaffective disorder, and bipolar i can overlap. this problem is especially apparent in the premorbid phase, as in the prodromal phase of schizophrenia, which typically occurs during teenage years ; the major symptoms of preschizophrenia can be loss of motivation, social withdrawal, and lack of focused attention and can overlap with the symptoms of depression. a misdiagnosis can have treatment implications because what is considered optimal treatment for each major mental illness category currently differs. additionally, some individuals with schizophrenia will receive multiple diagnoses throughout their life depending on the clinician, as described above, in combination with different information disclosed by the patient and variation in symptom presentation over time. these problems suggest that a diagnostic biomarker to discriminate schizophrenia from other major mental illnesses especially early in the course of the disease (a prognostic one) may be particularly helpful. another argument in support of a prognostic biomarker for schizophrenia is that if the biomarker is present in an individual before any behavioural symptoms are present, then it could be used to initiate specific (antipsychotic) treatment early to prevent schizophrenia onset. however, another major problem with this approach is that the underlying biological root causes could cut across diagnostic boundaries and may not be expected to be discriminate based on dsm criteria. prototypical biomarkers for disease are molecular and would encompass targets generated in the omics arena ; these are dna based (genomics), mrna based (transcriptomics), protein based (proteomics), or metabolism based (metabolomics). however, the term biomarker as it applies to the field of schizophrenia is also used on a more macroscopic scale largely because the abnormal tissue (brain) is not easily sampled and while important biological information regarding schizophrenia can be derived from other organs or cells like liver, pituitary, fibroblasts, nasal epithelium, or blood cells, most schizophrenia researchers have focused on brain measures. thus, it is typical for a schizophrenia researcher to consider mri brain imaging or electrophysiological measures (eeg) as biomarkers as well. further, it may be that more macroscopic markers and molecular markers need to be combined to be informative, as many individual molecular abnormalities in the brains of people with schizophrenia are often within the range of individuals without schizophrenia. if we consider the definition of a biomarker as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological response, then more systems - based, like fmri and eeg, measures would be consistent with this definition. once a potential measure or group of measures are made, be they at the molecular (micro) level or systems (macro) level, techniques (i.e., algorithms) for patient classification and disease prediction have to be devised. this requires a solid bioinformatics approach involving multivariate data analysis, mathematical modelling approaches, statistical learning techniques, and a team of experts. there are two phases of developing a prediction task (developing a rule or algorithm), one is referred to as training which allows the construction of the model based on available data and the second is referred to as the testing phase which requires that the rule is applied to an independent dataset to predict an thus, even when a given marker or set of markers are identified to be of high predictive value in the training phase, they may not have real - world traction in the testing phase. most of the papers available on biomarkers in schizophrenia have been concentrating on making an initial prediction based on the training phase and often use approaches of resampling or regrouping their own data in a testing phase. this may be a reasonable first step but is not a rigorous one, and replication across laboratories with different sets of researchers and samples is required for a true test of biomarker performance. the number of possible predictive models that could be developed and tested for biomarkers in schizophrenia is limitless. predictive models are based on multiple measures with uncertain weighting and uncertain thresholds for making decisions at branch points used for categorization. when constructing prediction models, there are unspecified parameters on the number and/or type of biomarkers needed and there is no set guide as to how many or in what order questions with binary outcomes should be posed (in terms of construction of decision trees). while the goal seems simple, to make accurate predictions about category membership or disease risk, the construction of models can be, in fact, quite complex. most of the diagnostic biomarker models use a two - classification scheme outcome, for example, likelihood of having / developing schizophrenia or not. however, as briefly described above, the challenge in the clinical setting is related to the notion of diagnostic specificity : separating schizophrenia from other major mental illnesses, which, as highlighted above, may be a false standard as we suggest that biomarkers should define an underlying pathophysiology and be indicators of a biological process that has gone awry rather than a dsm - defined disease category. furthermore, the structure of the prediction models we use as clinical tools in schizophrenia will have to vary depending on what is being classified (diagnostic group, future risk, and relapse likelihood). since the number of assumptions (factors) entering into a model is infinite and the best result will always be a probability of outcome, it is unlikely that schizophrenia will be predicted with 100% accuracy. we suggest that rather than aiming for a biomarker identifying a diagnostic category, to be most beneficial, a biomarker should provide information about the underlying pathophysiology that may in fact cut across traditional diagnostic categories. since schizophrenia is best viewed as a syndrome, with various root causes, then a biomarker may be used to identify distinct biological processes involved in subsets of people with the diagnosis of schizophrenia. one assumption often made by some schizophrenia researchers centres around the notion that for biomarkers to be useful, biomarkers need to be stable over time or need to be trait markers. however, when one considers the characteristics of an ideal biomarker as outlined for use in alzheimer 's disease, the main concerns are that the test measures an underlying component of the disease, is valid and specific, and would be easily and reliably measured across laboratories. in the review, the authors point out that valuable biomarkers could be either trait dependant (apoe4 genotype) or state dependant (csf -amyloid level). it may be that some individuals support the notion that an optimal biomarker needs to be consistent with an invariant diagnostic label, in a similar way that, the term endophenotype may be used to indicate an intermediate step involved in risk for schizophrenia, as being stable over time in order to aid identification of associated genes. however, it is well known that the severity of symptoms of people with schizophrenia can vary considerably over time (for multiple reasons, including but are not limited to voluntary medication withdrawal, stress, or sex hormone fluctuations in females). thus, it may be that biological underpinnings best associated with the disease may depend on environmental conditions or other factors such as endocrine state of the individual. if one considers the biological changes in schizophrenia to be dynamic, then the concerns about the timing of sampling individuals (particularly in relevance to time since onset, the state of acute exacerbation or remission, trajectory within the course of illness, time since last menstrual cycle (for females), and time since medication) will be important factors to consider in research designs and in clinical applications. typically, the stability of markers over time and in relationship to symptom variation within individuals with schizophrenia is not considered in design of many biomarker discovery projects to date. the schizophrenia biomarker field does not appear to be as organized and systematic as needed in testing for and accounting for human biological variance and time of day in sampling biological markers. this adds a confounding source of methodological heterogeneity in the currently available literature on biomarkers in schizophrenia. it will be important for future studies to try to control / record as many factors as possible, that is, for example, time of day of sampling (for the mri scan or blood draw), day of the menstrual cycle and status of oral contraceptive intake (in women), time since last dose of antipsychotic, time since last cold / flu symptoms, body composition (bmi), diet or exercise level, and degree of symptoms present. it is unclear if biomarkers assessed in a stable state or in a more actively psychotic state will be more informative for clinical application and it may be that the degree of change for a given marker over time within the same person will be the most predictive. in sum, since many biomarker studies in schizophrenia have sampled chronic patients who are typically stable, studies incorporating more dynamic sampling strategies could provide a more insight and possibly a unique set of markers to pursue. while some may argue a diagnostic biomarker for schizophrenia does not yet exist, a company originally founded by professor sabine bahn, psynova, in collaboration with rules - based medicine generated a putative blood biomarker assay for the diagnosis of schizophrenia [7, 10 ]. this test measured 51 blood anolytes (small molecules and proteins) and was reported to be 83% specific and sensitive. although this test, called veripsych, was available in 2010, the use of this test did not gain widespread support and the assay has recently been withdrawn from the market. there is a certain amount of healthy skepticism in the field of psychiatry in relation to the use of biologically based diagnostic tests for schizophrenia mainly due to the fact that the veripsych early tests did not include tests of diagnostic specificity (schizophrenia versus other psychiatric disorders). although this criticism was addressed by later studies from the same group, this diagnostic blood test for schizophrenia still did not become widely used. according to one of the developers of veripsych there may have been at least two further reasons for the reluctance (1) market research found that most psychiatrists believe that they are very good at diagnosing schizophrenia patients using a basic clinical interview and (2) the $ 2500 (us) cost was considered high. indeed, the extensive training involved in using clinical interview skills to make a differential diagnosis is considered the gold standard to which all bioassays should be calibrated, so it is not clear as to what additional information a blood test would provide and finding additional funds to cover serum testing for each patient is often prohibitive. it is also argued that the ~83% sensitivity and specificity reported for this proposed diagnostic blood test has not been rigorously tested with independent cohorts (predictive), by independent teams, working in independent laboratories, so the predictive power is still uncertain. it would also be the case that any newly developed diagnostic instrument (even if 100% predictive, specific, and sensitive) would take time to gain clinical use due to time required to train on site medical staff and the high initial cost involved. if the result of a biomarker test can not provide any insight into the optimal therapeutic strategy, it will likely be of limited usefulness to psychiatrists. another point to consider is that using blood - based assays is going to be evolving for use in psychiatry. any type of maker used in isolation is not likely to be as informative as using a combination of approaches including biological brain scans and nonbiological interviews, self reports, and cognitive testing, as is done in alzheimer 's disease. in this way, it is possible that the number of markers to be screened from blood could be reduced potentially lowering costs. another important point worth emphasizing again is there is a large biological heterogeneity found in the brains of patients with schizophrenia at the molecular and cellular levels (in addition to the cognitive and clinical level) with many individuals with the diagnosis of schizophrenia falling into the normal range [1315 ]. also, it seems that molecular neuropathology of schizophrenia is commonly shared with bipolar disorder and/or depression [1620 ]. further, if there are definable subsets of people with a distinct neuropathological profile even within a dsm - defined illness (e.g., see), then it would follow that identifying a single biomarker test that reflects the underlying disease processes with a high degree of diagnostic sensitivity and specificity should be unlikely. rather, multiple biomarkers each reflecting various neuropathological processes or biological underpinnings in subsets of individuals would be required. another possible use for biomarkers in schizophrenia is to predict who will become ill prior to first break when symptoms meet current diagnostic criteria. the argument in favour of development of an early diagnostic test is that patients with schizophrenia who have a shorter duration of untreated psychosis (dup) tend to be more treatment responsive to currently available antipsychotics and are often less symptomatic later and require lower maintenance doses of antipsychotics. thus, if the diagnosis of schizophrenia could be predicted with certainty even in some individuals, then one could theoretically reduce their dup to zero. however, there are many clinical issues to consider when developing an ideal treatment for a prodromal person who does not clearly fit into any diagnostic category. even in cases that present early with more severe preschizophrenia, there is increased risk for metabolic side effects, cardiac disease, obesity, and diabetes with second - generation antipsychotics and motor side effects of akathesia, extrapyramidal symptoms, and tardive dyskinesia with first - generation antipsychotics that need to be considered. despite the fact that no biomarker currently exists and no optimal early treatment is yet available, many psychiatrists do prescribe antipsychotics to individuals deemed to be at high risk of developing schizophrenia based on family history and functional decline. at this stage, it is unclear what value a diagnostic blood test to predict schizophrenia would be when early optimal treatment has not yet been defined. one could also argue that for an early predictive test to be useful, more research into early clinical staging and treatment algorithms needs to be developed in parallel [24, 25 ]. we suggest that perhaps one of the most promising areas for investment into development of schizophrenia biomarkers is in the prediction of treatment response, not only to existing pharmacological therapies, but in particular to novel therapies. the ability to predict response to a drug (i.e., theranostics) can be subdivided into (1) beneficial symptom attenuation, (2) deleterious side effect occurrence, and (3) probability of relapse. while clinical decisions on choosing the correct antipsychotic medication are based on many variables and require clinical training and skill, there is still an element of trial and error in relation to which antipsychotic will produce symptom reduction with the least side effects for a given individual. once a diagnosis of schizophrenia is made and antipsychotics are chosen as the drugs of choice, there is also consideration of which type (first- or second - generation antipsychotic), what dose, and which route of administration will be optimal. there are many parameters at the biological (risk of metabolic side effects), psychological (patient insight), and social (level of family support) levels that need to be considered. if a biomarker test could be used to help determine the degree of symptom reduction and potential for treatment discontinuation in a given patient with a given antipsychotic at a given dose, this could also shorten the duration of untreated psychosis, help maintain compliance, and lead to a better outcome. in fact, a blood test already exists that predicts if a high, medium, or low dose of antipsychotics drug would be most effective in a particular person. this test uses a panel of genetic polymorphisms coding for liver enzymes important for degradation of psychiatric medication (both antipsychotics and antidepressants) to predict the rate of metabolism of antipsychotics. this genetic test has been proposed as a tool to help determine treatment dose ; for example, if someone is a high metaboliser, then the person would require a higher dose. however, despite the availability of this test since 2003, it has not been widely accepted possibly because only a few individuals fall into the ultrahigh metaboliser range. also, genetic factors alone may provide an incomplete picture, as, for example, smoking cigarettes can increase the activity of liver enzymes that break down clozapine resulting in a need for a higher effective dose in patients who smoke. thus, any genetic biomarker when used in isolation and in absence of critical behavioural or other environmental information will have limited clinical value. this is not to suggest that potential biomarkers should not be tested and eventually used, but instead they need to be understood in a context and interpreted by trained staff. the important nonbiological factors that should be entered into predictive programs or algorithms are still mostly undefined, suggesting that biomarker use in schizophrenia is perhaps best thought of as being in a very early, exploratory stage and may be beginning to move to the more iterative model building stage of development. another useful aspect of a biomarker in regard to prediction of treatment response is to inform the patient and doctor about which individuals would be at risk for deleterious side effects of available treatments. this has obvious benefits as those at high risk for metabolic side effects may be able to avoid the rapid weight gain associated with some antipsychotics. as a proof of this concept, serotonin transporter genetic polymorphisms may predict risk for the common weight gain associated with clozapine administration. unfortunately, there are not a lot of alternatives since most second - generation antipsychotics cause weight gain. another serious, but uncommon, side effect observed with high doses of antipsychotic drugs is a lengthening of the heart 's electrical cycle of activity. this can be detected by a prolonged qt interval on an electrocardiogram (ecg) and is itself a biomarker for increased risk of sudden death. if a biomarker could be developed to predict risk for qt elongation, then clinicians may be able to avoid high doses of certain antipsychotics in those individuals at risk for sudden cardiac arrest and death. increased risk of developing agranulocytosis, characterized by a decrease in white blood cells and increased risk of death, has resulted in the failure to widely prescribe clozapine, especially in the united states. this is despite the claims that clozapine is believed to be one of the most efficacious clinical antipsychotics available. however, agranulocytosis occurs in about 1% of patients with schizophrenia - prescribed clozapine (without monitoring), and while significant, this suggests that 99% may be at low risk for this side effect. if a biomarker could be developed to determine those likely and unlikely to develop agranulocytosis from clozapine, then clozapine could be prescribed as a first - line treatment in more people with schizophrenia rather than just using it in treatment - resistant patients or as a last - resort treatment. it also may reduce the costs of having weekly or monthly blood tests in patients determined to be at very low risk of agranulocytosis. in 2007, a pharmacogenetic test was launched to measure the probability of developing agranulocytosis by examining the hla - dqb1 gene. this test has been limited in its clinical usage possibly due to the reluctance to use clozapine in general. discontinuation (via drug holiday) and relapse are associated with poor prognosis, poor functional outcome, and increased disability. from the examples of biomarker tests in schizophrenia outlined in this paper, it is clear that in order for any biomarker to successfully transform practice they can not simply be identified and available. biomarkers need to predict something of value (e.g., diagnosis when there is uncertainty, treatment response, etc.) and need to be justified in terms of cost and benefit to the patient. once a valid and reliable biomarker has been identified, to reach full implementation will require shifts at the political and societal level such that prescribing, training, and compliance practices can be changed to successfully collect medical specimens and interpret biomarker results. any biomarker in schizophrenia will require more clinical research to gather evidence of validity and cost effectiveness before it will be in routine use. any biomarker measurement has varying degree of error that needs to be considered when making conclusions. perhaps most importantly the heterogeneity associated with schizophrenia will most likely need to be taken into account resulting in multiple biomarkers that identify the multiple underlying pathophysiological processes involved in schizophrenia. currently, uncertainty overrules the predicative ability of any biomarker assay(s) rendering them of questionable clinical value. therefore, much additional work will be required prior to obtaining any well - established biomarkers for schizophrenia. some of the real challenges of determining biomarkers in schizophrenia may be change over time that requires following patients longitudinally which is challenging as followup is hard, noncompliance is high, medication change is the rule, and multiple comorbidities exist. further, development of longitudinal assessment of schizophrenia over time with a goal of developing biomarkers will take thoughtful leadership, large multidisciplinary teams, well - organized research protocols, and grand - scale funding from government, corporate, and private sources. | biomarkers have been sought after in the field of schizophrenia research for decades. in this paper, we discuss some of the concepts around developing biomarkers in an effort to understand why the use of biomarkers for schizophrenia has not been realized. in particular, we address the following 4 questions. why would we need a diagnostic biomarker for schizophrenia ? how is a biomarker typically defined and how does that influence the discovery of biomarkers in schizophrenia ? what is the best use of biomarkers in schizophrenia ? do any biomarkers for schizophrenia currently exist ? thus, while we suggest that no biomarker currently exists for schizophrenia, the heterogeneity associated with schizophrenia will most likely need to be taken into account which will result in multiple biomarkers that identify the multiple underlying pathophysiological processes involved in schizophrenia. therefore, much additional work will be required prior to obtaining any well - established biomarkers for schizophrenia. |
the epicardium develops from the proepicardial organ located at the venous pole of the heart in continuity with the second heart field. the epicardium is a mesothelial layer that forms a continuum with the coelomic lining and as such expresses many genes that are epithelial in origin like cytokeratin and 4 integrin. the evolutionary link to the developing excretory tract is exemplified by the expression of proteins like wilms tumour 1 (wt1) and podoplanin [5, 6 ]. the guidance of the epicardium over the looping myocardial tube, which consists of both primary and second heart field derived myocardium is still not completely understood, but epicardial myocardial interaction including retinoic acid [4, 8 ] as well as members of the platelet - derived growth factor signalling are of relevance. after initial spreading of the epicardium over the myocardium, several waves of epithelial mesenchymal transition (emt) have been described with subsequent migration into the sub - endocardial and intramyocardial location [10, 11 ]. the epdcs that are essential for the isolation of atrial and ventricular myocardium, with the formation of the annulus fibrosis [12, 13 ] and the population of the atrioventricular cushions [10, 11 ], migrate into the heart relatively late. the epdcs have been traced by using the chicken quail chimera model as well as several transgenic mouse models. from these studies the epdcs could be followed into their definitive position and eventual differentiation state. unequivocally, differentiations of the cardiac fibroblast, coronary smooth muscle cell and adventitial fibroblast have been proven. different opinions exist on the origin of the coronary endothelial cell and the cardiomyocyte. as both epicardial and myocardial cells derive from a common second heart field progenitor cell (fig. 1a), the recent studies using the conditional cre - lox model of wt1 and tbx18 do not provide an absolute proof of the origin of cardiomyocytes from the epicardial lineage. of interest in this respect is the described regenerative potential of the zebra fish heart that shows the capacity of the epicardial lineage to stimulate the cardiac myocyte population to renewed growth without actual cellular contribution. the endothelial lineage derives from the sinus venosus and liver microvascular lining but not from the epicardium. whether there is a further link between the epicardium and other intracardiac (stem) cell populations, such as the recently described telocyte, needs further research. (a) schematic representation of the first and second heart field contribution to the developing heart. the venous pole (posterior heart field) is essential as source of epdcs and cmpcs, a differentiation in a myocardial and epicardial lineage from the progenitor population is guided by the balance of bone morphogenetic protein (bmp) and fibroblastic growth factor 2 (fgf2). (b) expression of wt1 in the surface epicardium (b1) and intramyocardially (b2) in a control adult mouse heart is very limited (arrows). two days after mi, with injection of medium, this is not substantially altered (c1,2) except for the cases with injection of epdcs (c3,4), where both the surface epicardium and the interstitial fibroblasts show marked wt1 expression. at day 4 after mi this phenomenon is also present in the medium injected heart (d1,2) and maintained in the heart after epdc transplantation (d3,4). injection of the lentiviral katushka construct into the epicardial cavity showed, after 4 days survival, staining of the squamous surface epicardium in the control heart (e, arrows). after mi the surface epicardium becomes cuboid (f, arrows) indicative of activation and cells can be traced intramyocardially (g, arrows) after emt, which is supported by double staining of katushka and wt1 (h). the epicardium and epdcs with their specific supporter role in myocardial differentiation and their homing to the fibrous annulus [12, 13 ] and valves could also indicate a role in development of pathology. it has been shown that inhibition of epicardial outgrowth can lead to deficient annulus fibrosus formation with electrophysiological evidence of the wolff - parkinson - white (wpw) syndrome. also a periostin disturbance in the epdc deficient models could be linked to non - delamination of the endocardial cushions resulting in an ebstein - like valve phenotype. the study of cardiomyopathies and the observation of early coronary vascular disease preceding myocardial pathology have lead to a unifying concept on the role of epdcs not only in non - compaction cardiomyopathies but also in a far broader spectrum of cardiac diseases. the convincingly proven essential functions of the epicardium and the epdcs during normal embryonic development fuelled the hypothesis that epdcs might recapitulate some of these functions in case of cardiac disease. the potential of adult human epdcs was investigated in vitro, comparing them with mesenchymal stem cells (mscs), and in vivo. in vitro culture epdcs and mscs showed many similarities, but the unique cardiac commitment of epdcs was established by the expression of ctnt and gata4, lacking all mature cardiomyocyte differentiation genes. we also showed that human epdcs after injection into the ischemic myocardium of an immune incompetent mouse significantly improved cardiac function, already within 2 days. the beneficial effect persisted after definitive scar formation, at 6-week follow - up. the additional results were an enhanced angiogenesis of mouse origin, less loss of infarcted wall thickness and an up - regulation of proliferating cell nuclear antigen (pcna) in the host tissue infarct and borderzone area. the role of the exogenous epdcs, which acquired a myofibroblast phenotype, was mainly instructive rather than constructive. the results of the beneficial effect of epdc injection was further enhanced when a mix of adult derived epdcs and cardiomyocyte progenitors (cmpcs) was injected. the cmpcs were also instructive and did not differentiate into cardiomyocytes as was shown for the human derived cpmcs in vitro. altogether the conclusion was drawn that the observed effects were most probably based on paracrine or cell cell interactions from the exogenous transplant with the surrounding host tissue. there are several reports on the activation of epicardium and the interstitial fibroblasts after myocardial infarction (mi) without additional stem cell transplantation. the group of riley, for example, reported on the activation and coronary vascular improvement through thymosine b4. the factors triggering these events are not easily understood because of the concomitant effect of the influx of inflammatory cells after mi. we observed that 4 days after mi the endogenous epicardium and the interstitial fibroblasts started to re - express wt1 (fig. 1, compare control b1,2 with 4 days after mi d1,2), an indication of activation and dedifferentiation. this was enhanced by 2 days if additionally exogenous epdcs were injected (fig. 1, compare medium injected c1,2 with epdc injected c3,4 both 2 days after mi). to substantiate whether there was also renewed endogenous epicardial emt and migration after ischemia, we injected a lentiviral katushka construct (a far - red fluorescent marker) into the epicardial cavity of mice with or without mi. this showed indeed that re - activation of this embryonic program took place and that surface epicardial cells had migrated into the borderzone myocardium (fig. the therapeutic potential of the endogenous epicardium is therefore a new field of investigation, supported by our data and the literature. in conclusion, the hypothesis that epicardium and epdcs based on their broad embryonic potential might be a valuable source of research into mi treatment strategies as well as new fields to discover cardiac disease related mechanisms, seems to hold great promise. | abstractthe proepicardial - derived epicardium covers the myocardium and after a process of epithelial mesenchymal transition (emt) forms epicardium - derived cells (epdcs). these cells migrate into the myocardium and show an essential role in the induction of the ventricular compact myocardium and the differentiation of the purkinje fibres. epdcs are furthermore the source of the interstitial fibroblast, the coronary smooth muscle cell and the adventitial fibroblast. the possible differentiation into cardiomyocytes, endothelial cells and the recently described telocyte and other cells in the cardiac stem cell niche needs further investigation. surgically or genetically disturbed epicardial and epdc differentiation leads to a spectrum of abnormalities varying from thin undifferentiated myocardium, which can be embryonic lethal, to a diminished coronary vascular bed with even absent main coronary arteries. the embryonic potential of epdcs has been translated to both structural and functional congenital malformations and adult cardiac disease, like development of ebstein s malformation, arrhythmia and cardiomyopathies. furthermore, the use of adult epdcs as a stem cell source has been explored, showing in an animal model of myocardial ischemia the recapitulation of the embryonic program with improved function, angiogenesis and less adverse remodeling. combining epdcs and adult cardiomyocyte progenitor cells synergistically improved these results. the contribution of injected epdcs was instructive rather than constructive. the finding of reactivation of the endogenous epicardium in ischemia with re - expression of developmental genes and renewed emt marks the onset of a novel therapeutic focus. |
the antiobesity effect of incretinbased medicines is mainly mediated by the central anorectic effect of glucagonlike peptide1 (glp1) and glp1 receptor agonist. they also provoke various effects that involve the central and peripheral nervous systems, which include regulation of glucose and lipid metabolism, cardiovascular functions, cognitive functions, and stress and emotional responses. in the present review, we focus on the properties and functions of gutderived vs brainderived glp1, distinct roles of humoral vs neural pathways linking peripheral glp1 to the brain, and different modes of action between endogenous glp1 vs glp1 receptor agonists. glp1 is released from the enteroendocrine l cells in response to meals, and enhances glucoseinduced insulin secretion from pancreatic islets1, being recognized as an incretin hormone. glp1 is also produced by preproglucagonexpressing neurons in the nucleus tractus solitarius (nts) of the brain stem2, and acts as a neurotransmitter. it is important to distinguish the central action of gutderived glp1 from that of brainderived glp1. generally, the peripheral factors send their information to the brain through two distinct pathways, the humoral and neural pathways (figure 1). brain barrier (bbb) and the circumventricular organ (cvo) that has a leaky bbb. it is reported that glp1 can enter the brain through the bbb3, and radiolabeled glp1 is bound to its receptors expressed in the cvos4, 5. however, endogenous glp1 derived from the gut is rapidly cleaved by dipeptidyl peptidase4 (dpp4), with its halflife being less than 2 min6. hence, it is reasonable that gutderived glp1 influences the brain mainly through the neural pathway, which is composed of the vagal afferent fibers at the intestinal or hepatic portal area. we previously reported that glp1 evokes action potentials and increases cytosolic ca concentration ([ca]i) in the neurons of nodose ganglion, where cell bodies of the vagal afferent fibers are located (figure 2)7. vagal afferents also directly sense cholecystokinin8, 9, peptide yy336 10, leptin11, oxytocin11 and nesfatin112, the hormones regulating feeding and metabolism. intraperitoneal coinjection of glp1 and leptin13 or coinjection of glp1 receptor agonist exendin4 and peptide yy336 14 synergistically suppresses food intake. glucagonlike peptide1 (glp1), released from the intestine, informs the brain by passing through blood brain barrier (humoral pathway) and by interacting with vagal afferent nerves (neural pathway). consequent activation of the hypothalamus, circumventricular organ, and brain stem including nucleus tractus solitarius (nts), regulates feeding, energy / glucose metabolism and the cardiovascular system. (a) glucagonlike peptide1 (glp1) at 10 mol / l increases cytosolic ca concentration ([ca]i) in single nodose ganglion neurons isolated from rats. (b) as a control, [ca]i is stable in nodose ganglion neurons without glp1 administration. injection of glp1 into the portal vein enhances insulin secretion, and this effect is attenuated by hepatic vagotomy15, suggesting that the insulinotropic effect of glp1 is mediated not only by direct action on the pancreas, but also through vagal afferents, being recognized as a neuroincretin effect. a recent study by krieger.16 used the bilateral nodose ganglion injection technique to deliver a lentiviral vector to knock down the glp1 receptor specifically in the vagal afferent neurons of rats, and found that postmeal glycemia was elevated and insulin release was blunted in glp1 receptor knockdown rats. these results suggest that the vagal afferent glp1 receptor is a physiological contributor to the neuroincretin effect after meals. furthermore, they found glp1 receptor knockdown increases meal size and accelerates gastric emptying, whereas it has little effect on the longterm maintenance of food intake and weight in normal eating conditions. these results highlight a crucial role for the vagal afferent neuron in mediating the effects of endogenous glp1 on glycemia and food intake. glp1 neurons in the nts widely project in the central nervous system including the hypothalamus : the paraventricular nucleus (pvn), supraoptic nucleus (son), arcuate nucleus (arc) and hypothalamic dorsomedial nucleus (dmh), and the brainstem the parabrachial nucleus, reticular formation of medulla and dorsal vagal nucleus, thalamus and intermediolateral cell column2, 17, 18, 19, 20, 21 where glp1 receptors are expressed22, 23, 24, 25. we reported that endogenous glp1 in the brain targets pvn to restrict feeding behavior, in which the projection from nts glp1 neurons and activation of corticotropinreleasing hormone and nesfatin1 neurons might be implicated (figures 3 and 4)26. nts glp1 neurons are activated by both central signals and peripheral signals from vagal afferents. as a central signal, oxytocin neurons in pvn project to the nts glp1 neurons, and central infusion of oxytocin activates cfos expression in nts glp1 neurons27. it is established that peripheral signals, such as gastric distension28, and intraperitoneal injection of lithium chloride that leads to conditioned taste aversion and nausea29, 30 cause cfos expression in nts glp1 neurons. in addition, using brain slices of the transgenic mice expressing the yellow fluorescent protein under control of the preproglucagon promoter, the electrical properties of nts glp1 neurons were reported. that study showed that leptin directly depolarizes nts glp1 neurons, and cholecystokinin modulates noradrenergic or glutamatergic neurons and thereby secondarily depolarizes nts glp1 neurons. in contrast, nts glp1 neurons failed to show electrical responses to peptide yy, glp1 and ghrelin31. glucagonlike peptide1 (glp1) concentrationdependently increases [ca]i in corticotropinreleasing hormone (crh) and nesfatin1 neurons of paraventricular nucleus (pvn). (b) glp1 at 10 mol / l increased [ca]i in a single neuron isolated from pvn (left panel), and this neuron was subsequently shown to be immunoreactive (ir) to crh (right panel). this neuron also responded to 30 mmol / l kcl with an increase in [ca]i. (c) glp1 at 10 increased [ca]i in a single pvn neuron that was subsequently shown to be ir to nesfatin1. superfusate contained 1 mmol / l glucose and the bars above the tracings indicate the periods of administration of agents in (b) and (c). (d) incidence of the neurons that express crh or nesfatin1 over those responded to glp1 in the pvn. the numbers above the bars indicate the number of neurons ir to crh or nesfatin1 over that responded to glp1 in the range of 1010 mol / l. intragastric glucose infusion, compared with intragastric water infusion, increased muscle glycogen synthesis and cfos expression in the nts, and decreased cfos expression in the neuropeptide ypositive neurons in the arc. these effects were diminished with simultaneous central infusion of the glp1 receptor antagonist exendin(939), and abolished in glp1 receptor knockout mice. because intragastric glucose infusion does not affect the blood levels of glucose, insulin and glp1, enteric glucose might send signals to the brain through vagal afferents and activate a glp1 receptordependent signal in the brain to control peripheral glucose metabolism (figure 4). nucleus tractus solitarius (nts) glp1 neurons project to paraventricular nucleus (pvn), and activate corticotropinreleasing hormone (crh) and nesfatin1 neurons to suppress feeding, and possibly regulate glucose metabolism. several glp1 receptor agonists have recently been used to treat type 2 diabetes patients. glp1 receptor agonists have dpp4resistance and a longer halflife of several hours or more because of their structural modification. unlike the gutderived endogenous glp1 that is inactivated within 2 min by dpp4, glp1 receptor agonists administered in the periphery are stable and highly likely to act on the brain through the humoral pathway in addition to the neural pathway. in addition, glp1 receptor agonist could remain in the brain for several hours, and hence exert effects similar to those induced by the brainderived endogenous glp1. indeed, it was reported that the glp1 receptor agonist, liraglutide, administered in the periphery can reach the brain and exert central effects33. that study investigated the distribution of fluorescencelabeled liraglutide in the mouse brain after peripheral administration. fluorescencelabeled liraglutide was observed in all cvos, the zona interna of the median eminence (me), the area postrema (ap), the sobfornical organ (sfo) and the organum vasculosum of the lamina terminalis (ovlt), and also in hypothalamic regions, the arc, pvn and son. these signals of fluorescencelabeled liraglutide in specific brain areas were blunted in glp1 receptor knockout mice. interestingly, no fluorescencelabeled liraglutide was observed in the nts, suggesting that peripheral liraglutide could not directly interact with the nts glp1 neurons. this study also showed that peripheral liraglutideinduced feeding suppression was abolished by infusion of glp1 receptor antagonist exendin(939) into the arc, but not the pvn. neither the ablation of the ap nor deafferentation of the subdiaphragmatic vagal afferent affected the peripheral liraglutideinduced feeding suppression. furthermore, electrophysiological measurements of murine brain slices showed that glp1 directly stimulates proopiomelanocortin / cocaine and amphetaminestimulated transcript neurons, and indirectly inhibits neurotransmission to neuropeptide y / agoutirelated peptide neurons through gammaaminobutyric aciddependent signals. taken together, it was concluded that peripheral liraglutide directly acts on the arc, most likely proopiomelanocortin / cocaine and amphetaminestimulated transcript neurons, through the humoral pathway to suppress feeding. differences between glp1 and glp1 receptor agonist exendin4 in their central anorectic effects have been reported. this is partly explained by their different sensitivity to dpp434, but might also involve other factors. the anorectic effect of intracerebroventricular (i.c.v.) injection of glp1 was attenuated by i.c.v. by contrast, the anorectic effect of i.c.v. injection of exendin4 was not attenuated by i.c.v. injection of glp1 receptor antagonist before exendin4, but was abolished in glp1 receptor knockout mice. in contrast, the anorectic effect of intraperitoneal (i.p.) injection of exendin4 was completely blocked by i.p. these data suggest a key difference between glp1 and exendin4 in their interaction with the brain glp1 receptor. there might be a difference in the glp1 receptor properties between the brain and the periphery, which could include posttranslational processing, binding to glp1 receptor antagonist, interaction with other peptides and signal transduction. a recent report by beiroa.35 investigated the effects of specific injection of liraglutide in the various hypothalamic sites that regulate food intake and brown adipose tissue thermogenesis. injection of liraglutide specifically into the hypothalamic arc, pvn and lateral hypothalamic area (lha), but not ventromedial nucleus (vmh), reduced food intake. conversely, injection of liraglutide specifically into the vmh, but not arc, pvn and lha, stimulated brown adipose tissue thermogenesis. this effect depends on ampk in the vmh, and involves expression of uncoupling protein 1 in the brown adipose tissue to higher levels. infusion of glp1 and glp1 receptor agonist transiently increases blood pressure and heart rate in normal rodents. the possible underlying mechanisms involve central cholinergic system36, neurohypophysial hormones36, 37 and medullary catecholamine neurons38. regarding heart rate regulation, it was reported that exendin4 inhibits neurotransmission to cardiac vagal neurons in the medullary nucleus ambiguus, leading to suppression of cardiac parasympathetic activity thereby increasing heart rate39. studies in hypertensive patients with type 2 diabetes or obesity showed that longterm treatment with glp1 receptor agonists reduced blood pressure40. in animal studies, injection of exendin4 twice daily for 12 weeks reduced blood pressure in db / db mice41. furthermore, twicedaily exendin4 attenuated the increase in blood pressure by angiotensin ii infusion for 2 weeks in mice41. underlying mechanisms for the chronic antihypertensive effect of glp1 receptor agonists has not yet been fully elucidated. it is postulated as possible mechanisms that glp1 receptor agonist directly acts on the kidney to induce natriuresis41, 42, and on the endothelial cells to induce vasodilatation43. additionally or alternatively, chronic peripheral injection of glp1 receptor agonists would possibly inform the brain through humoral and/or neural pathways to activate central glp1 receptors and/or neural circuits, thereby evoking these chronic antihypertensive effects. these studies suggest that central glp1 or glp1 receptor agonists exert different actions depending on acute vs chronic paradigms, and on physiological vs pathological subjects. the result from a clinical trial of glp1 receptor agonist have provided cardiovascular safety for clinical use, although risk reduction of cardiovascular events has not been observed44. the followup period so far is approximately 3 years, and further investigations will be required to evaluate longerterm efficacy and safety. in the past several years of clinical experience, incretinbased medicines have been shown to exhibit pleiotropic effects beyond glycemic control, which include the effects on feeding, weight, lipid metabolism, cardiovascular functions and cognitive functions. central effects of glp1 contribute to these extrapancreatic effects, regulating systemic homeostasis in physiological states, and providing beneficial responses to pathological states. this review has shown that gutderived glp1 and glp1 receptor agonist send their information to the brain through neural and humoral pathways. however, how their information is relayed to efferent nervous systems to control peripheral tissues and organs remains to be clarified. further investigations in basic and clinical studies will provide new insights about central actions and the underlying mechanisms of incretinbased medicines. | abstractglucagonlike peptide1 (glp1) is derived from both the enteroendocrine l cells and preproglucagonexpressing neurons in the nucleus tractus solitarius (nts) of the brain stem. as glp1 is cleaved by dipeptidyl peptidase4 yielding a halflife of less than 2 min, it is plausible that the gutderived glp1, released postprandially, exerts its effects on the brain mainly by interacting with vagal afferent neurons located at the intestinal or hepatic portal area. glp1 neurons in the nts widely project in the central nervous system and act as a neurotransmitter. one of the physiological roles of brainderived glp1 is restriction of feeding. glp1 receptor agonists have recently been used to treat type 2 diabetic patients, and have been shown to exhibit pleiotropic effects beyond incretin action, which involve brain functions. glp1 receptor agonist administered in the periphery is stable because of its resistance to dipeptidyl peptidase4, and is highly likely to act on the brain by passing through the blood brain barrier (bbb), as well as interacting with vagal afferent nerves. central actions of glp1 have various roles including regulation of feeding, weight, glucose and lipid metabolism, cardiovascular functions, cognitive functions, and stress and emotional responses. in the present review, we focus on the source of glp1 and the pathway by which peripheral glp1 informs the brain, and then discuss recent findings on the central effects of glp1 and glp1 receptor agonists. |
we review recent findings pertaining to several environmental agents (ethanol, phencyclidine, ketamine, nitrous oxide, barbiturates, benzodiazepines, halothane, isoflurane, and propofol) that have the potential to delete large numbers of neurons from the developing brain by a newly discovered mechanism involving interference in the action of neurotransmitters [glutamate and gamma - amino butyric acid (gaba) at (italic)n(/italic)-methyl - d - aspartate (nmda) ] and gaba(subscript)a(/subscript) receptors during the synaptogenesis period, also known as the brain growth - spurt period. transient interference (lasting > = 4 hr) in the activity of these transmitters during the synaptogenesis period (the last trimester of pregnancy and the first several years after birth in humans) causes millions of developing neurons to commit suicide (die by apoptosis). many of these agents are drugs of abuse (ethanol is a prime example) to which the human fetal brain may be exposed during the third trimester by drug - abusing mothers. ethanol triggers massive apoptotic neurodegeneration in the developing brain by interfering with both the nmda and gaba(subscript)a(/subscript) receptor systems, and this can explain the reduced brain mass and lifelong neurobehavioral disturbances associated with intrauterine exposure of the human fetus to ethanol (fetal alcohol syndrome). exposure of the immature brain in a medical treatment context is also of concern because many of these agents are drugs used frequently as sedatives, tranquilizers, anticonvulsants, or anesthetics in pediatric and/or obstetrical medicine. because this is a newly discovered mechanism, further research will be required to fully ascertain the nature and degree of risk posed by exposure of the developing human brain to environmental agents that act by this mechanism.imagesfigure 2figure 3 |
|
diaryl thioethers, and particularly those containing heterocyclic moieties, are a common structural motif in natural products and medicinal agents (figure 1). for example, thioethers have therapeutic potential for treatment of hiv, breast cancer, inflammatory diseases, diabetes, and alzheimer s disease. despite their abundance, until recently few general methods were available for their synthesis under mild conditions that would tolerate sensitive heterocycles. in this manuscript, we report synthesis of diaryl sulfides by in situ formation of highly reactive sulfenyl chlorides and subsequent trapping with arylzinc reagents. a series of heterocyclic diaryl thioethers were designed and prepared as combretastatin a-4 analogues ; two of these compounds demonstrated micromolar activity against the mcf-7 breast cancer cell line. representative bioactive diaryl sulfides. in the past two decades, there have been numerous advances in the synthesis of this class of compounds, and particularly in the field of metal - catalyzed carbon sulfur bond formation (figure 2a). copper- and palladium - catalyzed cross - coupling reactions offer a broad scope of reactivity ; however, these methods often require elevated temperatures. mechanism - based reaction design has been employed to accelerate transformations such that they occur under milder reaction conditions. for example, the fu and peters laboratories disclosed a copper - catalyzed, photoinduced synthesis of diaryl sulfides that proceeds at 0 c. recent reports detail reactions of disulfides with aryl iodides, boronic acids, or silanes in the presence of stoichiometric reducing agents (figure 2b). schlosser and co - workers demonstrated reactions of indoles with sulfenyl chlorides prepared in situ. the high reactivity of sulfenyl chlorides allows the reaction to occur at 0 c. recently, expansion of the scope of this reaction to include grignard reagents was reported by lee and co - workers (figure 2c). contemporaneously, on the basis of our work with n - chloroamines, we developed sulfenylation of organozinc reagents as a functional - group tolerant strategy for synthesis of diaryl sulfides. a variety of in situ - formed alkyl and aryl sulfenyl chlorides react with phenylzinc bromide to afford the respective thioethers in good yields (scheme 1). substrates containing ortho - disubstituted aryl rings pose a significant challenge for most metal - catalyzed methods, yet our reaction conditions furnish 9 in 93% yield. electron - rich thiols such as 4-methoxy thiophenol are competent in the transformation, although the desired product 12 is afforded in slightly diminished yield due to competitive formation of diaryl disulfide. 4-nitrothiophenol proved to be a challenging substrate, as it is prone to decomposition under the reaction conditions, thus affording 13 in a modest yield. one benefit of using aryl zinc reagents in contrast to aryl grignard reagents is the increased functional group compatibility. for example, 1-(4-mercapto - phenyl)-ethanone reacted smoothly to provide desired diaryl sulfide 14 with no observed competitive addition to the ketone. isolated yields after silica gel chromatography. in an effort to ensure that our method is compatible with the sensitive heterocyclic moieties frequently found in bioactive compounds a broad range of heterocycles react with phenylzinc bromide to provide good to excellent yields of the corresponding sulfides, including benzothiazole, benzoxazole, pyrimidine, tetrazole, oxadiazole, and imidazole functional groups (1520). notably, when phenylmagnesium bromide was used instead of phenylzinc bromide, compounds 15, 16, and 20 were obtained in diminished yields (65, 57, and 59% respectively). furthermore, both electron - rich and electron - poor grignard reagents react smoothly to afford the desired thioether products in good yields (21 and 22, respectively). we sought to synthesize combretastatin a-4 analogues using our method since it tolerates a diverse range of heterocycles and would further sar studies of these compounds. diaryl sulfide analogues of combretastatin containing n - heterocyclic moieties have been reported to be active against mcf-7 breast cancer cell lines (e.g., 2). we examined reactions of a variety of heteroaryl sulfides with 3,4,5-trimethoxyphenylzinc bromide, biasing our small library of analogues toward inclusion of the 3,4,5-trimethoxyphenyl scaffold, a privileged motif commonly found in anticancer compounds that target microtubules. we were pleased to see that the corresponding arylzinc bromide reacts with a variety of in situ - formed heteroaryl sulfenyl chlorides to afford the respective trimethoxyphenyl - substituted thioethers in modest to good yields (2427). having synthesized a variety of combretastatin a-4 analogues, we set out to evaluate these compounds for anti - breast - cancer activity. select products from schemes 1 and 2 were tested for anticancer activity against the mcf-7 breast cancer cell line relative to the normal mcf-10a stromal cell line using a proliferation - based procedure (figure 3). results are compared to activity of the estrogen receptor antagonist, faslodex (ici 182,780). diaryl sulfide 25, containing benzoxazole and 3,4,5-trimethoxyphenyl moieties, was a potent inhibitor of mcf-7 cell proliferation (ec50 = 4.5 m). in comparison, in contrast, trimethoxyphenyl - containing thioether 27 performed poorly, while its phenyl analogue 19 was a more potent cell proliferation inhibitor (ec50 = 7.9 m). anti - breast - cancer activity of compounds at 10 m screened against breast cancer (mcf-7) and normal breast cell lines (mcf-10a). cell proliferation is represented as relative cell numbers after treatment, where a low percentage indicates potent anticancer activity for that compound. we have developed a mild and efficient protocol for the synthesis of alkyl and diaryl sulfides. this method tolerates a wide array of heterocyclic moieties and is amenable to the construction of highly functionalized diaryl and diheteroaryl sulfides. biological studies of select compounds have identified two promising inhibitors of mcf-7 breast cancer cell proliferation. future efforts will focus on using this methodology to create a larger library of functionalized heterocyclic sulfides and investigating their biological activity against a broad range of cancer cell lines. all reactions were carried out under an atmosphere of n2 using glassware that was either oven- or flame - dried prior to use. dichloromethane (ch2cl2) and tetrahydrofuran (thf) were degassed with argon and then passed through two 4 36 in. columns of anhydrous neutral a-2 alumina (8 14 mesh ; activated under a flow of argon at 350 c for 12 h) to remove h2o. h nmr spectra were recorded on 500 mhz (500 mhz h, 125.7 mhz c) or 400 mhz (400 mhz h, 100 mhz c) spectrometers. proton chemical shifts are reported in ppm () relative to internal tetramethylsilane (tms, 0.00). data are reported as follows : chemical shift (multiplicity [singlet (s), broad singlet (br s), doublet (d), doublet of doublets (dd), triplet (t), doublet of triplets (dt), quartet (q), multiplet (m), apparent singlet (ap s), and apparent doublet (ap d) ], coupling constants [hz ], integration. carbon chemical shifts are reported in ppm () relative to tms with the respective solvent resonance as the internal standard (cdcl3, 77.16 ppm). unless otherwise indicated, infrared spectra (thin film or neat) are reported in terms of frequency of absorption (cm). analytical thin - layer chromatography (tlc) was performed using silica gel 60 f254 precoated plates (0.25 mm thickness). visualization was accomplished by irradiation with a uv lamp and/or staining with kmno4 solution. flash chromatography was performed using silica gel 60 (170400 mesh) from fisher scientific. 4-(trifluoromethyl)phenylmagnesium bromide and 4-methoxyphenylmagnesium bromide were prepared from their respective halide precursors in thf. 3-cyanophenylmagnesium bromide was prepared by magnesium - halogen exchange with isopropylmagnesium bromide in the presence of licl. molarities of organomagnesium and organozinc reagents were determined by titration.n - chlorosuccinimide (ncs) was recrystallized from benzene and stored in an amber vial for up to two weeks. to a solution of ncs (0.073 g, 0.55 mmol) in dcm (1.0 ml) was added thiol (0.50 mmol), and the solution was stirred for 30 min in the absence of direct light. the solution was taken up using a teflon needle and added dropwise to a solution of arylzinc reagent in thf (1.25 mmol). upon completion, as judged by tlc, the reaction mixture was quenched with meoh and concentrated in vacuo, and the residue was adsorbed onto 3 ml of silica gel and purified by flash column chromatography. to a solution of ncs (0.073 g, 0.55 mmol) in dcm (1.0 ml) was added thiol (0.50 mmol), and the solution was stirred for 30 min in the absence of direct light. the solution was taken up using a teflon needle and added dropwise to a solution of arylmagnesium reagent in thf (1.25 mmol). upon completion, as judged by tlc, the reaction mixture was quenched with meoh and concentrated in vacuo, and the residue was adsorbed onto 3 ml of silica gel and purified by flash column chromatography. title compound was prepared according to general procedure a from octane thiol (0.087 ml, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (3% etoac in hexanes) afforded the title compound as a colorless oil (0.091 g, 82%). spectral data were consistent with reported values : tlc rf = 0.7 (10% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 7.337.25 (m, 4h), 7.15 (t, j = 7.0 hz, 1h), 2.91 (t, j = 7.3 hz, 2h), 1.681.61 (m, 2h), 1.42 (m, 2h), 1.27 (m, 8h), 0.88 (t, j = 6.8 hz, 3h) ; c nmr (125 mhz, cdcl3) 137.2, 128.9 (2c), 125.7, 33.7, 31.9, 29.31, 29.27 (2c), 29.0, 22.8, 14.2. title compound was prepared according to general procedure a from benzyl mercaptan (0.059 ml, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (3% etoac in hexanes) afforded the title compound as a colorless oil (0.087 g, 87%). spectral data were consistent with reported values : tlc rf = 0.5(10% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 7.317.18 (m, 9h), 7.16 (t, j = 7.2 hz, 1h), 4.10 (s, 2h) ; c nmr (125 mhz, cdcl3) 137.6, 136.5, 129.9, 128.95, 128.94, 128.6, 127.3, 126.4, 39.1. title compound was prepared according to general procedure a from thio-2-naphthol (0.080 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (3% etoac in hexanes) afforded the title compound as a colorless oil (0.110 g, 93%). spectral data were consistent with reported values : tlc rf = 0.6 (10% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 7.81 (s, 1h), 7.757.66 (m, 3h), 7.447.34 (m, 5h), 7.287.18 (m, 3h) ; c nmr (125 mhz, cdcl3) 136.0, 133.9, 133.1, 132.4, 131.0, 130.0, 129.3, 129.0, 128.8, 127.8, 127.5, 127.1, 126.7, 126.3. title compound was prepared according to general procedure a from 2,6-dimethylthiophenol (0.069 ml, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (3% etoac in hexanes) afforded the title compound as a colorless oil (0.100 g, 93%). spectral data were consistent with reported values : tlc rf = 0.5 (10% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 7.227.14 (m, 5h), 7.04 (t, j = 7.2 hz, 1h), 6.92 (d, j = 7.6 hz, 2h), 2.42 (s, 6h) ; c nmr (125 mhz, cdcl3) 144.0, 138.1, 130.6, 129.4, 129.0, 128.6, 125.8, 124.7, 22.0. title compound was prepared according to general procedure a from 4-chlorobenzenethiol (0.072 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (3% etoac in hexanes) afforded the title compound as a colorless oil (0.094 g, 85%). spectral data were consistent with reported values : tlc rf = 0.7 (10% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 7.357.29 (m, 4h), 7.287.22 (m, 5h) ; c nmr (100 mhz, cdcl3) 135.2, 134.8, 133.1, 132.1, 131.4, 129.5, 129.4, 127.6. title compound was prepared according to general procedure a from pentafluorothiophenol (0.067 ml, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (3% etoac in hexanes) afforded the title compound as a colorless crystalline solid (0.114 g, 84%). spectral data were consistent with reported values : tlc rf = 0.6 (5% etoac in hexanes) ; mp 4548 c ; h nmr (400 mhz, cdcl3) 7.35 (m, 2h), 7.327.24 (m, 3h) ; c nmr (125 mhz, cdcl3) 148.7 (m), 146.7 (m), 143.2 (m), 141.2 (m), 139.0 (m), 136.9 (m), 133.1, 130.7, 129.6, 128.1, 109.1 (m) ; f nmr (376 mhz, cdcl3) 131.9 (dd, j = 24.7 hz, 7.0 hz, 2f), 151.6 (t, j = 20.9 hz, 1f), 160.6 (td, j = 22.2 hz, 6.7 hz, 2f) ; ir (neat) 1482, 1093, 971 cm ; hrms (tof ms ci+) m / z calcd for c12h5f5s (m) 276.0032, found 276.0025. title compound was prepared according to general procedure a from 4-methoxybenzenethiol (0.062 ml, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (10% etoac in hexanes) afforded the title compound as a colorless oil (0.071 g, 66%). spectral data were consistent with reported values : tlc rf = 0.5 (10% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 7.40 (d, j = 8.5 hz, 2h), 7.227.11 (m, 5h), 6.87 (d, j = 8.6 hz, 2h), 3.77 (s, 3h) ; c nmr (125 mhz, cdcl3) 159.8, 138.7, 135.4, 129.0, 128.2, 125.8, 124.3, 115.0, 55.3. title compound was prepared according to general procedure a from 4-nitrobenzenethiol (0.082 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (10% etoac in hexanes) afforded the title compound as a colorless oil (0.042 g, 37%). spectral data were consistent with reported values : tlc rf = 0.4 (5% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 8.06 (dt, j = 9.6 hz, j = 2.2 hz, 2h), 7.53 (m, 2h), 7.46 (m, 3h), 7.18 (dt, j = 9.6 hz, j = 2.2 hz, 2h) ; c nmr (125 mhz, cdcl3) 148.6, 145.4, 134.8, 130.5, 130.1, 129.7, 126.7, 124.1. title compound was prepared according to general procedure a from 1-(4-sulfanylphenyl)ethan-1-one (60 l, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 2.6 ml). purification by flash column chromatography (5% etoac in hexanes) afforded the title compound as a pale yellow solid (0.072 g, 63%). spectral data were consistent with reported values : tlc rf = 0.3 (5% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 7.81 (d, j = 8.5 hz, 2h), 7.507.47 (m, 2h), 7.407.38 (m, 3h), 7.20 (d, j = 8.5 hz, 2h), 2.54 (s, 3h) ; c nmr (125 mhz, cdcl3) 197.3, 145.0, 134.5, 134.0, 132.1, 129.8, 129.0, 128.9, 127.5, 26.5 ; ir (neat) 2922, 1677, 1589, 690 cm. title compound was prepared according to general procedure a from 2-mercaptobenzothiazole (0.084 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (15% etoac in hexanes) afforded the title compound as a colorless oil (0.087 g, 71%). compound 15 was also prepared from phmgbr according to general procedure b to afford 65% yield (determined by h nmr in comparison to the internal standard phenyltrimethylsilane). spectral data were consistent with reported values : tlc rf = 0.5 (30% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 7.87 (d, j = 8.4 hz, 1h), 7.72 (m, 2h), 6.63 (d, j = 8.0 hz, 1h), 7.527.43 (m, 3h), 7.38 (m, 1h), 7.25 (m, 1h) ; c nmr (125 mhz, cdcl3) 169.8, 154.0, 135.6, 135.4, 130.6, 130.01, 129.98, 126.2, 124.4, 122.0, 120.9. title compound was prepared according to general procedure a from 2-mercaptobenzoxazole (0.076 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (15% etoac in hexanes) afforded the title compound as a colorless oil (0.093 g, 82%). compound 16 was also prepared from phmgbr according to general procedure b to afford 57% yield (determined by h nmr in comparison to the internal standard phenyltrimethylsilane). spectral data were consistent with reported values : tlc rf = 0.5 (30% etoac in hexanes) ; h nmr (500 mhz, cdcl3) 7.70 (m, 2h), 7.59 (m, 1h), 7.477.42 (m, 3h), 7.39 (m, 1h), 7.277.21 (m, 2h) ; c nmr (100 mhz, cdcl3) 163.4, 152.0, 142.1, 134.5, 130.0, 129.8, 127.3, 124.5, 124.4, 119.2, 110.2. title compound was prepared according to general procedure a from 2-mercaptopyrimidine (0.056 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (20% etoac in hexanes) afforded the title compound as a colorless oil (0.081 g, 86%). spectral data were consistent with reported values : tlc rf = 0.3 (30% etoac in hexanes) ; h nmr (500 mhz, cdcl3) 8.47 (d, j = 5.0 hz, 2h), 7.63 (m, 2h), 7.44 (m, 3h), 6.95 (t, j = 5.0 hz, 1h) ; c nmr (125 mhz, cdcl3) 172.9, 157.7, 135.4, 129.45, 129.43, 129.3, 117.1. title compound was prepared according to general procedure a from 1-phenyl-1h - tetrazole-5-thiol (0.089 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (30% etoac in hexanes) afforded the title compound as a colorless crystalline solid (0.102 g, 80%) : tlc rf = 0.4 (30% etoac in hexanes) ; mp 129133 c ; h nmr (400 mhz, cdcl3) 7.577.53 (m, 7h), 7.427.36 (m, 3h) ; c nmr (125 mhz, cdcl3) 153.7, 134.0, 133.7, 130.5, 130.1, 129.86, 129.85, 126.9, 124.5 ; ir (neat) 3067, 2922, 1498, 1412, 1389, 1240 cm ; hrms (tof ms es+) m / z calcd for c13h10n4s (m + na) 277.0524, found 277.0524. title compound was prepared according to general procedure a from 5-phenyl-1,3,4-oxadiazaole-2-thiol (0.089 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (2050% etoac in hexanes) afforded the title compound as a white solid (0.089 g, 70%). spectral data were consistent with reported values : tlc rf = 0.5 (20% etoac in hexanes) ; h nmr (500 mhz, cdcl3) 7.94 (d, j = 7.0 hz, 2h), 7.67 (m, 2h), 7.517.39 (m, 6h) ; c nmr (125 mhz, cdcl3) 166.4, 162.9, 133.7, 131.9, 129.9, 129.8, 129.1, 127.1, 126.8, 123.5. title compound was prepared according to general procedure a from 2-mercapto-1-methylimidazole (0.057 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and phznbr (1.3 mmol, 1.7 ml). purification by flash column chromatography (10% etoac in hexanes) afforded the title compound as a colorless oil (0.081 g, 95%). compound 20 was also prepared from phmgbr according to general procedure b to afford 59% yield (determined by h nmr in comparison to the internal standard phenyltrimethylsilane). spectral data were consistent with reported values : tlc rf = 0.2 (30% etoac in hexanes) ; h nmr (500 mhz, cdcl3) 7.25 (m, 2h), 7.187.13 (m, 4h), 7.06 (d, j = 1.0 hz, 1h), 3.62 (s, 3h) ; c nmr (125 mhz, cdcl3) 138.1, 135.0, 130.2, 129.3, 128.0, 126.6, 123.9, 33.9. title compound was prepared according to general procedure b from 2-mercaptopyrimidine (0.056 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and (4-ome)phmgbr (1.3 mmol, 1.8 ml). purification by flash column chromatography (2030% etoac in hexanes) afforded the title compound as a white solid (0.082 g, 75%). spectral data were consistent with reported values : tlc rf = 0.4 (30% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 8.47 (d, j = 4.4 hz, 2h), 7.54 (d, j = 8.0 hz, 2h), 6.95 (m, 3h), 3.83 (s, 3h) ; c nmr (100 mhz, cdcl3) 173.5, 160.6, 157.6, 137.1, 120.0, 116.8, 114.9, 55.4. title compound was prepared according to general procedure b from 2-mercaptopyrimidine (0.056 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and (4-cf3)phmgbr (1.3 mmol, 2.1 ml). purification by flash column chromatography (1030% etoac in hexanes) afforded the title compound as a colorless oil (0.110 g, 86%) : tlc rf = 0.6 (30% etoac in hexanes) ; h nmr (400 mhz, cdcl3) 8.51 (d, j = 4.8 hz, 2h), 7.76 (d, j = 8.0 hz, 2h), 7.67 (d, j = 8.0 hz, 2h), 7.02 (t, j = 4.8 hz, 1h) ; c nmr (100 mhz, cdcl3) 171.7, 157.8, 135.1, 134.6 (ap d, j = 1.4 hz, 1c), 131.1 (q, j = 32.8 hz, 1c), 126.1 (q, j = 3.7 hz, 1c), 124.0 (q, j = 272.4 hz, 1c), 117.6 ; f nmr (376 mhz, cdcl3) 63.0 ; ir (thin film) 3039, 2927, 1566, 1389, 1329, 1170, 1122 cm ; hrms (tof ms ci+) m / z calcd for c11h7f3n2s (m + h) 257.0360, found 257.0353. title compound was prepared according to general procedure b from 2-mercaptopyrimidine (0.056 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and (3-cn)phmgbr (1.3 mmol, 2.3 ml). purification by flash column chromatography (1030% etoac in hexanes) afforded the title compound as a white solid (0.074 g, 69%) : tlc rf = 0.4 (30% etoac in hexanes) ; mp 7173 c ; h nmr (400 mhz, cdcl3) 8.51 (d, j = 4.4 hz, 2h), 7.95 (s, 1h), 7.86 (d, j = 8.0 hz, 1h), 7.71 (d, j = 7.6 hz, 1h), 7.55 (t, j = 7.6 hz, 1h), 7.06 (t, j = 4.2 hz, 1h) ; c nmr (100 mhz, cdcl3) 171.1, 157.7, 139.2, 138.3, 132.5, 131.7, 129.8, 118.1, 117.7, 113.4 ; ir (thin film) 3066, 2927, 2231, 1560, 1379, 1182 cm ; hrms (tof ms ci+) m / z calcd for c11h7n3s (m + h) 214.0439, found 214.0433. title compound was prepared according to general procedure a from 2-mercaptopyrimidine (0.056 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and 3,4,5-trimethoxyphenylzinc bromide (1.3 mmol, 2.8 ml). purification by flash column chromatography (2050% etoac in hexanes, 1% et3n) afforded the title compound as a white solid (0.059 g, 43%) : tlc rf = 0.1 (30% etoac in hexanes) ; mp 103104 c ; h nmr (400 mhz, cdcl3) 8.52 (d, j = 4.8 hz, 2h), 6.99 (t, j = 4.8 hz, 1h), 6.88 (s, 2h), 3.90 (s, 3h), 3.87 (s, 6h) ; c nmr (125 mhz, cdcl3) 173.1, 157.8, 153.6, 139.2, 123.8, 117.1, 112.4, 61.0, 56.3 ; ir (neat) 2945, 2851, 1547, 1375, 1117 cm ; hrms (tof ms es+) m / z calcd for c13h14n2o3s (m+na) 301.0623, found 301.0616. title compound was prepared according to general procedure a from 2-mercaptobenzoxazole (0.076 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and 3,4,5-trimethoxyphenylzinc bromide (1.3 mmol, 2.8 ml). purification by flash column chromatography (515% etoac in hexanes, 1% et3n) afforded the title compound as a white solid (0.117 g, 74%) : tlc rf = 0.4 (30% etoac in hexanes) ; mp 129130 c ; h nmr (400 mhz, cdcl3) 7.62 (dd, j = 7.6 hz, j = 5.6 hz, 1h), 7.44 (dd, j = 8.8 hz, j = 6.0 hz, 1h), 7.27 (m, 2h), 6.94 (s, 2h), 3.90 (s, 3h), 3.88 (s, 6h) ; c nmr (125 mhz, cdcl3) 163.6, 153.8, 152.0, 142.1, 139.8, 124.54, 124.45, 121.2, 119.2, 112.0, 110.2, 61.0, 56.4 ; ir (neat) 2931, 2837, 1489, 1451, 1406, 1232, 1129, 1121 cm ; hrms (tof ms es+) m / z calcd for c16h15no4s (m + na) 340.0620, found 340.0620. title compound was prepared according to general procedure a from 1-phenyl-1h - tetrazole-5-thiol (0.089 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and 3,4,5-trimethoxyphenylzinc bromide (1.3 mmol, 2.8 ml). purification by flash column chromatography (3050% etoac in hexanes, 1% et3n) afforded the title compound as a white solid (0.105 g, 61%) : tlc rf = 0.3 (30% etoac in hexanes) ; mp 110111 c ; h nmr (400 mhz, cdcl3) 7.57 (m, 5h), 6.78 (s, 2h), 3.86 (s, 3h), 3.82 (s, 6h) ; c nmr (125 mhz, cdcl3) 153.9, 153.8, 139.9, 133.8, 130.5, 129.9, 124.7, 120.7, 111.7, 61.0, 56.4 ; ir (neat) 3042, 2951, 2860, 1585, 1408, 1231, 1125 cm ; hrms (tof ms es+) m / z calcd for c16h16n4o3s (m + na) 367.0841, found 367.0836. title compound was prepared according to general procedure a from 5-phenyl-1,3,4-oxadiazaole-2-thiol (0.089 g, 0.50 mmol), ncs (0.073 g, 0.55 mmol) and 3,4,5-trimethoxyphenylzinc bromide (1.3 mmol, 2.8 ml). purification by flash column chromatography (525% etoac in hexanes, 1% et3n) afforded the title compound as a white solid (0.100 g, 58%) : tlc rf = 0.3 (30% etoac in hexanes) ; mp 142 c ; h nmr (400 mhz, cdcl3) 7.98 (d, j = 6.8 hz, 2h), 7.51 (m, 3h), 6.93 (s, 2h), 3.88 (s, 9h) ; c nmr (100 mhz, cdcl3) 166.5, 163.3, 153.9, 139.8, 132.0, 129.2, 126.9, 123.6, 121.0, 111.5, 61.1, 56.5 ; ir (neat) 3009, 2943, 2850, 1582, 1463, 1128 cm ; hrms (tof ms es+) m / z calcd for c17h16n2o4s (m + na) 367.0728, found 367.0722. biological experiments were performed according to a modified procedure by sigman. the following reagents were obtained from commercial sources as indicated : dulbecco s modified eagle s medium (dmem)/high glucose containing 4.5 g / l glucose and 4.0 mm l - glutamine (hyclone) ; fetal bovine serum (fbs), heat - inactivated (omega scientific) ; l - glutamine, 200 mm (gibco) ; penicillin / streptomycin solution 50 (mediatech) ; dmem / ham s nutrient mixture f12 containing 2.5 mm l - glutamine, 3151 mg / l dextrose, and 55 mg / l sodium pyruvate (sigma - aldrich) ; horse serum (sigma - aldrich) ; 50 m hydrocortisone solution (sigma - aldrich) ; human insulin solution (sigma - aldrich) ; cholera toxin (sigma - aldrich) ; human epidermal growth factor (egf), recombinant (sigma - aldrich) ; 0.25% trypsin - edta (gibco) ; nuclease - free sterile water (fisher scientific) ; molecular biology grade dmso (sigma - aldrich) ; ici 182,780 (faslodex) (tocris bioscience). mcf-7 cells were maintained in dmem / high glucose supplemented with 10% fbs, l - glutamine, and penicillin / streptomycin. experiments with mcf-7 cells were performed in dmem / high glucose supplemented with 2% fbs, l - glutamine, and penicillin / streptomycin. mcf-10a cells were maintained in standard medium according to a modified recipe by brugge. : dmem / f12 supplemented with 5% horse serum, 10 g / ml of human insulin, 0.5 g / ml of hydrocortisone, 10 ng / ml of egf, 100 ng / ml of cholera toxin, and penicillin / streptomycin. mcf-7 cells were centrifuged in 1 pbs for 20 min, and then the pellet was resuspended in dmem supplemented with 10% fbs and filtered through a 40 m nylon cell strainer (fisher scientific) to prevent clumping. the cells were seeded at 1500 cells per well in 96-well flat bottom plates suitable for fluorimetry, using 175 l per well of dmem supplemented with 10% fbs, and grown for 24 h in 5% co2 at 37 c. the compounds (including the faslodex positive control) were dissolved in molecular biology grade dmso to achieve a 3.5 mm stock solution and then sterile filtered through a 0.45 m pvdf syringe filter unit (fisher scientific). the 3.5 mm stock solutions were subsequently diluted to a final concentration of 10 m in dmem supplemented with 2% fbs. after 24 h of growth, the cells were treated by replacing the normal media with fresh media containing the individual compounds or vehicle control (day 0). the outer rows of wells were not used to eliminate the possibility of effects due to evaporation of media. the cells were incubated with compound for 48 h and then treated again by aspirating the media and adding fresh media containing the compounds and controls (day 2). after incubating a final 24 h, the 96-well plates were rinsed with 1 pbs, blotted dry, and then frozen at 78 c overnight (day 5). on day 6, cell proliferation was measured using the fluorescence - based cyquant cell proliferation assay kit (invitrogen). fluorimetry analysis was performed according to a modified procedure by mcgowan. cells were stained with 200 l / well of 1 cyquant gr dye in cell lysis buffer for 10 min in the dark at room temperature and quantified by fluorimetry at 535 nm with 485 nm excitation. the normalized values were plotted as an average standard deviation of 6 wells per compound. mcf-10a cells were centrifuged in 1 pbs for 20 min, and then the pellet was resuspended in dmem / f12 and filtered through a 40 m nylon cell strainer (fisher scientific) to prevent clumping. the cells were seeded at 9000 cells per well in 96-well flat bottom plates suitable for fluorimetry, using 175 l per well of dmem / f12, and grown for 24 h in 5% co2 at 37 c. the 3.5 mm stock solutions of compound in dmso were subsequently diluted to a final concentration of 10 m in dmem / f12. fluorimetry analysis was performed as specified above for mcf-7 cells, with the exception of staining mcf-10a cells with 200 l / well of 5 cyquant gr dye in cell lysis buffer for 10 min in the dark at room temperature before quantification by fluorimetry. the normalized values were plotted as an average standard deviation of 6 wells per compound. mcf-7 cells were centrifuged in 1 pbs for 20 min, and then the pellet was resuspended in dmem supplemented with 10% fbs and filtered through a 40 m nylon cell strainer (fisher scientific) to prevent clumping. the cells were seeded at 1500 cells per well in 96-well flat bottom plates suitable for fluorimetry, using 175 l per well of dmem supplemented with 10% fbs, and grown for 24 h in 5% co2 at 37 c. the compounds 19 and 25 were dissolved in molecular biology grade dmso to achieve a 42 mm stock and then sterile filtered through a 0.45 m pvdf syringe filter unit (fisher scientific). the 42 mm stock solutions in dmso were subsequently diluted to 120 m in dmem supplemented with 2% fbs and then serially diluted to achieve 10 different concentrations. additionally, the corresponding dmso vehicle controls for each concentration were serially diluted using the same medium. fluorimetry analysis was performed as specified above for the evaluation of compounds against mcf-7 cells. the fluorescence values were normalized to the dmso vehicle controls corresponding to each concentration. the normalized values were plotted as an average standard deviation of 4 wells per concentration, and these data were analyzed using the dose response nonlinear regression fitting function (log[inhibitor ] vs response with variable slope (four parameters)). | a mild protocol for the synthesis of diaryl and heteroaryl sulfides is described. in a one - pot procedure, thiols are converted to sulfenyl chlorides and reacted with arylzinc reagents. this method tolerates functional groups including aryl fluorides and chlorides, ketones, as well as n - heterocycles including pyrimidines, imidazoles, tetrazoles, and oxadiazoles. two compounds synthesized by this method exhibited selective activity against the mcf-7 breast cancer cell line in the micromolar range. |
for most common and intermediate incidence cancers a firm histological diagnosis is available prior to staging investigations. with cancer of the ovary a diagnosis is uncommon before major surgery and is presumed from a characteristic clinical presentation of elevated levels of the tumour marker ca-125 and a typical pattern of imaging showing a complex pelvic mass with or without evidence of peritoneal spread. unfortunately there is overlap between the imaging appearances of ovarian cancer, benign ovarian masses and metastasis to the ovary and peritoneum from other primary sites. further peritoneal carcinomatosis can be mimicked by curable diseases such as tuberculosis and even lymphoma. the size of the problem is illustrated by the radiology diagnostic oncology group (rdog) major multicentre diagnostic imaging study of women prior to ovarian cancer surgery [13 ]. these studies compared us, ct and mr imaging in 280 women, evaluating these modalities for cancer diagnosis and staging. in the study 189 only 114 of the 280 women had ovarian cancer and of these 27 were not primary ovarian cancer but other malignancies metastatic to the ovary, some of which demand entirely different cancer management. women with a history of malignancy were excluded so the risk of confusion between primary ovarian cancer and other cancers metastatic to the ovaries was less than in routine clinical practice where women with previously treated breast and gi tract cancer are encountered. the rdog studies also showed that the ct appearance of ovarian metastases may be indistinguishable from that of primary ovarian cancer. both primary ovarian cancer and metastases may produce the bilateral solid masses considered typical of krukenberg tumours. this was a judgement that could only be made using us or mr imaging and was not a significant feature for ct. conversely ct well illustrates the stomach, colon, appendix and pancreas and these should be inspected as potential primary cancer sites within the abdomen. thus, in this study of women with complex ovarian masses, the majority did not have primary ovarian cancer. although we are not told whether cytoreductive cancer surgery was carried out in these women or was more limited by findings at surgery, the data re - emphasise the challenge faced by physicians and radiologists in defining appropriate management strategies for women with ovarian masses on a case by case basis. two clear messages emerge from these and other recent data. a rational and evidenced based imaging pathway for women suspected to have ovarian cancer the first is that mr imaging is a powerful tool to investigate the woman with an indeterminate mass and no signs of peritoneal spread the second is that a firm histological diagnosis should be sought in all cases when there is uncertainty that the diagnosis of malignancy actually represents primary ovarian cancer. the ready availability of ct makes it the investigation of choice for planning surgery in women believed to have metastatic spread of ovarian cancer. in the presence of bulky metastatic tumour ct predicts when cytoreductive surgery is likely to be incomplete by defining sites of unresectable tumour (fig. bulky disease in the supracolic compartment around the spleen and stomach, within suprarenal lymph nodes, and affecting the subdiaphragmatic recesses and parenchyma of the liver is usually beyond the scope of surgery. ct provides the surgeon with the detail required to discuss surgical and other therapeutic options with the patient and her carers. a variety of schemes have been devised to judge the tumour extent at key sites. when radical cytoreductive surgery is not considered appropriate for women with bulky disease, with poor performance status or with a history of malignancy which can mimic primary ovarian cancer, ct can help further. image guided needle core biopsy is an effective, safe and well tolerated alternative to surgery (mini - laparotomy, laparoscopy) in providing a definitive histological diagnosis when cancer surgery is not considered appropriate. there is current interest in neoadjuvant chemotherapy followed by intervention (interval) debulking surgery (ids) for women with primary ovarian cancer unable to undergo radical surgery at initial diagnosis. with this therapeutic approach primary chemotherapy is administered in the hope that tumour, as monitored by serial ct examination, can be debulked and downstaged to allow subsequent surgery. a firm histological diagnosis is essential when there is concern that peritoneal carcinomatosis results from disease metastatic to the ovary from other primary sites. with a history of breast cancer image guided biopsy of peritoneal disease is mandatory as in our experience there is a greater likelihood of a new gynaecological primary or primary peritoneal carcinoma (ppc), which is treated in the same way, than of recurrent breast cancer. simple cytological assessment of ascitic fluid infrequently results in distinction of primary ovarian cancer from disease metastatic from other sites. fluid can be spun down to form a pellet from which a cell block can be processed but this lacks the cellular architecture of a core biopsy specimen. peritoneal core biopsy using ct (or us) guidance is a valuable and useful alternative to laparoscopy or exploratory surgery in several circumstances (table 1). thus in women with undiagnosed peritoneal carcinomatosis both ppc and secondary ovarian cancer require consideration. peritoneal biopsy should precede an exhaustive (and potentially hazardous and unpleasant) series of investigations of potential primary sites such as upper and lower bowel endoscopy. needle core biopsy findings can focus the search for the primary tumour when appropriate. in the majority of women undergoing image guided needle core biopsy standard haematoxylin and eosin (h&e) staining is diagnostic, and this can be compared with historical material in women with prior malignancy. in women with poorly differentiated tumours further special immunohistochemical stains may be required which identify specific tumour markers and other cellular proteins such as cytokeratins. criteria for image guided biopsy are : (a) the presence of omental, peritoneal or pelvic mass identified on a staging ct study allowing core biopsy ; (b) no bleeding diathesis with platelet count > 1010/l and inr (international normalised ratio) < 1.4 ; and (c) a decision made after multidisciplinary review that obtaining a definitive diagnosis by non - surgical means was required to plan further treatment. the image guided biopsy is performed as a separate procedure after multidisciplinary review of the diagnostic studies. with ct guidance the study is performed with only oral contrast preparation beginning with a limited number of localising sections planned from the prior staging study. an 18 g cutting needle using a spring loaded device is used (temno, bauer medical international sa, dominican republic). the number of needle passes made is judged by the supervising radiologist to provide the equivalent of two full needle cores of solid material for the pathologist. for us guidance a decision is made from review of the staging ct that the omental cake or other peritoneal mass is likely to be visible with us. it requires some experience to confidently identify omental cake as a structure distinct from bowel loops. aftercare includes bed rest for 6 h with measurement of blood pressure and pulse half hourly for 2 h and then hourly for 4 h after which the patient may eat and drink and become ambulant. us guidance is our preferred technique for bulky disease with ct reserved for more inaccessible sites or with small volume infiltrates. needle core biopsies are formalin fixed, embedded in paraffin wax and sections cut at 34 micrometer thickness. there can be significant overlap between the histological patterns of primary ovarian cancer and metastatic disease, especially with mucinous carcinoma. further immunohistochemical analysis of the biopsy material is performed as deemed necessary for confirmation of diagnosis using the labelled streptavidin - biotin peroxidase system. most commonly used are monoclonal antibodies to cea - m, cytokeratin 7 (ck7), ck20 and ca-125. in selected cases additional monoclonal antibodies are used at the discretion of the reporting pathologist in women with previous breast cancer for oestrogen receptor and progesterone receptor and to gross cystic disease protein-15 which is a marker commonly positive in but not specific for primary breast cancer. isotypes reacting with these antibodies are lineage specific and this characteristic is retained during malignant transformation and progression. the distinction of metastasis from colorectal cancer from ovarian endometrioid adenocarcinoma remains problematical. as well as antibodies to ck7 and ca-125 react with ovarian epithelia but rarely with colonic ; antibodies to ck20 and cea - m react conversely [1012 ]. only strong and widespread positive staining finally the technique is simple, safe and effective. in an audit of 149 biopsy procedures performed with us (69) and ct (80) guidance there were no significant complications. with a variety of operators including supervised trainee radiologists there was a site - specific primary tumour diagnosis in 93% of cases. in the remainder the pathologist was able to diagnose adenocarcinoma but no primary site with only three patients requiring a surgical biopsy. when used in the context of multidisciplinary team discussion image guided peritoneal core biopsy is a valuable tool in initial diagnosis and management of ovarian cancer, especially prior to neoadjuvant chemotherapy and in patients where there are considerations of cancer metastatic to the ovary from other sites. when integrated into the investigative pathway of women with suspected ovarian cancer it offers improvements in case selection for ovarian cancer surgery and chemotherapy and more effective service delivery. ct showing inoperable ovarian cancer in the left supracolic compartment involving the spleen and stomach. | when used in the context of multidisciplinary team discussion, image guided biopsy using ultrasound (us) or computed tomography (ct) guidance is of value in planning management of women with suspected ovarian cancer and peritoneal carcinomatosis (pc) of uncertain aetiology. it is essential in women believed to have ovarian cancer but with poor performance status or with advanced disease believed beyond the scope of primary cytoreductive surgery for whom staging surgical pathology will not be obtained. it provides a site - specific primary tumour diagnosis in 93% of cases and it should replace diagnostic laparoscopy or laparotomy for this purpose. it allows provision of primary (neoadjuvant) chemotherapy based on a firm histological diagnosis. it is mandatory in women with a history of cancer whose metastases may mimic ovarian cancer (e.g. breast, gi tract, melanoma). more women with prior breast cancer who re - present with peritoneal cancer will have a new gynaecological primary than recurrence of their original primary tumour ; the two options require radically different therapies. finally it is a valuable problem solving tool in situations of diagnostic uncertainty, e.g. unusual imaging patterns of disease such as pc with bilateral solid ovarian masses or non - enlarged ovaries and with an unusual tumour marker profiles suggesting primary tumours outwith the ovary. the technique is simple, safe and effective and can be combined with palliative drainage of ascites at the same procedure. |
epilepsy is the most common serious neurological condition in the world, with an estimated prevalence of 1% of the population. traditional epilepsy management includes pharmacological treatment, epilepsy surgery, and vagal nerve stimulation. despite these therapies, the ketogenic diet (kd), which has been in use since 1921, is a treatment option for many of these epilepsy patients. this combination of energy results in a sustained ketosis that somehow serves to abate seizures through an unknown mechanism. in the following years, with the use of anti - epileptic drugs such as phenytoinum natricum, ketogenic diet is not widely used in epilepsy treatment. while in the recent years, with the increase of drug resistance and adverse effect of anti - epileptic drugs, ketogenic diet was newly considered and promoted in epilepsy treatment. recently, a review suggests that in children, the ketogenic diet results in short to medium term benefits in seizure control, the effects of which are comparable to modern antiepileptic drugs. in this article, existing studies (retrospective studies and prospective studies) were collected and used to systematically evaluate therapeutic success of ketogenic diet at different duration. as the basis for our analysis, we performed a medline search of the literature from 2000 to december 2011. the search themes were combined as (ketogenic diet or ketosis or ketone) and (epilepsy seizures or focal seizures or seizures or epilepsy or refractory seizures or generalized seizures). two investigators independently reviewed titles and abstracts, and selected articles addressing ketogenic diet and epilepsy. disagreements were resolved by discussion and consensus. on a second sift, we selected original studies on ketogenic diet and epilepsy with the following inclusion criteria : addressing ketogenic diet and epilepsy.the study should provide sample size.the study should provide the percentage of seizure reduction with the corresponding ketogenic diet duration.kd type including johns hopkins protocol, modified atkins diet, classical ketogenic diet. addressing ketogenic diet and epilepsy. the study should provide the percentage of seizure reduction with the corresponding ketogenic diet duration. kd type including johns hopkins protocol, modified atkins diet, classical ketogenic diet. for the purpose of meta - analysis, therapeutic success was defined as 50% seizure reduction at follow - up. for each study included, the full text was retrieved and the following data were extracted : authors, year of publication, size, gender and mean (or range) of age of the sample, and ketogenic diet duration. for each included study, we extracted the percentage of seizure reduction with the corresponding ketogenic diet duration. the percentage of seizure free, > 90% decrease, > 50% decrease were always selected when available ; otherwise the number of seizure free, > 90% decrease, > 50% decrease were recorded. we pooled studies that present percentages or the subjects that could be defined as therapeutic success. given the high number of potential ketogenic diet duration, we restricted our analysis to those for which the therapeutic success was assessed by at least 3 studies. the statistical heterogeneity of the included studies was assessed by using the test and the i index. the latter examines the percentage of total variation across studies that are due to heterogeneity between studies rather than by randomness. a value of i greater than about 70% indicates a high level of heterogeneity. since the results showed no statistical heterogeneity, we estimated therapeutic success rate for ketogenic diet as a treatment option for epilepsy and its 95% confidence interval (95%) cis using generic inverse variance method. using generic inverse variance method, each study estimate of the success rate is given a weight that is equal to the inverse of the variance of the effect estimate. as the basis for our analysis, we performed a medline search of the literature from 2000 to december 2011. the search themes were combined as (ketogenic diet or ketosis or ketone) and (epilepsy seizures or focal seizures or seizures or epilepsy or refractory seizures or generalized seizures). two investigators independently reviewed titles and abstracts, and selected articles addressing ketogenic diet and epilepsy. disagreements were resolved by discussion and consensus. on a second sift, we selected original studies on ketogenic diet and epilepsy with the following inclusion criteria : addressing ketogenic diet and epilepsy.the study should provide sample size.the study should provide the percentage of seizure reduction with the corresponding ketogenic diet duration.kd type including johns hopkins protocol, modified atkins diet, classical ketogenic diet. addressing ketogenic diet and epilepsy. the study should provide the percentage of seizure reduction with the corresponding ketogenic diet duration. for the purpose of meta - analysis, therapeutic success was defined as 50% seizure reduction at follow - up. for each study included, the full text was retrieved and the following data were extracted : authors, year of publication, size, gender and mean (or range) of age of the sample, and ketogenic diet duration. for each included study, we extracted the percentage of seizure reduction with the corresponding ketogenic diet duration. the percentage of seizure free, > 90% decrease, > 50% decrease were always selected when available ; otherwise the number of seizure free, > 90% decrease, > 50% decrease were recorded. we pooled studies that present percentages or the subjects that could be defined as therapeutic success. given the high number of potential ketogenic diet duration, we restricted our analysis to those for which the therapeutic success was assessed by at least 3 studies. the statistical heterogeneity of the included studies was assessed by using the test and the i index. the latter examines the percentage of total variation across studies that are due to heterogeneity between studies rather than by randomness. a value of i greater than about 70% indicates a high level of heterogeneity. since the results showed no statistical heterogeneity, we estimated therapeutic success rate for ketogenic diet as a treatment option for epilepsy and its 95% confidence interval (95%) cis using generic inverse variance method. using generic inverse variance method, each study estimate of the success rate is given a weight that is equal to the inverse of the variance of the effect estimate. review of titles and abstracts resulted in the selection of 55 articles, among which 38 articles met the inclusion criteria. summary of 38 studies investigating ketogenic diet and epilepsy nr indicates not reported table 2 provides the mean and range of success, and weighted success rate and 95% ci of ketogenic diet. in retrospective studies, the weighted success rate of the patients who take the kd as a treatment option for epilepsy was 58.4% (95% confidence interval (95%ci)=48.7% 69.9%) at 3 months (n=336) ; 42.8% (95%ci = 36.3% 50.3%) at 6 months (n=492), and 30.1% (95%ci = 24.3% 37.2%) at 12 months (n=387) ; in prospective studies, weighted success rate was 53.9% (95%ci 45.5% 63.8%) at 3 months (n=474) ; 53.2% (95%ci = 44.0% 64.2%) at 6 months (n=321), and 55.0% (95%ci = 45.9% 65.9%) at 12 months (n=347). in addition, weighted success rate for 1 month and 12 months in prospective study was 51.6% (95% ci = 41.0% 65.1%) at 1 month (n=212), 58.5% (95% ci = 48.1% 71.2%) at 12 months (n=278). the therapeutic success of ketogenic diet with the corresponding ketogenic diet duration figs. 1 and 2 show the mean weighted therapeutic success rate of ketogenic diet in included retrospective and prospective study respectively. the mean weighted therapeutic success rate according to the duration of ketogenic diet in included retrospective study the mean weighted therapeutic success rate according to the duration of ketogenic diet in included prospective study the pearson 's correlation coefficient (rho) of therapeutic success rate and age in ketogenic diet treatment is shown in figs. 3 and 4. there was a negative correlation between therapeutic success rate and age at 3 months in retrospective study. the relationship of success rate of ketogenic diet and age at 3 month in included retrospective study the relationship of success rate of ketogenic diet and age at 3 month in included prospective study over the past 90 years since its introduction, the ketogenic diet has been shown to be extremely successful in existing studies. although many studies have investigated the therapeutic success of ketogenic diet, a comprehensive and quantitative summary at different diet duration has been lacking. in most instances, however, results were fairly consistent in the direction of therapeutic success of ketogenic diet, even though studies differed in the estimation of effect extent. in this meta - analysis, compliance of patients it is reported that the initial 3 months on the diet are typically considered a trial period. so the surveillance for the compliance should be carried out especially in the initial 3 months. vining reported that 71% of patients with 50% seizure at 3 months could stay on the diet until the kd duration reached one year. in this study, this method is applied in meta - analysis when the results show no statistical heterogeneity. each study estimate of the success rate is given a weight that is equal to the inverse of the variance of the effect estimate. using this method, we found that in retrospective study, the success rate for patients who achieved therapeutic success (> 50% reduction in seizure frequency) were 58.4% at 3 month, 42.8% at 6 month, and 30.1% at 12 month respectively ; and in prospective study, the success rates were 53.9% at 3 month, 53.2% at 6 month, and 55.0% at 12 month respectively. it is consistent with reported efficacy of ketogenic diet in controlling seizures that 56% had a > 50% reduction. when the ketogenic diet duration prolonged, the efficacy was found decreased. in addition, the influence of lost to follow up may be considered when in the designing period, especially for long observation period, and measures to control the loss of follow up may be carried out in retrospective studies. therefore, the reported rates were lower in retrospective studies than in prospective studies at 12 months. furthermore, we can not calculate the efficacy for ketogenic diet considering the anti - epilepsy drugs treatment when taking the ketogenic diet, so there is a need for further studies to explore the interaction effect. guidance on the duration of dietary treatment is limited, and one study looked into the effect of the kd after its discontinuation, observing reduced benefit following cessation of treatment. levy rg also point out the problem of poor persistence. the education level and care of the patient 's family, the knowledge of epilepsy treatment, and the complicated recipe for daily diet treatment as well as the side effect of ketogenic diet result in poor compliance. common side effects of the ketogenic diet are gastrointestinal complaints and unfavorable lipid profiles. but kessler reported that side effects are usually transient and the most common reason for discontinuation of treatment is lack of effectiveness. anyway, for the ketogenic diet to be clinically meaningful, persistence or compliance would be quiet important. and to improve therapeutic success of the ketogenic diet, some measures such as effectiveness evaluation, health education, supervision of compliance, awareness of epilepsy treatment, side effect minimizing, user - friendly recipe developing should be taken into consideration. the ketogenic diet has been used with infants as young as 3 months and adults as old as 58 years. although our analysis indicates the negative correlation between therapeutic success rate and age, this relationship was only found statistically significant at 3 month in retrospective study. there is a tendency for younger children to have higher success rate although this trend was not statistically significant. ketogenic diet is currently used mainly for children who continue to have seizures despite treatment with antiepileptic drugs. children 's stomach capacity is small and the liver glycogen storage quantity is limited, so the energy storage level of children is lower than that of adults. due to the higher energy storage level of adults and comparable stable blood glucose levels, the results on less than 20% seizure free after treating with ketogenic diet for epileptic patients may be related to the age factor. this meta - analysis provides formal statistical support for the efficacy of the ketogenic diet in the treatment of epileptic patients. there is a tendency for younger age patients to have higher success rate although this trend was not statistically significant. it shows the need for further research on interaction effect considering both ketogenic diet and anti - epilepsy drugs. | objectiveto systematically evaluate therapeutic success of the ketogenic diet (kd) as a treatment option for epilepsy.methodsusing medline and google scholar search, we searched for studies investigating the therapeutic success of ketogenic diet for epilepsy. we estimated therapeutic success rate for ketogenic diet as a treatment option for epilepsy and its 95% cis using generic inverse variance method.findingsa total of 38 studies met the inclusion criteria. in retrospective studies, the weighted success rate of the patients who take the kd as a treatment option for epilepsy was 58.4% (95% confidence interval (95%ci)=48.7% 69.9%) at 3 months (n=336) ; 42.8% (95%ci = 36.3% 50.3%) at 6 months (n=492), and 30.1% (95%ci = 24.3% 37.2%) at 12 months (n=387) ; in prospective studies, weighted success rate was 53.9% (95%ci 45.5% 63.8%) at 3 months (n=474) ; 53.2% (95%ci = 44.0% 64.2%) at 6 months (n=321), and 55.0% (95%ci = 45.9% 65.9%) at 12 months (n=347).conclusionthis meta - analysis provides formal statistical support for the efficacy of the ketogenic diet in the treatment of epileptic patients. |
a 45-year - old female patient was referred to the vitreoretina unit of our tertiary eye care hospital with a history of diminished vision in the left eye of one week duration following blunt trauma to the left eye. on examination, the vision in the left eye was perception of light with good projection in all quadrants. slit lamp biomicroscopy showed a clear cornea with few descemet folds, a deep anterior chamber, iridodonesis, and aphakia. the vitreous face was broken, and tobacco dusting was present in the anterior vitreous. indirect ophthalmoscopy showed presence of a dislocated crystalline lens in the posterior vitreous with intact capsule and early cataractous changes. there was a total retinal detachment with a large peripheral retinal tear just short of 90 degrees extending from 12 to 2:30 o clock. the patient underwent a routine 3 port 20 g pars plana vitrectomy with peribulbar anesthesia after being explained the guarded prognosis. the procedure was done using the accurus vitrectomy system and visualization through a biom coupled to visu microscope. lensectomy was performed using a phacofragmatome in 3d mode, and all visible nuclear and cortical material was emulsified and/or aspirated in the mid vitreous cavity. iridotomy was performed at 6o clock. at the end of fge intra - operatively, the surgeon noticed a fluffy piece of hydrated lens cortical material lying sub - retinal, approximately 1.5 mm in diameter, just within the inferotemporal arcade. removal of the sub - retinal cortex would have involved turning over the flap and searching for the cortex. the risk to benefit of successfully locating and removing the cortex, without causing surgical trauma, was weighed. the surgeon decided to go ahead with the remaining steps of surgery as planned without attempting to remove the cortex [fig. 1 ]. immediate post - op day 3 fundus photo showing sub - retinal lens cortex just within inferotemporal arcade the patient had an uneventful post - operative course with no undue inflammation or raised intraocular pressure. there was complete absorption of the lens cortex over a period of 3 months [fig. 2 ]. reflexes from oil over foveal area subsequently, silicone oil removal was performed, and the patient had a best corrected visual acuity of 20/40 with an attached retina [fig. rpe changes in the form of stippling were visible over the underlying rpe [fig. 20 months after surgery, the patient had a flat retina with best corrected visual acuity of 20/40 and presence of rpe changes at the site of the cortex. fundus photo silicone oil removal showing flat retina and presence of rpe stippling in affect area ffa early phase confirms presence of changes in underling rpe in affected area it is not common to encounter sub - retinal lens cortical material under the retina. the presence of a large retinal tear coupled with a posterior dislocated lens requiring fragmentation resulted in this unique condition. during phacofragmentation, it is not uncommon to have cortical and nuclear pieces of the lens to be dispersed in the vitreous cavity. in this clinical setting with a detached retina and almost giant retinal tear, use of perfluorocarbon heavy liquid after vitrectomy and prior to starting phacofragmentation could have minimized the chances of the same, as prevention is the only viable option. the mechanism of spontaneous absorption is similar to the removal of sub - retinal blood. this is usually associated with permanent changes in the underlying rpe, as was confirmed in this case on ffa. it is important to note that the decision to leave behind the cortex near or under the fovea would have resulted in poor visual recovery due to underlying rpe changes and attendant overlying photo receptor damage. while this presentation was unique and visual recovery good, the possibility of this complication should be borne in mind in such clinical situations and suitable intra - operative preventive measures taken. | we report a rare case of retained sub - retinal cortical material, which underwent spontaneous resorption. patient presented with a left eye traumatic retinal detachment with a large retinal tear and posteriorly dislocated cataractous lens. vitrectomy, lensectomy, silicone oil injection, and endolaser were performed. a good visual result was achieved. the report draws attention to this condition and highlights possible technique for minimizing risk of this complication in similar cases. |
polyanionic molecules were shown to be effective inhibitors of the human immunodeficiency virus type 1 (hiv-1), not long after the recognition of hiv-1 as the viral agent responsible for the acquired immune deficiency syndrome (aids). dextran sulfate and heparin were two of the first sulfated compounds identified as compounds with antiviral activities against hiv-1 [1, 2 ]. polyanionic molecules are generally considered to be entry inhibitors, with the activities of these compounds stemming from their abilities to inhibit virus adsorption to the surface of the target cell membrane. the main mechanism of action associated with this family of compounds involves electrostatic interactions with hiv-1 gp120. interactions between these negatively charged molecules and the positively charged v3 loop of gp120 interfere with receptor - mediated binding and inhibit host cell infection [46 ]. it was also established that their activities were dependent on the level of sulfation, with two sulfate groups per monosaccaride required to achieve maximal anti - hiv-1 activity. these studies demonstrated that the negative charges on these polymeric molecules play important roles in inhibition of hiv-1 infection. a number of polyanionic compounds and their formulations have been developed as inhibitors of hiv-1 binding and entry [7, 8 ]. among these are pro 2000 (naphthalene 2-sulfonate polymer), pss [poly(sodium 4-styrene sulfonate ], hpmct (hydroxypropyl methylcellulose trimellitate), and cap (cellulose acetate phthalate). these compounds have been evaluated in preclinical studies or have been advanced into clinical trials for their use in microbicides, which are products designed to reduce or eliminate the risk of hiv-1 sexual transmission [7, 12 ]. although they have distinct chemical formulae, these compounds have three common structural and chemical characteristics : (i) they are polymeric in nature (high molecular weight) ; (ii) they possess a level of hydrophobicity associated with incorporated ring structures ; (iii) they bear multiple anionic charges along the length of the molecule. these characteristics (particularly the anionic charges) presumably play roles in defining the biological activities of these molecules, specifically their capacities to inhibit infection by hiv-1 and other enveloped viruses and their effects on cellular viability. we hypothesized that a molecule with similar structural characteristics would have comparable activity against hiv-1. poly(styrene - alt - maleic acid) is an alternating copolymer (alt - psma) comprised of styrene and maleic acid at a 1 : 1 ratio. like pro 2000, pss, hpmct, and cap, alt - psma is characterized by the presence of multiple anionic charges and hydrophobic, aromatic rings (figure 1(a)). however, in alt - psma (120,000 average mw), the hydrophobic phenyl side group, contributed by the styrene unit, is directly attached to the hydrocarbon backbone. in addition, unlike the sulfated or sulfonated molecules pss (figure 1(b)), pro 2000, or carrageenan, alt - psma derives its anionic charge from two free carboxyl groups (pka 1.9 and 6.0) of maleic acid, which are also directly attached to the hydrocarbon backbone instead of the aromatic ring. the properties of alt - psma prompted an exploration of the antiviral potential of this polyanionic molecule. in vitro assays of compound cytotoxicity and antiviral activity demonstrated that alt - psma combines low cytotoxicity with considerable antiviral activity against r5 and x4 viruses of multiple subtypes. furthermore, in washout assays typically used to explore compound mechanism of action and examine compound residence time after removal from target cells, alt - psma had no impact on subsequent hiv-1 infection, whereas pss caused increased levels of infection. these experiments compare and contrast alt - psma with other polyanionic compounds and provide the foundation for the further development of alt - psma as an inhibitor of hiv-1 infection. 662631, lot 71k1378) and dextran sulfate (ds ; dextralip 50, catalog no. d8787, lot 71k1378) were purchased from sigma - aldrich (st. louis, mo). polystyrene sulfonate (pss, lot dfs-1951) was kindly provided by national starch and chemical company (chattanooga, tn). p4-r5 magi cells (nih aids research and reference reagent program # 3580) were maintained in dulbecco 's modified eagle 's media (dmem) supplemented with 10% fetal bovine serum (fbs), sodium bicarbonate (0.05%), antibiotics (penicillin, streptomycin, and kanamycin at 40 g / ml each), and puromycin (1 g / ml). tzm - bl cells (nih aids research and reference reagent program # 8129) were maintained in dulbecco 's modified eagle 's media (dmem) supplemented with 10% fetal bovine serum (fbs), sodium bicarbonate (0.05%), and antibiotics (penicillin, streptomycin, and kanamycin at 40 g / ml each). human peripheral blood mononuclear cells (pbmcs) were isolated from whole blood (biological specialty corp., colmar, pa) using ficoll - hypaque (amersham biosciences, piscataway, nj) density gradient centrifugation and were subsequently cultured in rpmi supplemented with 10% fbs, sodium bicarbonate (0.05%), antibiotics (penicillin and streptomycin at 90 g / ml each), pha - p (sigma - aldrich ; 5 g / ml), and il-2 (nih aids research and reference reagent program # 11697 ; 20 u / ml). after 48 h, the pbmcs were washed and incubated for an additional 24 h in the absence of pha - p prior to infection. pbmcs (4 10 cells stimulated as described above) were treated with polybrene (sigma - aldrich) at a concentration of 2 g / ml for 30 min. the cells were then pelleted and 1 ml of virus stock was used to resuspend the cells. cells and virus were incubated at 37c with 5% co2 for 2 h with gentle mixing every 30 min. after the incubation interval, the cells were again pelleted and subjected to a second incubation with virus (1 ml). following the second incubation, the cells were again washed and resuspended in pbmc media (10 ml) supplemented with 2 g / ml of polybrene (co - culture media), and the volume was equally divided into two t-75 flasks. the cultures were assessed for p24 content on days 3 through 7 postinfection, and cells were split whenever the p24 level exceeded 100 ng / ml. on day 7, the cell suspension was clarified by centrifugation twice, initially at 1400 rpm for 10 min at 4c, followed by centrifugation of the supernatant at 2400 rpm for 10 min at 4c. after clarification, the viral suspension was aliquoted and stored at 85c until use. the clinical isolates (all r5 coreceptor phenotypes obtained from the nih aids research and reference reagent program) included two strains of subtype a (knh1144, catalog no. 112460 ; knh1207, catalog 7686) [1820 ], and two subtype c strains (93mw965, catalog no. p4-r5 magi cells were cultured at a density of 1.2 10 cells / well in a 96-well plate 18 h prior to infection. cells were incubated for 2 h with hiv-1 laboratory strains bal or iiib (advanced biotechnologies inc., columbia, md) in the absence or presence of alt - psma or ds. after 2 h, cells were washed, cultured for an additional 46 h, and subsequently assayed for hiv-1 infection using the galacto - star -galactosidase reporter gene assay system for mammalian cells (applied biosystems, bedford, ma). in a variation of this experiment, cell - free virus was preincubated with alt - psma or ds for 10 min at 37c, followed by a 1 : 100 dilution of the virus : compound mixture in complete rpmi media and addition to p4-r5 magi cells. alt - psma was evaluated for its ability to inhibit infection by hiv-1 iiib - infected sup - t1 t lymphocytes. compounds were incubated with 1 10 hiv-1 iiib - infected supt1 cells for 10 min followed by a 1 : 10 dilution in rpmi media and incubation for 2 h with p4-r5 magi cells at a density of 8 10 cells / well in a 12-well plate. in a variation of this experiment, mitomycin - treated hiv-1 iiib - infected supt1 cells (0.2 mg / ml mitomycin c for 30 min) were incubated with compounds for 2 min followed by a 1 : 10 dilution in rpmi media and incubation for 2 h with p4-r5 magi cells. cells were then washed and incubated for 46 h and assessed for -galactosidase production as described above. p4-r5 magi cells were seeded at a density of 4 10 cells / well in a 96-well plate approximately 18 h prior to experiment. cells were then exposed to the indicated concentrations of alt - psma or ds for 10 min, 2 h, or 24 h. following the exposure period, cells were washed and assessed for viability using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (mtt) assay of viability [21, 22 ]. for pbmcs, cells were seeded at a density of 1 10 cells / well in a 96-well plate. cells were then exposed to the indicated concentrations of alt - psma, pss, or ds for 6 h (to mirror the duration of compound exposure in the antiviral assay). following the exposure period, cells were washed and assessed for viability using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium (mts) assay of viability [23, 24 ]. cc50 values (concentration at which exposure to the compound resulted in a 50% decrease in cell viability relative to mock - treated cells) were subsequently calculated from the results. human pbmcs, stimulated as described above, were seeded at a density of 1 10 cells / well in a 96-well plate. cells were then incubated for 6 h with hiv-1 bal or iiib in the absence or presence of alt - psma, pss, or ds. after 6 h, cells were washed and subsequently cultured for 3 days, at which time the cells were washed and supplied with new media supplemented with il-2. the cells were then incubated for an additional 3 days and subsequently assayed for hiv-1 production by determining the level of p24 core antigen in the supernatant using an hiv-1 p24 antigen elisa assay (zeptometrix, buffalo, ny). tzm - bl cells were cultured at a density of 1.2 10 cells / well in a 96 well plate 18 h prior to infection. cells were incubated for 2 h with hiv-1 clinical isolates in the absence or presence of alt - psma or pss. after 2 h, cells were washed, cultured for an additional 46 h, and subsequently assayed for hiv-1 infection using the galacto - star -galactosidase reporter gene assay system for mammalian cells (applied biosystems, bedford, ma). ic90 concentrations for inhibition of cell - free hiv-1 infection by alt - psma, pss, or ds were determined using procedures as described above, except that the infection duration was shortened to 1 h. in the washout assay, p4-r5 magi cells were incubated with ic90 concentrations of compound for 1 h. cells were then washed extensively, provided with new media without compound, and then infected at 2 h or 4 h after compound removal with hiv-1 bal or iiib for 1 h at 37c. as a control, compound, virus, and cells were incubated together for 1 h. following infection, cells were washed again, provided with media without compound, and assayed 46 h postinfection as described above. data for all experiments are shown as mean values and calculated standard deviations from two independent assays in which each concentration was examined in triplicate. calculations of ic50/ic90 concentrations (concentrations that resulted in 50% or 90% decreases in infection relative to mock - treated, hiv-1-infected cells) and cc50 concentrations (concentrations that resulted in a 50% reduction in cell viability relative to mock - exposed cells) were calculated using the forecast function of microsoft excel. in the context of microbicide development, the importance of inhibiting infection by r5 viruses is underscored by the prevalence of r5 variants early in infection and the presumed preference for r5 viruses during the process of hiv-1 transmission [8, 25 ]. experiments were conducted to determine the ability of alt - psma to inhibit infection by hiv-1 bal, which has an r5 phenotype (figure 2(a)). alt - psma was an effective inhibitor of infection, achieving complete inhibition at 0.1 mg / ml and above and an ic50 value of 0.009 mg / ml during 2 h incubation with virus and p4-r5 magi cells. similar experiments, in which cell - free hiv-1 bal and compounds were incubated for 10 min prior to introduction to the target p4-r5 magi cells, were conducted to determine the direct effect of each compound on virus infectivity. a 10-min preincubation of cell - free virus with alt - psma followed by 1 : 100 dilution of the virus : compound mixture prior to addition to cells had no impact on the ability of alt - psma to inhibit infection by hiv-1 bal. these and subsequent experiments also included the control compound ds, which was used as a prototypical polyanionic compound with high antiviral activity and low cytotoxicity. when compared to ds (ic50 value of 0.03 mg / ml), alt - psma appeared to be modestly more effective against hiv-1 bal. preincubation of ds with cell - free virus had no impact on its antiviral activity. similar experiments were performed using the x4 hiv-1 strain iiib (figure 2(b)). relative to its activity against hiv-1 bal, alt - psma had slightly higher inhibitory activity against hiv-1 iiib (ic50 value of 0.005 mg / ml). similar differences in coreceptor usage - dependent activity have been demonstrated using other polyanions, including pss and hpmct. interestingly, a 10-min preincubation of alt - psma with cell - free hiv-1 iiib resulted in an approximately 6-fold increase in antiviral activity (ic50 = 0.0008 mg / ml) relative to its activity in the absence of preincubation (ic50 = 0.005 mg / ml). in contrast, the antiviral activity of ds (ic50 = 0.0006 mg / ml) was unaffected by preincubation (ic50 = 0.0005 mg / ml). since inhibitors and potential microbicide agents must also be effective against cell - associated infectivity, experiments were conducted to demonstrate the ability of alt - psma to inhibit infection by hiv-1-infected cells (figure 3). infection of p4-r5 magi target cells by hiv-1 iiib - infected sup - t1 t lymphocytes (figure 3(a)) was inhibited in a dose - dependent manner by alt - psma (ic50 = 0.03 mg / ml). inhibition by ds was comparable (ic50 = 0.01 mg / ml). additionally, to look solely at the ability of alt - psma to inhibit viral transfer from infected cells, mitomycin c - treated hiv-1 iiib - infected supt1 cells were used as the source of infectivity. alt - psma again inhibited infection in a dose - dependent manner (ic50 = 0.12 mg / ml) comparable to that of ds (figure 3(b)). in vitro cytotoxicity experiments were performed to determine the effect of alt - psma on cellular viability, to evaluate the potential safety of alt - psma, and to demonstrate that compound cytotoxicity had no impact on the results of the antiviral assays. exposures to alt - psma for 10 min or 2 h had no effect on p4-r5 magi cell viability at concentrations at or below 3.16 mg / ml (figure 4(a)). after a 24-h exposure to alt - psma, p4-r5 magi cell viability was reduced only at the highest concentration (62% viability at 3.16 mg / ml). similarly, cell viability was reduced by exposure to ds only after a 24-h exposure at the highest concentration examined (figure 4(b)). these results indicate that (i) the demonstrated antiviral efficacy of alt - psma was not affected by changes in target cell viability, since alt - psma exposure was not cytotoxic at the concentrations and exposure durations used in the antiviral experiments ; (ii) alt - psma may have a considerable margin of safety, with an in vitro therapeutic index (cc50/ic50) in excess of 350. in vitro experiments were also used to demonstrate the ability of alt - psma to inhibit hiv-1 infection of primary human immune cell populations (figure 5). secondarily, these experiments provided a comparison between alt - psma and pss, which are similar in that both compounds have repeating anionic charges and aromatic rings along their backbones. in experiments in which pbmcs were infected by hiv-1 bal (figure 5(a)), inhibition of infection by alt - psma (ic50 = 0.02 mg / ml) was concentration - dependent and comparable to levels of inhibition achieved in the presence of pss (ic50 = 0.04 mg / ml) or ds (ic50 = 0.04 mg / ml). similar experiments using hiv-1 iiib (figure 5(b)) demonstrated that alt - psma (ic50 = 0.003 mg / ml), pss (ic50 = 0.002 mg / ml), and ds (ic50 = 0.005 mg / ml) again had similar capacities for inhibiting hiv-1 infection. again, all three compounds were approximately one log more effective against hiv-1 iiib compared to their activities against bal. in assays of in vitro cytotoxicity using pbmcs, all three compounds were noncytotoxic (figure 5(c)) at concentrations shown to inhibit hiv-1 infection, with cc50 values of 9.1 mg / ml for alt - psma and in excess of 10 mg / ml for pss and ds. these results indicated that alt - psma and pss, which have similar structural features (anionic charge, aromatic rings), have similar capacities for inhibiting hiv-1 infection. furthermore, these results demonstrated, at least in the context of this experimental design, that the identity (carboxylic or sulfonic acid) or position (backbone or aromatic ring) of the anionic charge had no apparent effect on antiviral activity and a negligible effect on in vitro cytotoxicity. to be effective in the context of the global hiv / aids epidemic, inhibitors of hiv-1 infection must have antiviral activity against the range of regional hiv-1 subtypes found throughout the world. to this end, alt - psma was evaluated for its activities against clinical isolates from subtypes a, b, and c. these isolates included two subtype a strains (knh1144 and knh1207), one subtype b virus (us1), and two strains from subtype c (93mw965 and sm145). in antiviral assays using tzm - bl indicator cells, alt - psma was an effective inhibitor of all viral subtypes examined, with ic50 concentrations ranging from 0.003 to 0.009 mg / ml (table 1). furthermore, alt - psma appeared to be more effective against the subtype a and b isolates compared to pss ; pss ic50 concentrations were approximately 4- to 7-fold higher against these three isolates relative to the ic50 concentrations of alt - psma. a washout assay, in which antiviral compounds are removed from cells or tissues prior to hiv-1 infection, can be used to explore compound mechanism of action or, in the case of an agent that acts on or in the host cell, examine compound residence time after removal from the culture medium. preparative assays used to establish ic50 and ic90 concentrations for alt - psma, pss, and ds during a 1-h infection demonstrated that all three compounds were effective hiv-1 inhibitors, with ic50 values against bal of 0.009 mg / ml, 0.04 mg / ml, and 0.0058 mg / ml and ic50 values against iiib of 0.003 mg / ml, 0.01 mg / ml, and 0.00052 mg / ml for alt - psma, pss, and ds, respectively. in the washout assays using ic90 concentrations of each compound, co - incubation of compound, cells, and hiv-1 bal (figure 6(a)) or iiib (figure 6(b)) produced the expected results : approximately 90% inhibition of each virus was achieved. in contrast, removal of alt - psma and subsequent infection by either virus at 2 h or 4 h after compound removal had no effect on hiv-1 infection (compared to cells infected in the absence of compound). since polyanionic compounds have been shown to inhibit hiv-1 infection by interactions with gp120 [5, 6, 2833 ], abrogation of the inhibitory effects of psma and ds by compound washout was anticipated, since the compounds were not available to bind to gp120 and thus inhibit viral binding and entry. unexpectedly, pss washout caused increases in hiv-1 infection dependent on time of infection and coreceptor usage by the infecting virus. bal infection after pss washout was increased by approximately 39% over controls at the 2-h time point but unaffected by 4-h postwashout. in contrast, hiv-1 iiib infection was persistently enhanced at both 2 h (55% increase) and 4 h (67% increase) after pss removal. these results suggest functional interactions between pss and host cells that serve to enhance hiv-1 infection and/or replication. in addition, these experiments provide a clear functional distinction between alt - psma and pss despite their structural similarities. these in vitro studies confirmed the opening hypothesis, which stated that alt - psma would be an effective inhibitor of hiv-1 infection as a consequence of its gross structural similarities to the anti - hiv-1 compound pss. alt - psma was shown to have equipotent activities against laboratory - adapted r5 and x4 hiv-1 strains, activity against cell - free and cell - associated virus, demonstrable antiviral activity in the context of hiv-1 infection of primary human immune cells, and activity against a range of hiv-1 subtypes. the activity of alt - psma against hiv-1, which we first reported at the 2005 international meeting of the institute of human virology, was recently confirmed in structure - activity studies of poly(styrene - alt - maleic anhydride) derivatives. presumably, alt - psma inhibits hiv-1 attachment and entry in a manner comparable to pss. the specific mechanism of action of alt - psma, like polyanionic dextran sulfate, likely involves electrostatic interactions with the v3 loop and coreceptor binding site. demonstrated differences, however, between alt - psma and other polyanionic compounds, particularly in the compound washout experiments, suggested that subtle structural differences between these compounds may be functionally important under specific conditions. despite general similarities between members of the polyanionic compound family, alt - psma is distinguished from pss and other polyanionic molecules by three charge - related characteristics. first, the anionic charges of alt - psma are provided by carboxylic groups instead of the sulfonate side groups found on pss (charge identity). second, the negatively charged carboxylic groups of alt - psma are located on the backbone and are separated from the hydrophobic phenyl side chains of the styrene moiety (charge location). in contrast, the negatively charged sulfonate group of pss is itself a side group of the styrene repeat unit (figure 1). similarly, the sulfonate group of pro 2000, which provides its anionic charge, is a side chain of the naphthalene ring that is part of the polymeric structure. third, alt - psma includes two anionic charges per repeat instead of the single anionic side group found in each repeat of pss (charge density). as previously shown, differences in the number of charges per repeat unit, which affect the linear charge density of the molecule, can impact antiviral activity. these distinctions in charge identity, location, and density may be important factors in differentiating alt - psma from other polyanionic molecules. the present studies provide the starting point for future structure - function experiments that will examine the effects of charge identity, placement, and density on compound activity and will provide new insights into the mechanisms that underlie the efficacy and safety of this family of compounds. results of these investigations also indicated that variations in antiviral efficacy were evident not only as a consequence of differences in inhibitors, but also because of differences between infecting viruses and between target cell types. for example, the differential effect of virus - compound preincubation (figures 2(a) and 2(b)) on antiviral activity indicated both compound- and virus - specific differences in inhibitor retention on cell - free virions. specifically, enhanced antiviral activity against hiv-1 iiib but not bal following alt - psma preincubation with virus may be attributed to the more basic nature of x4 gp120 molecules, which would theoretically promote increased compound retention on the virus surface. furthermore, because of fundamental differences in backbone structure between alt - psma and ds, this mechanism of electrostatic retention of ds may not be possible. in addition, assays using pbmcs as target cells (figure 5) demonstrated that alt - psma, pss, and ds were more effective inhibitors of hiv-1 iiib (x4) relative to their activities against the r5 strain bal. similar differences in antiviral activity were previously reported in assays involving the polyanions pss and hpmct. differences in antiviral activity related to coreceptor usage could be attributed to the more basic nature of the x4 gp120 v3 region, which may promote a greater electrostatic affinity for anionic inhibitors and, thus, greater antiviral efficacy. however, this effect may be offset by other factors (such as host cell type), since alt - psma had equipotent activity against bal and iiib in experiments using p4-r5 magi cells as hosts for infection. in addition, differences in efficacy against viruses that differ in subtype but not in coreceptor usage (table 1) suggested that other genotypic or phenotypic differences between viruses affect antiviral efficacy. the unexpected enhancement of hiv-1 infection by pss suggested an activity that involves perturbation of the host cell, since, in this type of assay, only the target cells are exposed to significant concentrations of the compound. this activity appeared to compound - specific, since neither alt - psma nor ds produced this effect. in the context of the experimental design, first, low levels of pss may be retained on the cell surface despite extensive cell washing. this explanation appears to be consistent with a previous report of enhanced hiv-1 infection at low concentrations of the polyanionic compound cellulose sulfate. at low cellulose sulfate concentrations that are insufficient to provide antiviral activity, mechanisms that enhance hiv-1 infection the removal of pss from the media and the retention of small amounts of pss on the cell surface may have caused enhanced hiv-1 infection. however, the mechanisms at work in these situations may be similar but not identical, since the magnitude of enhancement in the washout assays was considerably higher (figure 6 and pirrone and krebs, manuscript in preparation) than increases in infection observed at low compound concentrations. in either case, low concentrations of compound on the cell surface may effect changes on the cell surface that facilitate the binding and entry steps of the hiv-1 replication cycle. alternatively, enhanced hiv-1 infection may be the result of intracellular changes initiated by interactions between pss and the cell surface. for example, incubation with pss may activate signaling pathways that affect hiv-1 gene expression. cellular activation mediated by transient exposure to pss would persist for several hours and would not require the continued presence of pss. interestingly, there is precedence for this type of activity among polyanionic hiv-1 inhibitors. previous studies have shown that activation of macrophages by -carrageenan, which is used to induce nonspecific inflammation in murine models of footpad edema and pleurisy, was dependent on toll - like receptor 4 (tlr4) and the downstream factor myd88. cellular signaling pathways, such as those activated by tlr stimulation, may stimulate hiv-1 gene expression through nf-b - mediated activation of the hiv-1 long terminal repeat (ltr). enhancement of hiv-1 infection by select polyanionic compounds may be relevant to microbicide clinical trials, particularly to the failure of ushercell (cellulose sulfate) in recent trials [39, 40 ]. for example, in clinical trials involving ushercell, compound leakage was reported by female users, and product leakage had a negative effect on product acceptability [41, 42 ]. in a scenario in which product leakage has sufficiently reduced the concentration of the active compound (i.e., cellulose sulfate) within the cervicovaginal environment, hiv-1 transmission may be enhanced rather than inhibited. this effect could explain the increased risk of hiv-1 infection associated with ushercell use in one of two clinical trials [39, 40 ]. to date, the clinical failures of cellulose sulfate, carrageenan, and pro 2000, despite clear demonstrations of in vitro efficacy and in vivo safety, have not been satisfactorily explained. two reports that presented possible explanations for the failures of two of these compounds [36, 43 ] were published after their respective clinical trials had been initiated. in light of the results of washout experiments described above we demonstrated using cell lines as well as primary human immune cells that other polyanionic molecules, including cellulose sulfate and carrageenan, also enhanced hiv-1 infection if the compounds were removed prior to the introduction of virus (pirrone and krebs, manuscript in preparation). in these experiments, demonstrated levels of enhancement in primary immune cells equaled or exceeded levels of enhancement achieved in the hela - based p4-r5 magi indicator cell line. we also demonstrated that enhancement of hiv-1 infection was dependent on a number of variables, including compound identity and concentration, time of application with respect to hiv-1 infection, and coreceptor usage. in light of the clinical failures of polyanionic molecules such as dextran sulfate, cellulose sulfate [40, 46 ], carrageenan, and pro 2000 [48, 49 ], a shift away from any further investigations focused on polyanionic hiv-1 inhibitors as drug or microbicide candidates might be perceived as an appropriate course of action. studies of existing polyanion hiv-1 inhibitors may contribute to a better understanding of the clinical failures of these compounds and provide much - needed links between preclinical investigations and clinical trials. in a similar manner, studies of new and effective polyanionic inhibitors, such as alt - psma, may further our understanding of these agents. finally, continued studies may provide useful information about molecular structures and charge characteristics that should be incorporated into future generations of hiv-1 inhibitors destined for systemic or microbicide use. | an alternating copolymer of styrene and maleic acid (alt - psma) differs from other polyanionic antiviral agents in that the negative charges of alt - psma are provided by carboxylic acid groups instead of sulfate or sulfonate moieties. we hypothesized that alt - psma would have activity against human immunodeficiency virus type 1 (hiv-1) comparable to other polyanions, such as the related compound, poly(sodium 4-styrene sulfonate) (pss). in assays using cell lines and primary immune cells, alt - psma was characterized by low cytotoxicity and effective inhibition of infection by hiv-1 bal and iiib as well as clinical isolates of subtypes a, b, and c. in mechanism of action assays, in which each compound was added to cells and subsequently removed prior to hiv-1 infection (washout assay), alt - psma caused no enhancement of infection, while pss washout increased infection 70% above control levels. these studies demonstrate that alt - psma is an effective hiv-1 inhibitor with properties that warrant further investigation. |
from 20022004, i had the privilege of working with a diverse group of patient advocates, communications professionals, political consultants, scientists, and professional politicians to conceive, nurture, design, and secure the passage of california 's landmark proposition 71 ballot initiative, which established the california institute for regenerative medicine (cirm). cirm was designed to award $ 3 billion in competitive, peer - reviewed grant funds to stem cell research running from basic science to clinical application. the proposition 71 ballot initiative that created cirm passed with almost 60% of the voters supporting it, primarily because of the medical promise of stem cell research, the unique political pressures and ethical debate surrounding stem cell research in the united states, and the potential and recognized benefit that biomedical research brings to california 's biotechnology industry and economy. much has been said and written about the merits and challenges of this landmark effort and its current function. i want to focus this essay, however, on a number of valuable lessons i learned about the role that scientists and rigorous scientific advice can play in the political process, in discussions with the public and patient advocates, and in the crucible of a high - visibility political and scientific initiative. in particular, i learned that, while the public political process can be messy, practicing research scientists not only can provide valuable information and advice, but can gain a great deal of valuable experience. i also learned that, while the world of science is not the only data - driven group, it nonetheless contributes a much longer - term perspective on issues than do other groups. do n't crush our hopes ! overhype ! too much pessimism ! i heard all of these conflicting statements and more from my patient advocate friends and colleagues during the proposition 71 campaign. the lesson i learned is that one has to walk a fine line between too much pessimism, which some patient advocates will tell you has a negative impact, because it kills their hope, as opposed to too much optimism, which contributes to the problem of overhyping or promising more than can be delivered in the time frame that you or someone else imagines. in short, i learned that i should paint a reasonable picture of what success would bring, while describing reasonable expectations and likely obstacles. for example, it is and was clear that success with stem cell approaches for type 1 diabetes will potentially bring complete insulin independence. but a number of important technical and safety problems will have to be solved on the path to achieving that goal, which leads to an uncertain time frame. i have often been asked, both during the proposition 71 process and since then, how long it will take a particular area of research to reach clinical application or, even harder to answer, when will my wife 's, child 's, or parent 's disease be cured ? these are difficult, if not impossible, questions to answer, because the point of research is to find answers to unanswered questions, often with little accurate information available at the outset. the answers one gives thus require an explanation, not a number, since one can not possibly give an accurate number. i also learned that it is appropriate to describe what success might look like, to talk about past experience with success in generating new therapies or discoveries, and to describe how broad portfolios of parallel opportunities will yield some that will proceed more quickly and some that will go more slowly. i would describe being a scientist in this (and other) fields as being like an explorer at the edge of a new continent, where i could see mountains and forests in the distance and knew it was likely that there were riches to be had. thus i could argue it was worth proceeding and there would be great rewards, but predicting exactly how long it would take to yield results would be problematic. but i also learned that it is compelling to note that not starting a long journey leads to no benefit, or adds delay to what will be a long journey, no matter what. nonetheless, the issue of time is hard and can also be difficult to communicate. thus i also learned one can talk about short - term and long - term returns and describe what the process will look like. finally, i learned that telling people that scientists are working as rapidly as possible to find better therapies is helpful. many people respect this honesty and find it reassuring that the scientific community does care about trying to find therapies for people 's diseases, even if success will take time. the media and the communications industry are essential if scientists are to communicate with the public. in fact, the media is a megaphone that allows us to talk to more than just a few people at a time. in that context, the media is an essential part of how scientists work with other groups in political settings. but i often hear my colleagues say that reporters never get it right or that some reporter did n't quote them accurately or that they talked to a reporter for half an hour or more and they only used a one - sentence quote or a 15- to 20-s segment in the final radio or tv report. first, my experience is that the vast majority of people i interacted with in the media are intelligent, hard - working professionals with little scientific background, who are nonetheless trying very hard to get the facts straight and to be as objective as possible. second, my experience is that the point of a 20- or 30-min interview is that this is time a scientist can use to educate a reporter about the topic being covered, so the overall story is accurate, including the parts not directly based on the interview. furthermore, my experience is that the journalist will be more likely to choose the right quote and/or get it right if the scientist has done a good job of providing an understandable explanation of the topic under discussion. to achieve this goal, however, requires that scientists learn to explain complex topics in plain everyday english with a minimum of jargon or latinate language, which of course, is the same as good teaching. for example, i learned to say blood - forming stem cell instead of hematopoietic stem cell to nonscientific audiences. any coordinated action of a group with media and the public will inevitably require teamwork, as well as consolidation and discipline around shared messages. this can get tricky when the shared messages fail to agree with any one scientist 's view regarding correctness of information imparted or focus on the proper priorities. but if every member of a group chooses to broadcast his or her own version of a message, instead of uniting around common themes, what will emerge is cacophony, confusion, and likely failure. the question is how to balance scientific and personal integrity with teamwork, shared goals, and shared messages. my own solution was to work vigorously in private to ensure that the common message themes were rigorously correct, so that i, and my colleagues on the team, were always saying things that to the best of our knowledge were scientifically accurate and defensible. but i also chose to agree to focus on shared message points publicly, even if my own view was that these points were not as important scientifically as other messages. similarly, i had to accept that the public at large and the scientific community sometimes use the same words differently. my personal and scientific view is that few diseases other than infectious diseases are ever truly cured and that what we offer are therapies that relieve, reduce, or manage a disease. in the public arena, however, what i regard as short- to medium - term therapies seem to be thought about as cures. in this case, my solution was to speak about therapies in my own interviews and public talks, but not to publicly debate the issues around the word cure with my own team. in this situation, the crucial role of a scientist is to work as hard as possible privately to get the substance of the messages absolutely correct, and then to work with the team on promoting them in a coordinated and agreed - upon way. any coordinated action of a group with media and the public will inevitably require teamwork, as well as consolidation and discipline around shared messages. this can get tricky when the shared messages fail to agree with any one scientist 's view regarding correctness of information imparted or focus on the proper priorities. but if every member of a group chooses to broadcast his or her own version of a message, instead of uniting around common themes, what will emerge is cacophony, confusion, and likely failure. the question is how to balance scientific and personal integrity with teamwork, shared goals, and shared messages. my own solution was to work vigorously in private to ensure that the common message themes were rigorously correct, so that i, and my colleagues on the team, were always saying things that to the best of our knowledge were scientifically accurate and defensible. but i also chose to agree to focus on shared message points publicly, even if my own view was that these points were not as important scientifically as other messages. similarly, i had to accept that the public at large and the scientific community sometimes use the same words differently. my personal and scientific view is that few diseases other than infectious diseases are ever truly cured and that what we offer are therapies that relieve, reduce, or manage a disease. in the public arena, however, what i regard as short- to medium - term therapies seem to be thought about as cures. in this case, my solution was to speak about therapies in my own interviews and public talks, but not to publicly debate the issues around the word cure with my own team. in this situation, the crucial role of a scientist is to work as hard as possible privately to get the substance of the messages absolutely correct, and then to work with the team on promoting them in a coordinated and agreed - upon way. building broad coalitions to support biomedical and other fields of scientific research is clearly an area where scientists, politicians, and other interested groups will have to work together more than ever to protect publicly funded science in the coming years. there is increasing scrutiny by politicians and the public, who want to know how the investment of their funds benefits them. responding to that legitimate concern, and finding ways to continue to expand scientific research that supports healthy vigorous societies, will require scientists to be part of these teams of diverse interest groups. i can virtually guarantee from my experiences that decisions about funding and science policy made in the absence of scientific input will likely be unpalatable to scientists and ultimately not in the best interest of our broader societies. thus a key lesson from the proposition 71 experience is that engagement of scientists with diverse nonscientific groups can make a big difference and that scientists must actively engage with the public in the future if we are to contribute robustly to the medical and economic health of our communities. | i describe a number of valuable lessons i learned from participating in california 's proposition 71 effort about the role that scientists and rigorous scientific advice can play in a public political process. i describe how scientists can provide valuable information and advice and how they can also gain a great deal from the experience that is valuable to a practicing research scientist. finally, i argue that in the future, building similar broad coalitions to support biomedical and other areas of scientific research will be essential to protect publicly funded science. thus, a key lesson from the proposition 71 experience is that engagement of scientists with diverse nonscientific groups can make a big difference and that scientists must actively engage with the public in the future if we are to contribute robustly to the medical and economic health of our communities. |
brain - derived neurotrophic factor (bdnf) is a member of the neurotrophin family, a group of structurally related polypeptide growth factors. the family also includes nerve growth factor (ngf), neurotrophin-3 (nt-3), and neurotrophin-4 (nt-4/5). other neurotrophins have also been identified, nt-6 and nt-7 ; however, these likely do not exist in mammals [24 ]. neurotrophins activate one or more of the high - affinity tropomyosin - receptor kinase (trk) receptor family [1, 5, 6 ], as well as the low - affinity p75 neurotrophin receptor (p75ntr). neurotrophins direct growth and differentiation in the developing nervous system [1, 3, 6, 8 ]. levels of the different neurotrophins relate in a predictable pattern to stages of embryonic development. infusion of bdnf into the adult brain promotes neurogenesis [9, 10 ], and dendritic spine reorganization in the rat hippocampal formation. bdnf gene transfection triggers dendritic and axonal branching in dentate gyrus (dg) granule cell cultures while mice with a targeted gene deletion show substantial impairment in normal cerebellar development, among other developmental and behavioral deficits. trk receptors precipitate the activation of many signaling cascades, including phospholipase c (plc), phosphoinositide 3-kinase (pi3k), and ras [14, 15 ], which are involved in different aspects of neuronal survival and neurite outgrowth [1719 ]. the p75ntr is implicated in both pro- and antitrophic processes, such as neurite outgrowth and apoptosis [14, 21, 22 ]. bdnf regulates synaptic transmission and activity - dependent plasticity [23, 24 ] and promotes long - term potentiation (ltp). bdnf exerts presynaptic effects in a retrograde or paracrine fashion [27, 28 ] and postsynaptic effects by modulating nmda receptor subunit expression. experimental reductions in bdnr or trkb activity significantly impair ltp [25, 30, 31 ]. previous research has demonstrated that exercise increases hippocampal bdnf mrna [32, 33 ] and protein [34, 35 ]. the effect of exercise to augment bdnf activity depends on camp - response - element binding protein (creb) activation, as creb suppression mitigates this effect. increases in hippocampal bdnf mrna have been detected after a period of running as short as 6 hours, to as long as two months, signifying a sensitive yet sustainable effect. physical activity leads to greater activation of cellular signaling pathways associated with survival and neurogenesis, and can enhance hippocampal ltp induction, all of which strongly implicate bdnf in their effects. this review will describe how bdnf and neurotrophins are involved in nervous system development, the signal transduction mechanisms associated with neurotrophin activity, and the modulating role bdnf plays in synaptic plasticity and ltp. the influence of exercise on bdnf activity, especially in the hippocampus, and the relationship between bdnf and excitotoxicity will also be examined. bdnf and nt-4/5 bind to trkb receptors [41, 42 ], as does nt-3 to a lesser extent. when a ligand binds to a trk receptor (see figure 1), the receptor dimerizes and autophosphorylates tyrosine residues to yield docking sites for the src homology 2 domain - containing adapter protein (shc) and phospholipase c- (plc-), which are coupled to intracellular signaling cascades such as ras, pi3k [45, 46 ], and, as mentioned, plc- [14, 15, 47, 48 ]. shc regulates protein interactions by binding, with a high - affinity, tyrosine - phosphorylated sites, and in this way regulates intracellular signaling such as ras. once shc is docked with the receptor and bound to the adapter protein grb2, ras promotes activation of the mitogen - activated protein kinase (mapk)/extracellular signal regulated kinase (erk) cascade, as well as the pi3k cascade [3, 15 ]. mapk / erk is essential for neurogenesis and promotes survival by two ways, by induction of prosurvival genes and inhibition of proapoptotic proteins. ras also suppresses apoptosis via pi3k ; pi3k activates akt, which sequesters pro - apoptotic proteins in the cytoplasm away from their transcriptional targets. the pi3k cascade can be activated two ways by trk receptors, through ras as well as through adapter proteins shc, grb2, and grb2-associated binder-1 (gab1) [15, 45 ]. in some neurons, bdnf phosphorylates the insulin receptor substrate, which can activate pi3k as well. vaillant and colleagues demonstrated that inhibition of any portion of the ras - pi3k - akt pathway significantly reduced survival of sympathetic neurons in culture in the presence of ngf. the adapter proteins shc and plc- are phosphorylated once they dock with the trk receptor [3, 14, 47 ]. once activated, plc- then catalyzes the breakdown of lipids to inositol 1,4,5 triphosphate (ip3), which promotes the release of calcium from intracellular stores, and a subsequent increase in intracellular calcium concentration and diacylglycerol (dag) occurs. dag regulates protein kinase c (pkc-), which may be required for the mapk / erk signal, and both dag and mapk / erk have been implicated in neurite outgrowth [18, 19 ]. interestingly, ltp is inhibited when plc- signaling is blocked, which may be related to reduced ip3 formation and subsequent reduction in intracellular calcium release. the other neurotrophin - responsive receptor, the p75ntr, can bind to each factor and regulate the trk receptor affinity for its ligand. the trk receptors and the p75ntr are often present in close proximity on the cellular membrane. while trkb responds to nt-3, nt-4/5, and bdnf, the receptor selectively binds only bdnf when colocalized with p75ntr. the largely presynaptic p75ntr serves dual roles in that it modulates trk receptor binding and is involved in prolonged ras - mediated activation of erk and neurite outgrowth, though p75ntr - induced neurite outgrowth ceases after prolonged p75ntr activation. the p75ntr also activates c - jun n - terminal kinase (jnk) and causes apoptosis in a variety of neurons [14, 21, 22 ]. although all trk receptors are assumed to behave in much the same fashion, differences do exist. kaplan and miller demonstrated, using trkb receptors with mutations at different binding sites, that trkb receptors use both pi3k and mapk cascades for cell survival while trka receptors depend mainly on pi3k. this demonstrates some comparative flexibility for trkb receptors with regard to pro - survival function. importantly, two truncated forms of trkb receptors exist that lack tyrosine kinase function and bind bdnf to negatively modulate trkb activity. given that bdnf - associated signaling modulates neuron survival and apoptosis, much attention has been focused on the role of bdnf (and other neurotrophins) in development. indeed, the critical involvement of trk receptors in neuronal survival [16, 5052 ], neurogenesis, and neurite outgrowth [12, 18, 19 ] implicates bdnf in developmental processes. throughout development, neurotrophins influence and guide neuronal differentiation in the central nervous system (cns) [6, 8 ] and peripheral nervous system (pns) [1, 3 ]. barnab - heider and miller demonstrated that cortical progenitor cells express bdnf and nt-3 and their associated trkb and trkc receptors, and that these neurotrophins were essential to the survival of the progenitor cells. bdnf mrna expression is lowest in the brains of early rat embryos and increases into adulthood. it demonstrates a reciprocal relationship with nt-3 mrna, which is at the highest levels in early embryos. maisonpierre and colleagues characterized distribution of bdnf, nt-3, and ngf mrna during rat development and found that the amount of all three dramatically increased between embryonic days 11 and 12, with transcripts widely distributed by embryonic day 13. this timing corresponds directly to the onset of robust neurogenesis in the peripheral and central nervous systems [60, 61 ]. examined bdnf protein concentration during postnatal development in rats and found that levels in the septum, cerebellum, and hippocampus increased over time and then stabilized (although hippocampal protein levels took much longer to plateau) while hypothalamic bdnf protein levels increased for a period following birth and then decreased. kim. further investigated bdnf - immunoreactivity (ir) in the rat forebrain and upper brainstem during postnatal development. the investigators defined three groups of bdnf cells : those that are detected early, increase in number, and remain stable during adulthood (e.g., piriform cortex, cingulate gyrus), those that increase with age then decrease in adulthood (e.g., basolateral nucleus of the amygdala), and those that appear briefly before dramatic reductions in adulthood (e.g., substantia nigra, various hypothalamic nuclei). in the hippocampus, bdnf - ir cells appeared in the pyramidal layer of ca2 and ca3 early on (postnatal day 7) and staining increased through 28 days following parturition but then decreased, with comparatively reduced cell staining detected in 56-day - old adults. the bdnf - ir cell decrease detected in ca2 and ca3 in adulthood was completely absent in colchicine - treated adults, which instead showed an increase in bdnf - ir cells in these areas, possibly reflecting increased accumulation of bdnf in the cell body associated with inhibited axonal transport. bdnf - ir fibers were detected in the stratum lucidum layer of ca2 and ca3 and the polymorph layer of the dg also early on during the intial 28-day postnatal period, and these bdnf - ir fibers actually continued to increase into adulthood. bdnf serves a unique role among neurotrophins in development, especially with regard to the cerebellum. an examination of the cerebella of mice with a targeted bdnf gene deletion (bndf mice) revealed that bdnf is essential for normal development and function of the cerebellar cortex. the study showed reduced trk activation in purkinje cell bodies and dendrites in the mutant mice, indicating a lack of redundancy among neurotrophins with regard to bdnf in these cells. in bndf mice, there was a dramatic increase in cell death among developing granule cells, impaired development of the layers of the cerebellar cortex, and irregular foliation in the middle and posterior cerebellum ; defects were apparent in the declival sulcus (absent), the intercrural fissure (absent), the prepyramidal fissure (nearly absent), and uvular sulcus (nearly absent). bndf is crucial to postnatal survival, as most bndf mice die shortly after birth, but some do survive for a month or more [64, 65 ]. mice that lack bdnf demonstrate severe deficiencies in pns development, especially with regard to afferent neurons, but exhibit comparatively mild deficiencies in cns development [64, 66 ]. bndf mice show an abnormal gait with uncoordinated movements, and their stance is substantially wider (distance between left and right paws), despite their smaller size compared to wildtype mice. this evidence further implicates the importance of bdnf not just to neuron development in the pns but also to cerebellar development and function. intraventricular bdnf application encourages neurogenesis in several parts of the rat brain, such as striatum, septum, thalamus, and hypothalamus, and infusion directly into the hippocampus increases the number of granule cells in the dg. danzer and colleagues transfected hippocampal cells in culture with either bdnf or ngf ; dg granule cells exhibited considerable axonal and dendritic branching following bdnf, but not ngf transfection. this effect was abolished with the application of a trk receptor tyrosine kinase inhibitor, demonstrating that bdnf and trk signaling promote neurogenesis both within and outside of the context of development. the involvement of p75ntr in refining trk receptor selectivity may serve to modulate targeting of growing neurons (see figure 2), with neurotrophic factors acting as markers. target tissues for growing pns neurons (e.g., skeletal muscle) express neurotrophins, and bdnf is released postsynaptically to influence cns neurons in a retrograde fashion [27, 69 ]. in a study of rat sympathetic neurons that largely express ngf and trka receptors, bdnf activated p75ntr - mediated apoptosis in conditions with low available ngf. for example, if a predominantly ngf / trka - associated neuron were to grow to network with a predominantly bdnf / trkb - associated neuron, the growing neuron would quickly become apoptotic. postsynaptic bdnf release and activation of the presynaptic p75ntr, coupled with the lack of ngf / trka signaling, would reduce the survivability of the growing neuron. thus p75ntr serves a regulatory role with regard to trk receptor behavior and neuronal proliferation. in summary, neurotrophins influence neuronal differentiation and are important to neuronal survival [16, 5052 ]. bdnf mrna is present in relatively low levels early in life and generally increases through to adulthood [59, 62 ], especially in certain hippocampal neurons. studies of mice lacking bdnf demonstrate how it is essential for proper pns [64, 66 ] and cerebellar development. bdnf is directly involved in the regulation of synaptic transmission and activity - dependent synaptic plasticity by both pre- and postsynaptic mechanisms [1, 23 ] (see figure 3). these mechanisms underlie the critical role of bdnf in ltp [2426, 29, 65 ]. in a study with cultured hippocampal and cerebellar granule cells, caldeira and colleagues demonstrated that bdnf increased the levels of nmda receptor subunits in the plasma membrane of hippocampal cells. they recorded a correlated increase in intracellular calcium concentration and described an increased calcium entry through the additional nmda receptors. interestingly, hartmann and colleagues stimulated cultured hippocampal neurons presynaptically and detected a postsynaptic bdnf release, dependent upon calcium influx, thereby suggesting a bdnf - dependent positive feedback loop between presynaptic and postsynaptic signaling in the generation of ltp.. found that bdnf is essential for activity - induced potentiation of presynaptic vesicle cycling in cultured embryonic neocortical neurons of bdnf - knockout mice. this phenomenon was dependent upon nmda receptor activation, and was significantly reduced in bdnf - deficient neurons, indicating a possible retrograde messenger or paracrine role for bdnf. in support of these findings, previous work in bdnf knockout mice showed a pronounced impairment of vesicle docking in hippocampal synapses while direct application of bdnf reversed the deficits. estrogen promotes hippocampal plasticity, and in ovariectomized rats, the loss of estrogen reduces cytoskeletal reorganization in hippocampal dendritic spines and produces deficits in ltp. kramr and colleagues recently demonstrated that application of bdnf to hippocampal sections restored spine actin polymerization and ltp stability in these rats. backpropagating (retrograde) action potentials modulate short- and long - term changes to synaptic activity and also serve to relieve the mg block on nmda receptors. the extent of backpropagation in a neuron is dependent upon dendritic morphology, ion channel distribution, and synaptic input. importantly, application of an endoplasmic reticulum ca - atpase inhibitor stops this dendritic bdnf release, suggesting a calcium - dependent mechanism for retrograde release. these findings thus reveal several mechanisms for bdnf - mediated promotion of ltp in the hippocampus. bdnf is released in an activity - dependent manner, as demonstrated in both hippocampal neurons and peripheral neurons. while it may not be the only mediator of ltp, mice lacking bdnf demonstrated reduced tetanus - induced ltp, a deficit that disappeared with the return of bdnf function. as described above, bdnf works through both pre- and postsynaptic mechanisms to support ltp, and a simultaneous block of pre- and postsynaptic plc- activity, but not either by itself, is required to reduce ltp in mouse hippocampal cultures. interestingly, the involvement of trkb in ltp is not completely dependent on bdnf signaling ; nmda receptors can activate trkb by way of zinc influx, and adenosine 2a receptors can also influence trkb. in brief, bdnf is involved in trafficking nmda receptor subunits to the membrane, thereby increasing the potential for calcium influx. a sufficient local buildup of calcium causes postsynaptic bdnf release, which then leads to an increase in presynaptic vesicle cycling. exercise leads to substantial changes in bdnf and nmda receptor activity in the hippocampus, and these changes in large part underlie the effects of exercise on learning and synaptic plasticity. in rats with free access to activity wheels for one to six weeks, increases in hippocampal bdnf mrna [32, 33 ] and bdnf protein [34, 35 ] have been detected. berchtold, castello, and cotman demonstrated that hippocampal bdnf protein remained elevated for two weeks following three weeks of access to cage wheels (protein levels were 186% of controls at the end of exercise, 137% one week followup, and 133% at two weeks). physical activity increases hippocampal bdnf expression after a relatively short amount of time, as voluntary wheel running induced bdnf mrna after only 6 hours of running. a substantial increase in bdnf mrna in the dg has also been described after 2 weeks, 3 weeks, 4 weeks, and 2 months of voluntary running. bdnf mrna and protein induction due to exercise varies over the life - span, however. younger mice (2 months) exhibit a larger increase compared to older mice (15 and 24 months) over four weeks of running. forced treadmill running for 30 minutes per day for six weeks increased hippocampal bdnf protein and brdu positive proliferating cells in younger rats (5 months), an effect that was less robust but still significant in older rats (24 months). exercise influences bdnf even under conditions of homeostatic perturbations such as stress or dysfunction in energy metabolism. in chronically stressed sprague - dawley rats, access to cage wheels for six weeks only during dark cycle (active period) yielded increased levels of bdnf protein in the striatum compared to sedentary rats. the characteristic lower levels of hippocampal bdnf protein and dendritic spine density in type ii diabetic mice (db / db) were significantly enhanced with free access to cage wheels. under a condition of food deprivation every other day, six weeks of wheel running still yielded significantly increased hippocampal bdnf protein compared to normally fed and food - deprived sedentary wistar rats, similar in magnitude to the levels observed in normally fed exercising rats. running is associated with a robust activation of survival pathways and vesicular proteins, effects that are linked to bdnf. shen and colleagues showed prolonged hippocampal mapk signaling following only a week of voluntary running. chen and russo - neustadt demonstrated a significant increase in hippocampal pi3k protein in rats after two weeks of wheel running. in another study, chen, and russo - neustadt found a similar consequence of running on akt protein, an effect dependent upon creb signaling ; in fact, the researchers showed that exercise - induced increases in hippocampal bdnf protein and mrna are also creb activation dependent, indicating the essential nature of creb signaling to exercise and bdnf - dependent cellular survival effects. these pathways are likely directly associated with the neurogenesis detected in the dg of exercising rats [80, 81 ]. voluntary exercise selectively increases hippocampal synapsin i and synaptophysin mrna [35, 82 ], a phenomenon blocked by a recombinant human trkb - igg chimera, a highly specific antagonist of bdnf. synapsin i tethers vesicles to the cytoskeleton while synaptophysin may modulate vesicle recycling during high - intensity activity. indeed, this bdnf - mediated increase in synapsin following exercise may in part explain how exercise enhances ltp, along with the other mechanisms for bdnf - facilitated ltp described above. voluntary running also yielded better performance on the morris water maze test, an ltp - associated effect abolished with the selective blocking of bdnf. importantly, farmer and colleagues also identified an increase in nmda receptor subunit nr2b mrna in the dg following activity wheel running, which may represent yet another mechanism explaining how exercise promotes ltp and learning and memory. a complicated relationship exists between bdnf and epileptogenic hyperexcitability, but this relationship may be fundamentally characterized as a positive feedback loop. in general, enhanced bdnf signaling is associated with increased excitability, presumably through increased synaptic plasticity. for example, mrna and protein levels for bdnf are increased in the hippocampus after seizure [66, 87 ], and bdnf knockout mice demonstrate reduced seizure behavior in response to kindling. in transgenic mice with an overexpression of the truncated trkb receptor, the reduced bdnf signaling led to reduced seizure activity after systemic kainic acid administration.. demonstrated development of hyperexcitable circuits due to abnormal mossy fiber sprouting, in part caused by activity - induced bdnf release and trk receptor activity. this sprouting did not occur in the presence of a trk receptor tyrosine kinase inhibitor or anti - bdnf antibody. such abnormal remodeling is, in part, responsible for dentate hyperexcitability [89, 90 ]. glutamatergic excitotoxicity, thought to result from excessive calcium influx via nmda receptors, is a major contributor to neuronal damage associated with anoxia, ischemia, and seizure. in cultured cerebellar granule cells, high glutamate concentrations cause immediate necrosis. following withdrawal to normal medium, hippocampal cell cultures, a high concentration of glutamate caused mainly necrosis while a lower concentration of glutamate led almost exclusively to delayed apoptosis. glazner and mattzon evaluated the effect of bdnf application on nmda receptor subunits, and found that nr1 and nr2a protein levels are increased whereas nr2b levels are decreased ; associated effects included increased calcium response to nmda activation, increased necrosis (cell swelling), and decreased apoptosis (cell shrinkage and chromatin fragmentation). bdnf - mediated enhancement of nmda receptor functions are consistent with the effects of exercise on increasing nmda receptor expression described above, further supporting the role of bdnf in exercise - enhanced synaptic plasticity. these results further illustrate how bdnf overexpression may lead to hyperexcitable circuitry through multiple mechanisms. bdnf thus encourages neurogenesis [9, 10 ], axonal, and dendritic sprouting, and nmda receptor - mediated transmission in a trk - b - dependent manner, which are generally considered to be beneficial effects on neuronal function. however, these same mechanisms may lead to hyperexcitable circuits in the dg [89, 90, 97 ], which may eventually lead to spreading excitotoxicity. this condition can therefore become a self - sustaining cycle of sprouting and hyperexcitability, fueled in part by activity - dependent bdnf release [69, 73, 74 ]. in a kainic acid model of temporal lobe epilepsy utilizing transgenic mice, increased trkb signaling facilitated significantly shorter time to epileptogenesis and more severe epileptiform electroencephalogram activity compared to wild - type mice, and epileptogenesis was delayed in reduced trkb (truncated form) mice. based on this evidence for a prolonged state of hyperexcitability coupled with a shorter facilitation resulting from enhanced expression of bdnf or trkb signaling in the hippocampus, it follows that exercise - induced plasticity could leave the brain more vulnerable to epileptic seizures and excitotoxicity. microinjections of kainic acid into the hippocampi of anaesthetized rats produce more damage in subjects that were provided with running wheels for several weeks compared to sedentary controls. this enhanced excitotoxicity in exercising rats was interpreted by the investigators as a consequence of exercise - induced upregulation of hippocampal bdnf expression. on the other hand, a large body of evidence from both clinical studies in humans and basic research in animal models reveals that physical exercise produces a wide range of neuroprotective effects [100103 ]. for example, exercise in humans decreases risk of neurological disease and stroke, and it may improve recovery after brain trauma. the brain damage caused by experimental infarct is diminished in exercising rats compared to sedentary controls. exercise also protects against oxidative stress, and the neurotoxic effects of 6-ohda [104, 105 ] and mptp. since neural hyperexcitability is a mechanism for the injury and death that occurs in all of these neurological insults and/or disorders [107, 108 ], determining how exercise protects against excitotoxicity should have broad implications for understanding its beneficial effects on the brain. nonetheless, these conditions are only indirectly linked to excitotoxicity, and a more direct examination of the influence of exercise on hyperexcitability is required to understand the mechanisms for its neuroprotective effects. in contrast to the findings of ramsden and colleagues described above, in which exercise led to increased excitotoxicty when kainic acid was injected into the hippocampus of anaesthetized rats, some evidence suggests that exercise may protect against hyperexcitability. experiments specifically designed to this hypothesis have revealed that exercise protects against kainic acid - induced seizures when studied in awake, freely behaving rats. reiss. reported that three weeks of voluntary exercise decreases the effects of kainic acid on seizure behaviors and hippocampal c - fos expression. further supporting the hypothesis that exercise diminishes hyperexcitability are similar reports of exercise protecting against experimentally induced seizures using other convulsant manipulations [110, 111 ]. exercise also diminishes the excitotoxic effects of systemic domoic acid in mice and reduces the learning deficits caused by systemic kainic acid administration in rats. taken together, the conflicting findings of exercise effects on hyperexcitability and excitotoxicity suggest that an exercise - induced compensatory mechanism may counter the hyperexcitable state associated with upregulation of bdnf. importantly, this compensatory mechanism may only function in awake, freely behaving subjects. in the report by ramsden and colleagues, the exercise - associated increase in neurotoxicity caused by kainic acid injected directly into the hippocampi if such a mechanism depended on extrahippocampal circuitry, then it is reasonable to assume that it may not engage in an anaesthetized state. though it is likely that a variety of mechanisms exist to counter the hyperexcitable state in the hippocampus caused by bdnf, elucidation of such a mechanism related to exercise should be based on specific criteria. this mechanism should involve some neural factor that (1) is upregulated by exercise, (2) diminishes neural hyperexcitability acutely, and (3) exerts latent neuroprotective effects. research from this laboratory suggests that hippocampal afferents originating from the noradrenergic locus coeruleus may be a critical component of this mechanism. more specifically, experimental evidence points to the neuropeptide galanin as a critical factor in dampening hippocampal excitability. galanin is a 29 - 30 amino acid neurotransmitter and putative trophic factor that regulates neural activity in several brain structures, most notably the hypothalamus and hippocampus. retrograde tracing / double labeling experiments reveal that the hippocampus receives galaninergic innervation via projections from the locus coeruleus. at least three g - protein coupled galanin receptor subtypes have been confirmed, designated gal r1 - 3. galaninergic transmission is predominantly inhibitory, producing hyperpolarization through increased k or decreased ca conductance. several lines of evidence reveal that galanin functions as an endogenous neuroprotective factor for hippocampal neurons [116118 ]. infusion of galanin into the hippocampus inhibits seizures provoked by a variety of methods, and the galanin receptor antagonists m-35- and m-40 block the antiseizure effects of galanin. transgenic galanin knock - out mice show increased susceptibility to kainic acid - induced seizures whereas transgenic mice overexpressing galanin display a resistance to these seizures. hippocampal electrophysiological activity is similarly exaggerated in the galanin knockouts and decreased in galanin overexpressers. in vitro studies in hippocampal cultures from transgenic mice previous work from this laboratory has demonstrated that either three weeks of voluntary wheel running or treadmill training increases galanin gene expression in locus coeruleus neurons in rats [120123 ]. no exercise - induced changes in neuropeptide - y or tyrosine hydroxylase were observed in these studies, suggesting that locus coeruleus neurons respond to exercise by specifically upregulating galanin. the critical role of galanin in mediating the protective effects of exercise against convulsant - induced seizures was demonstrated by the administration of the galanin receptor antagonist m-40, which largely blocked the neuroprotection as measured by behavioral seizure severity. these results suggest that exercise - induced upregulation of galanin mediates the enhanced inhibitory tone that may counteract bndf - mediated hyperexcitability. to conclude, bdnf receptor activity encourages neurogenesis [9, 10 ], suppresses apoptosis, and modulates synaptic activity via a variety of signaling cascades [45, 48, 50 ]. these effects are beneficial with regard to development [1, 8 ] and synaptic plasticity [25, 26, 29, 65 ], but can be harmful in conditions sensitive to hyperexcitability. dendritic bdnf release is activity - dependent, based on calcium influx [27, 69 ]. this release causes an increase in presynaptic vesicle cycling, which further stimulates synaptic activity. the bdnf - induced increase in nmda receptor subunits creates more opportunity for calcium influx, supporting additional bdnf release. the increased trkb signaling boosts ip3 activity and intracellular calcium release, contributing to the enhancement of the cycle even further. these processes feed off of one another to overwhelm any modulatory effects of truncated trkb receptors (which bind bdnf but lack tyrosine kinase function) or p75ntr, the latter of which will act more to refine the actions of the increased available bdnf to yet further potentiate the cycle. since bdnf encourages neurogenesis [9, 10 ] and neurite outgrowth, the effects of these processes are further compounded with the establishment of additional excitatory synapses (see figure 4). thus hyperexcitable circuits in the dg can develop due to excessive bdnf and nmda receptor activity [89, 90 ], activity that maintains a self - supporting cycle of neurotransmitter release in the hippocampus that can be overwhelmed to promote excitotoxic vulnerability. considering the maladaptive nature of this condition, selection for hippocampal circuitry that regulates this hyperexcitability would provide clear evolutionary benefits. furthermore, environmental stimuli that promote hippocampal bdnf expression, such as exercise, would be expected to engage this compensatory mechanism. evidence suggests that a circuit including galaninergic projections from the lc to the hippocampus provides this function [116, 118, 120, 121, 123 ]. | the present paper examines the nature and function of brain - derived neurotrophic factor (bdnf) in the hippocampal formation and the consequences of changes in its expression. the paper focuses on literature describing the role of bdnf in hippocampal development and neuroplasticity. bdnf expression is highly sensitive to developmental and environmental factors, and increased bdnf signaling enhances neurogenesis, neurite sprouting, electrophysiological activity, and other processes reflective of a general enhancement of hippocampal function. such increases in activity may mediate beneficial effects such as enhanced learning and memory. however, the increased activity also comes at a cost : bdnf plasticity renders the hippocampus more vulnerable to hyperexcitability and/or excitotoxic damage. exercise dramatically increases hippocampal bdnf levels and produces behavioral effects consistent with this phenomenon. in analyzing the literature regarding exercise - induced regulation of bdnf, this paper provides a theoretical model for how the potentially deleterious consequences of bdnf plasticity may be modulated by other endogenous factors. the peptide galanin may play such a role by regulating hippocampal excitability. |
answer : intracranial calcification associated with isolated hypoparathyroidism. a 75yearold man with no history of systemic disease presented with an inability to walk. computed tomography (ct) of the brain revealed bilateral calcification of the lenticular nuclei and cerebellum (fig. biochemical test results indicated low serum total calcium (5.6 mg / dl), elevated serum phosphorus (5.9 mg / dl), inappropriately low levels of intact parathyroid hormone (13 pg / ml), and normal serum creatinine (0.94 mg / dl). although chronic hypocalcemia is often asymptomatic, longlasting chronic hypocalcemia may have accounted for the patient 's difficulty in walking. untreated cases may affect function in multiple systems 1, rendering early diagnosis utilizing ct imaging essential. computed tomography (ct) of the brain demonstrates dense calcification of the bilateral basal ganglia and dentate nuclei. written informed consent was obtained from the patient for the publication of this case and the accompanying images. a copy of the written informed consent is available for review by the editorinchief of clinical case reports. | key clinical messagehypocalcemia due to chronic hypoparathyroidism presents with nonspecific symptoms. however, if untreated, hypocalcemia may affect neurological, cognitive, muscular, and cardiac function. computed tomography (ct) findings may confirm a diagnosis of chronic hypoparathyroidism. although autoimmune acquired hypoparathyroidism is a rare disease, early diagnosis and treatment are critical for avoiding severe complications. |
glioblastoma (gbm) is the most frequent and aggressive malignant primary brain tumor with only about 12% of patients surviving beyond 36 months (long - term survivors). according to the latest central brain tumor registry of the usa statistical report the incidence of gbm increases with age and peaks at 7584 years (14.93/100,000), being more common in males (3.97/100,000). the current treatment strategy for gbm patients combines maximal surgical resection, followed by radiotherapy (rt) with concomitant and adjuvant temozolomide (tmz). complete surgical resection is virtually impossible due to the infiltrative nature of these tumors, yet gross total resection is still a positive prognostic marker. concurrent adjuvant rt in combination with tmz represents the standard of care for patients with newly diagnosed gbm, but still < 5% of patients survive for longer than 5 years after diagnosis. decades of molecular studies have identified key genetic abnormalities in human gbms, including the following : (1) dysregulation of growth factor signaling pathways via amplification and mutational activation of receptor tyrosine kinase (rtk) genes ; (2) activation of the phosphatidylinositol-3-oh kinase (pi3k) pathway ; and (3) inactivation of the p53 and retinoblastoma tumor suppressor pathways. during recent years, large - scale research efforts spearheaded by the cancer genome atlas (tcga) and chinese glioma genome atlas (cgga) have made rapid advances in understanding gbm tumor genetics. the discovery of new genetic alterations and their mapping against clinical outcome will trigger an avalanche of novel perceptions of the genomic and epigenomic landscape, biological subgroups and putative cells of origin of gbm, which has encouraged hopes for more effective treatment strategies in the near future. this review mainly discusses the recent advances in gbm molecular research and current trends in personalized therapy of this disease. malignant gliomas are histologically heterogeneous and invasive tumors that are derived from glia. the world health organization (who) classification system groups gliomas into 4 histological grades defined by increasing degrees of undifferentiation, anaplasia, and aggressiveness. malignant gliomas, the most common form of gliomas, consist of who grade iv tumors (gbm) and grade iii tumors (anaplastic astrocytoma, oligodendroglioma, and oligoastrocytoma). gbms account for approximately 6070% of malignant gliomas and is characterized histologically by considerable cellularity and mitotic activity, microvascular proliferation, necrosis and they are also recalcitrant to radio / chemotherapy. primary (de novo, approximately 95% of cases) gbms manifest rapidly, without evidence of less malignant precursor lesions, after a short clinical history. secondary gbms (approximately 5% of cases) develop more slowly by progression from low - grade diffuse astrocytoma and anaplastic astrocytoma. gbm and other malignant gliomas are highly invasive, infiltrating surrounding brain parenchyma, yet they are typically confined to the central nervous system (cns) and do not metastasize. unfortunately, who morphological classification is based on subjective criteria, lacks reproducibility, and remains imperfect in its ability to predict individual outcomes. the first genome - wide exon sequencing effort for glioma identified heterozygous hotspot mutations at codon 132 (most commonly r132h) in isocitrate dehydrogenase 1 (idh1) in 12% of gbm. these homologous enzymes decarboxylate isocitrate to -ketoglutarate (kg), and this neomorphic mutation renders the idh enzyme to reduce kg into 2-hydroxyglutarate in the nicotinamide adenine dinucleotide phosphate - dependent manner. mutant idh1 or idh2 are correlated with increased histone methylation, causing epigenetic alterations in both dna and histones, altering gene expression and promoting oncogenic transformation. nowadays, mutations in idh1 are commonly established as a hallmark molecular feature of secondary gbm (~70% of secondary gbm, compared with 520% in primary gbm) who have predominant localization of gbm in the frontal and temporal lobes. since primary gbm is a clinically defined entity and the presence of idh1/2 mutations has been shown to be inversely related to or even mutually exclusive of epidermal growth factor receptor (egfr) and phosphatase and tensin homolog (pten) abnormalities, which are hallmarks of primary gbm, idh - mutated gbm lesions may represent genetically secondary gbm tumors. moreover, the idh mutation status is stable during the progression of lower - grade gliomas to secondary gbms. mutations in the idh genes are thought to cause glioma - cpg island methylator phenotype (g - cimp) within the proneural gbm subgroup. idh mutations seem to require cooperating mutations in tp53 and atrx, and they are less frequently detected in primary gbms. the o(6)-methylguanine - dna methyltransferase (mgmt) is a dna repair enzyme, preventing errors during dna replication. abnormal methylation of the mgmt promoter caused its silencing, a reduction of the mgmt enzyme expression, and subsequently to less repair activity of dna damage, including that induced by tmz. mgmt promoter methylation in gbm is a prognostic and predictive biomarker indicating a response to chemoradiation. the trial of the effect of tmz on newly diagnosed gbm showed that mgmt promoter methylation was an independent favorable prognostic factor. patients with tumors with methylated mgmt promoter had a survival benefit when treated with tmz and rt, compared to those who received rt only factor. a recent report from the neuro - oncology working group (noa) of the german cancer society confirmed a predictive value of mgmt methylation for benefit from chemotherapy in patients with a wild - type idh, independent of tumor grade. recently, novel somatic mutations in the promoter region of telomerase reverse transcriptase (tert) have been identified in malignant melanomas, as well as being associated with increased telomerase expression and activity. the tumors derived from cell populations with low self - renewal capacity generally depend on alterations that keep telomerase activity, while epigenetic alteration maintains telomerase activity in tumor types arisen from self - renewing stem cells. the two most common mutations are located at c228 t and c250 t, with identical hotspots also found in gliomas. the highest incidence was identified among most tumors harboring 1p/19q co - deletion and idh mutations (98%), as well as idh wild - type (idh wt) tumors with egfr amplification (92%). the frequency of tert mutations is relatively low in diffuse and anaplastic astrocytomas (19% and 25%, respectively). in the study by killela., patients with tert promoter mutations alone (i.e., no idh mutation) had the poorest overall survival (os) (median 11.3 months), patients with tumors without tert or idh1/2 mutations had a slightly better survival (median 16.6 months), while patients with only idh mutant gbm had the best survival (median 42.3 months). although another study with 358 patients found no significant difference in os between tert mutant and tert wild - type (idh wt) gbm, the role of tert promoter mutations may provide a tool to identify non - idh mutant gbms. the range of high - amplitude focal copy - number aberrations in adult gbm highlights a key role of egfr amplifications (43% of cases) which co - occurred with egfr intragenic deletions and/or point mutations. egfr mutations were accompanied by regional dna amplification in the majority of cases, leading to a wide range of mutation allelic frequencies. the prominent intragenic deletions in gbm target parts of the gene encoding either the extracellular domain of egfr (exons 27, the deletion of which forms egfr variant iii) or the carboxyl terminus, and these deletions are always correlated with amplification and co - expression of the wild - type egfr. egfr was recently shown to be activated by recurrent translocations in 7% of gbm samples : it was most frequently fused in - frame to septin 14 or phosphoserine phosphatase as the 3 gene segment. overall, 57% of gbm showed evidence of mutation, rearrangement, altered splicing, and/or focal amplification of egfr. loss of heterozygosity (loh) at chromosome 10q23 occurs at high frequency in a variety of human tumors. pten is a negative regulator of the phosphoinositide 3-kinase pathway, a major signaling pathway that stimulates cellular proliferation in response to growth factor stimulation. pten deletions were more common in gbm, except classical grade ii / iii gliomas. pten deletions were fairly common across all gene expressions subtypes, but absent in idh1 mutant tumors. several studies demonstrated that patients with loss of function mutations of pten generally had shorter survival than patients with pten retention. however, pten loss was not associated with worse os in newly diagnosed gbm patients of the tmz era. in a smaller fraction of primary gbms (about 3%), a fusion of the tyrosine kinase coding region of fibroblast growth factor receptor 1 (fgfr1) to the transforming acidic coiled - coil (tacc) coding domain of tacc1 (or fusion of fgfr3 to tacc3) results in constitutive kinase activity. in transcriptome profiling of 272 gliomas from cgga, 67 in - frame fusion transcripts were identified, including three recurrent fusion transcripts : fgfr3-tacc3, rnf213-slc26a11, and ptprz1-met (fusion transcript involving the protein tyrosine phosphatase, receptor - type, z polypeptide 1 gene and the met proto - oncogene, zm). exogenous expression of the zm fusion in the u87 mg gbm line enhanced cell migration and invasion. clinically, patients afflicted with zm fusion harboring gbms survived poorly relative to those afflicted with non - zm - harboring. therefore, recurrent fusion events that involve rtk - encoding genes might be a promising therapeutic target and provide a strong rationale for the inclusion of these patients in future stratified clinical trials using different rtk inhibitors. table 1 summarizes all of the above described and other genetic alterations and related altered signaling pathways in primary versus secondary gbm. genetic abnormalities and the major signaling pathways involved in the pathogenesis of gbm idh : isocitrate dehydrogenase ; cdkn2a : cyclin - dependent kinase inhibitor 2a ; pten : phosphatase and tensin homolog ; nf1 : neurofibromatosis 1 ; rb1 : retinoblastoma 1 ; tert : telomerase reverse transcriptase ; ampl. : amplification ; egfr : epidermal growth factor receptor ; pdgfra : platelet - derived growth factor receptor alpha ; fgfr3 : fibroblast growth factor receptor 3 ; tacc3 : transforming acidic coiled - coil 3 ; rtk : receptor tyrosine kinase ; gbm : glioblastoma ; mapk : mitogen - activated protein kinase. the first genome - wide exon sequencing effort for glioma identified heterozygous hotspot mutations at codon 132 (most commonly r132h) in isocitrate dehydrogenase 1 (idh1) in 12% of gbm. these homologous enzymes decarboxylate isocitrate to -ketoglutarate (kg), and this neomorphic mutation renders the idh enzyme to reduce kg into 2-hydroxyglutarate in the nicotinamide adenine dinucleotide phosphate - dependent manner. mutant idh1 or idh2 are correlated with increased histone methylation, causing epigenetic alterations in both dna and histones, altering gene expression and promoting oncogenic transformation. nowadays, mutations in idh1 are commonly established as a hallmark molecular feature of secondary gbm (~70% of secondary gbm, compared with 520% in primary gbm) who have predominant localization of gbm in the frontal and temporal lobes. since primary gbm is a clinically defined entity and the presence of idh1/2 mutations has been shown to be inversely related to or even mutually exclusive of epidermal growth factor receptor (egfr) and phosphatase and tensin homolog (pten) abnormalities, which are hallmarks of primary gbm, idh - mutated gbm lesions may represent genetically secondary gbm tumors. moreover, the idh mutation status is stable during the progression of lower - grade gliomas to secondary gbms. mutations in the idh genes are thought to cause glioma - cpg island methylator phenotype (g - cimp) within the proneural gbm subgroup. idh mutations seem to require cooperating mutations in tp53 and atrx, and they are less frequently detected in primary gbms. the o(6)-methylguanine - dna methyltransferase (mgmt) is a dna repair enzyme, preventing errors during dna replication. abnormal methylation of the mgmt promoter caused its silencing, a reduction of the mgmt enzyme expression, and subsequently to less repair activity of dna damage, including that induced by tmz. mgmt promoter methylation in gbm is a prognostic and predictive biomarker indicating a response to chemoradiation. the trial of the effect of tmz on newly diagnosed gbm showed that mgmt promoter methylation was an independent favorable prognostic factor. patients with tumors with methylated mgmt promoter had a survival benefit when treated with tmz and rt, compared to those who received rt only factor. a recent report from the neuro - oncology working group (noa) of the german cancer society confirmed a predictive value of mgmt methylation for benefit from chemotherapy in patients with a wild - type idh, independent of tumor grade. recently, novel somatic mutations in the promoter region of telomerase reverse transcriptase (tert) have been identified in malignant melanomas, as well as being associated with increased telomerase expression and activity. the tumors derived from cell populations with low self - renewal capacity generally depend on alterations that keep telomerase activity, while epigenetic alteration maintains telomerase activity in tumor types arisen from self - renewing stem cells. the two most common mutations are located at c228 t and c250 t, with identical hotspots also found in gliomas. the highest incidence was identified among most tumors harboring 1p/19q co - deletion and idh mutations (98%), as well as idh wild - type (idh wt) tumors with egfr amplification (92%). the frequency of tert mutations is relatively low in diffuse and anaplastic astrocytomas (19% and 25%, respectively). in the study by killela., patients with tert promoter mutations alone (i.e., no idh mutation) had the poorest overall survival (os) (median 11.3 months), patients with tumors without tert or idh1/2 mutations had a slightly better survival (median 16.6 months), while patients with only idh mutant gbm had the best survival (median 42.3 months). although another study with 358 patients found no significant difference in os between tert mutant and tert wild - type (idh wt) gbm, the role of tert promoter mutations may provide a tool to identify non - idh mutant gbms. the range of high - amplitude focal copy - number aberrations in adult gbm highlights a key role of egfr amplifications (43% of cases) which co - occurred with egfr intragenic deletions and/or point mutations. egfr mutations were accompanied by regional dna amplification in the majority of cases, leading to a wide range of mutation allelic frequencies. the prominent intragenic deletions in gbm target parts of the gene encoding either the extracellular domain of egfr (exons 27, the deletion of which forms egfr variant iii) or the carboxyl terminus, and these deletions are always correlated with amplification and co - expression of the wild - type egfr. egfr was recently shown to be activated by recurrent translocations in 7% of gbm samples : it was most frequently fused in - frame to septin 14 or phosphoserine phosphatase as the 3 gene segment. overall, 57% of gbm showed evidence of mutation, rearrangement, altered splicing, and/or focal amplification of egfr. loss of heterozygosity (loh) at chromosome 10q23 occurs at high frequency in a variety of human tumors. pten is a negative regulator of the phosphoinositide 3-kinase pathway, a major signaling pathway that stimulates cellular proliferation in response to growth factor stimulation. pten deletions were more common in gbm, except classical grade ii / iii gliomas. pten deletions were fairly common across all gene expressions subtypes, but absent in idh1 mutant tumors. several studies demonstrated that patients with loss of function mutations of pten generally had shorter survival than patients with pten retention. however, pten loss was not associated with worse os in newly diagnosed gbm patients of the tmz era. in a smaller fraction of primary gbms (about 3%), a fusion of the tyrosine kinase coding region of fibroblast growth factor receptor 1 (fgfr1) to the transforming acidic coiled - coil (tacc) coding domain of tacc1 (or fusion of fgfr3 to tacc3) results in constitutive kinase activity. in transcriptome profiling of 272 gliomas from cgga, 67 in - frame fusion transcripts were identified, including three recurrent fusion transcripts : fgfr3-tacc3, rnf213-slc26a11, and ptprz1-met (fusion transcript involving the protein tyrosine phosphatase, receptor - type, z polypeptide 1 gene and the met proto - oncogene, zm). exogenous expression of the zm fusion in the u87 mg gbm line enhanced cell migration and invasion. clinically, patients afflicted with zm fusion harboring gbms survived poorly relative to those afflicted with non - zm - harboring. therefore, recurrent fusion events that involve rtk - encoding genes might be a promising therapeutic target and provide a strong rationale for the inclusion of these patients in future stratified clinical trials using different rtk inhibitors. table 1 summarizes all of the above described and other genetic alterations and related altered signaling pathways in primary versus secondary gbm. genetic abnormalities and the major signaling pathways involved in the pathogenesis of gbm idh : isocitrate dehydrogenase ; cdkn2a : cyclin - dependent kinase inhibitor 2a ; pten : phosphatase and tensin homolog ; nf1 : neurofibromatosis 1 ; rb1 : retinoblastoma 1 ; tert : telomerase reverse transcriptase ; ampl. : amplification ; egfr : epidermal growth factor receptor ; pdgfra : platelet - derived growth factor receptor alpha ; fgfr3 : fibroblast growth factor receptor 3 ; tacc3 : transforming acidic coiled - coil 3 ; rtk : receptor tyrosine kinase ; gbm : glioblastoma ; mapk : mitogen - activated protein kinase. the phenotype of a tumor is the result of the genotype and the influence of the tumor 's environment on the tumor. one would expect that molecular diagnostics will contribute to a better classification of brain tumors [tables 24 ]. phillips described three subclasses of high - grade gliomas (termed proneural, mesenchymal, and proliferative) associated with different outcomes ; specifically, prolonged survival of the proneural subclass. similar classification of gbms was also detected in a larger cohort of mixed gliomas. in 2010, unsupervised clustering of gene expression data from adult gbm samples from the tcga identified four different molecular subtypes : proneural, neural, classical, and mesenchymal. proneural gbms were subdivided into g - cimp - positive and g - cimp - negative gbm subsets on the basis of characteristic dna methylation patterns that strongly correspond with idh1 mutation status. another later study, which compared dna methylation patterns across both pediatric and adult patients with gbm, found a similar clustering in tumors from adult patients and further identified three more distinct clusters that predominantly consisted of children and adolescents. recently, liu. profiled the genetic features of multifocal gbm and found that m - gbms had no idh1, atrx, or pdgfra mutations, significantly associated with the mesenchymal subtype. they also identified the cyb5r2 gene to be hypomethylated and overexpressed in m - gbms. phillips classifications of gbm based on transcription profiling egfr : epidermal growth factor receptor ; pten : phosphatase and tensin homolog ; gbm : glioblastoma. tcga classifications of gbm based on transcription and methylation profiling tcga : the cancer genome atlas ; gbm : glioblastoma ; g - cimp : glioma - cpg island methylator phenotype ; ampl. : amplification ; idh : isocitrate dehydrogenase ; nf1 : neurofibromatosis 1 ; rb1 : retinoblastoma 1. dkfz classifications of gbm based on methylation profiling dkfz : deutsches krebsforschungszentrum (german cancer research center) ; gbm : glioblastoma ; rtk : receptor tyrosine kinase ; pdgfra : platelet - derived growth factor receptor alpha ; egfr : epidermal growth factor receptor ; idh : isocitrate dehydrogenase ; ampl. : the recent reports published on the nature genetics and nejm were comprehensively analyzed by whole - exome sequencing and/or targeted deep sequencing as well as array comparative genomic hybridization. in the nature genetics article, grade ii and iii gliomas were divided into and exhausted by the genetically well - defined type i iii subtypes. type iii tumors represented the idh wild - type grade ii and iii tumors in the current cohort, showing an os rate more similar to that of gbm. similarly, the report from tcga research network independently identified similar groups, using unsupervised clustering analyses of dna mutation, rna expression, dna copy number, and dna methylation data. the integration of genome - wide data from multiple platforms delineated three molecular classes of lower - grade gliomas (grade ii / iii gliomas) that were more concordant with idh, 1p/19q, and tp53 status than with histologic class. this multi - platform approach yielded three groups similar to those initially described by jiao 's model. the large majority of lower - grade gliomas without an idh mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary gbm. the report from mayo clinic and ucsf defined a priori groups that were based on the presence or absence of tert promoter mutations, idh mutations, and 1p/19q codeletion and found consistent associations between the molecular groups and age at diagnosis, survival, patterns of acquired alterations, and germline variants across the three data sets. the group with only tert mutations has a high prevalence of loss of chromosome 4 and acquired pik3ca or pik3r1 mutations. these tests (for idh mutations, 1p/19q codeletion, and tert promoter alterations) can be used to define five principal groups of gliomas with characteristic distributions of age at diagnosis, clinical behavior, acquired genetic alterations, and associated germline variants. over the past decade, insights into the molecular pathology of gliomas have significantly improved both our biological understanding of neoplasms as well as our abilities to diagnose tumors and estimate their prognosis and likelihood of response to specific therapies. to discuss the inclusion of molecular information into the next who classification of cns tumors, a meeting under the sponsorship of the international society of neuropathology (isn) has been held in haarlem, the netherlands. according to the isn - haarlem consensus, integrated diagnosis was established through the usage of atrx, idh1-r132h ihc, 1p/19q analyses, and idh sequencing in the diagnosis of diffuse gliomas. rt plus concomitant and adjuvant tmz chemotherapy is the current standard of care for patients with gbm. several clinical trials have displayed that mgmt promoter methylation correlated with prolonged progression - free and os in patients with gbm receiving alkylating drug chemotherapy. in 2012, two independent randomized trials in elderly patients with gbm assessed rt alone versus tmz chemotherapy alone as an initial treatment. subgroup analyses of both trials showed better outcome for chemotherapy in patients with mgmt promoter methylated tumors but reduced survival in patients with unmethylated tumors. recently, the cgga project delineated that patients with idh wild - type gbm who underwent rt + tmz exhibited significantly longer survival times compared to the patients who were assigned to the rt alone treatment. however, among patients with idh mutation tumors, the survival pattern of patients undergoing rt + tmz or rt was comparable. these results strongly suggest that treatment strategies for elderly patients with gbm should be individualized dependent on idh and mgmt. in addition, due to the high heterogeneity of gbm, each of which may respond differently to one targeted therapy, there has been considerable interest in identifying molecular markers that predict response to a specific molecular targeted therapy. bevacizumab, a monoclonal antibody against vascular endothelial growth factor, being granted approval by the us food and drug administration for treating recurrent gbm in 2009. however, it does not benefit os in either recurrent gbm or newly diagnosed gbm. the presence of egfr overexpression and egfr mutations in gbm could predict the activity of egfr - targeted drugs in patients with these aberrations. however, these potential treatment approaches still have not been clear with contradictory findings in previous clinical trials. it was demonstrated that a point mutation in idh1r132h, expressed in gliomas and other tumors, is presented on human major histocompatibility complex (mhc) class ii and induces a mutation - specific cd4 antitumor t - cell response in patients and a syngeneic tumor model in mhc - humanized mice. conceptually, patients with low - grade and anaplastic gliomas, secondary gbm with a high prevalence of the idh1 (r132h) mutation represent a patient population that may particularly benefit from an idh1r132h specific tumor vaccine. these recurrent genetic aberrations occur in a specific context of cellular origin, co - oncogenic hits and are present in distinct patient populations. primary and secondary gbms are distinct disease entities that affect different age groups of patients and develop through distinct genetic aberrations. these differences are important, especially because they may affect sensitivity to radio- and chemo - therapy and should thus be considered in the identification of targets for novel therapeutic approaches. the biological distinction of gbm subgroups should therefore guide the design of future clinical trials. | objective : to summary the recent advances in molecular research of glioblastoma (gbm) and current trends in personalized therapy of this disease.data sources : data cited in this review were obtained mainly from pubmed in english up to 2015, with keywords molecular, genetics, gbm, isocitrate dehydrogenase, telomerase reverse transcriptase, epidermal growth factor receptor, ptprz1-met, and clinical treatment.study selection : articles regarding the morphological pathology of gbm, the epidemiology of gbm, genetic alteration of gbm, and the development of treatment for gbm patients were identified, retrieved, and reviewed.results:there is a large amount of data supporting the view that these recurrent genetic aberrations occur in a specific context of cellular origin, co - oncogenic hits and are present in distinct patient populations. primary and secondary gbms are distinct disease entities that affect different age groups of patients and develop through distinct genetic aberrations. these differences are important, especially because they may affect sensitivity to radio- and chemo - therapy and should thus be considered in the identification of targets for novel therapeutic approaches.conclusion:this review highlights the molecular and genetic alterations of gbm, indicating that they are of potential value in the diagnosis and treatment for patients with gbm. |
parkinson 's disease (pd) is a frequent neurological disorder of the basal ganglia characterized by the progressive loss of dopaminergic neurons, mainly in the substantia nigra pars compacta (snpc), cytoplasmic inclusions of aggregated proteins, and neuroinflammation [2, 3 ]. several hypotheses have been postulated regarding the possible causes for neuronal degeneration in patients with pd. these include genetic factors, environmental toxins, mitochondrial dysfunction, and free radical - mediated cell death [46 ]. although there is less evidence suggesting that neuroinflammation is the primary trigger causing neurodegeneration, preclinical and epidemiological data now strongly suggest that chronic neuroinflammation may be a slow and steady reason for neuronal dysfunction during the asymptomatic stage of pd. neuroinflammation induced by exposure to either infectious agents or toxicants with proinflammatory characteristics is now increasingly recognized as a major contributor to the pathogenesis of pd. whitton in 1988 initially suggested the involvement of inflammation in pd by describing upregulation of major histocompatibility complex (mhc) molecules in patients with pd. the hallmarks of neuroinflammation are the presence of activated microglia and reactive astrocytes in the parenchyma (neurons, astrocytes, and endothelial cells) of the central nervous system (cns), direct participation of the adaptive immune system, increased production of cytokines, chemokines, prostaglandins, a cascade of complement proteins, and reactive oxygen and nitrogen species (ros / rns), which in some cases can result in disruption of the blood - brain barrier (bbb). the extent to which neuroinflammation and peripheral immune responses contribute towards the development of pd or modify its course is not exactly known. in fact, dysregulation of the neuroimmune system has been postulated by many to be one of the underlying causes of the chronic nature of pd. several lines of evidence support the hypothesis that glial reactive and inflammatory processes participate in the cascade of events leading to neuronal degeneration. one of the earlier studies reporting neuroinflammation in pd involved a quantitative confirmation of the astroglial reaction using glial fibrillary acidic protein (gfap) immunostaining in the substantia nigra (sn) of patients with pd., over two decades ago, first identified significantly increased levels of human leucocyte antigen - dr - positive microglia in the postmortem brains of patients with pd. following these reports, an increased number of activated microglial cells had consistently been reported in the neuroinflammatory pathogenesis of pd. initially, the pathological role played by these glial cells was not fully understood, but activated microglial cells have a deleterious effect on dopaminergic neurons. microglial cells represent resident brain macrophages that are transformed into activated immunocompetent antigen - presenting cells during the pathological process. microglia in pd have been observed to grow densely in the striatum and sn with increased expression of proinflammatory mediators, including tumor necrosis factor alpha (tnf-), interleukin-1 (il-1), il-2, il-4, il-6, transforming growth factor- (tgf-), cyclooxygenase-2 (cox-2), and inducible nitric oxide synthase (inos). tgf-1 and 2 have also been detected by several investigators in the cerebrospinal fluid (csf) and brain parenchyma of patients suffering from pd. taken together, these data indicate that glial cells are one of the most important cells involved in mediating neuroinflammatory events in pd. in addition to glial cells, other cells may also participate in the neuroinflammatory processes in pd. increasing evidence now demonstrates the involvement of both innate and adaptive immune responses in the pathophysiology of pd [1921 ]. innate immunity does not require the presence of a specific antigen to elicit an immune response, whereas adaptive immunity is activated when specific antigens are presented and recognized by lymphocytes. in contrast, endogenous pathological antigens that normally do not occur in physiological conditions may also initiate adaptive immune responses. indeed, hirsch., in their various experiments, reported a small number of cd8-positive t lymphocytes in the vicinity of degenerating neurons in the sn of a patient with pd. in line with this observation, an increased density of interferon-(ifn-) -positive cells with lymphocytes in the sn of patients with pd has also been reported. taken together, these data indicate that injured dopaminergic neurons release immunogenic mediators which have the potential to provoke detrimental innate and adaptive immune responses thereby amplifying the neuroinflammatory process in pd. furthermore, mounting evidence suggests that bbb permeability may be modulated under neuroinflammatory conditions, and trafficking of leukocytes and peripheral macrophages into the brain becomes a normal process that must be tightly regulated to promote brain homeostasis and avoid the neuronal demise [10, 23 ]. these pathomechanisms not only produce complex cross - talk between the peripheral immune system and cns but also highlight the interactions between microglial cells and other brain parenchymal cells. therefore, identifying and understanding the nature and role of the neuroinflammatory mediators involved in the pathogenesis of pd might provide us with various options to target these neuroinflammatory pathways to help curb neuronal death in pd. this review describes various cellular and molecular mediators of neuroinflammation which occur in response to or as part of the ongoing disease progression in pd. glia are composed of three distinct cells types named as microglia, astrocytes, and oligodendrocytes. several populations of macrophages are present in different compartments of healthy brain tissue, each with a distinct phenotype and morphology. the most abundant of these macrophages are the microglia, the resident macrophages of the brain parenchyma. microglia maintains homeostasis and performs immune surveillance by continuously examining their environment by extending cellular protrusions. with a plentitude of ion channels, cytokines, toll - like receptors, and chemokine receptors, microglia promptly reacts in response to subtle alterations in their microenvironment such as alterations in ion homeostasis and brain insults, ranging from aggregated proteins to pathogens. microglial cells are generally quiescent in the normal brain, with their cell bodies barely visible and few detectable fine ramified processes. however, resting microglial cells quickly proliferate, become hypertrophic, and persistently increase expression of a large number of marker molecules such as cd11b, cd68, and mhc - i and ii molecules and are further transformed to macrophage - like cells in patients with pd [11, 29, 30 ]. microglia may be transformed into m1 or m2 macrophages depending upon the type of stimulus [31, 32 ]. it is now apparent that microglia occur in many different phenotypes that can not be readily divided into a small number of discrete subsets following tissue injury. the sn is relatively rich in microglia compared with other brain regions [34, 35 ]. in addition, a reduced level of intracellular glutathione in the sn dopaminergic neurons makes them much more susceptible to a variety of insults, including activated microglial - mediated injury and oxidative stress. this observation indicates that localization of microglia in the sn predisposes dopaminergic neurons to immunological insult in patients with pd. the selective acute degeneration of dopaminergic cells in the sn can be induced by toxins such as 6-hydroxydopamine (6-ohda), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (mptp), and rotenone. in all of these rodent toxin models, dopaminergic cells of the sn a recent study comparing the commonly used medial forebrain or intrastriatal injection of 6-ohda showed rapid degeneration induced by the toxin, which was accompanied by activation of microglia as assessed by the upregulation of the complement type 3 receptor. similarly, mptp - induced neurodegeneration is associated with activated microglia [38, 39 ]. it has become increasingly apparent that there are various triggers through which microglia are activated to elicit their neurotoxic response. interestingly, while these diverse toxins exhibit several mechanisms of microglial activation, toxin - induced activation of nadph oxidase is the most common pathway through which microglia exerts neurotoxicity. apart from this toxin - induced microglial activation, various other triggers involved in microglial activation include immunological insults such as ifn-, lipopolysaccharide (lps), chemokines (ccl5, ccl2, and cxcl10), neurotransmitters, gangliosides, the cd40 ligand, proteases such as thrombin, tissue plasminogen activator, matrix metalloproteinase-3 (mmp-3), endogenous disease proteins, and neuronal injury itself. among these activators, lps - induced neuroinflammation is one of the most accepted and widely used endotoxin models that induces a strong neuroinflammatory response in bv-2 microglial cells [43, 44 ] or when injected directly into the vicinity of the sn. recent findings demonstrate that neurons are not simply passive targets of microglia but rather control microglial activation [3, 46, 47 ]. a variety of signals that neurons use to modulate microglia can be categorized into excitatory and inhibitory signals. inhibitory signals from neurons constitutively maintain microglia in their quiescent state and antagonize proinflammatory activity, whereas excitatory signals are inducible and incite activation of microglia under pathological conditions towards a beneficial or detrimental phenotype. thus, various neuronal signaling molecules actively modulate microglial functions and contribute to the inflammatory milieu of pd. the two subclasses of neuronal inhibitory signals are called released and membrane - bound signals. released inhibitory signals can be cx3cl1, cd22, tgf-, brain derived neurotrophic factor (bdnf), neurotrophin-3, nerve growth factor, or neurotransmitters. the release site for neuronal inhibitory signals is not yet known but is thought to be related to synaptic activity [48, 49 ]. membrane - bound neuronal inhibitory signals consist of several molecules from the immunoglobulin superfamily including cd200, cd22, and cd47, which are expressed or secreted by neurons that bind to receptors on microglia [48, 51, 52 ]. these immunoreceptors, so - called tyrosine - based inhibitory motif receptors, contain a cytoplasmic motif that inhibits activation of microglia. this mechanism of maintaining microglia in their silent state also depends upon the density of the ligands expressed by neurons, which are reduced during pd and will thus shift the level of inhibitory tone to activation in microglia. in parallel with the inhibitory signals, the released excitatory signals control various aspects of microglia function and can be listed as chemokines (cx3cl1, ccl21, and cxcl10) [5355 ], glutamate, purines (atp and utp) [57, 58 ], or mmp-3 [59, 60 ] which modulate various aspects of microglia function. taken together, microglia are maintained in a quiescent state under normal physiological conditions by the orchestrated action of neurons and astrocytes ; however, microglia are rapidly activated when integrity of a neuron is disrupted in pd, probably as a result of both direct activation signals from neurons or loss of inhibitory signals by neurons. while it is clear that microglia becomes activated upon neuronal damage, proteases known to modify the extracellular matrix (ecm) may also be a critical mechanism through which damaged neurons activate microglia to produce extracellular superoxide. earlier reports from chang. emphasized the critical role of ecm proteins in the interactions between microglia and neurons. later, it was found that mmp-3, a proteinase that degrades ecm components, is released following damage to dopaminergic neurons exposed to 1-methyl-4-phenylpyridinium (mpp), exerting neurotoxicity to the dopaminergic neurons. it was also observed that exposure of mesencephalic neuron / glia cultures to mpp results in a dose - dependent increase in mmp-3 protein expression, both in conditioned media and in cell lysates, indicating that death of dopaminergic neurons upregulates mmp-3 expression. these data suggest that mmp-3 is a crucial mediator released upon damage to dopaminergic neurons and that it activates microglia to further propagate neuronal cell death in pd. besides mmp-3, damaged or dying dopaminergic neurons release neuromelanin to activate microglia in the sn of patients with pd [62, 63 ]. neuromelanin has the potential to be neurotoxic, because excess neuromelanin inhibits the function of dopaminergic neurons, proteasomes and induces the production of toxic factors such as tnf-, il-6, and nitric oxide (no) [64, 65 ]. among the proinflammatory mediators released during microglial activation, some act synergistically to produce inflammation - related neuronal damage. hence, the identification of several potential mediators of microglia activation has allowed a general classification of how microglia respond to stimuli. microglia can also be activated by products of the classical complement cascade and by chromogranin a [66, 67 ], which has been reported to occur in the pd sn. among the array of mediators released, superoxide is necessary for both the induction and amplification of neurotoxicity in pd [69, 70 ]. activation of phox results in translocation of its cytosolic subunits to the cellular membrane to form a functional enzyme that not only generates superoxide but also controls the levels of other proinflammatory neurotoxic mediators produced by microglia in pd. it has been revealed that phox is closely paired with mac-1 and plays an important role in microglia - mediated neuroinflammation and neurotoxicity. therefore, coupling between phox and mac-1 might be a central mechanism responsible for the oxidative damage induced by reactive microgliosis that results in progressive neuroinflammation in pd. to date, one of the best elucidated cytotoxic mechanisms induced by proinflammatory cytokines in pd is activation of inos. inos mediates the synthesis of high levels of no, which is toxic to dopaminergic neurons. the density of glial cells expressing inos increases significantly in the sn of patients with pd compared with that in control subjects. the induction of nos produces high levels of no and superoxide radicals for a prolonged period of time. prostaglandins and their synthesizing enzymes, such as cox-2, represent a second group of potential culprits in pd. expression of cox-2 along with the levels of its product, prostaglandin e2 (pge2), increases significantly in glial cells of the snpc, which are responsible for many of the cytotoxic effects to dopaminergic neurons in pd. many reports have demonstrated increased expression of cox-2 in pd [77, 78 ]. in fact, several studies have observed upregulation of cox-2 in animal models of pd [7981 ]. increased cox-2 expression has also been shown in the sn of postmortem pd specimens compared to that in normal controls [75, 82 ]. inhibiting cox-2 [80, 81, 83, 84 ] and transgenic mice lacking cox-2 expression in models of pd has been demonstrated to increase survival of dopaminergic neurons. release of -synuclein (-syn) from neuronal damage could also incite the production of proinflammatory mediators such as pge2 from microglia, thus, contributing to the progression of nigral neurodegeneration. it has recently been observed that modifying ubiquitin carboxy - terminal hydrolase l1 by cyclopentenone prostaglandins causes protein unfolding and aggregation. hence, the deleterious effect of cox-2 in pd could be due to the production of cyclopentenone prostaglandins. although the exact causal link between neuronal injury and microglial activation in pd remains controversial, one of the earliest reported harmful effects demonstrated to cause demise of dopaminergic neurons was microglial - mediated release of proinflammatory cytokines, including ifn- il-1, tnf-, il-2, and il-6 with elevated levels of tnf- receptor r1 (p55), bcl-2, soluble fas, caspase-1 and caspase-3 in postmortem striatum, sn, and csf of patients with pd [89, 90 ]. these cytokines, in turn, propagate and intensify neuroinflammation and cause irreversible destruction of sn dopaminergic neurons by a number of mechanisms, including upregulating isoforms of phospholipases a2, generating platelet - activating factor, stimulating nos, and activating calpain [9, 92 ]. we have briefly summarized some of the major mediators of neuroinflammation during the pathogenesis of pd in figure 1. the brain has long been considered an immune privileged system, as it is protected by the bbb. however, recent findings demonstrate that both the innate and adaptive immune systems play a very critical role disrupting bbb permeability and mediating the pathogenesis of pd via their ability to supply the required signals for antigen presentation and to act as final effectors by t cells [25, 93, 94 ]. for example, infiltration of t cells has been found in the brains of patients with pd as well as significant infiltration of adoptively transferred immune splenocytes into the brains of mptp - intoxicated mice and localization within the inflamed sn. similarly, a recent study of mptp model demonstrated the necessity of t cells to mediate degeneration of dopaminergic neurons and that dopaminergic neuronal loss is exacerbated by t cells. increased mutual coexpression of cd4 and cd8 by cd45r0 t cells with increased expression of cd25, tnf- receptors, and diminished expression of ifn- receptors suggests that subsets of t cell are indeed activated in patients with pd. the influence of infiltrating t cells on dopaminergic neurons has been demonstrated in mice lacking t lymphocytes, wherein death of dopaminergic neurons was significantly attenuated in both types of mice as compared to that in wild - type animals. it was also observed that a subset of cd4 t cells, rather than cd8 t cells, mediate the cytotoxic effects on dopaminergic neurons, as survival of dopaminergic neurons after mptp administration increases in cd4-deficient mice but not in cd8-deficient mice. further analysis showed that cd4 t cells exert their cytotoxicity through the fas - fasl pathway rather than through ifn- secretion. early evidence from the sn by autopsy of patients with pd showed increased numbers of cd8 t cells in close proximity with activated microglia and degenerating neurons. more recently, both cd4 and cd8 t cells have been discovered within the sn of patients with pd. significant level of unrepaired single - strand dna breaks and a number of micronuclei are more also observed in lymphocytes and activated t cells from patients with pd due to inflammation and exposure to ros than those in age - matched controls [100, 101 ]. cd8 t cells were the first type of peripheral t lymphocytes to be located in the postmortem brain from a pd patient. later, infiltration of cd4 and cd8 t cells was found in the sn and striatum of mptp - intoxicated mice [93, 102 ]. a more recent report provided substantial evidence of significant infiltration of cd4 and cd8 t cells in the sn of patients with pd and in mptp - intoxicated mice. increased frequencies of activated cd4 t cells expressing fas, increased ifn--producing th1 cells, decreased il-4-producing th2 cells, and a decrease in cd4, cd25 t cells have been found in the peripheral blood of patients with pd. these data suggest complex roles for cd4 subsets of t cells in mediating the development of pd. compelling evidence suggests the possible involvement of the bbb, including changes in lymphocytic subpopulations in the blood and csf of patients with pd [91, 103 ]. moreover, an increased proportion of -positive lymphocytes, thought to play a role in infections and autoimmunity, have also been reported in the csf and blood of patients with pd. these data suggest that infiltration of immune cells across the bbb into the brain participates in the pathophysiology of pd. this infiltration of peripheral lymphocytes into the brain through the bbb occurs mainly because activated microglia and monocytes in the brains of patients with pd release proinflammatory cytokines and chemokines that act on the vascular endothelium to induce upregulation of cell adhesion molecules, including vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 (icam-1) that disrupt the bbb and attract lymphocytes expressing counterreceptors such as leukocyte function antigen-1 (lfa-1) to the neuronal injury site. activated t cells further release proinflammatory cytokines such as ifn- and tnf-, which positively induce the expression of costimulatory molecules and mhc - ii on microglial cells. when these activated microglial molecules bind to their respective receptors on t cells, they are transformed into effector t cells. among the activated t cells, cd8 t lymphocytes mediate direct cytotoxic lysis of target cells, recruit and activate accessory cells via proinflammatory mediators, whereas activated cd4 t lymphocytes induce b cells to produce high - affinity antibodies. nitrated--syn (n--syn), a misfolded protein present in intraneuronal inclusions or lewy bodies in patients with pd, can be released to the extraneuronal space and cross the bbb to enter the cervical lymph nodes, where it can activate antigen - presenting cells. this phenomenon has also been observed in mptp - intoxicated mice, wherein -syn drains to cervical lymph nodes. as a novel epitope, n--syn can be processed and presented to naive t cells, thus, stimulating them to expand into different subsets of effector t cells. peripherally activated effector t cells, such as th1 or th17 cells, can also cross the bbb and reach inflammatory sites within the brain where they activate microglia and release proinflammatory cytokine il-17 and secrete cytolytic enzymes such as granzyme b. although a dysfunctional bbb in patients with pd may show some leakiness, it may not be sufficient to allow unrestricted lymphocyte infiltration because cd4/cd8 ratios are 1 : 4.8 compared with the typical 2 : 1 ratio expected for peripheral t cells performing surveillance functions. thus, the mechanisms by which these t cells gain access to the sn, their activation state, and their functions are questions that remain to be answered. in summary, infiltrating subsets of t cells may induce excessive microglial - mediated inflammation and oxidative stress that exacerbate neuroinflammation in pd. further study is needed to identify the exact roles of specific subsets of cd4 t cells on the pathogenic progression of pd. studies suggest that the adaptive immune system similar to the innate immune system not only responds to but also actively participates in the pathogenesis of pd. however, more work needs to be done to determine if and how they could serve as a potential target for pd therapy. the majority of findings from research on pd point toward microglia as the major mediator of neuroinflammation, but the astrocytic reaction is another well - known neuropathological characteristic in the sn of patients with pd. astrocytes function as supportive cells for neurons and maintain homeostasis and other neuronal functions. compared to microglia, nonetheless, astrocytes form the glia limitans around blood vessels, preventing entry of immune cells via the bbb into the cns parenchyma. emerging evidence has focused upon the importance of astrocytes in the regulation of neuroinflammation in pd [113, 114 ]. the snpc of many postmortem pd cases had been observed to have an increased number of astrocytes and gfap immunoreactivity with a decrease in glial - derived neurotrophic factor, bdnf, and ciliary neurotrophic factor. the amount of gfap - positive astrocytes is inversely proportional to the demise of dopaminergic neurons, indicating that dopaminergic neurons are more susceptible to the degenerative process wherein there are fewer astrocytes. activation of astrocytes is characterized by the formation of hypertrophic and glial scars, which hinder axonal regeneration, enlarged cell bodies, and projections into the injured area that seem to be mediated by proteoglycans. during inflammatory conditions, astrocyte - derived granulocyte macrophage - colony stimulating factor, il-6, ccl2, and ccl5 regulates migration, activation, and proliferation of microglia. astrocytes may detect neuron - derived -sys as a degenerative marker released by neurons and get activated to protect neurons. however, such reactive astrocytes are exposed to increasing toxicity from -sys oligomers and/or protofibrils, until they no longer serve a protective function. astrocytes play a major role mediating mptp toxicity, as the active metabolite of mptp, mpp is extruded into the extracellular space from astrocytes and further enters into dopaminergic neurons and induces neurotoxicity by inhibiting complex i in the mitochondrial electron transport chain. astrocytic activation parallels the development of dopaminergic cell death in the snpc and striatum, whereas gfap expression remains high even after most dopaminergic neurons have died due to administration of mptp. these findings suggest that the astrocytic reaction occurs after neuronal cell death in pd. in a recent study, it was demonstrated that -syn released from neuronal cells can also be transferred to and accumulate in astrocytes and induce expression of genes associated with immune functions. proinflammatory cytokines that are differentially expressed in astrocytes in response to extracellular -syn include il-1, il-1, il-6, il-18, and colony - stimulating factors-1, 2, and 3, suggesting a strong inflammatory response from astrocytes upon exposure to neuron - derived -syn. exposure to neuron - derived -syn also causes dramatic changes in chemokine expression in astrocytes, including cc - type (ccl-3, 4, 5, 12, 20), cxc - type (cxcl-1, 2, 5, 10, 11, 12, 16), and cx3c - type (cx3cl1) chemokines. these chemokines are involved in a variety of functions, such as recruitment of monocytes and macrophages, migration of microglia and neural progenitors, regulation of microglial activity, proliferation and survival of astrocytes, and synaptic plasticity and transmission. these released chemokines, including cxcl12 and ccl5, induce glutamate release and restart the synthesis of cytokines and chemokines in astrocytes, suggesting their role in glia - glia and glia - neuron communication. other than cytokines, inflammatory oxidants have emerged as key contributors to pd and mptp - related neurodegeneration. in this context, myeloperoxidase (mpo), a key oxidant - producing enzyme, which is mostly expressed by reactive astrocytes during inflammation, is upregulated in the ventral midbrain of human patients with pd and in mptp mice. mpo oxidizes nonreactive nitrite whose concentration is increased in parkinsonism to reactive nitrite (no2) and, thus, nitrosylates many proteins. reactive nitrites also contribute towards the production of the nonradical oxidant, hypochlorous acid (hocl), which can damage macromolecules indirectly by fuelling hydroxyl freeradicals or directly by converting amines into chloramines, phenols, and unsaturated bond chlorination. apart from the direct release of proinflammatory cytokines, including tnf- and il-6, astrocytes can also be activated by cytokines such as il-1 and tnf- released from microglia, thus, producing ros and rns. in support of this observation, a recent study has reported that microglial inflammatory responses are enhanced by astrocytes through a nuclear factor-b - dependent mechanism leading to increased dopaminergic toxicity. astrocytes also produce mediators which play a vital role mediating the inflammatory reaction that occurs in the sn of patients with pd. for example, icam-1-positive astrocytes are seen in the sn in patients with pd and attract reactive microglia to the area because such microglia carries the lfa-1 counterreceptor. the action of -syn on astrocytes is thought to be through receptors, but the identity of these receptors is currently unknown. activated cd4 t cells express and release several inflammatory factors such as the fas ligand, a cell - surface molecule in the tnf- family. fas expression increases in patients with pd and in mice exposed to mptp [129, 130 ]. this fas ligand binds with the fas receptor expressed on astrocytes and causes a release of various cytokines such as il-6 and il-8 and chemokines such as monocyte chemoattractant protein-1. the detrimental consequence of activating the fas - fas ligand pathway in pd has recently been established by several investigators. it has also been reported that mice deficient in fas are more resistant to mptp exposure than wild - type controls. the current literature certainly suggests that astrocytes have the ability to modulate the function and survival of dopaminergic neurons in pd. the complement system is believed to amplify the effectiveness of both the specific and nonspecific immunological defense system. the complement system destroys invading pathogens, encourages inflammation, and supports phagocytosis of waste materials. the complement system has the full ability to recognize molecular patterns associated with injured tissues and dying cells or molecular patterns on pathogens. various complement proteins, mostly present in tissue fluids and blood, are in the form of soluble monomers. one such molecule, which is elevated in the sn of patients with pd, is c - reactive protein. similar evidence about the involvement with the complement system in pd has also been reported wherein membrane attack complex (mac) together with all complement protein components has been identified on oligodendroglia of the sn and intracellularly in lewy bodies of patients with sporadic pd [135, 136 ] and familial pd. additionally, elevated levels of mac, c - reactive protein, and complement 3 have been observed in the sn and csf of patients with pd [138, 139 ]. increased mrna levels of complement components have also been found in affected brain regions of pd models. activation of the complement system leads to a cascade of events ultimately leading to the destruction of cell surface with three different recognized pathways, which share a common juncture at the level of the c3 protein [140, 141 ]. products of the activated complement cascade include opsonizing components (c3b, ic3b, and c4b), which stain material for phagocytosis, mac, and anaphylatoxins (c3a, c4a, and c5a). the opsonins perform a clearance function, whereas anaphylatoxins are involved in generation of the neuroinflammatory response. in contrast, mac induces cell death by entering cell membranes and causing organelles to leak. although mac destroys foreign cells and viruses, nearby host cells are at a significant risk of lysis if they are not protected by mac [135, 140 ]. the complement system also contributes to the secretion of inflammatory cytokines from activated microglial cells. very recent evidence has demonstrated involvement of the complement system in the pathogenesis of pd, wherein the only cells in the sn and other brain areas that express c1q are microglia. one of the important features of pd is that degeneration of dopaminergic neurons is accompanied by the deposition of extracellular neuromelanin. degenerating neurons along with neuromelanin granules are opsonized by c1q and phagocytosed by c1q - positive microglia and macrophages in the perivascular spaces and parenchyma. furthermore, the luminal surfaces of blood vessels in the sn of patients with pd have attached neuromelanin - laden c1q - positive cells. thus, microglia are capable of clearing cellular debris from degenerating neurons of the sn and phagocytosing cells through the c1q - mediated pathway in pd. pentraxin is one of the mediators which activates the complement system by binding to the collagen tail of c1q. pentraxin is an acute - phase protein that is involved in innate immunity and inflammatory response. glial cells may be the major cellular source of this protein in the cns. under the inflammatory milieu of pd, pentraxin proteins secreted by reactive glial cells are detected in the plasma and csf of patients with pd [146, 147 ]. overall, it seems clear that there is a role for the complement system in inflammation - mediated neurodegeneration in pd [138, 140 ] ; hence, research aiming at developing effective inhibitors targeting these sites appears to be worthwhile. pd is one of the most common neurodegenerative diseases with a well - established group of symptoms. although a number of different mechanisms have been considered responsible for the development of pd, none are absolute. growing evidence from patients and experimental models of pd has indicated that neuroinflammation is one of the driving forces in the pathogenesis of pd. the cns had been thought to be an immunologically protected organ, but this notion has now undergone considerable reassessment. it has become apparent from a number of reports that various neuronal injury signals from different neuronal cell types in response to environmental insults, involving many mediators, incite and disseminate the ongoing neuroinflammation in pd. we have summarized the evidence wherein neuroinflammatory mediators play a key role in the pathogenesis of pd. neuroinflammatory mediators have a profound action on cns cells that differently affect the progress of inflammation and neuronal death. therefore, regulating the production of neuroinflammatory mediators or their action on respective receptors would be an effective approach to mitigate the inflammatory processes in pd. thus, further studies are required to form a more comprehensive idea about the role of these neuroinflammatory mediators in pd. furthermore, it is of significant interest for ongoing research to identify and target various neuroinflammatory mediators released in response to various toxins to help explain how neuronal damage can signal inflammation and propagate neuronal cell death. this knowledge might serve to develop pharmacological strategies for treating the neuroinflammation in pd. | neuroinflammation is a host - defense mechanism associated with restoration of normal structure and function of the brain and neutralization of an insult. increasing neuropathological and biochemical evidence from the brains of individuals with parkinson 's disease (pd) provides strong evidence for activation of neuroinflammatory pathways. microglia, the resident innate immune cells, may play a major role in the inflammatory process of the diseased brain of patients with pd. although microglia forms the first line of defense for the neural parenchyma, uncontrolled activation of microglia may directly affect neurons by releasing various molecular mediators such as inflammatory cytokines (tumor necrosis factor-, interleukin [il]-6, and il-1), nitric oxide, prostaglandin e2, and reactive oxygen and nitrogen species. moreover, recent studies have reported that activated microglia phagocytose not only damaged cell debris but also intact neighboring cells. this phenomenon further supports their active participation in self - enduring neuronal damage cycles. as the relationship between pd and neuroinflammation is being studied, there is a realization that both cellular and molecular mediators are most likely assisting pathological processes leading to disease progression. here, we discuss mediators of neuroinflammation, which are known activators released from damaged parenchyma of the brain and result in neuronal degeneration in patients with pd. |
the metastable tc widely applied as radioactive tracer in medical diagnostic procedures currently is mainly obtained from the molybdenum-99 (mo) in its radioactive decay. the mo is produced by irradiation of enriched u with flux of neutrons provided by research reactors. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$^{235}{\text{u } } + { \text{n } } \,{\rightarrow}\,^{99}{\text{mo}}\, { \mathop{\rightarrow}\limits^{\upbeta}}\,^{99\text{m } } { \text{tc } } \,{\mathop{\rightarrow}\limits^{\upgamma}}\,^{99}{\text{tc}}$$\end{document}235u+n99mo99mtc99tc the mo produced in the above reaction is extracted from target and after purification is delivered to hospitals where is used as generator of tc. the reactors used to supply the mo were build 4050 years ago and recently world customers had to face not only planned but as well unexpected shut downs of some of reactors what caused shortages in the mo and thus tc supply. mo, source of tc, can be produced also by neutron capture in mo inserted into the core of a nuclear reactor so this method, although considered as alternative for use of heu, requires reactors as well what is a significant drawback when assessing usefulness of tc production this way. thus, the growing problem with operationality of research reactors (interruptions of their work) stimulated search for alternative ways of tc production either via production of mo or direct production of tc [3, 4 ] although the last solution due to the isotope half - life can be seen as alternative for local supplies only. both isotopes can be produced in accelerators providing protons, deuterons, alpha projectiles using various mo isotopes as targets (table 1), but direct tc production in reaction of mo with protons is considered as the most promising (due to its cross section, production energy range) alternative way of tc production. advantages and drawbacks of this solution are presented in many publications [e.g. 3, 5 ] and they will not be discussed in this paper as it is aside of the work objective.table 1possible accelerator based reactions to produce tc or mo tc mo mo(p,2n)tc mo(p, x)mo mo(d,3n)tc mo(d, x)mo mo(p,)tc mo(,n)mo mo(d, n)tc mo(p,2p)nb mo mo(d,)tc mo(d, p)mo mo(,p)tc mo(,2p)mo zr(,n)mo possible accelerator based reactions to produce tc or mo the excitation function of mo(p,2n)tc reaction has been studied by many authors for decades ([69 ] just to list few) but nevertheless the value of cross section of this reaction is still not well defined. the measured values of the excitation function of the proton - induced reactions on molybdenum obtained by different researchers are presented in fig. 1. as can be seen from the plot, values presented by different authors differ even by factor of 2. it is difficult to point out all sources of this inconsistence but it is not excluded that one of them is related to the fact that in the most cases the cross section studies were completed with natural material. the excitation function expected for isotopically enriched mo was then estimated based on the results obtained for mo (mo14.84 %, mo9.25 %, mo15.92 %, mo16.68 %, mo9.55 %, mo24.13 %, mo9.63 %) [e.g. 8].fig. 1excitation function of the mo(p,2n)tc reaction excitation function of the mo(p,2n)tc reaction this scatter of the cross section values motivated us to study the cross section of the mo(p,2n)tc reaction by direct measurement of the excitation function using targets of the enriched mo to avoid errors resulting from estimation of the cross section for mo based on results obtained with natural mo. isotopically enriched molybdenum is available in powder form and thus studying the excitation function of the discussed reaction required conversion of this material into a foil of relatively low thickness, while studies of the reaction yield require thick targets. there are many methods applied to prepare thick targets of molybdenum on backings for tc production for medical use : electrophoretic deposition plus high temp (1 600 c) sintering in h2 atmosphere (described in), powder pressing as self standing pellet followed by sintering, brazing or pressing into backing, or powder pressing into backing [12, 13],foil forming by direct powder rolling, thermal cladding laser plating, forming low melting mo alloys. electrophoretic deposition plus high temp (1 600 c) sintering in h2 atmosphere (described in), powder pressing as self standing pellet followed by sintering, brazing or pressing into backing, or powder pressing into backing [12, 13 ], foil forming by direct powder rolling, thermal cladding laser plating, forming low melting mo alloys. however, tc production with target in solid metallic form could be favourable considering its better thermal conductivity comparing to powdered targets, what may allow the use of higher beam intensity. taking into account the form of the available enriched mo, our procedure of preparation of the metallic foils consists of powder consolidation by melting and then the bead conversion into a foil by mechanical reshaping. the powdered material in the amount corresponding to the target thickness and its size (up to about 1 300 mg) was pelletized with use of a die allowing the air removal during pellet forming (fig. 2), and a hydraulic press.fig. 2the pellet die with air evacuation option the pellet die with air evacuation option the obtained pellet was melted into a droplet in the vacuum of ~10 mbar with e - beam gun. before reaching the melting temperature, pellet was carefully heated with e - beam, both for outgassing, i.e. removing the air residual, and evaporating the molybdenum oxide (tevap = ~1 155 c). the e - beam intensity was increased gradually until stable pressure of ~10 mbar was reached. only then the e - beam intensity was increased to melt the mo pellet into a droplet. in case of thicker pellets only the upper part was melted in the first run and formation of droplet was completed after breaking the vacuum and turning the half melted pellet upside - down (fig. 3partially melted molybdenum pellet made of 1 350 mg of the mo powder partially melted molybdenum pellet made of 1 350 mg of the mo powder further re - melting of the received droplet is required to prepare a bead of good for rolling quality (smooth, without deformation that can act as a starting point of droplet cracking when rolled). re - melting of the material has to be done with changing of its position in the crucible of e - beam gun, i.e. turning the bead to expose each side to the electron beam. it is important especially in case of droplets made of big amount of material (few hundreds milligram). droplet produced by powder melting was placed between stainless steel sheets (rolling pack) and passed through the rolling mill. the applied rolling speed was of about 10 rpm (125 cm min) and thickness reduction was not greater than 45 m at the initial steps irrespective the size of the droplet / disc. higher reduction of the thickness would result with inevitable droplet crack at first pass through the rolling mill (as reported by and others), see fig. 4.fig. 4molybdenum bead after first pass through the rolling mill set too tightly molybdenum bead after first pass through the rolling mill set too tightly below 0.5 mm the thickness reduction was not bigger than ~2.5 m, otherwise the rolled material emerged as disc / foil with many cracks or as small, inutile pieces, too small to produce even the thin (10 m) foils. during rolling process material, after each change of the rollers distance, was passed 45 times through rolling mill. to remove stresses from the rolled foils they were annealed in vacuum for ~1015 min at temperature of ~1 200 c. the influence of the annealing on the foils properties can be seen in fig. 5the 10 m thick mo foils prepared with (left) and without (right) annealing. not only the foil bending but as well the small cracks on the edge of not annealed foil can be observed the 10 m thick mo foils prepared with (left) and without (right) annealing. not only the foil bending but as well the small cracks on the edge of not annealed foil can be observed described procedure allows production of thin (10 m) foils. the production of sufficient area of these foils (to prepare stacked foil target composed of 10 mo pieces) took about 1 week of the whole day work. annealing useful at preparation of thin foils (below 100 m) was not significantly helpful in production of thick ones (400600 m). the amount of cracks was lower but, when appearing, they propagated through the foil area preventing production of the foil of the required size (fig. 6example of crack passing through the disc of ~1 mm thick example of crack passing through the disc of ~1 mm thick lipski suggests that slow reduction of the e - beam intensity should reduce the stresses in the material and decrease crackability but such relation was not observed in case when big droplets needed for thick target preparation were produced. slow cooling of big droplets, as mainly described in this work, resulted with brittle material. n. y. kheswa in her paper reports production of malleable, not cracking molybdenum droplet just by thorough melting but the amount of material used in (only 75 mg of the starting amount) is incomparably smaller than the amount required by our needs (one target of 1.4 cm 1.4 cm of 600 m thick requires ~1 3001 400 mg of molybdenum). thorough melting, at a single run, of the amount of mo as used by kheswa seems to be easier. the cold flattening recommended by k. zell, applied by him to the droplet of ~2 mm in diameter most probably does not stress the material at the same level as in case of droplet of 67 mm in diameter made of 1 3001 400 mg of starting amount of mo. substantial material loss (40 %) reported in is not acceptable as well in case of thick targets of expensive material such as mo. there is also no information on thickness and size of the produced foils so the final result can not be compared to our work. expecting improvement of the purity of the melted material, and thus its malleability, the mo powder was heated in the reducing atmosphere (1 h at 1 600 c at h2 atmosphere) for removing the oxide residues before pellet forming. at other approach the pellet was sintered under mentioned condition but no improvement of the molybdenum malleability was observed. on the contrary, the droplet resulting from the pre - treated powder was less malleable. 7 shows the foil prepared using the droplet produced from the powder sintered in the above listed condition.fig. 7the 80 m thick foil produced from the droplet obtained by melting the mo pellet beforehand sintered at 1 600 c under the hydrogen atmosphere the 80 m thick foil produced from the droplet obtained by melting the mo pellet beforehand sintered at 1 600 c under the hydrogen atmosphere to produce thick foils, the relatively big droplets (67 mm diameter) were flattened in high temperature before rolling. molybdenum, oxygen resistant metal at ambient temperature, oxidises easily at temperature above 600 c. to protect molybdenum from oxidation at elevated temperature, the mo droplet was packed into the stainless steel packet (envelope) under argon atmosphere (fig. 8a, b).fig. 8to flatten the mo droplet in the high temperature, droplet prepared by powder melting was packed into the stainless steel envelope (a) and sintered tightly under the argon atmosphere (b). the packet was heated up to 1 100 c and when hot, pressed under the hydraulic press (c) to flatten the mo droplet in the high temperature, droplet prepared by powder melting was packed into the stainless steel envelope (a) and sintered tightly under the argon atmosphere (b). the packet was heated up to 1 100 c and when hot, pressed under the hydraulic press (c) the packed droplets of ~67 mm in diameter were heated at temperature of 1 100 c for 35 min and when hot were flattened with the use of hydraulic press as quickly as possible to preserve the high temperature (see fig. the height of the droplet and further of the disc was reduced by 2025 % at initial steps and by ~15 % in consecutive steps until disc was about 11.5 mm thick. example of the forces used for flattening is given in the table 2.table 2examples of droplets reshaped by hot flatteningsample no ; initial amount (% material loss at melting)pressure (bar)commentssample 6/1 mo1 041.46 mg(~20 %) 1st 602nd 803rd 130size after hot press : 7 7.5 2.5 mm, slightly oxidised, after oxide removal by e - beam heating, additional cold press was applied : 8.65 8.35 1.81.4 mm (small crack on the rim)part of this sample was rolled down to 250 nm with thickness reduction by 2.53 msample 8/3 mo708.73 mg(~15 %) 1st 1402nd 1803rd 180no visible oxidation ; only hot press, appears to be easier to roll than sample 6/1 but final thickness similar to sample 6/1, ~280 nmsample e4/1 mo1 387.182 1.51.65 mm, size : 0.95 1 cm, very malleable, rolled only down to required target thickness i.e. 600 m, the foil of 600 m (1.5 2.5 cm) obtained with only one crack on the edgesample e5/2 mo1 337.494 mg(~15 %) 1st 1402nd + 3rd 1804th 2005th 220only hot press ; thickness : 1.51.3 mm, size : 1 1.2 cm, cracking easier than e4/1, rolled down to 410 m (was there too high force at flattening ?) examples of droplets reshaped by hot flattening after last flattening, the packet was left under argon atmosphere for cooling down. when cold, disc was removed from the envelope and rolled down to the required thickness of few hundreds micrometer. the fig. 9 shows the foil of 320 m with crack free area (~1.5 cm 1.6 cm) sufficient for the target. but as can be seen in table 2 (sample e4/1), foils of 600 m of bigger area (1.5 cm 2.5 cm) with only single crack, 23 mm long, were prepared from later produced droplets of mo.fig. 9 mo after hot flattening and cold rolling down to 320 m mo after hot flattening and cold rolling down to 320 m the foil was prepared from droplet of 7 mm diameter. the upper part of the presented foil was used to produce thinner, 10 m thick foils needed to build stacked foil target. the powdered material in the amount corresponding to the target thickness and its size (up to about 1 300 mg) was pelletized with use of a die allowing the air removal during pellet forming (fig. 2), and a hydraulic press.fig. 2the pellet die with air evacuation option the pellet die with air evacuation option the obtained pellet was melted into a droplet in the vacuum of ~10 mbar with e - beam gun. before reaching the melting temperature, pellet was carefully heated with e - beam, both for outgassing, i.e. removing the air residual, and evaporating the molybdenum oxide (tevap = ~1 155 c). the e - beam intensity was increased gradually until stable pressure of ~10 mbar was reached. only then the e - beam intensity was increased to melt the mo pellet into a droplet. in case of thicker pellets only the upper part was melted in the first run and formation of droplet was completed after breaking the vacuum and turning the half melted pellet upside - down (fig. 3partially melted molybdenum pellet made of 1 350 mg of the mo powder partially melted molybdenum pellet made of 1 350 mg of the mo powder further re - melting of the received droplet is required to prepare a bead of good for rolling quality (smooth, without deformation that can act as a starting point of droplet cracking when rolled). re - melting of the material has to be done with changing of its position in the crucible of e - beam gun, i.e. turning the bead to expose each side to the electron beam. it is important especially in case of droplets made of big amount of material (few hundreds milligram). droplet produced by powder melting was placed between stainless steel sheets (rolling pack) and passed through the rolling mill. the applied rolling speed was of about 10 rpm (125 cm min) and thickness reduction was not greater than 45 m at the initial steps irrespective the size of the droplet / disc. higher reduction of the thickness would result with inevitable droplet crack at first pass through the rolling mill (as reported by and others), see fig. 4molybdenum bead after first pass through the rolling mill set too tightly molybdenum bead after first pass through the rolling mill set too tightly below 0.5 mm the thickness reduction was not bigger than ~2.5 m, otherwise the rolled material emerged as disc / foil with many cracks or as small, inutile pieces, too small to produce even the thin (10 m) foils. during rolling process material, after each change of the rollers distance, was passed 45 times through rolling mill. to remove stresses from the rolled foils they were annealed in vacuum for ~1015 min at temperature of ~1 200 c. the influence of the annealing on the foils properties can be seen in fig. 5the 10 m thick mo foils prepared with (left) and without (right) annealing. not only the foil bending but as well the small cracks on the edge of not annealed foil can be observed the 10 m thick mo foils prepared with (left) and without (right) annealing. not only the foil bending but as well the small cracks on the edge of not annealed foil can be observed described procedure allows production of thin (10 m) foils. the production of sufficient area of these foils (to prepare stacked foil target composed of 10 mo pieces) took about 1 week of the whole day work. annealing useful at preparation of thin foils (below 100 m) was not significantly helpful in production of thick ones (400600 m). the amount of cracks was lower but, when appearing, they propagated through the foil area preventing production of the foil of the required size (fig. 6example of crack passing through the disc of ~1 mm thick example of crack passing through the disc of ~1 mm thick lipski suggests that slow reduction of the e - beam intensity should reduce the stresses in the material and decrease crackability but such relation was not observed in case when big droplets needed for thick target preparation were produced. slow cooling of big droplets, as mainly described in this work, resulted with brittle material. n. y. kheswa in her paper reports production of malleable, not cracking molybdenum droplet just by thorough melting but the amount of material used in (only 75 mg of the starting amount) is incomparably smaller than the amount required by our needs (one target of 1.4 cm 1.4 cm of 600 m thick requires ~1 3001 400 mg of molybdenum). thorough melting, at a single run, of the amount of mo as used by kheswa seems to be easier. the cold flattening recommended by k. zell, applied by him to the droplet of ~2 mm in diameter most probably does not stress the material at the same level as in case of droplet of 67 mm in diameter made of 1 3001 400 mg of starting amount of mo. substantial material loss (40 %) reported in is not acceptable as well in case of thick targets of expensive material such as mo. there is also no information on thickness and size of the produced foils so the final result can not be compared to our work. expecting improvement of the purity of the melted material, and thus its malleability, the mo powder was heated in the reducing atmosphere (1 h at 1 600 c at h2 atmosphere) for removing the oxide residues before pellet forming. at other approach the pellet was sintered under mentioned condition but no improvement of the molybdenum malleability was observed. on the contrary, the droplet resulting from the pre - treated powder was less malleable. 7 shows the foil prepared using the droplet produced from the powder sintered in the above listed condition.fig. 7the 80 m thick foil produced from the droplet obtained by melting the mo pellet beforehand sintered at 1 600 c under the hydrogen atmosphere the 80 m thick foil produced from the droplet obtained by melting the mo pellet beforehand sintered at 1 600 c under the hydrogen atmosphere to produce thick foils, the relatively big droplets (67 mm diameter) were flattened in high temperature before rolling. molybdenum, oxygen resistant metal at ambient temperature, oxidises easily at temperature above 600 c. to protect molybdenum from oxidation at elevated temperature, the mo droplet was packed into the stainless steel packet (envelope) under argon atmosphere (fig. 8a, b).fig. 8to flatten the mo droplet in the high temperature, droplet prepared by powder melting was packed into the stainless steel envelope (a) and sintered tightly under the argon atmosphere (b). the packet was heated up to 1 100 c and when hot, pressed under the hydraulic press (c) to flatten the mo droplet in the high temperature, droplet prepared by powder melting was packed into the stainless steel envelope (a) and sintered tightly under the argon atmosphere (b). the packet was heated up to 1 100 c and when hot, pressed under the hydraulic press (c) the packed droplets of ~67 mm in diameter were heated at temperature of 1 100 c for 35 min and when hot were flattened with the use of hydraulic press as quickly as possible to preserve the high temperature (see fig. the height of the droplet and further of the disc was reduced by 2025 % at initial steps and by ~15 % in consecutive steps until disc was about 11.5 mm thick. example of the forces used for flattening is given in the table 2.table 2examples of droplets reshaped by hot flatteningsample no ; initial amount (% material loss at melting)pressure (bar)commentssample 6/1 mo1 041.46 mg(~20 %) 1st 602nd 803rd 130size after hot press : 7 7.5 2.5 mm, slightly oxidised, after oxide removal by e - beam heating, additional cold press was applied : 8.65 8.35 1.81.4 mm (small crack on the rim)part of this sample was rolled down to 250 nm with thickness reduction by 2.53 msample 8/3 mo708.73 mg(~15 %) 1st 1402nd 1803rd 180no visible oxidation ; only hot press, appears to be easier to roll than sample 6/1 but final thickness similar to sample 6/1, ~280 nmsample e4/1 mo1 387.182 : 1.51.65 mm, size : 0.95 1 cm, very malleable, rolled only down to required target thickness i.e. 600 m, the foil of 600 m (1.5 2.5 cm) obtained with only one crack on the edgesample e5/2 mo1 337.494 mg(~15 %) 1st 1402nd + 3rd 1804th 2005th 220only hot press ; thickness : 1.51.3 mm, size : 1 1.2 cm, cracking easier than e4/1, rolled down to 410 m (was there too high force at flattening ?) examples of droplets reshaped by hot flattening after last flattening, the packet was left under argon atmosphere for cooling down. when cold, disc was removed from the envelope and rolled down to the required thickness of few hundreds micrometer. the fig. 9 shows the foil of 320 m with crack free area (~1.5 cm 1.6 cm) sufficient for the target. but as can be seen in table 2 (sample e4/1), foils of 600 m of bigger area (1.5 cm 2.5 cm) with only single crack, 23 mm long, were prepared from later produced droplets of mo.fig. 9 mo after hot flattening and cold rolling down to 320 m mo after hot flattening and cold rolling down to 320 m the foil was prepared from droplet of 7 mm diameter. the upper part of the presented foil was used to produce thinner, 10 m thick foils needed to build stacked foil target. droplet produced by powder melting was placed between stainless steel sheets (rolling pack) and passed through the rolling mill. the applied rolling speed was of about 10 rpm (125 cm min) and thickness reduction was not greater than 45 m at the initial steps irrespective the size of the droplet / disc. higher reduction of the thickness would result with inevitable droplet crack at first pass through the rolling mill (as reported by and others), see fig. 4molybdenum bead after first pass through the rolling mill set too tightly molybdenum bead after first pass through the rolling mill set too tightly below 0.5 mm the thickness reduction was not bigger than ~2.5 m, otherwise the rolled material emerged as disc / foil with many cracks or as small, inutile pieces, too small to produce even the thin (10 m) foils. during rolling process material, after each change of the rollers distance, was passed 45 times through rolling mill. to remove stresses from the rolled foils they were annealed in vacuum for ~1015 min at temperature of ~1 200 c. the influence of the annealing on the foils properties can be seen in fig. 5the 10 m thick mo foils prepared with (left) and without (right) annealing. not only the foil bending but as well the small cracks on the edge of not annealed foil can be observed the 10 m thick mo foils prepared with (left) and without (right) annealing. not only the foil bending but as well the small cracks on the edge of not annealed foil can be observed described procedure allows production of thin (10 m) foils. the production of sufficient area of these foils (to prepare stacked foil target composed of 10 mo pieces) took about 1 week of the whole day work. annealing useful at preparation of thin foils (below 100 m) was not significantly helpful in production of thick ones (400600 m). the amount of cracks was lower but, when appearing, they propagated through the foil area preventing production of the foil of the required size (fig. 6example of crack passing through the disc of ~1 mm thick example of crack passing through the disc of ~1 mm thick lipski suggests that slow reduction of the e - beam intensity should reduce the stresses in the material and decrease crackability but such relation was not observed in case when big droplets needed for thick target preparation were produced. slow cooling of big droplets, as mainly described in this work, resulted with brittle material. n. y. kheswa in her paper reports production of malleable, not cracking molybdenum droplet just by thorough melting but the amount of material used in (only 75 mg of the starting amount) is incomparably smaller than the amount required by our needs (one target of 1.4 cm 1.4 cm of 600 m thick requires ~1 3001 400 mg of molybdenum). thorough melting, at a single run, of the amount of mo as used by kheswa seems to be easier. the cold flattening recommended by k. zell, applied by him to the droplet of ~2 mm in diameter most probably does not stress the material at the same level as in case of droplet of 67 mm in diameter made of 1 3001 400 mg of starting amount of mo. substantial material loss (40 %) reported in is not acceptable as well in case of thick targets of expensive material such as mo. there is also no information on thickness and size of the produced foils so the final result can not be compared to our work. expecting improvement of the purity of the melted material, and thus its malleability, the mo powder was heated in the reducing atmosphere (1 h at 1 600 c at h2 atmosphere) for removing the oxide residues before pellet forming. at other approach the pellet was sintered under mentioned condition but no improvement of the molybdenum malleability was observed. on the contrary, the droplet resulting from the pre - treated powder was less malleable. 7 shows the foil prepared using the droplet produced from the powder sintered in the above listed condition.fig. 7the 80 m thick foil produced from the droplet obtained by melting the mo pellet beforehand sintered at 1 600 c under the hydrogen atmosphere the 80 m thick foil produced from the droplet obtained by melting the mo pellet beforehand sintered at 1 600 c under the hydrogen atmosphere to produce thick foils, the relatively big droplets (67 mm diameter) were flattened in high temperature before rolling. molybdenum, oxygen resistant metal at ambient temperature, oxidises easily at temperature above 600 c. to protect molybdenum from oxidation at elevated temperature, the mo droplet was packed into the stainless steel packet (envelope) under argon atmosphere (fig. 8a, b).fig. 8to flatten the mo droplet in the high temperature, droplet prepared by powder melting was packed into the stainless steel envelope (a) and sintered tightly under the argon atmosphere (b). the packet was heated up to 1 100 c and when hot, pressed under the hydraulic press (c) to flatten the mo droplet in the high temperature, droplet prepared by powder melting was packed into the stainless steel envelope (a) and sintered tightly under the argon atmosphere (b). the packet was heated up to 1 100 c and when hot, pressed under the hydraulic press (c) the packed droplets of ~67 mm in diameter were heated at temperature of 1 100 c for 35 min and when hot were flattened with the use of hydraulic press as quickly as possible to preserve the high temperature (see fig. the height of the droplet and further of the disc was reduced by 2025 % at initial steps and by ~15 % in consecutive steps until disc was about 11.5 mm thick. example of the forces used for flattening is given in the table 2.table 2examples of droplets reshaped by hot flatteningsample no ; initial amount (% material loss at melting)pressure (bar)commentssample 6/1 mo1 041.46 mg(~20 %) 1st 602nd 803rd 130size after hot press : 7 7.5 2.5 mm, slightly oxidised, after oxide removal by e - beam heating, additional cold press was applied : 8.65 8.35 1.81.4 mm (small crack on the rim)part of this sample was rolled down to 250 nm with thickness reduction by 2.53 msample 8/3 mo708.73 mg(~15 %) 1st 1402nd 1803rd 180no visible oxidation ; only hot press, appears to be easier to roll than sample 6/1 but final thickness similar to sample 6/1, ~280 nmsample e4/1 mo1 387.182 mg(~13 %) 1st 1202nd 1603rd 1804th 180only hot press, thick. : 1.51.65 mm, size : 0.95 1 cm, very malleable, rolled only down to required target thickness i.e. 600 m, the foil of 600 m (1.5 2.5 cm) obtained with only one crack on the edgesample e5/2 mo1 337.494 mg(~15 %) 1st 1402nd + 3rd 1804th 2005th 220only hot press ; thickness : 1.51.3 mm, size : 1 1.2 cm, cracking easier than e4/1, rolled down to 410 m (was there too high force at flattening ?) examples of droplets reshaped by hot flattening after last flattening, the packet was left under argon atmosphere for cooling down. when cold, disc was removed from the envelope and rolled down to the required thickness of few hundreds micrometer. the fig. 9 shows the foil of 320 m with crack free area (~1.5 cm 1.6 cm) sufficient for the target. but as can be seen in table 2 (sample e4/1), foils of 600 m of bigger area (1.5 cm 2.5 cm) with only single crack, 23 mm long, were prepared from later produced droplets of mo.fig. 9 mo after hot flattening and cold rolling down to 320 m mo after hot flattening and cold rolling down to 320 m the foil was prepared from droplet of 7 mm diameter. the upper part of the presented foil was used to produce thinner, 10 m thick foils needed to build stacked foil target. big molybdenum beads (67 mm in diameter made of more than 1 g of the material), prepared for rolling by powder melting with e - beam gun and hot flattening of the received droplet, demonstrated better malleability than only thoroughly melted material. it was possible to produce the thin foils in much shorter time than in the case of material prepared by melting only. the thickness reduction per pass was of similar value but number of passes per reduced thickness required to get no size changeable foil significantly dropped down. described procedure allows not only production of thick foils free from cracks but makes also possible to produce the thin foils of big area (fig. 10the 4.55.5 m foil of 3 cm 15 cm produced from natural mo the 4.55.5 m foil of 3 cm 15 cm produced from natural mo the thinnest foil produced at this work was of ~250 nm (thickness measured by alpha particle energy loss method). below this thickness the material starts sticking to the rolling pack and tries to further reduce the foil thickness were not undertaken. the main aim of this work was to develop the procedure of production of thick (few hundreds micrometer) and thin (10 m) mo foils / plates of area of ~1.5 1.5 cm, thus the possibilities of further thinning of the foil were not investigated. it is not excluded that an application of anti - adhesive agent such as e.g. teflon as rolling pack lining would allow reduction of the foil thickness. the hot reshaping of the mo droplet in the way described above, applied before cold rolling, is relatively simple. the mo material after cooling down can be easily removed from the envelope and sticking to the stainless steel as reported by karasek at hot rolling applied by him was not observed. | targets required to determine the parameters of the 100mo(p, xn)99mtc reaction and to estimate the yield of the 99mtc production were prepared starting with powder material. material, melted with electron beam gun into solid bead, was reshaped into foil mechanically. targets were prepared by powder melting and hot flattening of the droplet followed by cold rolling. procedure allowed preparation of thick (in the range of hundreds of microns) and thin (down to 250 nm) foils. |
psoriasis is a chronic systematic inflammatory disease which is connected with the metabolic syndrome (1). metabolic syndrome which is also called syndrome of insulin resistance, or syndrome x, is a combination of factors of cardio vascular risk, including central obesity, increased blood pressure, glucose intolerance and dyslipidemia (2). although the connection between psoriasis and metabolic syndrome is known, what is trying to be discovered is the precise mechanism of that connection, that is, precise pathogen mechanisms. it is considered that a chronic inflammation and inflammatory mediators are the initiators for the development of metabolic syndrome. a term psoriasis march is described where the state of chronic inflammatory in the patient affected by psoriasis leads to insulin resistance, which leads to endothelial dysfunction and atherosclerosis, and consequently to myocardial infarction and stroke (3). in those affected by psoriasis, inflammatory cytokines, tnf, il-1 and il-6 they can have a wide specter of effects on insulin signalization, metabolism of lipids and adipogenesis. besides that, inflammatory induced resistance to insulin can lead to a development of systematic resistance to insulin (5). cytokines which are important in pathogenesis of psoriasis il-1, il-4, il-6, il-8, il-12 and tnf are also identified in those affected by metabolic syndrome (6). similarly to psoriasis, metabolic syndrome is characterized by increase of immunologic activity type 1 of helping t lymphocytes (7). psors2, psors3 and psors4 are related to psoriasis and they are also connected with metabolic ailments (4). it is considered that next to the stated role of chronic inflammation, genetic factors also play a role in the development of metabolic syndrome in those affected by psoriasis, a significant role is also played by life style, including improper nutrition, physical inactivity, smoking and consummation of alcohol, stress, which leads to obesity and development of metabolic syndrome (8,9). importance of metabolic syndrome is also in its frequency in those affected by psoriasis compared to the general population. many, differently designed studies have shown connection between psoriasis and metabolic syndrome. compared to the general population, those affected by psoriasis have a higher frequency of metabolic syndrome, and patients with a more severe form of psoriasis have greater odds at developing metabolic syndrome, compared to those with mild psoriasis (10). in usa, patients affected by psoriasis have nearly double prevalence of metabolic syndrome. based on this data, it is estimated that from 6,6 millions of adults affected by psoriasis, age ranging from 20 to 59 in usa, 2,7 million of them have metabolic syndrome, or nearly a million more people than expected from individuals without psoriasis (11). the aim of the research is to determine the frequency between metabolic syndrome in those affected by psoriasis, and connection between severity of psoriasis and occurrence of metabolic syndrome. it enveloped 70 consecutively chosen patients, affected by psoriasis, both genders, over the age of 18. average age being 47,14 (sd= 15,41), from that there were 36 men, or 51,43 % and 34 women or 48,57%. research also included patients with documented cases of diagnosed psoriasis, ambulatory or hospital reports from dermatologist, and pathophysiological report, of those who were treated at the clinic for skin diseases of university clinical center tuzla. research did not include patients which besides psoriasis suffered from some other skin disease, and those which suffered from psoriasis for less than a year. research has been approved from the side of ethics committee of university clinical center tuzla. firstly, all examinees were explained the purpose of the research, asked for a written consent to participate in the research, taken anamnesis, got their skin and visible mucous membranes dermatologically tested and had their pasi score values determined, after which they were subjected to the research, it was established if they suffered from metabolic syndrome. for diagnosing the metabolic syndrome, national cholesterol education program adult treatment panel iii (ncep atp iii) was used, and metabolic syndrome was diagnosed in the presence of 3 or more criteria. central obesity;waist line (by ethnically affiliation)women > 80 cmmen > 94 (for europe)increased level of triglyceride150 mg / dl (1,7 mmol / l)or treatment of increased triglycerideslowered hdl - cholesterol 130 mmhgdiastolic > 85 mmhgor treatment of previously diagnosed high blood pressure.increased glucose starvationglucose in plasma > 100mg / dl (5,6 mmol / l)or previously diagnosed diabetes type 2. waist line (by ethnically affiliation) men > 94 (for europe) increased level of triglyceride 150 mg / dl (1,7 mmol / l) or treatment of increased triglycerides lowered hdl - cholesterol 100mg / dl (5,6 mmol / l) or previously diagnosed diabetes type 2. for determining the severity and outspread of psoriasis, psoriasis area and severity index (pasi) was used. in four regions body - head, torso, upper and lower extremities. in the four regions of the body - head, torso, upper and lower extremities evaluated the characteristics of the disease, severity of erythema, infiltration and desquamation with the score 1 - 4, and the affected area of skin psoriatic changes with the score 1 - 6 (table 1). in assessing the severity of erythema scales we calculate : buttocks as a part of lower extremities, that is, region of the leg (l)armpits and shoulders as a part of upper extremities, that is, region of the arm (a)the neck is calculated as a part of the head (h)torso (t) buttocks as a part of lower extremities, that is, region of the leg (l) armpits and shoulders as a part of upper extremities, that is, region of the arm (a) the neck is calculated as a part of the head (h) for calculating the pasi, the summation of erythemas, infiltrations and desquamations of single region is multiplied with the numerical value of the region of the body and with the percentage by which the lesions have spread at a single region. form for calculating pasi pasi=0,1x(eh+ih+dh)xah+0,3x(et+it+dt)xat+0,2x-(ea+ia+da)xaa+0,4x(el+il+dl)xal legend : e- erythema ; i infiltration ; d desqumation ; a area ; h head ; t torso ; a -arm ; l leg for data processing and hypothesis testing was used statistical package arcus quickstat biomedical version 1.0. the relationship between variables was estimated correlation test. as the minimum value of the level of significance was taken as p < 0,05. frequency of metabolic syndrome in those affected by psoriasis was 38,57%, average age of those affected by metabolic syndrome was 55,84 years. (sd= 9,61), and average duration of basic disease was 19,09 years (sd= 12,46). clinical characteristics of the patients were estimated based on the index of severity of psoriasis on the affected area (pasi). although the exact mechanism of connection between psoriasis and metabolic syndrome is not determined, an increased occurrence in metabolic syndrome in those affected by psoriasis has been detected. metabolic syndrome which is defined as a presence or treatment of at least three of the following five categories : increased blood pressure, insulin resistance, lowered hdl cholesterol, hypertriglyceridemia, and central obesity. frequency of metabolic syndrome in this research was 38,57% and results were similar to those from other studies. more studies have found an increased frequency of metabolic syndrome in those affected by psoriasis, compared to the control group. so in research conducted in india, metabolic syndrome was present in around 30% of those affected by psoriasis and was around three times more frequent compared to the control group (13). in research conducted in our country, in mostar, in 60 people affected by psoriasis occurrence of metabolic syndrome was 46,7% (14). this research had a positive correlation between the severity of psoriasis, as measured by pasi score and metabolic syndrome (r=0,35, p=0,0001), the higher the pasi, the higher the occurrence of metabolic syndrome. as far the relation between severity of psoriasis and metabolic syndrome, studies have shown different results. in research conducted in great britain, which included over 4.000 people affected by psoriasis, metabolic syndrome was more often present in more severe cases of psoriasis (32% had a metabolic syndrome with mild psoriasis, 36% with medium - severe psoriasis and 40% with severe psoriasis) (15). also, in a research conducted by sommer. (16), severity of psoriasis was connected with prevalence of metabolic syndrome, that an increased occurrence of metabolic syndrome in the patients with mild and severe form of psoriasis. and in the research conducted in italy, there was no connection between the severity of psoriasis and occurrence of metabolic syndrome. patients with psoriasis and metabolic syndrome were older compared to the average age of all examinees (55,84 ; sd = 9,61 compared to 47,14 ; sd=15,41) and they also carried the disease for a somewhat longer period of time 19,0 years (sd=12,46) compared to the average duration of psoriasis which is 15,52 years (sd= 12,54). in research in italy, metabolic syndrome was more often present in those affected by psoriasis which were over 40 years of age, and those affected with psoriasis and metabolic syndrome were older and carried psoriasis for a longer period of time compared to those that were affected by psoriasis without metabolic syndrome (17). in a research conducted by nisa and qazi (18) metabolic syndrome was found in 28% of those affected by psoriasis, and in the control group the number was 6%. those affected by psoriasis with metabolic syndrome had psoriasis for a longer period of time 13,67 11,87 years, compared to 6,46 5,80 years of those who were affected by psoriasis without metabolic syndrome. in korea, those affected with psoriasis with metabolic syndrome were older than those affected with psoriasis without metabolic syndrome (53,3 years compared to 39,5 years, p < 0,001), and they also had a more severe form of the disease, that is, age and severity of the disease were a risk factor for development of metabolic syndrome (19). so, psoriasis is related to metabolic syndrome, and the severity of psoriasis, age, and the length from which the patient has suffered from psoriasis all contributed to the occurrence of metabolic syndrome in those affected by psoriasis. psoriasis is connected with metabolic syndrome, and further research of the relation between them is necessary, in the goal of prevention, screening and treatment of metabolic syndrome in those affected by psoriasis. | introduction : psoriasis is a chronic skin ailment which can be connected with an increased occurrence of other illnesses, including the metabolic syndrome.examinees and methods : a prospective study has been conducted which included 70 patients affected by psoriasis, both genders, older than 18 years. average age being 47,14 (sd=15,41) years, from that there were 36 men or 51,43 and 34 women or 48,57%. the average duration of psoriasis was 15,52 (sd= 12,54) years. for purposes of diagnosing the metabolic syndrome, the criteria of national cholesterol education program adult treatment panel iii, (ncep atp iii) were used. for purposes of detecting the severity and spread of psoriasis, psoriasis area and severity index (pasi) was used.results:the incidence of metabolic syndrome in patients with psoriasis was 38,57%. average values of pasi score were 16,65. the increase in values of pasi score and metabolic syndrome were statistically highly connected. (r=0,3, p=0,0001).conclusion : psoriasis is connected with metabolic syndrome, there is a positive correlation between the severity of psoriasis and frequency of metabolic syndrome. |
obstructive fibrinous tracheal pseudomembrane (oftp) is a rare condition associated with endotracheal intubation in which a pseudomembrane that encircles the tracheal wall is formed. little is known about the mechanisms involved in the development of this fibrinous pseudomembrane, but it requires early detection and urgent management, as it causes a life - threatening tracheal obstruction. here, we present a case of oftp that developed 3 days after extubation in a patient who was intubated for 3 days. a previously healthy, nonsmoking 66-yr - old woman (height 160 cm, weight 58 kg) was admitted to the hospital because of altered mental status on november 8, 2007. according to her family, she was vomiting and becoming increasingly lethargic when found, and empty sleeping pill bottles were discovered beside her. on arrival to the hospital, the patient was somnolent and agitated, and unable to follow commands. her blood pressure was 110/70 mmhg, pulse rate 80/min, body temperature 36, and respiration rate 24/min. the arterial ph was 7.23, pao2 35.3 mmhg, paco2 62.7 mmhg, and sao2 55.8% while the patient was breathing room air. after several unsuccessful and traumatic attempts, her trachea was intubated with a 7.5-mm cuffed tracheal tube (hi - lo, mallinckrodt medical, athlone, ireland) to treat acute respiratory distress. after mechanical ventilation, the patient 's condition improved rapidly. suctioning via the endotracheal tube revealed scanty amount of whitish sputum that showed no evidence of microorganisms. seventy - two hours after intubation, the trachea was extubated. while intubated, the intra - cuff pressure was monitored and maintained below 15 cmh2o., she was afebrile and tachypneic. despite treatment with a short - acting bronchodilator and steroids, there were no signs suggestive of infection on a chest radiography, blood cultures, urinalysis and sputum cultures. but chest ct revealed an irregular luminal narrowing in the proximal trachea just below the vocal cords (fig. a white, rubbery membrane encircling the trachea was seen just below the vocal cords, which resulted in narrowing of the tracheal lumen by approximately 80% (fig. 2). this pseudomembrane was tubular in shape, 4 cm long, and adherent to the tracheal wall. we tried to remove the pseudomembrane using bronchoscopic forceps, but the lesion was cut to pieces. subsequently, rigid bronchoscopy was performed under general anesthesia and the pseudomembrane was removed entirely with mechanical forceps (fig., no findings suggestive of tracheomalacia or granulation tissue formation in the tracheal wall were detected. tracheal complications associated with endotracheal intubation include tracheal stenosis, ulcers, granuloma, and tracheomalacia (1). of these, tracheal stenosis is one of the most dire complications and requires appropriate evaluation and management, such as endotracheal stenting or tracheal surgery. (2) reported a rare, fatal case involving the development of a tubelike formation in the upper trachea after intubation. harbison. (3) also described a patient who developed a tracheal fibrinoid membrane 3 days after extubation. (4) reported a series of ten patients with tubular, rubberlike pseudomembranes molding the trachea after a short duration of intubation. in these patients, the mean duration of endotracheal intubation was 6.21.8 days, but in four cases it was less than 24 hr. seven patients developed symptoms of acute airway obstruction and these symptoms occurred shortly after extubation (5927 hr). as in the reported cases, our patient was intubated for 72 hr and developed dyspnea and stridor 3 days after extubation. most reported cases of pseudomembrane formation in the trachea are associated with an infectious cause. diphtheria is a well - known cause of pseudomembrane formation in the respiratory tract and other infectious etiologies have also been implicated, including fungi, bacteria, and viruses (5). membranous laryngotracheobronchitis (membranous croup) may be considered in the differential diagnosis of oftp, but it is predominantly associated with signs of infection including high fever, a greater degree of toxicity and presence of pathogenic bacteria in tracheal secretions or membrane (6). the mechanism involved in the development of a pseudo - membrane following endotracheal intubation is not clear, although tracheal ischemia due to cuff pressure injury of the endotracheal tube has been suggested as the etiology (4). when the cuff pressure exceeds 30 mmhg, it can result in mucosal ischemia, which causes ulceration and shedding of the tracheal epithelium. the pathological findings of tracheal pseudomembranes, such as superficial abrasions of the mucosa and desquamated necrotic tracheal epithelium, support this hypothesis (4). however, we suggest that a pseudomembrane can be formed independently of cuff pressure. indeed, the cuff pressure in our case was maintained below 15 cmh2o while she was intubated. considering the history of vomiting and several traumatic attempts to intubate the trachea, acid injury or traumatic injury to the tracheal mucosa, rather than ischemic injury, may have triggered the development of a pseudomembrane in our patient. when we tried to intubate the trachea, the endotracheal tube might have been smeared with gastric acid in her oral cavity, which may eventually have damaged the tracheal mucosa in contact with the cuff, and trauma might have increased the damage to the tracheal mucosa. several papers support our hypothesis that the formation of a pseudomembrane is associated with subglottic epithelial trauma or caustic injuries to the trachea caused by aspirated gastric contents during intubation (7, 8). if left untreated, oftp may cause acute tracheal obstruction, which can be life - threatening. once a diagnosis of oftp is established, the lesion should be removed immediately. in many cases, the only successful treatment is excision of the lesion via rigid bronchoscopy (4). in our case, we tried to remove the pseudomembrane with flexible forceps, but they were cumbersome and the procedure took too long. subsequently, we performed rigid bronchoscopy and removed the pseudomembrane easily. in summary, we described a case of oftp that was removed via rigid bronchoscopy. oftp is a very rare condition, but should be considered in every patient who develops symptoms of acute airway obstruction shortly after extubation. tracheal ischemia has been considered as the main etiology in the development of a tracheal pseudomembrane, but we postulate that acidic or traumatic injury to the tracheal mucosa was a more likely cause in our case. physicians should be alert to this fatal complication and bronchoscopy should be performed for the precise diagnosis to allow early detection. | obstructive fibrinous tracheal pseudomembrane is a rare, but potentially fatal complication associated with endotracheal intubation. it has been known that the formation of tracheal pseudomembrane is related with intracuff pressure during endotracheal intubation or infectious cause. but in the patient described in this case, pseudomembrane formation in the trachea was associated with subglottic epithelial trauma or caustic injuries to the trachea caused by aspirated gastric contents during intubation rather than tracheal ischemia due to high cuff pressure. we report a patient with obstructive fibrinous tracheal pseudomembrane after endotracheal intubation who presented with dyspnea and stridor and was treated successfully with mechanical removal using rigid bronchoscopy. |
tetralogy of fallot (tof) is a common cyanotic cardiac lesion, and may be associated with aortic arch anomalies. double aortic arch (daa) is one vascular ring which completely encircles the trachea and oesophagus. the most common cyanotic congenital heart lesions associated with daa are tof and transposition of great arteries. most cases of daa with or without congenital heart disease (chd) present in infancy or early childhood. further examination included a single second heart sound, and a grade 2/6 ejection systolic murmur at the upper left parasternal area. twelve lead electrocardiography confirmed sinus rhythm, a qrs axis of + 120, right ventricular dominance with early transition of the qrs complex in v2. the chest x - ray was usual for tof, except that there was an indentation of the tracheal shadow on both sides, more prominent on the right (figure 1). chest x ray (pa view) showing no cardiomegaly, right ventricular type apex, decreased pulmonary blood flow. suprasternal short axis view demonstrated two circles one on each side of the trachea, suggesting a double aortic arch (figure 2). suprasternal short axis view showing two circles of both arches on both sides of trachea. raa- right aortic arch, laa- left aortic arch, tr - trachea. to define the anatomy further, high - resolution ct, and cardiac catheterization were carried out which revealed a double aortic arch, with the right arch being smaller and more cranially situated (figs 3 - 6). aortogram : injection in ascending aorta in frontal view showing double aortic arch, dominant left arch, right arch is higher then left, descending aorta on left. raa- right aortic arch, laa- left aortic arch, tr - trachea, rpa- right pulmonary artery. three dimensional reconstruction of aortic arch showing daa, left descending aorta, dominant left arch and superior position of right arch. raa- right aortic arch, laa- left aortic arch, aao - ascending aorta, lv - left ventricle, rv - right ventricle. three dimensional reconstruction of daa from posterior view showing same findings as described in figure 4b. two neck vessels arose from each arch, and there was no pull - back pressure gradient on either arch. having reviewed these investigations the patient admitted to direct questioning that he had suffered swallowing difficulties especially for solid foods for many years but attributed this to his cyanotic heart disease. the surgical plan was to perform single stage correction of the tof through a midline sternotomy, together with relief of the vascular ring, by division of the minor arch. at operation, the ventricular septal defect (vsd) was closed, the infundibular muscle resected with trans annular enlargement of the right ventricular (rv) outflow, together with insertion of a pulmonary valve prosthesis (23 mm epic valve). as visualization of the aortic arches from the midline approach was difficult because of the size of the ascending aorta, relief of the daa was deferred. following uneventful recovery from this initial procedure, on the 7th post operative day via a right thoracotomy, the minor (right) component of the double arch was ligated and divided distal to the origin of 2 arch branch. this arch was non dominant, higher, and more posteriorly positioned, clearly compressing the oesophagus. recovery continued uneventfully. at the six month postoperative review, he was doing well with complete disappearance of the dysphagia. echocardiography confirmed no residual postoperative intracardiac issues and normal flow in the remaining left arch. tof is known to have arch anomalies especially due to its association with digeorge syndrome. although very rare, several cases of tof associated with daa have been reported so far.. however most of these patients belonged to the infant age group or they were detected during early childhood. to the best of our knowledge probably only one adult patient has been reported previously who had tof associated with daa and underwent a successful surgical repair. daa is very often missed on echocardiography but we had a strong suspicion of daa on suprasternal view. two circle appearance on suprasternal view strongly suggest the possibility of double aortic arch. in our patient, images of this view were not satisfactory due to poor quality resulting from limited acoustic windows which is not uncommon in many of adult patients. ct and mri are the imaging modalities of choice in a patient who is thought to have a vascular ring. the advantage of the use of ct angiography is that the modality is a noninvasive technique that enables evaluation of vascular anomalies and the status of tracheal or esophageal compression in the same study. this information often eliminates the more traditional multistage work - up, which includes barium study, bronchoscopy, echocardiography, and angiography. in our case too, anatomy of the arch and its branches were confirmed and well profiled in relation to adjoining structures by ct scan. catheter - directed cardiac angiography was done to rule out any additional vsd in our case of an adult with tof. for an isolated daa catheterization compared with ct, cardiac catheterization has a higher complication rate owing to its invasive nature, typically requires a larger volume of intravascular contrast material and often imparts greater radiation dose. although echocardiography and angiography are the dominant imaging modalities in patients with congenital heart disease, ct is an extremely valuable noninvasive adjunct which is effective in demonstrating the complex cardiovascular morphology, especially the extracardiac morphology. compared with the ct scanners of the past, newer scanners yield images with better temporal and spatial resolution, greater anatomic coverage per rotation, and more consistent enhancement with a lesser volume of intravascular contrast material, and higher - quality two - dimensional reformation and three - dimensional reconstruction owing to the acquisition of an isotropic data set. rapid imaging with these ct scanners requires less patient sedation which makes it even superior to mri. ct can be used to determine which arch is dominant and to establish the relations between the great vessels and each arch. this information is important because thoracotomy is typically performed on the side of the smaller arch. chest x - ray in our patient was not of very classical of tracheal indentation. use of high kv roentgenograms or barium swallow could have shown compression of trachea and esophagus better but these additional investigations were not required in view of available ct reconstructed images. daa usually manifests early in the life with the symptoms of stridor, wheezing and choking episodes with feeds. those with loose rings can present with dysphagia during adulthood as is the case with our patient. our patient was so ignorant about this symptom that it could be elicited in the history retrospectively only. to plan effective management of congenital heart disease, a thorough clinical history is the basic need. all patients with tof, child or adult, must be looked thoroughly for aortic arch anomalies meticulously by suprasternal echocardiography to avoid misses. a collaborative approach by cardiologists, surgeons, and radiologists in reviewing ct imaging data of daa maximizes the diagnostic yield. a thorough preoperative understanding of complex cardiovascular anatomy in patients with congenital heart disease facilitates a directed and prepared surgical approach. | double aortic arch (daa) is a common vascular ring. it may occur in isolation or coexist with various types of congenital heart disease. the anomaly usually presents in early infancy. this reports a 23yr old male presenting with dysphagia, who was found to have a double aortic arch and tetralogy of fallot. both lesions were successfully corrected surgically. |
a 42 year old male patient with a complaint of intermittent abdominal pain lasting for 5 - 7 days presented to the internal medicine outpatient clinic of ankara education and research hospital in august 2010. he was not smoking. on physical examination, he was alert and oriented with a temperature of 36.8c, a pulse of 82 bpm and a blood pressure of 120/60 mmhg. cardiac exam was normal with regular heart rate and rhythm, normal heart sounds and no murmurs. laboratory data included a hemoglobin of 13.3 g / dl (normal range : 11.7 - 15.5), white blood count of 10,500 cells / mm (normal range : 4.5 - 11) and platelets of 131,000 cells / mm (normal range : 150 - 450). biochemical tests were all within normal limits except a slight elevation in aminotransferases (aspartate aminotransferase : 54 u / l (normal range : 0 - 35), alanine aminotransferase : 57 u / l (normal range : 0 - 35)). chest x ray film did not show any acute process and electrocardiogram showed normal sinus rhythm and no st and t wave changes. abdominal ultrasonography (usg) revealed portal vein thrombosis. in order to ascertain the hematological status of the patient, after the patient was accepted in our clinic, treatment was commenced with low molecular weight heparin (enoxaparin, 0.4 cc, once a day). on the eighth day of his admission, abdominal pain worsened and abdominal defence was detected. a new usg was done and all branches of the portal vein, inferior mesenteric vein and proximal part of splenic vein were found to be thrombotic. the patient was transferred to the surgery department for an emergent operation. during the operation, after the operation, the patient did not experience short bowel syndrome, his status stayed stable and his abdominal pain subsided. extensive laboratory workup for thrombophilia including measurements of factor v leiden, factor vr2, prothrombin g20210a and lupus anticoagulants showed no abnormality. however, the patient was found to be heterozygous for the mtfhr gene both in c677 t and a1298c alleles. thrombophilic parameters of the patient on admission in order to exclude any malignancy or ibd, colonoscopy was performed and caecum was reached. in the regions of rectum, rectosigmoid junction, descending colon, splenic flexure, and approximately through the middle of transverse colon, hyperemic fragile, oedematous mucosa and decreased vascular pattern were observed. these findings revealed uc. while the patient had chronic portal vein thrombosis and had been operated because of acute splenic and mesenteric venous thrombosis, subcutaneous enoxaparin (0.6 cc, twice a day) and oral warfarin treatment (2.5 mg, once a day) were started. after three days, according to suitable international normalized ratio (inr) levels, enoxaparin was stopped and warfarin dose was adjusted to 5 mg. nowadays, it is defined more usefully covering conditions not directly related to homeostatic system, such as hyperhomocysteinemia. thrombophilia has congenital and acquired causes (table 2).1 although ibd is a rare cause of thrombophilia, it has been accepted as an independent risk factor for thromboembolism.11 the frequency of thromboembolic events has varied between 1.3%12 to 6.2%11 among patients with uc. in a review of 7199 patients with ibd (half of whom had uc) over the course of 11 years, 1.3% had a thromboembolic event.12 the mortality of those who had thromboembolism was 25% during the acute thrombotic event. this emphasizes the importance of understanding the risk for thromboembolism in this group of patients and the need to identify those who would benefit from pharmacological prophylaxis. causes of thrombophilia it was stated that thrombotic complications increased in active periods of uc.19 our patient had neither symptoms nor diagnosis of uc before the thromboembolic events. like patients of irving,26 increased platelet aggregation was accepted to be responsible, at least in part, for thromboembolism that occurs in the setting of active inflammation.26 webberley. found that spontaneous platelet aggregation occurred in vitro in platelets isolated from 30% of patients with active ibd but not in platelets from control subjects.27 increased platelet aggregation in ibd may be related to an enhanced cd40/cd40l system which is a key regulator and amplifier of immune reactivity and some investigators had shown that both uc and crohn disease (cd) patients with clinically active disease have significantly greater expression of this immunoregulatory and pro - inflammatory molecule when compared with healthy controls.28 however, it can not be predicted whether or not increased platelet aggregation occurs in patients that are in clinical remission with no evidence of systemic inflammation. interestingly, two authors observed that 56%6 and 77%12 of their patients with ibd developed peripheral venous thromboses during a period of clinical remission. these authors suggested that clinically detectable systemic inflammation had little role in the pathogenesis of thrombosis, although undetected inflammation could not be excluded. the discrepancy between their results and ours may be explained by the difference in the site of thrombotic event. in our patient, thrombosis occurred in portal, splenic and mesenteric veins, while their patients had thrombotic peripheral veins which are accepted as common places of thrombosis. it was also observed that the extent and distribution of colonic disease correlated with thromboembolic risk.29 jackson. found all the subjects with cd to have ileocolonic disease and those with uc to have pancolonic disease.6 unlike these observations, moreover, in our patient, the perioperative period was not a risk factor for development of thromboembolism in contrast to studies of hatoum5 and fischera.30 there is evidence indicating that genetic factors such as factor v leiden, factor ii (prothrombin, g20210a), mthfr, plasminogen activator inhibitor type 1 (pai-1) gene mutation and factor xiii (val34leu) contribute to the thrombotic manifestations of ibd.21233132 mthfr is involved in the one - carbon cycle, which is of importance for nucleotide synthesis and methylation of dna, membranes, proteins and lipids. mutations in this gene result in a decrease of the enzyme activity that leads to mild hyperhomocysteinemia.33 a study suggested mutations in this gene for allele c677 t and a1298c to be a tendency to associate with coronary artery disease, in both homozygous and heterozygous carriers even when blood homocysteine levels were not elevated.34 our patient, whose homocysteine level was not elevated, was heterozygous for both c677 t and a1298c alleles of mthfr gene. usually in uc, venous thromboembolism manifests as lower extremity deep venous thrombosis and pulmonary embolism.814 rare cases of uc patients have been described with thrombus formation at uncommon sites such as the portal or mesenteric vein.581112 splenic thromboembolism has been even rarer.17 the majority of reported cases of mesenteric thrombosis in association with uc have been arterial, although venous thromboembolism has also been described.1427 mesenteric, splenic and portal vein thrombosis were predominantly encountered as postsurgical complications.56121735 in our patient with uc, three rare thromboembolic phenomena, including portal vein, inferior mesenteric vein and splenic vein, were observed without a history of surgery. the overall mortality of such complications in patients with uc has been estimated to be very high. we therefore recommend that patients with ibd should be managed with high index of suspicion of thromboembolism. in conclusion, our patient had a rare cause of thrombophilia, i.e. uc. he was exceptional for the sites of thrombosis, and for combined portal, mesenteric and splenic venous thrombosis, which were seen together. he was not a usual case, as thromboembolic complication of uc was seen in an inactive period without any history of operation. in addition, his disease was not extensive. since uniqueness of the presented symptoms contribute to the late diagnosis in these patients, an increased awareness of the association between ibd and thromboembolism in usual sites may help to diagnose a thrombophilic case in an early stage, when treatment options show a better outcome. it is important to recognize that even in the absence of clinically significant inflammation, devastating thrombosis may occur. furthermore, our patient was also heterozygous for two alleles of mthr gene without hyperhomocysteinemia. we also recommend screening a patient with thrombophilia for predisposing genetic factors, especially in young age. | thrombophilia is a rare but potentially catastrophic phenomenon occurring in patients having tendency of thrombosis. it may lead to serious complications. the etiology of thrombophilia is thought to be multifactorial and related to both acquired and inherited factors. inflammatory bowel disease is an acquired cause of thrombophilia. thromboembolic events are seen during inflammatory bowel disease, especially during the active period of the disease. in inflammatory bowel disease, thrombus formation in portal, splenic and mesenteric veins are not common. besides, the association of genetic disorders related to metabolism of homocysteine with inflammatory bowel disease has been evidenced, especially in crohn disease and rarely in ulcerative colitis. we present a rare case of ulcerative colitis in association with combined portal, splenic and mesenteric vein thrombosis. the patient was recently diagnosed with the disease which was in the inactive period. interestingly, our patient was also heterozygous for the mutation in methylenetetrahydrofolate reductase (mthfr) gene. |
protein structure determination has contributed greatly to our understanding of structural biology by providing the three - dimensional protein structure and the role of the associated conformational dynamics of the protein. despite the importance of protein structures, three - dimensional protein structure determination has been limited to x - ray crystallography in the solid state (single crystals) or nuclear magnetic resonance (nmr) spectroscopy in solution. recently, 9049 macromolecular structures obtained by nmr spectroscopy were available in the protein data bank (pdb) (1,2). nmr is uniquely suited to the characterization of protein structure and dynamics in solution, and it is not hampered by the ability or inability of a protein to crystallize. despite these advantages, nmr protein structures are usually not a first choice for studies of protein structure and function. because nmr structures can be of lower quality, less reliable and less suitable for structural analysis than crystallography, they are often excluded (35). because of the uncertainty in nmr structures, numerous refinement protocols and force fields have been developed to improve the quality of nmr structures (3,57). even with the recent advancements in protocols and force fields, the structures obtained can still be of unsatisfactory quality, indicating that there is room for further improvement. numerous studies have advised that the poor quality of the ramachandran plot, backbone conformation and/or side - chain packing were caused by the low quality of the nmr structures (810). to address these weaknesses, re - refinement and other protocols have been introduced in several projects [dress (11), recoord (12), etc. the re - refinement in this study is focused on 2405 of the 9049 selected solution nmr structures deposited in the pdb. we developed a new refinement protocol to refine the solution nmr structure by correcting the backbone polypeptide torsion angles. we illustrate the clear overall improvement of the structures in our database, statistical torsion angle potentials (stap) refinement of the nmr database, compared to the currently available refinement databases. unlike crystallography, nmr structure determination uses very heterogeneous geometrical information : distances, dihedral angles and orientations (12). among this geometrical information torsion angles are assumed to be a very important factor in the quality of nmr structures (810). its deviation sways the inter - atomic distance and greatly influences the nuclear overhauser effect (noe) signals in experiments (13,14). torsion angle population analysis provides a firm ground for deriving knowledge - based energy functions. stap was developed with 18 353 high - resolution x - ray crystallographic structures with a resolution below 2.0 from the pdb. from that, we removed the structures with redundancies and built two - dimensional histograms as a function of the two backbone torsion angles (and) with a grid point for every 15. by applying a log transformation to these histograms (15), we obtained the two - dimensional knowledge - based potentials on / torsion angles for favourable conformational isomerism. most of the experimental structures available from the pdb were loaded directly into the automatic pipeline for refinement. each noe distance restraint was downloaded from the biological magnetic resonance bank (bmrb) (16). briefly, the refinement protocol used is as follows : (i) after stap refining potentials are applied to all of the structures, the implicit solvation model is applied (17) and energy - minimized ; (ii) the system is heated from 100 to 500 k using 1000 steps of molecular dynamics ; (iii) 2000 steps of molecular dynamics at 500 k are performed ; (iv) cooling down to 25 k runs during 4000 steps ; and (v) a short minimization with 200 steps is performed. 8.0) (9) was used to measure the root mean square (rms) z - score distribution of several parameters of the protein structure. there are three optimal energy properties for protein structures : discrete optimised protein energy (dope, ver. 9v7) (20) and dipolar distance - scaled, finite - ideal gas reference (ddfire, ver. statistical rms z - score distribution, rms noe violations and ndope scores for the original and refined structure are shown in figure 1. as we expected, the rms z - score for the ramachandran plot appearance (figure 1d) showed a great improvement because it shows two separate gaussian distributions. in addition, the result of the rms z - score distribution for the 12 rotamer normality (figure 1e) also appears similar to that of the ramachandran plot appearance. other parameters show remarkable improvement with a stable low energy profile for ndope and 2nd - generation packing quality. interestingly, the influence of the torsion angle conformation on the nmr structure may cause rms noe violations to provide better quality, indicating that noe violations directly affect the geometrical conformation of the torsion angle (23,24). figure 1.distribution of the protein quality assessment of the original (red) and refined structures (blue bars). the figures present the distributions of the 2405 selected nmr structures as follows : (a) the rms value of the noe violations, (b) the normalized dope score, (c) the 2nd - generation packing quality (all backbone and side chain contacts), (d) the ramachandran plot appearances, (e) the 12 rotamer normality and (f) the backbone conformation. the values were measured by ddfire, aqua and what_check. distribution of the protein quality assessment of the original (red) and refined structures (blue bars). the figures present the distributions of the 2405 selected nmr structures as follows : (a) the rms value of the noe violations, (b) the normalized dope score, (c) the 2nd - generation packing quality (all backbone and side chain contacts), (d) the ramachandran plot appearances, (e) the 12 rotamer normality and (f) the backbone conformation. the values were measured by ddfire, aqua and what_check. the average values of the original and refined parameters pertaining to the structural quality are presented in table 1. evaluation of the structural quality indicated that our protocol, stap, provides improved quality in the polypeptide backbone conformation. remarkably, the number of rms noe violations was closer to zero in the refined structure than in the original structure, which is shown above in the histogram depicted in figure 1. table 1.summary of the average quality indicators of the original and refined nmr structuresoriginalrefined (stap)steric clash score33.16 39.521.57 5.90rms noe distance violations0.20 0.350.13 0.24optimal protein energy dope score8050.74 4572.848587.34 4817.02 normalized dope score0.62 1.021.02 0.97 ddfire score163.12 89.44179.07 96.77ramachandran indicators (%) molprobity favourable83.50 10.0995.51 4.83 allowed12.95 7.363.80 3.92 disallowed3.54 4.040.69 1.48 procheck favourable75.66 12.0889.83 7.45 allowed21.04 9.658.01 5.80 generously2.35 3.310.84 1.55 disallowed0.96 1.721.32 2.01structure z - score distribution 1st - generation packing quality3.14 1.271.52 1.55 ramachandran plot appearance4.50 1.860.65 1.95 12 rotamer normality5.25 2.362.07 1.68 backbone conformation0.93 1.280.80 1.33rms z - score distribution bond lengths0.48 0.340.84 0.12 bond angles0.70 0.391.04 0.16 angle restraints0.36 0.511.30 0.43 side - chain planarity0.59 0.920.68 0.45 improper dihedral distribution0.64 0.390.79 0.25 inside / outside distribution1.05 0.101.03 0.10bold font indicates the best scores.positive is better than average.rms z - score should be close to 1.0. summary of the average quality indicators of the original and refined nmr structures bold font indicates the best scores. rms z - score should be close to 1.0. from analysis of the stap data, there are 70 structures present in the two public nmr refined databases (dress and recoord databases), as shown in table 2. the refined data of dress (refined pdb files) and recoord (cnw and cyw pdb files) the quality improvement by the three protocols (stap, dress and recoord) is shown in table 2. the stap refinement protocol has a great improvement on the ramachandran backbone conformation, especially on the percentage of favourable indicators by molprobity and procheck. the z - score distributions by what_check indicated that our protocol provides slightly better quality than the other two protocols [steric clash score, ndope score, rms z - score distributions on the structure (2nd - generation packing quality, 12 rotamer normality, ramachandran plot appearance and backbone conformation) and rms z - score distributions (bond angles and inside / outside distribution) ]. the result of the optimized energy comparison indicated that the other databases (recoord and dress) are slightly better than our approach. with the many positive attributes of stap, it can be confidently concluded that stap provides comparable performance to justify the geometric consensus for torsion angles and shows comparable or slightly better performance than the two known protocols. table 2.comparison of the 70 nmr structures that are common among the stap, dress and recoord databasesoriginalstapdressrecoord - cnwrecoord - cywsteric clash score68.26 57.861.03 2.4416.79 9.5616.31 8.6216.56 8.46optimal protein energy dope score6098.15 4586.336941.80 5130.946996.31 5244.707274.30 5902.437330.00 6150.59 normalized dope score0.21 1.171.00 1.061.01 1.130.95 1.110.96 1.10 ddfire score125.95 88.72147.52 102.79143.07 101.01146.95 114.19148.66 120.39ramachandran indicators (%) molprobity favourable75.02 12.3794.80 4.5186.38 8.1884.69 9.0083.59 9.35 allowed18.76 8.874.27 3.6410.71 6.7111.89 7.0312.75 7.58 disallowed6.22 5.250.92 1.552.90 2.923.43 3.343.66 3.29 procheck favourable67.02 15.1488.45 7.6077.92 11.7075.33 13.0273.73 13.88 allowed27.18 11.958.73 6.0818.52 9.7920.20 10.7321.48 11.54 generously4.38 4.101.00 1.592.06 2.492.70 3.002.83 2.99 disallowed1.42 1.931.82 2.191.50 2.121.77 2.261.97 2.29structure z - score distribution 1st - generation packing quality3.48 1.712.41 1.732.14 1.722.37 1.972.44 2.03 2nd - generation packing quality2.96 1.191.28 1.501.89 1.122.09 1.272.08 1.26 ramachandran plot appearance5.85 1.920.98 2.034.13 1.444.31 1.604.43 1.59 12 rotamer normality6.35 2.351.68 1.682.58 1.592.50 1.422.77 1.40 backbone conformation1.67 1.501.07 1.421.50 1.401.64 1.371.63 1.33rms z - score distribution0.83 0.340.85 0.060.84 0.140.84 0.130.86 0.13 bond lengths1.06 0.421.03 0.120.78 0.120.78 0.110.80 0.11 bond angles0.16 0.271.31 0.370.75 0.150.74 0.150.75 0.14 angle restraints1.71 1.420.70 0.370.81 0.181.27 0.541.31 0.58 side - chain planarity0.92 0.390.79 0.171.01 0.161.19 0.361.23 0.38 improper dihedral distribution1.07 0.131.06 0.121.07 0.121.08 0.131.08 0.13bold font indicates the best scores.positive is better than average.rms z - score should be close to 1. comparison of the 70 nmr structures that are common among the stap, dress and recoord databases bold font indicates the best scores. we have discussed the statistical analysis of the original and refined structures and compared our refined structure quality with that of other known nmr refined databases. based on these facts, we believe that the quality of our 2405 nmr structure database is significantly improved, and this database will be a good start for further development of research and validation tools, structure - related studies and modelling in many fields of research. the stap refinement of the nmr database can be accessed using a web - portal (http://psb.kobic.re.kr/stap/refinement) that provides a 2405 refined nmr structure database, which runs on the centos operation system (ver. the web server uses a standard web browser as the graphic user interface to the front - end server based on php (ver. the following five website features are described in detail : (i) the main page, (ii) the general structure information page, (iii) the results page, (iv) the visualization page and (v) the download page. (i) the front page allows the user to enter the selected pdb i d to access the database. on the main page, there are three different ways to access the database (pdb list, lucky and manual). the pdb list is somewhat similar to the ms windows file explorer interface, which is one of the search engines using the tree structure as the user interface, sequentially listing pdb ids. if a user clicks the pdb list button, a tree browser layer will open at the upper left side of the window on the main page. the top - level directory consists of the first letter of each pdb i d. + button placed on the left side of the directory image, the next lower level of the tree node appears. when the user clicks first two letters of id, the user can see the list of pdb ids. the second way is using lucky, which is known as the online demonstration, and uses the pseudo - random number generator algorithms (25) to select a random target. lucky provides visual information with a mouse rollover tooltip layer, so that a user can learn our system quickly. finally, for the manual search, a user types the pdb i d to access the database. (ii) general information on the target nmr structure (figure 2) is provided, including information on the protein, experimental information, visualization of the structure using the jmol viewer (ver. 11.4.rc4) (26) and links to the pdb, bmrb, pubmed (dependent on pdb data ; if the pdb data do not include the pubmed information then the pubmed icon will be disabled), and the ncbi taxonomy browser. (iii) the results page shows the calculated results for the structural quality assessment based on the original and refined structures. it sometimes provides information on the evaluation results with dress and recoord with a link to the target protein sites and an overall summary table for the target structure. (iv) visualization information on the details of the target structure and ramachandran plot for protein quality assessment are provided. both the original and refined structures are displayed (detailed view of ramachandran indicators, location of steric clash sites, etc.), and the location of the ramachandran indicator is shown on the plot with king display software (27). (v) a download area is available for various data, for example, the result of the protein quality assessment for the original and refined structures, both pdb files, and the visualization inputs for the king display. figure 2.general information on the target nmr structure : jmol viewer, link - out to other databases, comparison of original and refinement structures. general information on the target nmr structure : jmol viewer, link - out to other databases, comparison of original and refinement structures. unlike crystallography, nmr structure determination uses very heterogeneous geometrical information : distances, dihedral angles and orientations (12). among this geometrical information, we focused on the dihedral angle. torsion angles are assumed to be a very important factor in the quality of nmr structures (810). its deviation sways the inter - atomic distance and greatly influences the nuclear overhauser effect (noe) signals in experiments (13,14). torsion angle population analysis provides a firm ground for deriving knowledge - based energy functions. stap was developed with 18 353 high - resolution x - ray crystallographic structures with a resolution below 2.0 from the pdb. from that, we removed the structures with redundancies and built two - dimensional histograms as a function of the two backbone torsion angles (and) with a grid point for every 15. by applying a log transformation to these histograms (15), we obtained the two - dimensional knowledge - based potentials on / torsion angles for favourable conformational isomerism. most of the experimental structures available from the pdb were loaded directly into the automatic pipeline for refinement. each noe distance restraint was downloaded from the biological magnetic resonance bank (bmrb) (16). briefly, the refinement protocol used is as follows : (i) after stap refining potentials are applied to all of the structures, the implicit solvation model is applied (17) and energy - minimized ; (ii) the system is heated from 100 to 500 k using 1000 steps of molecular dynamics ; (iii) 2000 steps of molecular dynamics at 500 k are performed ; (iv) cooling down to 25 k runs during 4000 steps ; and (v) a short minimization with 200 steps is performed. 8.0) (9) was used to measure the root mean square (rms) z - score distribution of several parameters of the protein structure. there are three optimal energy properties for protein structures : discrete optimised protein energy (dope, ver. 9v7) (20) and dipolar distance - scaled, finite - ideal gas reference (ddfire, ver. statistical rms z - score distribution, rms noe violations and ndope scores for the original and refined structure are shown in figure 1. as we expected, the rms z - score for the ramachandran plot appearance (figure 1d) showed a great improvement because it shows two separate gaussian distributions. in addition, the result of the rms z - score distribution for the 12 rotamer normality (figure 1e) also appears similar to that of the ramachandran plot appearance. other parameters show remarkable improvement with a stable low energy profile for ndope and 2nd - generation packing quality. interestingly, the influence of the torsion angle conformation on the nmr structure may cause rms noe violations to provide better quality, indicating that noe violations directly affect the geometrical conformation of the torsion angle (23,24). figure 1.distribution of the protein quality assessment of the original (red) and refined structures (blue bars). the figures present the distributions of the 2405 selected nmr structures as follows : (a) the rms value of the noe violations, (b) the normalized dope score, (c) the 2nd - generation packing quality (all backbone and side chain contacts), (d) the ramachandran plot appearances, (e) the 12 rotamer normality and (f) the backbone conformation. distribution of the protein quality assessment of the original (red) and refined structures (blue bars). the figures present the distributions of the 2405 selected nmr structures as follows : (a) the rms value of the noe violations, (b) the normalized dope score, (c) the 2nd - generation packing quality (all backbone and side chain contacts), (d) the ramachandran plot appearances, (e) the 12 rotamer normality and (f) the backbone conformation. the values were measured by ddfire, aqua and what_check. the average values of the original and refined parameters pertaining to the structural quality are presented in table 1. evaluation of the structural quality indicated that our protocol, stap, provides improved quality in the polypeptide backbone conformation. remarkably, the number of rms noe violations was closer to zero in the refined structure than in the original structure, which is shown above in the histogram depicted in figure 1. table 1.summary of the average quality indicators of the original and refined nmr structuresoriginalrefined (stap)steric clash score33.16 39.521.57 5.90rms noe distance violations0.20 0.350.13 0.24optimal protein energy dope score8050.74 4572.848587.34 4817.02 normalized dope score0.62 1.021.02 0.97 ddfire score163.12 89.44179.07 96.77ramachandran indicators (%) molprobity favourable83.50 10.0995.51 4.83 allowed12.95 7.363.80 3.92 disallowed3.54 4.040.69 1.48 procheck favourable75.66 12.0889.83 7.45 allowed21.04 9.658.01 5.80 generously2.35 3.310.84 1.55 disallowed0.96 1.721.32 2.01structure z - score distribution 1st - generation packing quality3.14 1.271.52 1.55 ramachandran plot appearance4.50 1.860.65 1.95 12 rotamer normality5.25 2.362.07 1.68 backbone conformation0.93 1.280.80 1.33rms z - score distribution bond lengths0.48 0.340.84 0.12 bond angles0.70 0.391.04 0.16 angle restraints0.36 0.511.30 0.43 side - chain planarity0.59 0.920.68 0.45 improper dihedral distribution0.64 0.390.79 0.25 inside / outside distribution1.05 0.101.03 0.10bold font indicates the best scores.positive is better than average.rms z - score should be close to 1.0. summary of the average quality indicators of the original and refined nmr structures bold font indicates the best scores. from analysis of the stap data, there are 70 structures present in the two public nmr refined databases (dress and recoord databases), as shown in table 2. the refined data of dress (refined pdb files) and recoord (cnw and cyw pdb files) were loaded directly from their websites to measure the protein structure qualities. the quality improvement by the three protocols (stap, dress and recoord) is shown in table 2. the stap refinement protocol has a great improvement on the ramachandran backbone conformation, especially on the percentage of favourable indicators by molprobity and procheck. the z - score distributions by what_check indicated that our protocol provides slightly better quality than the other two protocols [steric clash score, ndope score, rms z - score distributions on the structure (2nd - generation packing quality, 12 rotamer normality, ramachandran plot appearance and backbone conformation) and rms z - score distributions (bond angles and inside / outside distribution) ]. the result of the optimized energy comparison indicated that the other databases (recoord and dress) are slightly better than our approach. with the many positive attributes of stap, it can be confidently concluded that stap provides comparable performance to justify the geometric consensus for torsion angles and shows comparable or slightly better performance than the two known protocols. table 2.comparison of the 70 nmr structures that are common among the stap, dress and recoord databasesoriginalstapdressrecoord - cnwrecoord - cywsteric clash score68.26 57.861.03 2.4416.79 9.5616.31 8.6216.56 8.46optimal protein energy dope score6098.15 4586.336941.80 5130.946996.31 5244.707274.30 5902.437330.00 6150.59 normalized dope score0.21 1.171.00 1.061.01 1.130.95 1.110.96 1.10 ddfire score125.95 88.72147.52 102.79143.07 101.01146.95 114.19148.66 120.39ramachandran indicators (%) molprobity favourable75.02 12.3794.80 4.5186.38 8.1884.69 9.0083.59 9.35 allowed18.76 8.874.27 3.6410.71 6.7111.89 7.0312.75 7.58 disallowed6.22 5.250.92 1.552.90 2.923.43 3.343.66 3.29 procheck favourable67.02 15.1488.45 7.6077.92 11.7075.33 13.0273.73 13.88 allowed27.18 11.958.73 6.0818.52 9.7920.20 10.7321.48 11.54 generously4.38 4.101.00 1.592.06 2.492.70 3.002.83 2.99 disallowed1.42 1.931.82 2.191.50 2.121.77 2.261.97 2.29structure z - score distribution 1st - generation packing quality3.48 1.191.28 1.501.89 1.122.09 1.272.08 1.26 ramachandran plot appearance5.85 1.920.98 2.034.13 1.444.31 1.604.43 1.59 12 rotamer normality6.35 2.351.68 1.682.58 1.592.50 1.422.77 1.40 backbone conformation1.67 1.501.07 1.421.50 1.401.64 1.371.63 1.33rms z - score distribution0.83 0.340.85 0.060.84 0.140.84 0.130.86 0.13 bond lengths1.06 0.421.03 0.120.78 0.120.78 0.110.80 0.11 bond angles0.16 0.271.31 0.370.75 0.150.74 0.150.75 0.14 angle restraints1.71 1.420.70 0.370.81 0.181.27 0.541.31 0.58 side - chain planarity0.92 0.390.79 0.171.01 0.161.19 0.361.23 0.38 improper dihedral distribution1.07 0.131.06 0.121.07 0.121.08 0.131.08 0.13bold font indicates the best scores.positive is better than average.rms z - score should be close to 1. comparison of the 70 nmr structures that are common among the stap, dress and recoord databases bold font indicates the best scores. we have discussed the statistical analysis of the original and refined structures and compared our refined structure quality with that of other known nmr refined databases. based on these facts, we believe that the quality of our 2405 nmr structure database is significantly improved, and this database will be a good start for further development of research and validation tools, structure - related studies and modelling in many fields of research. the stap refinement of the nmr database can be accessed using a web - portal (http://psb.kobic.re.kr/stap/refinement) that provides a 2405 refined nmr structure database, which runs on the centos operation system (ver. the web server uses a standard web browser as the graphic user interface to the front - end server based on php (ver. the following five website features are described in detail : (i) the main page, (ii) the general structure information page, (iii) the results page, (iv) the visualization page and (v) the download page. (i) the front page allows the user to enter the selected pdb i d to access the database. on the main page, there are three different ways to access the database (pdb list, lucky and manual). the pdb list is somewhat similar to the ms windows file explorer interface, which is one of the search engines using the tree structure as the user interface, sequentially listing pdb ids. pdb list button, a tree browser layer will open at the upper left side of the window on the main page. the top - level directory consists of the first letter of each pdb i d. when the user clicks the + button placed on the left side of the directory image, the next lower level of the tree node appears. when the user clicks first two letters of id, the user can see the list of pdb ids. the second way is using online demonstration, and uses the pseudo - random number generator algorithms (25) to select a random target. lucky provides visual information with a mouse rollover tooltip layer, so that a user can learn our system quickly. finally, for the manual search, a user types the pdb i d to access the database. (ii) general information on the target nmr structure (figure 2) is provided, including information on the protein, experimental information, visualization of the structure using the jmol viewer (ver. 11.4.rc4) (26) and links to the pdb, bmrb, pubmed (dependent on pdb data ; if the pdb data do not include the pubmed information then the pubmed icon will be disabled), and the ncbi taxonomy browser. (iii) the results page shows the calculated results for the structural quality assessment based on the original and refined structures. it sometimes provides information on the evaluation results with dress and recoord with a link to the target protein sites and an overall summary table for the target structure. (iv) visualization information on the details of the target structure and ramachandran plot for protein quality assessment are provided. both the original and refined structures are displayed (detailed view of ramachandran indicators, location of steric clash sites, etc.), and the location of the ramachandran indicator is shown on the plot with king display software (27). (v) a download area is available for various data, for example, the result of the protein quality assessment for the original and refined structures, both pdb files, and the visualization inputs for the king display. figure 2.general information on the target nmr structure : jmol viewer, link - out to other databases, comparison of original and refinement structures. general information on the target nmr structure : jmol viewer, link - out to other databases, comparison of original and refinement structures. funding for open access charge : a grant from the korean research institute of bioscience and biotechnology research initiative program ; and the korean ministry of education, science and technology (mest) under grant numbers 20110002321 and 20110019747, respectively. | according to several studies, some nuclear magnetic resonance (nmr) structures are of lower quality, less reliable and less suitable for structural analysis than high - resolution x - ray crystallographic structures. we present a public database of 2405 refined nmr solution structures [statistical torsion angle potentials (stap) refinement of the nmr database, http://psb.kobic.re.kr/stap/refinement ] from the protein data bank (pdb). a simulated annealing protocol was employed to obtain refined structures with target potentials, including the newly developed stap. the refined database was extensively analysed using various quality indicators from several assessment programs to determine the nuclear overhauser effect (noe) completeness, ramachandran appearance, 1-2 rotamer normality, various parameters for protein stability and other indicators. most quality indicators are improved in our protocol mainly due to the inclusion of the newly developed knowledge - based potentials. this database can be used by the nmr structure community for further development of research and validation tools, structure - related studies and modelling in many fields of research. |
mycosis fungoides (mf) represents the prototype of cutaneous t - cell lymphoma, which is defined as clonal expansion of skin - homing t lymphocytes. the natural history of mf is characterized by an indolent progression through four stages : patch, plaque, tumor, and visceral involvement. the disease begins with lightly erythematous patches that subsequently evolve into well - demarcated scaling plaques. these plaques may then progress to tumor lesions and subsequently spread to the viscera, but this progression is not necessarily seen in all patients. previous studies of prognostic indicators have shown that the skin (t) stage and the presence or absence of extracutaneous disease are the most important determinants of outcome. patients with limited skin involvement (t1) have a favorable prognosis, whereas patients with tumor (t3) or erythrodermic mf (t4) have an unfavorable prognosis [3, 4 ]. usually, mf is characterized by an infiltrate of / t helper memory lymphocytes (f1 +, cd3 +, cd4 +, cd5 +, cd8, and cd45ro+). however, in a minority of cases the neoplastic cells exhibit a t - cytotoxic (cd4cd8 +), / (f1, cd3 +, cd4, cd5 +, and cd8 +) or a cd4/cd8 double - negative phenotype, that show no clinical and/or prognostic differences. recently, a previously unrecognized phenotype characterized by coexpression of cd4 and cd8 has been described [7, 8 ], but the influence of such a phenotype on prognosis of mf has not been evaluated. therefore, although techniques of immunophenotyping have been successful in characterizing the cells in the cutaneous infiltrates of mf little evidence suggests that variations in the phenotypic characterization correlate with prognosis. in this preliminary prospective, single - centre study on a limited number of patients with mf we correlated the t - cell phenotype in cutaneous lesions with the progression of the disease to determine whether the coexpression of cd4 and cd8, compared to conventional cd4+cd8 phenotype, has an impact on prognosis. patients with mf were prospectively included in this study between january 2005 and december 2012, after giving informed consent according to the declaration of helsinki. the diagnosis of mf was made according to the criteria of the who - eortc classification for cutaneous lymphomas. patients received a comprehensive history and physical examination, complete blood cell count including peripheral smear for szary cells, and general chemistry panel. lesional cutaneous biopsies were obtained in all patients upon admission into the study before the start of therapy. patients with clinically significant adenopathy had their nodes evaluated by fine needle aspiration or lymph node biopsy. when indicated, patient had an extensive staging evaluation, including bone marrow aspirate and biopsy, and appropriate radiologic studies to determine visceral involvement. we included in the study only newly diagnosed patients who had not previously received topical therapies (corticosteroids, topical nitrogen mustard, carmustine, and psoralen with ultraviolet a), interferon, chlorambucil, methotrexate, or polychemotherapy. after initial evaluation, the patients were included into a program of visits that were made every three months throughout the period of followup. according to the course of the disease and, more specifically, to their clinical status at the time of the last clinical update the first group was formed by patients with a progressive disease defined as the change to a more advanced stage. in the second group patients who underwent complete or partial remission and patients with stable disease defined as presence of mf without progression were included. factors associated with disease progression were studied with special emphasis to t - cell phenotype. indeed treatments were administered according to clinical stage and were relatively homogeneous within each study group for most of the patients, but no prospective therapeutic trial was simultaneously performed. at early stages (i - iia), most of the patients were treated by topical therapies (corticosteroids, topical nitrogen mustard, carmustine, and psoralen with ultraviolet a). at advanced stages, radiotherapy was performed on tumors, and interferon, chlorambucil, methotrexate, or polychemotherapy was administered as needed. formalin fixed and paraffin - embedded tissues of lesional cutaneous biopsies were obtained from all patients. the diagnosis of mf was established according to the criteria proposed by guitart.. immunostaining was performed on fixed, paraffin - embedded tissue sections as previously described using monoclonal antibodies specific for t - cell associated antigens (cd3, cd4, and cd8) and for b cell - associated antigens (cd20). statistical analysis was performed with dedicated stata se 12 (stata corporation, tx, usa). the fisher 's exact test and the pearson -test were used to identify the differences between the variables. logistic regression is a variation of ordinary regression which predicts the probability of the occurrence of a specific event as a function of two or more independent variables. the results obtained were expressed in terms of the odds ratio (or) associated with each predictor value, defined as the probability of the event occurring divided by the probability of the event not occurring. in all analyses, the cut - off level of statistical significance was set at 0.05. thirty patients (17 men and 13 women) with clinical and histologic diagnosis of mf were included in the study. the mean age at diagnosis was 64.5 years (range 4978 years). according to the tnm classification 8 patients were in stage i, 13 in stage ii, 6 in stage iii, and 3 in stage iv. the thirteen patients in stage ii were classified as 6 stage iia and 7 stage iib. stages i - iia were considered as early stages and stages iib iv as advanced stages. at the time of diagnosis, mf was in a more advanced stage in women than men. indeed, 52.9% of men (versus 38.4% of women) were in early stages whereas 61.6% of women (versus 47.1% of men) were in advanced stages. immunophenotypic analysis demonstrated that 93.3% (28 out 30) of the patients with mf were cd4 positive. the cytotoxic t - cell phenotype cd4/cd8 + was present in only one patient as well as the cd4/cd8 double - negative phenotype. ten out of 30 patients (33.3%) had an immunophenotypic profile characterized by the coexpression of cd4 and cd8. when we compared the distribution of patients with the coexpression and those with the conventional cd4+/cd8 phenotype according to the tnm staging we could not find any association between phenotypes and clinical stage. indeed the percentages of the patients with advanced stage, as compared with those in early stage, were not significantly different in both groups (group cd4+/cd8 50% versus 50% ; group cd4+/cd8 + 40% versus 60%). the median follow - up time in the study was 42 months (ranging from 12 to 70 months) and was comparable among the different clinical stages and phenotypes. none of the patients died during the study period. at the end of the followup 23 of 30 patients underwent complete or partial remission or presented a stable disease without extension of the lesions or increase of the number of lesions. seven patients showed a progressive disease defined as the change to a more advanced stage. the age of patient at diagnosis (65 years), the sex, the duration of symptoms before diagnosis of mf (60 months), and the tnm stage at presentation (early stages versus advanced stages) were not associated with disease progression in univariate as in multivariate analysis. none of the independent variables introduced in the logistic regression model was associated with disease progression defined as the change to a more advanced stage. however, by analyzing the distribution of t - cell phenotypes within the progressing and nonprogressing groups we found that patients with the coexpression cd4 and cd8 had a slightly lower rate of progressive disease as compared to patients with conventional phenotype (10.0% versus 27.8%). in the logistic regression model the value obtained by the independent variable phenotype has almost reached the statistical significance (odds ratio 17.03, p = 0.09, 95% confidence interval 0.62467.7). in this preliminary prospective, single - centre study, we found by conventional immunophenotyping a profile characterized by the coexpression of cd4 and cd8 in one - third of patients with mf. these patients showed a slightly lower rate of progressive disease, compared to patients with conventional cd4+/cd8 phenotype. these findings raise the possibility that the coexpression of cd4 and cd8 in cutaneous lesions may confer a better prognosis in mf. in our patients the conventional t - helper phenotype (cd4+/cd8) was the most common (60% of patients), thus confirming previous studies [1, 12 ]. two patients showed, respectively, the cytotoxic phenotype (cd4/cd8 +) and the double negative phenotype (cd4/cd8). the conventional immunostaining in the remaining 10 patients (33.3%) showed the coexpression of cd4 and cd8. the coexpression of cd4 and cd8 is an expected event on common thymocytes, but it is fairly infrequent on normal peripheral t lymphocytes. there is evidence that in certain situations the normal cd4 + cells may coexpress cd8 and interleukin-4 is able to induce the expression of cd8 on t cd4 + clones. peripheral t lymphocytes with cd4+/cd8 + phenotype have been described in some solid and hematologic malignancies [14, 15 ]. however the double positive cd4/cd8 phenotype is extremely rare in mycosis fungoides [7, 8 ]. the findings of our patients may not be due to the coexpression of both cd in the same neoplastic cell but to the presence in the cutaneous lesions of a mixture of neoplastic cells and inflammatory cd8 + tumor infiltrating lymphocytes as previously demonstrated by hoppe.. immunohistochemical study combined with confocal microscopy might clarify this issue as recently reported by tournier and coworkers in a 31-year - old woman. in their patient this technique revealed in lesional cutaneous biopsies the coexpression of cd4 and cd8 in a subset of atypical t lymphocytes. in typical lesional biopsy specimens of mf other nonneoplastic mononuclear cells the role that these cells play in etiopathogenesis and natural history of mf is still unclear. in our patients with mf the subgroup with the coexpression of cd4 and cd8 has a slightly lower rate of progressive disease in comparison to patients with conventional cd4+/cd8 phenotype (10.0% versus 27.8%), and in the logistic regression model the value obtained by the independent variable t - cell phenotype has almost reached the statistical significance. this data might be of interest taking into account that the limited number of patients and the median follow - up period of just 3.5 years, although similar to those of other investigations, may not allow adequate time for this indolent lymphoma to progress. the lower tendency to progression of disease in our patients with the coexpression of cd4 and cd8 might be related to increased activity of antitumor cd8 + lymphocytes infiltrating the lesions. our results may be consistent with previous findings by hoppe. who demonstrated that cd8 + tumor - infiltrating lymphocytes influenced the long - term survival of patients with mf. another problem is the possibility that cd8 + inflammatory cells can appear in the course of treatment. this is not the case of our patients because we have included in the present study only newly diagnosed patients who had not previously received topical therapies (corticosteroids, topical nitrogen mustard, carmustine, and psoralen with ultraviolet a), interferon, chlorambucil, methotrexate, or polychemotherapy. finally, in this preliminary prospective, single - centre study, conventional immunophenotyping demonstrates the coexpression of cd4 and cd8 in lesional cutaneous biopsies of one - third of patients with mf. these patients show a slightly lower rate of progressive disease, compared to patients with conventional cd4+/cd8 phenotype. a prospective multicenter study on a larger population of patients with a longer followup might be useful to confirm if the coexpression of cd4 and cd8 may confer a better prognosis in patients with mf. | background. although techniques of immunophenotyping have been successful in characterizing the cells in the cutaneous infiltrates of mycosis fungoides little evidence suggests that variations in the phenotypic characterization correlate with prognosis. objectives. in a preliminary prospective, single - centre, study we correlated the t - cell phenotype in cutaneous biopsies with the progression of the disease to determine whether the coexpression of cd4 and cd8 has an impact on prognosis. methods. skin biopsy specimens from 30 newly diagnosed patients were stained with immunoperoxidase techniques to determine their phenotypic characteristics. after a median followup of 42 months patients were divided into two groups with stable and progressive disease. results. eighteen patients had the conventional cd4+cd8 t - cell phenotype. ten patients showed the coexpression of cd4 and cd8 and had a slightly lower rate of progressive disease. conclusions. the coexpression of cd4 and cd8 in cutaneous lesions is not rare and is associated with a slightly lower rate of progressive disease. since double positive cd4/cd8 phenotype is rarely reported in mycosis fungoides the presence on conventional immunophenotyping of both cd may be due to a mixture of neoplastic cells and inflammatory cd8 + tumor infiltrating lymphocytes. immunohistochemical study combined with confocal microscopy could clarify this issue. |
to report a case of recurrent ocular inflammation after optimal therapy of bilateral syphilitic panuveitis responding to oral celecoxib. ocular examination disclosed bilateral panuveitis. serological testing confirmed blood and cerebrospinal fluid syphilitic involvement. ocular inflammation after healing of infectious uveitis is a rare ophthalmic sequela. in an immunocompetent patient non - steroidal therapeutic options, as celecoxib, could be a good option of treatment in such immune cases. syphilis is experiencing an extreme increase of its incidence in europe and the usa since the early 2000s. although penicillin is considered an excellent effective therapy against treponema pallidum, patients hiv positive with cerebrospinal fluid involvement can fail treatment. in immunocompetent patients, either immune uveitis or syphilitic re - infection should be evoked if ocular inflammation is observed after optimal therapy. we report a syphilitic panuveitis presenting recurrent ocular inflammation after correct treatment with penicillin, treated with celecoxib, a nonsteroidal anti - inflammatory drug selective for cyclooxygenase-2. a 76-year - old immunocompetent man presented with discomfort and blurry vision in both eyes for the last week. slit - lamp examination revealed 1 + cells and rare flare with fine keratic precipitates and 4 + cells with hypopyon in the right and left eye, respectively (fig. dilated fundus exam showed dense vitritis and hyperemic optic nerve with blurred margins in both eyes. extensive workup undertaken revealed a serum t. pallidum rpr titer of 1:256 and positive tpha. the patient completed a 2-week cycle of intravenous penicillin g and topical steroids, experiencing a complete resolution of panuveitis and bcva of 20/20 in both eyes. after 3 weeks of the end of treatment, the patient presented up to three anterior sclerouveitis episodes, responding to topical steroids. a re - infection of treponema was microbiologically and clinically ruled out, so an immune ocular response was evoked, starting with celecoxib 200 mg a day, with progressive improvement and no recurrences observed for 1 year of follow - up. b slit - lamp photograph showing a cellular reaction and keratic precipitates in the right eye. e and f bilateral episodic anterior sclerouveitis after penicillin treatment a slit - lamp photograph demonstrating anterior uveitis with hypopyon in the left eye. b slit - lamp photograph showing a cellular reaction and keratic precipitates in the right eye. interaction of t. pallidum with the host 's immune system stimulates humoral and cellular immunity. t. pallidum produces lipoproteins on the outer membrane that interact with lipopolysaccharide - binding proteins, inducing the expression of inflammatory mediators via cd14 and toll - like receptor 2 recognition. these glycolipids induce cellular activation by releasing pro - inflammatory cytokines, mainly tumoral necrosis factor alpha, even after successful penicillin treatment. recurrent ocular inflammation after infectious uveitis treatment is a rare ocular sequela. in the immunocompetent host, such as our patient, it can occur due to a syphilitic re - infection or to an immune process, which may be caused by antigenically inert treponema cell surface. to our knowledge, this is the first report of an immune response after optimal treatment of ocular syphilis at an immunocompetent host. systemic nsaids such celecoxib are known to control ocular inflammatory processes such chronic iridocyclitis or recurrent acute anterior uveitis [4, 5 ], being suitable and sparing - steroids option of therapy in these cases. in conclusion, the outer treponema surface may play a role in immune responses observed after syphilis treatment. we present the first case reported in literature of immune sclerouveitis after neurosyphilis treatment treated successfully with celecoxib. | purposeto report a case of recurrent ocular inflammation after optimal therapy of bilateral syphilitic panuveitis responding to oral celecoxib.methodsa case report was conducted.resultsa 76-year - old man presented with painful blurry vision in both eyes. ocular examination disclosed bilateral panuveitis. serological testing confirmed blood and cerebrospinal fluid syphilitic involvement. after 2 weeks of intravenous penicillin therapy, recurrent episodic sclerouveitis was observed.conclusionocular inflammation after healing of infectious uveitis is a rare ophthalmic sequela. in an immunocompetent patient, either re - infection or immune uveitis should be evoked. non - steroidal therapeutic options, as celecoxib, could be a good option of treatment in such immune cases. |
terpenoid natural products are generally derived from isoprenyl diphosphate precursors with trans double - bond configuration, and no diterpenoid derived from the cisoid precursor (z, z, z)-nerylneryl diphosphate (1) has yet been identified. here further investigation of a terpenoid biosynthetic gene cluster from tomato is reported, which resulted in identification of a biosynthetic pathway from 1, in a pathway featuring a number of interesting transformations. compound 1 is first cyclized to a tricyclene core ring structure analogous to that found in -santalene, with the resulting diterpene termed here lycosantalene (2). quantum chemical calculations indicate a role for the diphosphate anion coproduct in this cyclization reaction. subsequently, the internal cis double bond of the neryl side chain in 2 is then further transformed to an -hydroxy ketone moiety via an epoxide intermediate (3). oxygen labeling studies indicate 3 undergoes oxidative conversion to lycosantalonol (4). thus, in addition to elucidating the cisoid origins of 4, this work has further provided mechanistic insight into the interesting transformations required for its production. |
|
data available for this phase of the fnih sarcopenia project analysis included : the study of osteoporotic fractures, both the original cohort (study visit 6) (14) and african american cohort (study visit 1) (15) ; the osteoporotic fractures in men study (baseline visit) (16) ; the health, aging and body composition study (year 6 clinic visit) (17) ; the framingham study (both the offspring cohort [exam cycles 6 and 7, 19962001 ] (18) and original cohort [exam cycle 26, 19992001 ]) (19) ; the inchianti study (aging in the chianti area, year 3 visit) (20) ; the boston puerto rican health study (baseline visit) (21) ; the age, gene / environment susceptibility - reykjavik study (baseline visit) (22) ; and four clinical trials from the university of connecticut (uconn, randomization visit for all studies) (2326). to be included in these analyses, participants were required to have height and weight, grip strength, and gait speed measured at a single time point : of the 26,625 participants aged 65 and older in the pooled data, 1,403 were ineligible because they were in studies that did not collect the variables used in this analysis ; 1,978 were not eligible for assessment of key measures within their study ; and an additional 2,397 were excluded due to missing data, yielding a final sample size of 20,847 (9,897 men and 10,950 women). participants excluded due to missing data were older, slower, weaker, had lower body mass index (bmi), higher rates of chronic conditions, and were more likely to be women. walking speed less than 0.8 m / s was selected as the primary outcome for the fnih sarcopenia project because of its strong longitudinal associations with disability and mortality and because its use has been recommended by other experts (refs. detailed descriptions of gait speed assessment are available elsewhere (10). grip strength was selected as the primary measure of strength for several reasons. it is clearly related to mobility outcomes (4,6,27) and is easy to use in both clinical and community settings. standard protocols are available for use without a high level of investigator training, and similar protocols were used across project studies. preliminary analysis suggested that grip strength explained a similar amount of variance in walking speed compared with knee extension strength (r for grip strength =.01.16, r for knee extension strength =.04.17). the majority of studies (11 out of 13 cohorts) utilized jamar dynamometers. the maximum strength value in either hand was analyzed. our approach relied on identifying a level of grip strength below which older persons are more likely to have a mobility impairment (gait speed < 0.8 m / s). individual - level data from all cohorts were combined into a single, pooled data set. scatterplots with overlaid locally weighted scatterplot smoothing (loess) curves were used to describe the shape of the relationship between grip strength and gait speed. cart analysis was then performed as the primary method for deriving cutpoints for muscle strength (29). briefly, cart recursively partitions study participants into mutually exclusive groups defined by predictor cutpoints within which participants have similar outcome probabilities. cart is virtually free of modeling assumptions, which provides several advantages in this context : (i) it optimizes concurrent validity by identifying predictors and cutpoints with the strongest relationship with the outcome based on the criterion of minimum error sum of squares ; (ii) it does not require an a priori specified number of cutpoints ; and (iii) it can identify complex interactions (ie, nonlinear interactions involving multiple variables) with other potentially important variables (eg, bmi, height). cart has been used previously to study the association between strength and walking speed (5,30). cart analysis was performed using r version 2.10.1, and cross - validation was used to select the best - performing cutpoints. internal cross - validation is intended to avoid overfitting and development of sample - specific cutpoints. cross - validation was performed by randomly partitioning the pooled data into 10 equally sized mutually exclusive subsamples (ie, each sample excluded 10% of the original pooled data). the tree was then applied to the 10 subsamples each of which contained 90% of the data, and the error variance (called the prediction error, calculated using the error sum of squares) from each subsample was calculated. the 10 prediction errors were used to calculate the empirical standard error of the prediction error. following published guidelines (31), the tree was pruned to the most parsimonious tree within one standard error of the tree with the smallest prediction error. several sets of candidate predictors were included in cart models to identify the most appropriate model for the prediction of slow gait speed : maximum grip strength, body size indicators (bmi, height, weight), and the ratio of strength to body size (grip strength / height, grip strength / weight, grip strength / height, and grip strength / bmi), because previous research suggests that the association between strength and mobility may differ across strata of bmi (4). sensitivity analysis examined the predictive power of recommended cutpoints across cohorts and by characteristics such as age, bmi, height, and comorbidities. cutpoints were evaluated by predicting slow walking speed based on strength category in a mixed - effects logistic regression model including indicator functions for cutpoints as fixed effects and random intercepts accounting for heterogeneity between data sets. additional sensitivity analysis examined the stability of cart - derived cutpoints using alternative gait speed outcomes (< 0.6 m / s and continuous gait speed). although additional analyses were planned based on inability to rise from a chair (an alternative physical performance measure), only 3.8% of men and 6.0% of women met this criterion, providing insufficient sample size to implement those analyses. data available for this phase of the fnih sarcopenia project analysis included : the study of osteoporotic fractures, both the original cohort (study visit 6) (14) and african american cohort (study visit 1) (15) ; the osteoporotic fractures in men study (baseline visit) (16) ; the health, aging and body composition study (year 6 clinic visit) (17) ; the framingham study (both the offspring cohort [exam cycles 6 and 7, 19962001 ] (18) and original cohort [exam cycle 26, 19992001 ]) (19) ; the inchianti study (aging in the chianti area, year 3 visit) (20) ; the boston puerto rican health study (baseline visit) (21) ; the age, gene / environment susceptibility - reykjavik study (baseline visit) (22) ; and four clinical trials from the university of connecticut (uconn, randomization visit for all studies) (2326). to be included in these analyses, participants were required to have height and weight, grip strength, and gait speed measured at a single time point : of the 26,625 participants aged 65 and older in the pooled data, 1,403 were ineligible because they were in studies that did not collect the variables used in this analysis ; 1,978 were not eligible for assessment of key measures within their study ; and an additional 2,397 were excluded due to missing data, yielding a final sample size of 20,847 (9,897 men and 10,950 women). participants excluded due to missing data were older, slower, weaker, had lower body mass index (bmi), higher rates of chronic conditions, and were more likely to be women. walking speed less than 0.8 m / s was selected as the primary outcome for the fnih sarcopenia project because of its strong longitudinal associations with disability and mortality and because its use has been recommended by other experts (refs. detailed descriptions of gait speed assessment are available elsewhere (10). grip strength was selected as the primary measure of strength for several reasons. it is clearly related to mobility outcomes (4,6,27) and is easy to use in both clinical and community settings. standard protocols are available for use without a high level of investigator training, and similar protocols were used across project studies. preliminary analysis suggested that grip strength explained a similar amount of variance in walking speed compared with knee extension strength (r for grip strength =.01.16, r for knee extension strength =.04.17). the majority of studies (11 out of 13 cohorts) utilized jamar dynamometers. the maximum strength value in either hand was analyzed. our approach relied on identifying a level of grip strength below which older persons are more likely to have a mobility impairment (gait speed < 0.8 m / s). individual - level data from all cohorts were combined into a single, pooled data set. scatterplots with overlaid locally weighted scatterplot smoothing (loess) curves were used to describe the shape of the relationship between grip strength and gait speed. cart analysis was then performed as the primary method for deriving cutpoints for muscle strength (29). briefly, cart recursively partitions study participants into mutually exclusive groups defined by predictor cutpoints within which participants have similar outcome probabilities. cart is virtually free of modeling assumptions, which provides several advantages in this context : (i) it optimizes concurrent validity by identifying predictors and cutpoints with the strongest relationship with the outcome based on the criterion of minimum error sum of squares ; (ii) it does not require an a priori specified number of cutpoints ; and (iii) it can identify complex interactions (ie, nonlinear interactions involving multiple variables) with other potentially important variables (eg, bmi, height). cart has been used previously to study the association between strength and walking speed (5,30). cart analysis was performed using r version 2.10.1, and cross - validation was used to select the best - performing cutpoints. internal cross - validation is intended to avoid overfitting and development of sample - specific cutpoints. cross - validation was performed by randomly partitioning the pooled data into 10 equally sized mutually exclusive subsamples (ie, each sample excluded 10% of the original pooled data). the tree was then applied to the 10 subsamples each of which contained 90% of the data, and the error variance (called the prediction error, calculated using the error sum of squares) from each subsample was calculated. the 10 prediction errors were used to calculate the empirical standard error of the prediction error. following published guidelines (31), the tree was pruned to the most parsimonious tree within one standard error of the tree with the smallest prediction error. several sets of candidate predictors were included in cart models to identify the most appropriate model for the prediction of slow gait speed : maximum grip strength, body size indicators (bmi, height, weight), and the ratio of strength to body size (grip strength / height, grip strength / weight, grip strength / height, and grip strength / bmi), because previous research suggests that the association between strength and mobility may differ across strata of bmi (4). sensitivity analysis examined the predictive power of recommended cutpoints across cohorts and by characteristics such as age, bmi, height, and comorbidities. cutpoints were evaluated by predicting slow walking speed based on strength category in a mixed - effects logistic regression model including indicator functions for cutpoints as fixed effects and random intercepts accounting for heterogeneity between data sets. additional sensitivity analysis examined the stability of cart - derived cutpoints using alternative gait speed outcomes (< 0.6 m / s and continuous gait speed). although additional analyses were planned based on inability to rise from a chair (an alternative physical performance measure), only 3.8% of men and 6.0% of women met this criterion, providing insufficient sample size to implement those analyses. sample descriptive characteristics are available in the online supplementary appendix and generally resemble characteristics of the parent studies described elsewhere (10). figure 1 provides plots of the association between grip strength and gait speed in men and women. visual inspection of the loess curves provided little evidence of a clear threshold effect of strength on continuous gait speed. association of grip strength and gait speed in the fnih sarcopenia project : scatterplot and smoothed locally weighted moving averages. cart analysis predicting probability of slow walking (< 0.8 m / s) yielded somewhat different results for men and women. in men, grip strength alone was the strongest predictor of slow walking (ie, resulted in the lowest relative error compared to other predictors), and cart analysis did not identify differences in grip strength cutpoints by bmi. in women, grip strength / bmi was the strongest predictor of slow walking. in order to further explore these gender effects, we evaluated cutpoints based on grip strength alone and grip strength / bmi in both men and women. we conducted logistic regression using alternative definitions of weakness and comparing both model fit statistics and the strength of the associations between weakness and slowness. because the additional value of including bmi in the definition of weakness was unclear (ie, including bmi did not consistently improve model fit or result in stronger associations between weakness and slowness), we elected to use cutpoints based on grip strength alone as our primary analysis. the first cutpoint identified in men was based on having grip strength equal to or above 31.83 kg versus below 31.83 kg. within the low - strength group (< 31.83 kg), a second cutpoint was identified at 25.99 kg. among the weakest men (grip strength < 25.99 kg), 40.4% had slow gait speed, compared with 20.6% among men with intermediate strength (grip strength of 25.9931.82), and 5.7% among men in the highest strength group (grip strength 31.83). classification tree for gait speed < 0.8 m / s in the fnih sarcopenia project. the first cutpoint identified in women was based on having grip strength equal to or above 19.99 kg versus below 19.99 kg. within the low - strength group (< 19.99 kg), a second cutpoint was identified at 15.92 kg. among the weakest women (grip strength < 15.92 kg), 51.4% of women had slow gait speed, compared with 35.6% among women with intermediate strength (grip strength of 15.9219.98 kg), and 20.1% among women in the highest strength group (grip strength 19.99 kg). based on these results, we defined three strength categories for both men and women (normal strength, intermediate, and weak). table 1 provides the prevalence of strength categories in the full sample, accounting for heterogeneity across studies, and provides the relative odds of slow walking across strength groups. the majority of men were classified as normal strength (84%), 11% were classified as intermediate, and 5% as weak. men in the intermediate and weak groups, respectively, had 3.6 (odds ratio [or ] = 3.63, 95% ci : 3.014.38) and 7.6 (or = 7.62, 95% ci : 6.139.49) times greater odds of having slow gait speed relative to men in the normal strength group. over half of women were classified as normal strength (57%), 25% were classified as intermediate and 18% as weak. women in the intermediate and weak groups, respectively, had 2.4 (or = 2.44, 95% ci : 2.202.71) and 4.4 (or = 4.42, 95% ci : 3.944.97) times the odds of slow walking relative to women in the normal strength group. test statistics (sensitivity, specificity, positive predictive value) are provided in the supplementary appendix. sensitivity analysis : prevalence of categories of strength and likelihood of slowness across subsamples in the fnih sarcopenia project notes : chf = congestive heart failure ; copd = chronic obstructive pulmonary disease.odds ratio (or) from logistic regression predicting probability of gait speed < 0.8 m / s with random effect for study.. as expected, the prevalence of weakness varied across age, bmi, height, and diseases status. however, in most cases, the excess prevalence of slowness associated with weakness was similar across groups. for example, the prevalence of weakness was higher among women aged 80 and older (27%) than among women aged 6574 (12%). however, in both age groups, weakness was associated with an excess 3.53.7 times the odds of slow walking relative to normal strength. underweight women were the most likely to be weak (32%), whereas obese women were the least likely to be weak (14%). however, weakness was associated with an excess 4.44.8 times the odds of slow walking across all bmi groups. the association between weakness and slow walking was stronger in men in the 6579 age group (or = 7.42, 95% ci : 5.2810.43) than in men aged 80 and older (or = 4.17, 95% ci : 3.105.61). the association between weakness and slow walking was strongest in men in the tallest height tertile (or = 10.24, 95% ci : 5.9817.54) compared with the lowest tertile (or = 6.69, 95% ci : 4.939.09). additional analysis (supplementary appendix table 4) examined the prevalence of strength categories and low mobility across cohorts. although the prevalence of weakness and mobility limitations differed across cohorts, the relationship between weakness and mobility limitation was generally similar. relationships were less consistent in the clinical trials and in the boston puerto rican health study. sensitivity analysis also considered the stability of grip strength cutpoints based on alternative gait speed outcomes (results not shown). in men, use of gait speed less than 0.6 m / s produced results similar to the main analysis (< 26 kg), identifying a weak group with grip strength less than 25.90 kg. use of a continuous gait speed outcome resulted in selection of a slightly higher cutpoint for weakness (< 27.94 kg). in women, both alternative specifications yielded slightly higher cutpoints for grip strength than the cutpoint found in the main analysis (< 16 kg). models using gait speed less than 0.6 m / s as the outcome identified a weak group with grip strength less than 17.78 kg, and models using a continuous gait speed outcome identified a weak group with grip strength less than 19.99 kg, similar to the intermediate group in the main analysis. results for an alternative definition utilizing grip strength / bmi are reported in the supplementary appendix and briefly summarized here. using a definition of weakness based on grip strength alone, 14% of obese women were classified as weak, and weakness in obese women was associated with 4.4 times the odds of slow walking relative to normal strength (or = 4.41, 95% ci : 3.455.64). alternatively, using a definition based on grip strength divided by bmi (see supplementary appendix table 5), 33% of obese women were classified as weak, and weakness using this definition was associated with 4.8 times the odds of slow walking relative to normal strength (or = 4.84, 95% ci : 3.945.95). in contrast, underweight women were more likely to be classified as weak when using a definition based on grip strength alone (32%) than when using a definition based on grip strength divided by bmi (5%). the purpose of this analysis was to identify cutpoints that distinguish weakness associated with poor mobility performance using cross - sectional data. the cutpoints developed here and carried forward in additional analysis as part of the fnih sarcopenia project define grip strength less than 26 kg in men and less than 16 kg in women as weak. these cutpoints classified 5% of men and 18% of women as weak, and weakness based on this definition was associated with more than 7 times the odds of slow walking in men and more than 4 times the odds of slow walking in women relative to normal strength. cutpoints performed well across a range of subgroups defined by anthropometric characteristics (bmi, height) and the presence of chronic conditions. in addition to identifying a weak group, models identified a group with detectable, although less severe weakness in both men and women (termed intermediate). this intermediate level of weakness (< 32 kg in men and < 20 kg in women) was associated with 3.6 times the odds of slow walking in men and 2.4 times the odds of slow walking in women relative to normal strength. the analytic approach used here focused on maximizing concurrent validity between strength and mobility impairment. this approach resulted in a relatively conservative definition of weakness, based both on test statistics and on comparison to existing definitions. our aim was to maximize confidence that the criterion was detecting a clinically relevant degree of weakness. in men who were not slow (ie, walking speed 0.8 m / s), only 3% were weak, whereas 23% of men who were slow were weak. in women who are not slow, 13% were weak, compared with 31% of women who are slow. thus, the definition of weakness proposed here identifies a subgroup of older persons with a higher probability of combined weakness and slowness than expected in the older population. the european working group on sarcopenia in older persons defined weakness based on a grip strength less than 30 kg in men and less than 20 kg in women (9). this definition corresponds most closely to the cutpoints we identified to classify the intermediate level of weakness. weak are lower (< 26 kg in men and < 16 kg in women) and resulted in a smaller proportion of the population being classified as weak (13). importantly, the role of bmi differed by gender. in models including all potential variations on grip strength and body size (grip strength, body size, and grip strength / body size ratios), grip strength alone was the best predictor for men, whereas grip strength / bmi was the best predictor for women. after carrying forward both definitions for additional analysis and comparing the strength of the relationships between weakness and slowness, as well as model fit statistics, neither definition performed consistently better than the other. in the absence of clear evidence that including bmi in the definition of weakness added to our ability to predict mobility disability, we elected to use the simpler indicator of weakness unadjusted for bmi. although the two different definitions of weakness had similar relationships with slow walking (as indicated by the strength of the relationship and the model fit statistics), they characterized different subgroups of the population as weak a group with limited ability to generate strength and a group unable to generate sufficient strength relative to bmi. because the a priori focus of this analysis was on identifying a subgroup of older adults in whom weakness might be due to low lean mass, we selected the definition based on strength unadjusted for bmi. however, another group may exist in whom weakness is due to low muscle quality. men and women were drawn from different study populations (mros in men, sof in women, different uconn clinical trials available for each gender). it may also reflect sex differences in body composition or the association between strength and mobility. strength declines more rapidly in men at older ages (32), and the association between strength and physical function appears stronger in men than in women (5,33). a central finding of this analysis is the lack of a clear threshold effect in the association between grip strength and gait speed. previous research has reported nonlinearities in the relationship between strength and mobility (1,3), suggesting that there may a level of strength below which mobility becomes more difficult. however, we did not find evidence of a threshold in the relationship between grip strength and gait speed. although grip strength is well correlated with lower extremity strength and is associated with mobility outcomes, it may not detect subtle differences in the association between lower extremity strength and function. however, preliminary analysis of knee extension strength also did not demonstrate nonlinearities in the association of strength and gait speed. it is possible that other measures of function, such ability to rise from a chair, would exhibit threshold effects that we were not able to observe using gait speed as an outcome. however, we were unable to examine inability to rise from a chair as a mobility outcome, because of low prevalence in this sample. the populations included in the fnih sarcopenia project were all drawn from community - dwelling samples of older adults and may be underrepresentative of older populations with mobility limitations and weakness. notably, however, some other studies have also been unable to identify thresholds in the association between strength and functional performance (34). importantly, strength is one of many factors that influence gait speed, including balance, vision, cognition, and muscle power. we used cart to identify weakness cutpoints, but it is important to recognize limitations in this approach. first, cart, by definition, partitions data into groups, even when the underlying relationship between the predictor and outcome is linear. second, our cart models did not account for differences between studies included in the model, although we accounted for study in subsequent logistic models. finally, no statistical model alone can identify a disease state, thus further work is necessary to understand the physiological implications of these results. although grip strength was measured using a variety of protocols, the proposed strength cutpoints performed well across cohorts. unfortunately, data are unavailable from the present studies to directly compare measurement across protocols. by using the maximum measured grip strength from either hand, we sought to minimize variation based on these factors, but variation due to dynamometer used, hand position, and other factors may still exist (28). the analysis used a data - driven approach in a large and diverse pooled data set to identify weakness cutpoints associated with mobility impairment defined by gait speed. this is an important first step in identifying populations that may benefit from interventions to improve strength and muscle function. results from this analysis highlight the utility of an absolute measure of weakness across population groups but also point to the need to consider strength relative to body size, particularly in women. results also cast doubt on the existence of a strong threshold effect in the association between strength and gait speed, suggesting that increases in strength may have positive effects on physical function across the spectrum of strength observed in community - dwelling older adults. despite lack of strong evidence of threshold effects, criteria for the identification of weakness may help identify populations that would experience the greatest benefit from interventions to improve strength and help clinicians identify patients at risk of weakness - associated mobility limitations. funding support for the conference and the work of the consortium was provided by the national institute on aging (1u13ag041583 and p30 ag024827), the food and drug administration, and through grants from the foundation for the nih, made possible by funding from abbott nutrition, amgen, eli lilly, merck, novartis, and the dairy research institute. this research was supported in part by the intramural research program of the nih, national institute on aging. additional acknowledgements for each contributing cohort and members of the fnih sarcopenia project can be found in an online supplement. | background.weakness is common and contributes to disability, but no consensus exists regarding a strength cutpoint to identify persons at high risk. this analysis, conducted as part of the foundation for the national institutes of health sarcopenia project, sought to identify cutpoints that distinguish weakness associated with mobility impairment, defined as gait speed less than 0.8 m / s.methods.in pooled cross - sectional data (9,897 men and 10,950 women), classification and regression tree analysis was used to derive cutpoints for grip strength associated with mobility impairment.results.in men, a grip strength of 2632 kg was classified as intermediate and less than 26 kg as weak ; 11% of men were intermediate and 5% were weak. compared with men with normal strength, odds ratios for mobility impairment were 3.63 (95% ci : 3.014.38) and 7.62 (95% ci 6.139.49), respectively. in women, a grip strength of 1620 kg was classified as intermediate and less than 16 kg as weak ; 25% of women were intermediate and 18% were weak. compared with women with normal strength, odds ratios for mobility impairment were 2.44 (95% ci 2.202.71) and 4.42 (95% ci 3.944.97), respectively. weakness based on these cutpoints was associated with mobility impairment across subgroups based on age, body mass index, height, and disease status. notably, in women, grip strength divided by body mass index provided better fit relative to grip strength alone, but fit was not sufficiently improved to merit different measures by gender and use of a more complex measure.conclusions.cutpoints for weakness derived from this large, diverse sample of older adults may be useful to identify populations who may benefit from interventions to improve muscle strength and function. |
the 2 year mbbs students of 2013 batch of a tertiary care teaching hospital appearing for their third semester practical examinations were included in the study, after successfully completing the designated syllabus for the same and obtaining approval from the institutional ethics committee. the students were sensitized and oriented toward ospe beforehand by explaining the ospe pattern and discussing sample questions during their practical classes. the ospe questions were constructed and validated beforehand for each station along with the answer key and checklists as applicable. viva voce that evaluated theoretical knowledge was allowed a weightage of 15 marks in the practical examination. the questions were based on dose calculation, drug label, routes of drug administration, pharmacy exercises, interpretation of data and graphical records from animal experiments, checking of various parameters and interpretation of results in rabbit eye experiment, therapeutic problems, adverse drug reactions, fixed dose combinations, etc. the effectiveness of ospe was assessed through a student s feedback questionnaire that was prepared after the literature search and validation by the faculty in pharmacology and medical education. the results of ospe were compared with the results of the conventional practical examination (cpe) of the same batch during the 4 semester. the cpe in pharmacology consists of two components, total of 40 marks, including practical exercises of 25 marks and viva voce of 15 marks. comparison of the results was based on 25 marks allotted for ospe and practical exercises. the questions asked were in the form of short exercises related to the topics taught during the semester to different batches such as dose calculation, prescription writing, pharmacy exercise, drug labeling, simulation exercises, and management of diseases. the questions administered were checked for coefficient of reliability by cronbach s alpha, and it was found to be 0.71 with high internal consistency. the feedback given by the students were categorized into three domains, i.e., cognitive, psychomotor, and affective, and an assessment was made for its further use [table 1 ]. students feedback regarding objective structured practical examination majority of the students (74%) said that the pattern of questions asked was well correlated with the syllabus taught in the class. majority (94%) of the students rated ospe well (71% good / satisfactory, 23% excellent) and opined that instructions given at each station were clear and understandable. a total of 96% of students opined (66% good / satisfactory and 30% excellent) that the questions asked in the examination contributed to their learning and helped them improve their knowledge [figure 1 ]. cognitive domain response from the study group twenty - five percent of students said that this pattern of examination is excellent and 72.5% said that it is good or satisfactory in testing practical skills of the students. eighty - one percent of students agreed that it is an excellent method for assessing the applied part of the subject, i.e., applying knowledge to actual situation [figure 2 ]. seventy percent of the students opined that this pattern of examination encouraged them and created interest for learning and minimized their stress level during examination [figure 3 ]. psychomotor domain response from the study group affective domain response from the study group seventy - six percent (76%) of the students rated this methodology of practical examination as good / satisfactory and 23% as excellent in terms of obtaining better scores. students (67.5%) felt that time allotted at each station was good / satisfactory whereas 27.5% felt it to be excellent. 72.5% of the students felt that it would be an excellent idea to introduce this ospe for future assessments [figure 4 ]. assessment domain response from the study group eighty out of 100 students in the 2 year mbbs class appeared in both the examination patterns, i.e., ospe in the first semester and cpe in the second semester. the mean scores out of 25 were 19.94 6.94 and 14.48 10.98 for ospe and cpe, respectively. there was significant difference in the mean scores between ospe and cpe (p < 0.001, df = 158, confidence interval = 95%) [figure 5 ]. ospe has introduced for the 2 year mbbs students and was evaluated with the help of student s feedback questionnaire and a comparison of performance of the students in the two evaluation methods. student s feedback for educational methodologies is an important and useful basis for modifying and improving medical education. the basic aim of this feedback is to assess the areas of strength and/or lacunae of teaching methodologies to rectify the shortcomings and revise the curriculum suitably. in our study, about 79% of the students opined that ospe assessed the syllabus that has been taught earlier in the practical classes. majority of the students were able to understand and follow the instructions properly, due to prior sensitization to this novel method. students perceptions with regard to improvement in their practical were positive as majority of the students perceived ospe to be good or satisfactory, suggesting that it would be acceptable to a majority of students in the event that it replaced the cpe. previous studies also revealed similar findings in this regard. around 81% of students opined that ospe is an excellent tool for assessing the application of knowledge., where 74% students agreed to multiple modes of assessment to improve their knowledge and skills in anatomy. in another study by chandelkar., it was found that all the students accepted ospe because it helped them improve their practical skills and application in pharmacology. results of another study indicated better effect of objective structured assessment of technical skills on learning. any examination is a well - known source of stress and anxiety and ospes in particular considered as quite stressful. in contrast to this, around 80% of the students agreed that ospe is an examination with minimum stress level in our study. this could be possibly due to least or indirect interaction with the examiners as well as students undergoing limited number of stations., where they had limited number of stations and no direct interaction with examiners, but the students there agreed that this type of examination may be exhausting and stressful if number of stations will be increased. low level of stress can make student more alert and motivated. however, high level of stress can cause several difficulties, including reduced ability to prepare for and perform during their exams. 76.2% of the students felt ospe to be a better method of evaluation for future examinations as well. objectivity and more uniform evaluation are the documented advantages of this method. in our study, only 5% of the students were not satisfied with the time allotted for ospe., where only one student was not satisfied with the time allotted for ospe. in another study by ranjan. communication skill is one of the most important skills of a physician for patient s management. this limitation could be overcome in future studies by including clinical cases, where students may be asked to give verbal instructions regarding the use of various drugs or in the form of clinical pharmacological exercises. ospe when compared to cpe, a significant difference (p < 0.001) was found in the mean scores of the result showing better performance of the students [figure 5 ]. this was in accordance to a study by ranjan., where significant (p < 0.05) improvement was observed in the mean marks obtained in ospe as compared to cpe. in another study by nigam., ospe was found to be important in competency - based performance discrimination and also in improving students performance quality in laboratory exercises. this study confirmed the feasibility and students acceptability of ospe in evaluating the pharmacology skills in the undergraduate medical curriculum. most of participants in the study were in favor of using this assessment method in future also. in comparison to cpe thus, it can be concluded that use of ospe is a relevant, meaningful, and feasible tool for the assessment of practical skills in undergraduate training in pharmacology. | objective : assessment method can influence student learning. use of objective structured practical examination (ospe) has been reported in various institutes with great benefits. we evaluated ospe for the assessment of practical skills in pharmacology examination for undergraduate medical students and compared it with conventional practical examination (cpe).materials and methods : after sensitizing the 2nd year mbbs students to ospe, the students were divided into four batches with twenty students in each batch. students were assessed by attending five ospe stations, each for duration of 5 min. the effectiveness was assessed through a student s feedback questionnaire and was checked for its reliability by cronbach s alpha. the result of ospe was compared with that of cpe of the same batch.results:cronbachs alpha of the feedback questionnaire was 0.71, with high internal consistency. the feedback given was categorized into three domains : cognitive, psychomotor, and affective, and an assessment was also done for its further use. in cognitive domain, 74% of the students felt that the questions asked and the syllabus taught were well correlated. in psychomotor domain, 81% agreed that it is excellent for assessing the applied part of the subject. seventy percent of students opined that it was associated with lesser stress than cpe. on overall assessment, 76% rated this methodology as good / satisfactory and 23% as excellent in terms of better scoring. there was a significant difference in the mean score between the results of ospe and cpe (p < 0.001, df = 158, confidence interval = 95%).conclusion : ospe is a feasible and skill enhancing tool for the assessment in pharmacology examinations for undergraduate students. |
chiari malformation (cm) is characterized by ectopia of the hindbrain through the foramen magnum1216). typically, tonsillar descent of > 5 mm is defined as cm type 1. although the exact pathophysiology has not been determined, disturbance of free cerebrospinal fluid (csf) flow at the craniocervical junction is a common component of several theories6111221). treatment is indicated for symptomatic patients, particularly when cm is combined with syringomyelia in adult patients818). there are many secondary causes of cm, such as hydrocephalus, congenital vertebral anomaly and arachnoid scarring, and these factors may contribute to the variation in outcomes231015). in the case of idiopathic cm type 1, restoration of the cisterna magna by foramen magnum decompression (fmd) and duroplasty seems to be a sufficient treatment for restoration of free csf flow5). however, caudal migration of the medulla oblongata is present in certain patients with cm type 1, and this condition is referred to as cm type 1.5 in pediatric patients22). the surgical outcome is poorer in pediatric cm type 1.5 than in cm type 122). however, the significance of additional migration of the medulla oblongata in adult patients is unknown. we hypothesized that additional migration of the medulla oblongata would also be associated with disturbed csf flow and that the surgical outcomes would not differ between adult cm type 1 and cm type 1.5 if normal csf flow were restored. the objective of the present study was to analyze surgical outcomes of adult idiopathic cm type 1 with and without additional migration of medulla oblongata. the cases of patients with cm type 1, who were surgically treated at an adult hospital from 19882012, were retrospectively reviewed. we included patients with syringomyelia, and excluded patients with possible secondary causes, such as a history of meningitis, encephalitis, hydrocephalus, intracranial lesions, an occipitocervical anomaly, brain or spine surgery, or head trauma. 38 (81%) with traceable medical records and at least a 12-month follow - up period were included in the present study (m : f=11 : 27 ; mean age, 34.713.8 years ; median, 33.5 ; range, 1863). preoperatively, all patients underwent whole - spine magnetic resonance (mr) imaging, and the mid - sagittal t1- and t2-weighted mr images were reviewed. thirteen patients (34%) had additional migration of the medulla oblongata, which was indicated when the obex was located below the foramen magnum722). cm type 1 without herniation of the medulla oblongata was classified as group a, and cm type 1 with herniation of the medulla oblongata was classified as group b. syringomyelia was confined to the cervical spinal cord in 2 patients and extended to the thoracic spinal cord in 36 patients. the modified mccormick scale (mms) (table 1) was used to evaluate functional status of the patients before surgery and one year after surgery17). clinically, the presenting symptoms / signs were varied, and each patient had a variable combination of symptoms / signs. sensory disturbance in the upper limb(s) was the most common symptom, present in 24 patients, and lower - limb sensory disturbance was present in 9 patients. upper - extremity weakness was also observed in 5 patients, and lower - extremity weakness was observed in 4 patients. other symptoms included the following : neck pain, affecting 13 patients ; headache, 4 ; and dizziness, 1. the patients were encouraged to ambulate from the day of surgery and walked home at approximately postoperative day 7. the patients were scheduled to visit the outpatient clinic at 3, 6 and 12 months postoperatively and yearly thereafter. follow - up mr imaging was performed between postoperative 6 and 12 months and was repeated at 1224 months if the size of the syringomyelia cavities had not decreased sufficiently in the prior image. the patients were followed up for 72.755.6 months (median, 56.5 months ; range, 12232 months). suboccipital craniotomy (fmd) and removal of the posterior c1 ring were performed in all patients. additional removal of the c2 lamina was selectively performed if preoperative mr imaging demonstrated that the tip of the tonsil reached the c2 level. in the past, more recently, the arachnoid was left intact if there was no arachnoid pathology and if free csf flow was observed in the subtonsillar subarachnoid space. in patients with secondary causes, the wound was closed layer by layer, without a closed - suction drain19). we analyzed factors affecting radiological success, rather than clinical success, because comparison of the diverse clinical symptoms was inconsistent10). the ratio between the largest diameter of the spinal syrinx on each mr image and that of the spinal cord on preoperative mr imaging was used to evaluate the change in syringomyelia2). radiological success was defined as when the syrinx ratio decreased by more than 50% during the postoperative 12 months. clinical (age, sex, motor symptoms, duration of symptoms, and mms), radiological [the extent of the syrinx (cervical vs. cervicothoracic), sufficient postoperative restoration of the cisterna magna, and migration of the medulla oblongata (group a vs. group b) ] and surgical (arachnoid dissection, tonsillectomy, and syrinx - subarachnoid shunting) factors were included in the risk factors analysis. restoration of the cisterna magna was regarded as sufficient when the length of the dorsal csf pathway was 12.0 mm at the level of the foramen magnum in mid - sagittal t2-weighted mr images collected at postoperative 612 months5). all statistical analyses were performed using the commercially available software spss, version 17.0 (spss inc., non - parametric continuous values were compared using mann - whitney 's u test or the wilcoxon rank sum test, and dichotomous values were analyzed with the chi - square test. we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers / animals were followed during the course of this research. suboccipital craniotomy (fmd) and removal of the posterior c1 ring were performed in all patients. additional removal of the c2 lamina was selectively performed if preoperative mr imaging demonstrated that the tip of the tonsil reached the c2 level. in the past, more recently, the arachnoid was left intact if there was no arachnoid pathology and if free csf flow was observed in the subtonsillar subarachnoid space. in patients with secondary causes, the wound was closed layer by layer, without a closed - suction drain19). we analyzed factors affecting radiological success, rather than clinical success, because comparison of the diverse clinical symptoms was inconsistent10). the ratio between the largest diameter of the spinal syrinx on each mr image and that of the spinal cord on preoperative mr imaging was used to evaluate the change in syringomyelia2). radiological success was defined as when the syrinx ratio decreased by more than 50% during the postoperative 12 months. clinical (age, sex, motor symptoms, duration of symptoms, and mms), radiological [the extent of the syrinx (cervical vs. cervicothoracic), sufficient postoperative restoration of the cisterna magna, and migration of the medulla oblongata (group a vs. group b) ] and surgical (arachnoid dissection, tonsillectomy, and syrinx - subarachnoid shunting) factors were included in the risk factors analysis. restoration of the cisterna magna was regarded as sufficient when the length of the dorsal csf pathway was 12.0 mm at the level of the foramen magnum in mid - sagittal t2-weighted mr images collected at postoperative 612 months5). all statistical analyses were performed using the commercially available software spss, version 17.0 (spss inc., non - parametric continuous values were compared using mann - whitney 's u test or the wilcoxon rank sum test, and dichotomous values were analyzed with the chi - square test. we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers / animals were followed during the course of this research. the extent of tonsillar descent was greater in group b than in group a (p<0.01) (table 2). the obex was located 11.04.7 mm (range, 4.319) below the foramen magnum in group b. syringomyelia was decreased by more than 50% in 32 (84%) patients (21/25 in group a and 11/13 in group b). the preoperative syrinx ratio was 0.750.23, which decreased to 0.200.18 at the last follow - up (p<0.05). the extent of the decrease did not differ between group a (75.320.3%) and group b (68.127.4%) (p= 0.60). an analysis of factors related to radiological improvement revealed that sufficient restoration of the cisterna magna (p=0.01 ; or, 46.5 ; 95% ci, 3.2676.2) was the only significant factor (table 3). preoperative functional status were mms i in 11 patients and mms ii in 14 patients of group a and mms i in 8 and mms ii in 5 patients of group b. postoperatively, clinical symptoms were improved in 31 (82%) patients (21/25 in group a and 10/13 in group b). of patients with mms ii, 8 patients showed functional improvement (5/14 in group a and 3/5 in group b), from mms ii to i, and there was no case of deterioration from mms i and ii (table 4). although clinical improvement was noted, minimal change of clinical symptoms was not reflected in mms. complications occurred in 2 patients in group a (cerebellar contusion in 1 and subdural hemorrhage in 1) and 3 patients in group b (csf leakage in 2 and epidural hemorrhage in 1). reoperation was necessary in 2 patients due to epidural hemorrhage and subdural hemorrhage. during the follow - up period, intraoperative findings showed that the arachnoid was clear in all patients and was left intact in 11/25 patients in group a and 5/13 patients in group b (p=0.74). before december 2005, arachnoid dissection and/or tonsillectomy were performed in 15/17 patients, and they were performed in 7/21 patients after january 2006. the following two cases exemplify dynamic changes in hindbrain herniation in response to surgical treatment. a 21-year - old man presented with a 5-month history of decreased sensation in his right leg. mr imaging showed a cm type i and migration of the medullar oblongata with large syrinx on the cervical spine. the obex and tonsil were located 16.1 mm and 17.2 mm, respectively, below the foramen magnum (fig. cm type 1 with syringomyelia was diagnosed, and fmd, removal of the posterior c1 ring and duroplasty were performed. the arachnoid membrane was left intact because it was clear, and free csf flow was observed in the subtonsillar subarachnoid space. mr imaging performed 2 days after surgery showed similar hindbrain herniation and syringomyelia (fig. his sensation was normalized from postoperative 3 months, and mr imaging performed at 6 months showed a marked improvement in the syringomyelia and normalization of the hindbrain herniation. the size of the dorsal csf pathway was 14.5 mm, and rounding of the tonsil was also observed (fig. 2c). a 20-year - old female presented with a 7.5-year history of decreased sensation in her right upper extremity. mr imaging showed cm type 1, additional migration of the medulla oblongata and syringomyelia (fig. the tonsil and obex were located 6.5 mm and 5.7 mm, respectively, below the foramen magnum. although the arachnoid membrane was clear, arachnoid dissection, tonsilectomy and duroplasty were performed. mr imaging performed 3 months after the operation did not show rounding of the tonsil, restoration of the cisterna magna or improvement in the syringomyelia (fig. the herniation of the medulla oblongata progressed with time, and the locations of the obex at 9, 15, and 27 months were 7.2, 8.2, and 12.1 mm, respectively. mr imaging performed at 50 months showed further herniation of the tonsil (13 mm) and medulla oblongata (obex, 12.9 mm below the foramen magnum) (fig. the patient refused reoperation because her symptoms were stationary and similar to her preoperative state. intraoperative findings showed that the arachnoid was clear in all patients and was left intact in 11/25 patients in group a and 5/13 patients in group b (p=0.74). before december 2005, arachnoid dissection and/or tonsillectomy were performed in 15/17 patients, and they were performed in 7/21 patients after january 2006. the following two cases exemplify dynamic changes in hindbrain herniation in response to surgical treatment. a 21-year - old man presented with a 5-month history of decreased sensation in his right leg. mr imaging showed a cm type i and migration of the medullar oblongata with large syrinx on the cervical spine. the obex and tonsil were located 16.1 mm and 17.2 mm, respectively, below the foramen magnum (fig. cm type 1 with syringomyelia was diagnosed, and fmd, removal of the posterior c1 ring and duroplasty were performed. the arachnoid membrane was left intact because it was clear, and free csf flow was observed in the subtonsillar subarachnoid space. mr imaging performed 2 days after surgery showed similar hindbrain herniation and syringomyelia (fig. his sensation was normalized from postoperative 3 months, and mr imaging performed at 6 months showed a marked improvement in the syringomyelia and normalization of the hindbrain herniation. the size of the dorsal csf pathway was 14.5 mm, and rounding of the tonsil was also observed (fig. 2c). the syrinx had nearly disappeared at postoperative month 24. a 20-year - old female presented with a 7.5-year history of decreased sensation in her right upper extremity. mr imaging showed cm type 1, additional migration of the medulla oblongata and syringomyelia (fig. the tonsil and obex were located 6.5 mm and 5.7 mm, respectively, below the foramen magnum. although the arachnoid membrane was clear, arachnoid dissection, tonsilectomy and duroplasty were performed. mr imaging performed 3 months after the operation did not show rounding of the tonsil, restoration of the cisterna magna or improvement in the syringomyelia (fig. the herniation of the medulla oblongata progressed with time, and the locations of the obex at 9, 15, and 27 months were 7.2, 8.2, and 12.1 mm, respectively. mr imaging performed at 50 months showed further herniation of the tonsil (13 mm) and medulla oblongata (obex, 12.9 mm below the foramen magnum) (fig. the patient refused reoperation because her symptoms were stationary and similar to her preoperative state. a 21-year - old man presented with a 5-month history of decreased sensation in his right leg. mr imaging showed a cm type i and migration of the medullar oblongata with large syrinx on the cervical spine. the obex and tonsil were located 16.1 mm and 17.2 mm, respectively, below the foramen magnum (fig. cm type 1 with syringomyelia was diagnosed, and fmd, removal of the posterior c1 ring and duroplasty were performed. the arachnoid membrane was left intact because it was clear, and free csf flow was observed in the subtonsillar subarachnoid space. mr imaging performed 2 days after surgery showed similar hindbrain herniation and syringomyelia (fig. his sensation was normalized from postoperative 3 months, and mr imaging performed at 6 months showed a marked improvement in the syringomyelia and normalization of the hindbrain herniation. the size of the dorsal csf pathway was 14.5 mm, and rounding of the tonsil was also observed (fig. 2c). a 20-year - old female presented with a 7.5-year history of decreased sensation in her right upper extremity. mr imaging showed cm type 1, additional migration of the medulla oblongata and syringomyelia (fig. the tonsil and obex were located 6.5 mm and 5.7 mm, respectively, below the foramen magnum. although the arachnoid membrane was clear, arachnoid dissection, tonsilectomy and duroplasty were performed. mr imaging performed 3 months after the operation did not show rounding of the tonsil, restoration of the cisterna magna or improvement in the syringomyelia (fig. the herniation of the medulla oblongata progressed with time, and the locations of the obex at 9, 15, and 27 months were 7.2, 8.2, and 12.1 mm, respectively. mr imaging performed at 50 months showed further herniation of the tonsil (13 mm) and medulla oblongata (obex, 12.9 mm below the foramen magnum) (fig. the patient refused reoperation because her symptoms were stationary and similar to her preoperative state. herniation of the medulla oblongata was observed in 34% (13/38) of patients in the present study. syringomyelia and clinical symptoms were improved in 32 (84%) and 31 (82%) patients, respectively. the sufficient restoration of the cisterna magna (p=0.01) was the only significant factor. the reduction in syringomyelia after restoration of the cisterna magna was not dependent on herniation of the medulla oblongata (p=0.96). we demonstrated that hindbrain herniation changes dynamically, depending on whether successful restoration of the cisterna magna is achieved in case 1 and 2. cm type 1.5 is defined as a combination of brain stem herniation through the foramen magnum in addition to the presence of cm type 1 in pediatric patients22). the obex has been found to lie 144 mm below the foramen magnum (11.04.7 mm in the present study)22). in pediatric patients, although no differences in clinical symptoms or the incidence of syringomyelia are characteristic of cm type 1.52122), persistent syringomyelia after posterior decompressive surgery and duroplasty has been reported to be twofold higher in cm type 1.5 than in cm type 1 (14% vs. 7%), and elective resection of the tonsil has been recommended22). kim.7) reported a case of spontaneous progression of pediatric cm type 1 to cm type 1.5 over a 9-year follow - up. the incidence and significance of medulla oblongata herniation in adult idiopathic cm type 1 have not been reported. in the present study, although it was not specified, we found similar findings in other papers579). in those studies, if there were no arachnoid pathology, rapid csf flow through the narrow subarachnoid space resulted in a pressure difference between the cranial and the caudal compartments (the venturi effect), which caused hindbrain herniation, and further migration of the medulla oblongata could occur over time4). these findings suggest that the adverse consequences could be resolved by eliminating the venturi effect itself14). therefore, the goal of surgery is the restoration of unimpeded flow of the csf across the craniocervical junction by enlarging the retrocerebellar and subtonsillar cisterns (restoration of the cisterna magna), allowing the normal csf pulsations to be transmitted into the spinal subarachnoid space23131820). the value of opening the arachnoid membrane and performing tonsillectomy to restore the cisterna magna is questionable. many papers have supported arachnoid opening and/or tonsillectomy12310). however, a successful surgical outcome (100% normalization of hindbrain herniation) was reported without arachnoid opening by heiss.5). klekamp10) showed that combined arachnoid pathology was a strong risk factor for symptom recurrence (or, 7.4) in a large case series (n=371). the arachnoid membrane was normal only in 59/371 patients, and neurological deterioration was observed in 13% of cases within 5 years and in 19% within 10 years after the surgery.10) in contrast, alfieri and pinna1) reported that recurrence was observed in only 1.8% (2/109) of patients over a median of 12.7 years in a study that included only patients without arachnoid pathology. these results imply that arachnoid pathology might cause an unpredictable surgical outcome, most likely due to different pathophysiology. in the present study, the only significant prognostic factor was sufficient restoration of the cisterna magna ; arachnoid opening and tonsillectomy were not significant factors. for patients without arachnoid pathology, adequate restoration of the cisterna magna with fmd, decompression of c1/c2 and duroplasty may be sufficient, even for patients with additional herniation of the medulla oblongata. although the definition of " adequate " is not established, confirming free csf flow in the subtonsillar subarachnoid space during the operation may lead to adequate cisterna magna restoration1245). first, the present study was a retrospective analysis of 38 patients and the small number may have resulted in a lower statistical power. second, the surgical procedure was not uniform, and selection of the surgical procedure evolved over time. a prospective randomized controlled trial with long - term follow - up will be essential to establish the practical value of each surgical procedure. third, the follow - up period (72.755.6 months ; median, 56.5 months ; range, 12232 months) was not sufficient to draw a solid conclusion. cm type 1.5 is defined as a combination of brain stem herniation through the foramen magnum in addition to the presence of cm type 1 in pediatric patients22). the obex has been found to lie 144 mm below the foramen magnum (11.04.7 mm in the present study)22). in pediatric patients, although no differences in clinical symptoms or the incidence of syringomyelia are characteristic of cm type 1.52122), persistent syringomyelia after posterior decompressive surgery and duroplasty has been reported to be twofold higher in cm type 1.5 than in cm type 1 (14% vs. 7%), and elective resection of the tonsil has been recommended22). kim.7) reported a case of spontaneous progression of pediatric cm type 1 to cm type 1.5 over a 9-year follow - up. the incidence and significance of medulla oblongata herniation in adult idiopathic cm type 1 have not been reported. in the present study, although it was not specified, we found similar findings in other papers579). in those studies, if there were no arachnoid pathology, rapid csf flow through the narrow subarachnoid space resulted in a pressure difference between the cranial and the caudal compartments (the venturi effect), which caused hindbrain herniation, and further migration of the medulla oblongata could occur over time4). these findings suggest that the adverse consequences could be resolved by eliminating the venturi effect itself14). therefore, the goal of surgery is the restoration of unimpeded flow of the csf across the craniocervical junction by enlarging the retrocerebellar and subtonsillar cisterns (restoration of the cisterna magna), allowing the normal csf pulsations to be transmitted into the spinal subarachnoid space23131820). the value of opening the arachnoid membrane and performing tonsillectomy to restore the cisterna magna is questionable. many papers have supported arachnoid opening and/or tonsillectomy12310). however, a successful surgical outcome (100% normalization of hindbrain herniation) was reported without arachnoid opening by heiss.5). klekamp10) showed that combined arachnoid pathology was a strong risk factor for symptom recurrence (or, 7.4) in a large case series (n=371). the arachnoid membrane was normal only in 59/371 patients, and neurological deterioration was observed in 13% of cases within 5 years and in 19% within 10 years after the surgery.10) in contrast, alfieri and pinna1) reported that recurrence was observed in only 1.8% (2/109) of patients over a median of 12.7 years in a study that included only patients without arachnoid pathology. these results imply that arachnoid pathology might cause an unpredictable surgical outcome, most likely due to different pathophysiology. in the present study, the only significant prognostic factor was sufficient restoration of the cisterna magna ; arachnoid opening and tonsillectomy were not significant factors. for patients without arachnoid pathology, adequate restoration of the cisterna magna with fmd, decompression of c1/c2 and duroplasty may be sufficient, even for patients with additional herniation of the medulla oblongata. although the definition of " adequate " is not established, confirming free csf flow in the subtonsillar subarachnoid space during the operation may lead to adequate cisterna magna restoration1245). first, the present study was a retrospective analysis of 38 patients and the small number may have resulted in a lower statistical power. second, the surgical procedure was not uniform, and selection of the surgical procedure evolved over time. a prospective randomized controlled trial with long - term follow - up will be essential to establish the practical value of each surgical procedure. third, the follow - up period (72.755.6 months ; median, 56.5 months ; range, 12232 months) was not sufficient to draw a solid conclusion. normalization of csf flow was the most important surgical goal for adult patients with idiopathic cm type 1. migration of the medulla oblongata did not make a difference in the surgical outcome when the cisterna magna was restored. a long - term follow - up study with a large number of patients will be necessary to confirm these findings. | objectivethe pathophysiology of idiopathic chiari malformation (cm) type 1 is disturbance of free cerebrospinal fluid (csf) flow and restoration of normal csf flow is the mainstay of treatment. additional migration of the medulla oblongata in pediatric patients is referred to as cm type 1.5, but its significance in adult patients is unknown. this study is to compare surgical outcomes of adult idiopathic cm type 1.5 with that of type 1.methodsthirty-eight consecutive adult patients (m : f=11 : 27 ; median, 33.5 ; range, 1863) with syringomyelia due to idiopathic cm type 1 were reviewed. migration of the medulla oblongata was noted in 13 patients. the modified mccormick scale (mms) was used to evaluate functional status before and one year after surgery. all patients underwent foramen magnum decompression and duroplasty. factors related to radiological success (50% decrease in the diameter of the syrinx) were investigated. the follow - up period was 72.755.6 months.resultspreoperative functional status were mms i in 11 patients and mms ii in 14 of cm type 1 and mms i in 8 and ii in 5 of cm type 1.5. of patients with mms ii, 5/14 patients in group a and 3/5 patients in group b showed improvement and there was no case of deterioration. radiological success was achieved in 32 (84%) patients and restoration of the cisterna magna (p=0.01 ; or, 46.5) was the only significant factor.conclusionmigration of the medulla oblongata did not make a difference in the surgical outcome when the cisterna magna was restored. |
transverse myelitis is a neurological disorder of the spinal cord caused by inflammation that typically has an acute presentation. the etiology may be infectious, parainfectious, other systemic inflammatory disorders, a spinal form of multiple sclerosis or idiopathic. infectious myelitis presents with fever and paraparesis, most commonly affecting the thoracic spinal cord. sensory symptoms usually present as ascending paresthesias, with or without back pain at or near the level of the myelitis. motor symptoms often include weakness that preferentially affects the flexors of the legs and the extensors of the arms and can include sphincter dysfunction. autonomic involvement manifests with bowel and bladder dysfunction, temperature dysregulation or bouts of hypertension. we present a case of infectious transverse myelitis secondary to hsv-1 with an unusual presentation of descending paralysis, who despite appropriate therapy did not recover motor function. on arrival to the hospital, the patient was talking and moving his upper extremities. he was afebrile, tachycardic (106 bpm) and tachypneic (25 bpm). during initial evaluation, he remained fully conscious. on physical exam, strength was 0/5 in all four extremities. sensation was intact over the head and neck, decreased 3/5 in the right arm and absent over the rest of his body. lumbar puncture (lp) was done on admission and csf analysis showed 49 red blood cells, one white blood cell, normal protein and normal glucose. magnetic resonance imaging (mri) of the spine showed segmental increase in t2 and stir signal with associated mild expansion of the spinal cord (fig. tests for lupus, neuronal potassium channel antibodies, anti - calcium - channel antibodies, anti - purkinje - cell antibodies, anti - chromatin antibodies, anti - glial antibodies, anti - neuronal antibodies, rheumatoid arthritis latex turbid test, lyme serology, mycoplasma antibodies and quantitative hiv rt - pcr were all negative. lp was repeated after one week and csf analysis showed this time 92,000 red cells / cubic cm, 40 white cells / cubic cm with 88% neutrophils, glucose 72 mg / dl and protein 209 mg / dl. csf aerobic and fungal cultures, enterovirus rt - pcr, west nile virus igm and igg, vdrl, oligoclonal bands and nmo / aqp4 were negative. after repeat lp, intravenous dexamethasone 6 g every 4 h was initiated, followed by a taper. in addition patient received intravenous acyclovir 1 g every 8 h and was given also ivig, but his symptoms failed to improve. of note, during his hospital stay, patient also underwent plasma exchange every other day for 5 days without signs of improvement. this was done due to suspicion of possible guillain barre syndrome, prior to repeating the lp. repeat spine mri done 12 days from admission showed interval increase in abnormal signal intensity of cervical cord, acute cervical cord edema with associated diffuse intramedullary enhancement with complete effacement of the csf space surrounding the cervical spinal cord. a follow up mri done at 51 days showed marked improvement in the focal expansile appearance of upper cervical cord and abnormal t2 signal within the spinal cord and slightly decreased in craniocaudal extent, improved in cystic myelomalacia with t2 signal changes and minimal persistent enhancement. despite completing three weeks of intravenous acyclovir, high dose steroids several viruses have been associated with infectious transverse myelitis, most commonly enteroviruses, hsv type 2 and varicella - zoster virus. hsv, most frequently hsv type 2, has been reported since as the causative agent in several other cases of transverse myelitis. it can affect all ages, but seems to be more common in the10 - 19 years old and 3039 years old age groups. hsv myelitis has been described mostly in immunocompromised patients, but it has also been reported in immunocompetent patients. patients may or may not present with hsv skin manifestations at the time of neurological symptoms. it has been hypothesized that the pathogenesis of hsv myelitis starts with hsv latent infection of the dorsal root ganglion at the level of the lumbosacral spinal cord which reactivates. this leads to invasion of the spinal cord, forming of necrotizing lesions and sometimes spread to the cervico - thoracic spinal cord. related to the localization of the latent hsv infection, it has been described that hsv type 1 more often produces myelitis involving the cervical to the thoracic segment, while hsv type 2 involves the whole spinal cord or just the lumbosacral segment. the initial symptoms of hsv myelitis are sensory - motor disturbances of the lower limbs and urinary disturbance. the ascending necrotizing form often presents with encephalitis, quadriplegia or respiratory muscle paralysis which makes the prognosis poor. necrotic cord changes are common, and hemorrhagic lesions are occasionally seen. ascending paralysis has been described as the most common presentation of transverse myelitis secondary to hsv type 2 infection. in contrast, al described cases of transverse myelitis secondary to hsv type 1 infection where a non - ascending paralysis pattern was observed. magnetic resonance imaging is the most specific test and may reveal signs of demyelination with or without necrosis. histopathologic findings include marked necrosis of both gray and white matters, hemorrhage, perivascular lymphocyte infiltration and vascular necrosis. progression may be prevented by administration of antiviral agents and steroids which lower mortality and improve neurological symptoms. in this case, presentation was unusual with initial complaints of weakness in his upper extremity, followed by rapid progression to quadriplegia. the first csf hsv pcr was negative, but subsequent repeat csf hsv pcr was positive for hsv type 1. after administration of acyclovir, steroids, ivig and plasma exchange, mri of spine showed improvement of the lesions, but at four month follow - up, the patient had not recovered any of his motor function. | transverse myelitis is a neurological disorder of the spinal cord that can have a variety of etiologies. herpes simplex virus (hsv) infection has been described as one of the causes, most commonly hsv type 2.we report here a case of an 18 year old male who presented with weakness that started in his upper extremities and rapidly evolved to quadriplegia. magnetic resonance imaging of spine was consistent with transverse myelitis. hsv type 1 pcr testing on cerebrospinal fluid (csf) was positive. he was started on acyclovir and steroids, but despite therapy, patient did not recover motor function. |
marijuana is found to have many physiological and pathophysiological functions, including mood alteration, motor and co - ordination activities. there is no literature on molecular adsorbent recirculation system (mars) therapy for hepatotoxicity following marijuana drug abuse. we report a case of fulminant hepatic failure following marijuana drug abuse successfully managed with mars therapy. a 23-year - old male college student presented to the emergency department with a history of malaise, fatigue and vomiting of 1 week duration. he also had a history of yellowish discoloration of the sclera and breathlessness of 3 days and developed altered sensorium a day prior to presentation. his father gave a history of marijuana abuse for the past few months and denied history of smoking and alcohol. on examination, he was irritable pale and icteric, blood pressure was 130/80 mmhg. sequential laboratory parameters of patient tests for hepatitis a, hepatitis b, hepatitis c and e, human immunodeficiency virus, cytomegalovirus, parvovirus, wilson s disease, autoimmune hepatitis and acetaminophen induced hepatitis were all negative. peripheral smear for malaria parasite, anti - dengue antibodies and leptospira serology were all negative. the diagnosis of acute fulminant hepatitis secondary to marijuana abuse was considered as other causes of fulminant hepatic failure were all ruled out and blood and urine were positive for thc. his condition worsened over the next 2 days and developed respiratory distress requiring intubation and ventilator support. on the 3 day, he was initiated on extra - corporal liver - purification system ; mars. after three sessions of mars, each lasting 8 h, there was a remarkable improvement in level of sensorium and biochemical parameters. he was extubated on the 6 day and by 10 day his liver parameters came down remarkably. it was found to have many physiological and pathophysiological functions, including mood alteration, control of feeding and appetite, motor and co - ordination activities, analgesia, immune modulation and gut motility. marijuana toxicity usually produces drowsiness, disorientation, confusion, cognitive disorganization, impaired judgment, hallucinations, paranoia and rarely toxic psychosis. until 1969 the prevalence of hepatotoxicity, ranging from 17% to 66.7% is higher when consumed in combination with alcohol. in a recent study by borini., among users of only marijuana, hepatomegaly was observed in 57.7% and splenomegaly in 73.1% and slightly elevated aspartate transaminase, alanine aminotransaminase and alkaline phosphatase in 42.3%, 34.6% and 53.8% respectively. we report an unusual case of fulminant hepatic failure following marijuana drug abuse as other causes of fulminant hepatic failure were all ruled out and blood and urine for active metabolite of marijuana, thc was positive. activated charcoal may prevent further absorption from the gut if ingestion is within a couple of hours, but clinical data is lacking on efficacy. the effect of cannabinoid antagonist, 41716a [n-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1h - pyrazole-3-carboxamide hydrochloride ] in animal models of rat and rhesus monkey produced reversible, dose - dependent antagonism of the discriminative stimulus properties of delta 9-thc. however, the effect of sr141716a on blocking or reversing the cannabis intoxication in humans is lacking. the most important aspect of treatment of fulminant hepatic failure is to provide good intensive care support. monitoring of metabolic parameters, surveillance for infection, maintenance of nutrition, and prompt recognition of gastrointestinal bleeding are crucial. though artificial support systems for kidney failure have been widely available for the past several decades, it is only recently that they have become a promising treatment modality for fulminant hepatic failure. they are mainly used temporarily as a bridge to liver transplantation or until the liver regenerates and to date, have no impact on survival. mars is performed with an albumin - containing dialysate, which is recycled in a closed loop that contains a charcoal cartridge, an anion exchanger resin adsorber and a conventional hemodialyzer. it was 1 applied in the treatment of acute liver failure in 1993 by stange. it has also been used to treat various poisoning with or without liver failure such as acetaminophen, amanita phalloides, phenytoin, lamotrigine, theophylline and calcium channel blockers. to date there is no published literature on the use of mars therapy for hepatotoxicity following marijuana drug abuse. however, it is unclear whether thc is also removed by mars, which needs further investigation. mars is an effective and safe means of treating fulminant hepatic failure of any etiology. however, studies are required to focus on the optimal timing of initiation and intensity of mars albumin liver dialysis. | marijuana is used for psychoactive and recreational purpose. we report a case of fulminant hepatic failure following marijuana drug abuse who recovered following artificial support systems for acute liver failure. there is no published literature of management of marijuana intoxication with molecular adsorbent recirculation system (mars). mars is effective and safe in patients with fulminant hepatic failure following marijuana intoxication. |
myf5 and mrf4 (also known as myf6) are upstream of myod in muscle precursor differentiation ; alternate tbx1 and pax3 pathways can initiate myod expression in pharyngeal muscles and in body and limb skeletal muscles, respectively, but not in eoms. regulatory elements for mrf4 reside within the myf5 locus, and myf5-targeted alleles have variable eom hypoplasia depending on the degree of residual mrf4 expression, with complete loss of both myf5 and myf4 resulting in a specific loss of eoms. we purchased myf5 mice from jackson laboratory (b6.129s4-myf5/j, stock no. 007893 ; bar harbor, me, usa). this myf5 strain expresses cre recombinase at the endogenous myf5 locus, disrupting the myf5 basic helix - loop - helix domain, and is also predicted to disrupt transcription of the adjacent mrf4 gene because the myf5 strain, created by using a similar targeting vector, disrupts expression of both genes and lacks eoms (but not other muscle types). myf5 mice were crossed to transgenic isl : gfp reporter mice (mgi : j:132726 ; gift of sam pfaff, salk institute, san diego, ca, usa), which contain a farnesylated green fluorescent protein (gfp) that localizes to the membrane of motor neurons and axons and allows for visualization of the nerves during development. we refer to myf5::isl : gfp embryos as wild type and myf5::isl : gfp embryos as myf5. all animal work was approved and performed in compliance with boston children 's hospital institutional animal care and use committee protocols and the arvo statement for the use of animals in ophthalmic and vision research. wild - type and myf5 embryos were taken at embryonic day (e) 11.5 and fixed in 4% paraformaldehyde (pfa) overnight (o / n), washed, dehydrated, and placed in dent 's fixative (20% dmso, 80% methanol) o / n, rehydrated, and placed in blocking solution (5% heat - inactivated goat serum, 20% dmso, 75% 1 pbs) o / n. actin -smooth muscle - cy3 antibody (anti-sma ; sigma - aldrich corp., st. louis, mo, usa) and anti - gfp (invitrogen, carlsbad, ca, usa) for 5 days at room temperature (rt), washed, incubated in secondary antibody (488 goat anti - rabbit igg ; alexa fluor, life technologies, carlsbad, ca, usa) for 3 days at rt, washed, dehydrated, and cleared o / n in babb (one part benzyl alcohol and two parts benzyl benzoate). images were acquired with a zeiss lsm 710 laser scanning confocal microscope and zen software (zeiss, oberkochen, germany) and processed with imaris software (bitplane, zurich, switzerland). onward, orbital dissections provide superior image detail and quality as compared to whole mount preparations. wild - type and myf5 embryos were taken at the appropriate age and fixed in 4% pfa o / cortex and olfactory bulbs above the orbit were removed under a dissecting microscope, leaving the distal cranial nerves and eoms (if present) intact. orbits were incubated with anti-sma for 3 days at rt or 4c, washed, and incubated in secondary antibody for 3 days at rt. after washing with pbs, orbits were further dissected sagitally at e12.5 or, from e13.5 onward, either from the superior aspect, looking down on the orbit from above (superior view), or from the inferior aspect, looking up at the orbit from the palate (inferior view). the oculomotor nerve was measured at its widest diameter, which occurs where the superior division of cn3 forms in control embryos, adjacent to where cn4 crosses above cn3. the length of the cn3 branch to the io muscle was measured along its trajectory from the distal decision region to the distal nerve tip. statistical analyses of significance were performed by using unpaired t - tests in graphpad prism (graph pad, san diego, ca, usa). whole embryos (e11.5) or brain and brainstem (e18.5) were fixed in 4% pfa o / n, washed in pbs, equilibrated in 30% sucrose, and frozen in optimal cutting temperature (oct) media. serial coronal sections of 20-m thickness were cut on a cryostat and stained with rabbit anti - islet1 (isl1 ; abcam, cambridge, ma, usa). after blinding as to genotype, isl - positive motor neuron nuclei were counted in every fifth section through the rostral - caudal extent of the oculomotor and trochlear nuclei by using the cell - counter plug - in in fiji, and the total number of motor neurons counted was then multiplied by 5. there were n = 3 mutant and n = 3 control embryos at each of the two ages. myf5 and mrf4 (also known as myf6) are upstream of myod in muscle precursor differentiation ; alternate tbx1 and pax3 pathways can initiate myod expression in pharyngeal muscles and in body and limb skeletal muscles, respectively, but not in eoms. regulatory elements for mrf4 reside within the myf5 locus, and myf5-targeted alleles have variable eom hypoplasia depending on the degree of residual mrf4 expression, with complete loss of both myf5 and myf4 resulting in a specific loss of eoms. we purchased myf5 mice from jackson laboratory (b6.129s4-myf5/j, stock no. 007893 ; bar harbor, me, usa). this myf5 strain expresses cre recombinase at the endogenous myf5 locus, disrupting the myf5 basic helix - loop - helix domain, and is also predicted to disrupt transcription of the adjacent mrf4 gene because the myf5 strain, created by using a similar targeting vector, disrupts expression of both genes and lacks eoms (but not other muscle types). myf5 mice were crossed to transgenic isl : gfp reporter mice (mgi : j:132726 ; gift of sam pfaff, salk institute, san diego, ca, usa), which contain a farnesylated green fluorescent protein (gfp) that localizes to the membrane of motor neurons and axons and allows for visualization of the nerves during development. we refer to myf5::isl : gfp embryos as wild type and myf5::isl : gfp embryos as myf5. all animal work was approved and performed in compliance with boston children 's hospital institutional animal care and use committee protocols and the arvo statement for the use of animals in ophthalmic and vision research. wild - type and myf5 embryos were taken at embryonic day (e) 11.5 and fixed in 4% paraformaldehyde (pfa) overnight (o / n), washed, dehydrated, and placed in dent 's fixative (20% dmso, 80% methanol) o / n, rehydrated, and placed in blocking solution (5% heat - inactivated goat serum, 20% dmso, 75% 1 pbs) o / n. actin -smooth muscle - cy3 antibody (anti-sma ; sigma - aldrich corp., st. louis, mo, usa) and anti - gfp (invitrogen, carlsbad, ca, usa) for 5 days at room temperature (rt), washed, incubated in secondary antibody (488 goat anti - rabbit igg ; alexa fluor, life technologies, carlsbad, ca, usa) for 3 days at rt, washed, dehydrated, and cleared o / n in babb (one part benzyl alcohol and two parts benzyl benzoate). images were acquired with a zeiss lsm 710 laser scanning confocal microscope and zen software (zeiss, oberkochen, germany) and processed with imaris software (bitplane, zurich, switzerland). from e12.5 onward, orbital dissections provide superior image detail and quality as compared to whole mount preparations. wild - type and myf5 embryos were taken at the appropriate age and fixed in 4% pfa o / cortex and olfactory bulbs above the orbit were removed under a dissecting microscope, leaving the distal cranial nerves and eoms (if present) intact. orbits were incubated with anti-sma for 3 days at rt or 4c, washed, and incubated in secondary antibody for 3 days at rt. after washing with pbs, orbits were further dissected sagitally at e12.5 or, from e13.5 onward, either from the superior aspect, looking down on the orbit from above (superior view), or from the inferior aspect, looking up at the orbit from the palate (inferior view). the oculomotor nerve was measured at its widest diameter, which occurs where the superior division of cn3 forms in control embryos, adjacent to where cn4 crosses above cn3. the length of the cn3 branch to the io muscle was measured along its trajectory from the distal decision region to the distal nerve tip. statistical analyses of significance were performed by using unpaired t - tests in graphpad prism (graph pad, san diego, ca, usa). whole embryos (e11.5) or brain and brainstem (e18.5) were fixed in 4% pfa o / n, washed in pbs, equilibrated in 30% sucrose, and frozen in optimal cutting temperature (oct) media. serial coronal sections of 20-m thickness were cut on a cryostat and stained with rabbit anti - islet1 (isl1 ; abcam, cambridge, ma, usa). after blinding as to genotype, isl - positive motor neuron nuclei were counted in every fifth section through the rostral - caudal extent of the oculomotor and trochlear nuclei by using the cell - counter plug - in in fiji, and the total number of motor neurons counted was then multiplied by 5. there were n = 3 mutant and n = 3 control embryos at each of the two ages. in whole mount views of e11.5 wild - type embryos, the eom anlage can be seen adjacent to the globe (figs. 1a, 1c) and by e12.5 in contrast, homozygous myf5 embryos lack eoms but develop facial and skeletal muscles (figs. 1b, 1d). sagittal views of cranial nerve and extraocular muscle development in e11.5 and e12.5 myf5::isl : gfp and myf5::isl : gfp embryos. ocular motor nerves are genetically labeled (green), and eom and arteries are antibody labeled with -sma (red). extraocular muscles are absent in myf5 embryos (b, d, f), and thus the red muscle channel has been turned off in wild - type images (a, c, e), to permit equivalent comparison of nerve trajectories in the presence and absence of eoms. (a, a) whole mount wild - type embryo and (b) whole mount myf5 embryo at e11.5. the trajectories of the nerves from the brainstem appear nearly identical in mutant and wild - type embryos at e11.5. the dotted line denotes the area of the orbit imaged in greater detail in (c), (c), and (d). cn4 is defasciculated, while cn6 is fasciculated along their nerve trunks, and distal cn3 has formed a decision region. the developing muscle anlage stained with -sma can be seen in the wild type (c), and an artery within the orbit stained with -sma is visible in both mutant and wild - type (c, d). (e, e, f) orbital imaging at e12.5. in the myf5 orbit, distinct extraocular muscles have formed and share a common origin at the annulus of zinn (e, asterisk). the trunk of cn4 is now fasciculated, while the distal end forms a decision region adjacent to the so and sends terminal branches into the body of the muscle (e, e, arrowhead). by contrast, the distal end of cn4 in the myf5 orbit fails to form an equivalent distal decision region and has no terminal branches (f, arrowhead). in the wild - type orbit, the inferior division decision region has become more compact (e, long arrow) and extends a branch to the io muscle (e, caret). the myf5 cn3 terminal decision region is less compacted (f, long arrow) and the branch to the io muscle appears hypoplastic (f, caret). arrow in the lower left corner indicates direction relative to the eye. a, anterior (toward front of eye) ; s, superior ; isl, islet1 ; -sma, -smooth muscle actin. in e11.5 wild - type embryos, the ocular motor nerves follow stereotypical trajectories to reach the developing orbit (figs. the main trunk of cn3 is fasciculated, and its distal end has formed a decision region (figs. despite the absence of the eom anlage in the e11.5 myf5 embryos, the ocular motor nerves appear similar to wild - type (figs. 1e, 1e) ; the four rectus muscles and the so muscle (whose tendon travels through the trochlea before forming its muscle body) share a common origin at the annulus of zinn (denoted by an asterisk in fig. 1e), while the io muscle is located nearer the front of the orbit (see fig. the main trunk of cn4 is now fasciculated, and terminal axons are fanning out into the so muscle. the cn3 inferior division decision region is nestled adjacent to the mr and ir muscles, and the fasciculated branch to the io muscle is extending from or through it. thin branches extend from the cn3 superior and inferior divisions into the sr, mr, and ir muscles. the distal end of the abducens nerve crosses inferiorly to cn3 and extends toward the lr muscle. (a) schematic diagram showing semisagittal lateral view of mature mouse eye and orbital muscles. (b) horizontal top - down view showing orientation of mature mouse orbits relative to the head ; this orientation shifts during early development. (c) schematic of boxed area in (b), showing the muscles and nerves visible in a top - down, superior view. (d) schematic showing the muscles and nerves visible in a bottom - up, inferior view. the views in (c) and (d) are those used in figure 3. (mr, ir, so, io) are green, cn4 and the muscle it innervates (so) are blue, and cn6 and the muscles it innervates (lr and rb) are pink. beginning at e12.5, differences are noted in development of ocular motor nerves in the presence and absence of eoms (fig. distal cn4 does not form the extensive fan - like terminal branches seen in wild - type embryos. the cn3 inferior division decision region remains large ; its appearance is similar to wild - type at e11.5. a branch of cn3 extends toward where the io muscle should be located, but it appears thinner, straighter, and blunter than in wild - type embryos (fig. 1f, caret). in mouse, the orbits are oriented slightly laterally (fig. 2b), and the position changes slightly as the embryo grows, so further anatomic descriptions are in reference to the eye. by e13.5, orbit growth requires that orbital dissections be performed from either the superior (looking down from above) or inferior view (looking up from below ; see schematic figs. the distal aspect of cn4 widens considerably, and terminal branches emanate into the so muscle from one - half of the decision region, while branches from the other half are oriented away from the muscle (figs. multiple branches of the cn3 superior division extend from the main trunk and travel along and into the sr muscle (fig. the cn3 inferior division decision region contains multiple nerve branches extending directly into the ir and mr muscles, while a long fasciculated branch extends to and sends terminal branches into the io muscle. cn6 crosses inferiorly to cn3, extends along the lr muscle, and sends terminal branches into it (figs. orbital dissections of e13.5 to e15.5 myf5:isl : gfp and myf5:isl : gfp embryos. nerves and muscles orbits are imaged from a superior (a, a, b, e, e, f, i, i, j) or inferior (c, c, d, g, g, h, k, k, l) view, as per figures 2b through 2d schematics. (a d) at e13.5, myf5 eoms share a common origin at the annulus of zinn (a, asterisk ; c). from the superior view (a, a) the so muscle begins to curve at its insertion onto the globe, and the rectus muscles have further differentiated from each other. cn3 superior division has multiple branches extending to the sr muscle (a, triple - headed arrow). cn6 has crossed inferiorly to cn3 and extends terminal branches into the lr muscle. from the inferior view (c, c), cn3 inferior division sends terminal branches to the ir and mr muscles (c, long arrow), and a well - formed branch to the io muscle (c, caret). at e13.5, the myf5 orbit is devoid of eoms (b, d). from the superior view (b), cn4 has fasciculated but lacks an extensive distal decision region and terminal branches (arrowhead), and cn3 superior division is not visible. from the inferior view (d), cn3 distal decision region (d, long arrow) and the branch to the io muscle (d, caret) are hypoplastic and misoriented, and cn6 is stunted and barely crosses over cn3 (d, double - headed arrow). (e h) at e14.5, the myf5 eoms continue to grow and change in orientation (e, g) : the sr muscle is now a broad band inserting on the top of the globe, the lps muscle has appeared between the sr and so muscles, and the so muscle has obtained its stereotypic curved shape. there is extensive terminal branching of cn3 superior division into the sr muscle (e, e), and cn4 has developed a blunt - ended branch that is not in association with an eom (e, e). cn3 inferior division branch has continued extending terminal branches to the mr and ir muscles, and the branch to the io muscle extends into the muscle (g, g). in the absence of eom, the myf5 cn4 appears thin and has developed multiple aberrant branches (f), cn3 main trunk widens (h) where the absent superior branch should have formed (h, triple arrowhead), the inferior division decision region is small, with no visible branches to the ir and mr muscles, and the branch to the io muscle is now hypoplastic and aberrant (h). cn6 trajectory is aberrantly parallel to cn3 and its growth cones pathologically wander (h, double arrowhead). l) at e15.5, myf5 orbits reveal the near - final ocular motor innervation patterns, and each nerve sends terminal and intramuscular branches into the appropriate eom. cn3 superior division branches have coalesced into a single trunk, and the blunted branch of cn4 is retracting (i, i, k, k). by contrast, in myf5 orbits the nerves have failed to develop further since e14.5 and appear to be regressing, while the abducens nerve wanders (double arrowhead). although more of the oculomotor nerve appears visible in figure 2j than in figure 2f, this is a staining and imaging artifact. in both images, the oculomotor nerve can be seen traveling inferiorly to the trochlear nerve and descending out of view near the stained artery ; the embryo in (j) was imaged slightly deeper. cn4 terminates in a single blunt end adjacent to where the so muscle should be (fig. the cn3 superior division has not formed, and the nerve trunk is significantly wider where it should have formed, averaging 62.05 8.17 m in myf5 embryos and 35.92 8.43 m in wild type (p = 0.0052) (figs. the cn3 inferior division decision region is small and no terminal branches extend toward the region where the ir and mr should be. the branch extending toward where the io muscle should be is short, thin, and straight rather than arching as seen in wild - type embryos (figs. cn6 appears thinner and shorter than in wild type and ends bluntly just after passing inferiorly to cn3 (fig. the myf5 cn3 widens at the origin of the absent superior division and has a shorter branch to the io muscle as compared to the myf5 orbit. (a) orbit of an e13.5 myf5 from the inferior view as pictured in figure 2c with measurements illustrated. the diameter of cn3 was measured at the location where the superior division normally forms in control embryos. the length of the inferior division branch to the io muscle was defined as the distance from the termination of the distal decision region to the distal tip of the nerve and was measured in three dimensions by using two to three straight lines that approximated the curvature of the nerve. (b) cn3 superior division branch point is significantly wider in myf5 embryos from e13.5 to e15.5 (average from 62.05 8.17 m at e13.5 to 63.96 2.08 m at e15.5) than in the wild - type embryos (average 35.92 8.43 m at e13.5 to 38.26 2.89 m at e15.5). this difference in width was significant at each age measured (e13.5 : p = 0.0052 ; e14.5 : p < 0.0001 ; e15.5 : p < 0.0001). (c) at e13.5, cn3 inferior division branch to the io muscle is significantly longer in myf5 (373.8 138.7 m) than in myf5 embryos (138.7 56.6 m ; p = 0.0017). while this branch continues to lengthen in wt orbits, reaching an average length of 412.8 25.5 m at e14.5 and 477.6 57.9 m at e15.5, it fails to lengthen substantially in mutant orbits (155.3 87.7 m at e14.5 and 106.7 44.0 m at e15.5). the branch to the io muscle is significantly shorter in the mutant at each age (e14.5 : p = 0.0007 ; e15.5 : p < 0.0001). a second branch oriented away from the so terminates in a blunt end without visible muscle nearby (figs. the cn3 superior division branches have grown in length and are running along the body of the sr muscle (figs. 3e, 3e). the cn3 inferior division continues to send terminal branches into the mr and ir muscles, and the branch to the io muscle extends further into the muscle (figs. cn4 is thin and forms multiple aberrant distal nerve branches that appear incorrectly oriented relative to the artery visible within the orbit (fig. the cn3 superior division has not formed, and the nerve trunk widens where the superior division normally forms (figs. 3h, 4b). the cn3 inferior division does not extend branches to the area of the ir or mr muscles. the branch to the io muscle is only a thin wisp extending toward where the muscle would normally be located (figs. cn6, which had crossed inferiorly to the oculomotor nerve at e13.5, now has an aberrant trajectory parallel to the oculomotor nerve. at e15.5, the ocular motor nerves in wild - type embryos are close to their mature configuration (figs. so muscle has defined terminal branches associated with the muscle, while the second blunted branch appears thinner and less developed than at e14.5 (figs. the cn3 superior division has formed a single fasciculated main trunk and developed more extensive intramuscular nerve branches into the sr (figs. the cn3 inferior division sends longer terminal branches to the ir and mr muscles, and the io nerve branch has continued to elongate with its terminal branches in close association with the io muscle (figs. cn6 continues to expand into the lr muscle, and branches to the rb muscle are now visible (figs. cn4 maintains a few wandering branches that are mislocalized relative to the normal location of the so muscle (fig. the cn3 trunk where the cn3 superior division should have formed remains significantly wider in myf5 embryos (63.96 2.08 m) than in wild - type embryos (38.26 2.89 m, p < 0.001) (figs. cn3 inferior division branches to the mr and ir muscles are not visible and the branch to the io muscle has failed to elongate (155.3 87.7 m at e14.5, 106.7 44.0 m at e15.5 ; figs. this branch is significantly shorter than that in the wild type at e15.5 (477.6 57.9 m ; p < 0.001). the cn6 trajectory remains aberrantly parallel to cn3, and its terminal axons are defasciculated (fig. 3l, double - headed arrow). to determine if eoms are required for motor neuron survival, we examined the number of motor neurons in oculomotor and trochlear nuclei at e11.5 and e18.5. at e11.5, myf5 and control embryos had a similar number of isl1-positive motor neurons in the oculomotor and trochlear nuclei (4285 214 control, 4477 182 myf5, p = 0.84 ; figs. 5a c), indicating that there is normal initial proliferation of motor neurons. by e18.5, there is substantial normal developmental cell death in the control embryos, as reported previously. myf5 embryos had greater levels of cell death, however, and have significantly fewer oculomotor and trochlear neurons than control embryos (2465 271 vs. 868.2 81.7, p = 0.00049, figs. f). myf5 oculomotor and trochlear neuron number and position are similar to control at e11.5, but are significantly reduced in number by e18.5. (a, b) oculomotor nucleus motor neurons were labeled with anti - isl1 in control (a) or myf5 (b) brainstems at e11.5. (c) the number of motor neurons in the oculomotor and trochlear nuclei is equal in myf5 and control embryos at e11.5 (4285 214, n = 3, vs. 4477 182, n = 3, p = 0.84), indicating that motor neurons are formed in equal numbers. (d, e) oculomotor nucleus motor neurons were labeled with anti - isl1 in control (d) or myf5 (e) brainstems at e18.5. (f) the number of motor neurons is reduced by 2/3 in myf5 oculomotor nucleus (2465 271, n = 3, vs. 868.2 81.7, n = 3, p = 0.00049), indicating that extraocular muscles are necessary for oculomotor neuron survival. in whole mount views of e11.5 wild - type embryos, the eom anlage can be seen adjacent to the globe (figs. 1a, 1c) and by e12.5 in contrast, homozygous myf5 embryos lack eoms but develop facial and skeletal muscles (figs. 1b, 1d). sagittal views of cranial nerve and extraocular muscle development in e11.5 and e12.5 myf5::isl : gfp and myf5::isl : gfp embryos. ocular motor nerves are genetically labeled (green), and eom and arteries are antibody labeled with -sma (red). extraocular muscles are absent in myf5 embryos (b, d, f), and thus the red muscle channel has been turned off in wild - type images (a, c, e), to permit equivalent comparison of nerve trajectories in the presence and absence of eoms. (a, a) whole mount wild - type embryo and (b) whole mount myf5 embryo at e11.5. the trajectories of the nerves from the brainstem appear nearly identical in mutant and wild - type embryos at e11.5. the dotted line denotes the area of the orbit imaged in greater detail in (c), (c), and (d). cn4 is defasciculated, while cn6 is fasciculated along their nerve trunks, and distal cn3 has formed a decision region. the developing muscle anlage stained with -sma can be seen in the wild type (c), and an artery within the orbit stained with -sma is visible in both mutant and wild - type (c, d). (e, e, f) orbital imaging at e12.5. in the myf5 orbit, distinct extraocular muscles have formed and share a common origin at the annulus of zinn (e, asterisk). the trunk of cn4 is now fasciculated, while the distal end forms a decision region adjacent to the so and sends terminal branches into the body of the muscle (e, e, arrowhead). by contrast, the distal end of cn4 in the myf5 orbit fails to form an equivalent distal decision region and has no terminal branches (f, arrowhead). in the wild - type orbit, the inferior division decision region has become more compact (e, long arrow) and extends a branch to the io muscle (e, caret). the myf5 cn3 terminal decision region is less compacted (f, long arrow) and the branch to the io muscle appears hypoplastic (f, caret). arrow in the lower left corner indicates direction relative to the eye. a, anterior (toward front of eye) in e11.5 wild - type embryos, the ocular motor nerves follow stereotypical trajectories to reach the developing orbit (figs. 1a, 1c). the main trunk of cn3 is fasciculated, and its distal end has formed a decision region (figs. despite the absence of the eom anlage in the e11.5 myf5 embryos, the ocular motor nerves appear similar to wild - type (figs. by e12.5, distinct eoms are recognizable (figs. 1e, 1e) ; the four rectus muscles and the so muscle (whose tendon travels through the trochlea before forming its muscle body) share a common origin at the annulus of zinn (denoted by an asterisk in fig. 1e), while the io muscle is located nearer the front of the orbit (see fig. the main trunk of cn4 is now fasciculated, and terminal axons are fanning out into the so muscle. the cn3 inferior division decision region is nestled adjacent to the mr and ir muscles, and the fasciculated branch to the io muscle is extending from or through it. thin branches extend from the cn3 superior and inferior divisions into the sr, mr, and ir muscles. the distal end of the abducens nerve crosses inferiorly to cn3 and extends toward the lr muscle. schematic diagrams of orbital anatomy. (a) schematic diagram showing semisagittal lateral view of mature mouse eye and orbital muscles. (b) horizontal top - down view showing orientation of mature mouse orbits relative to the head ; this orientation shifts during early development. (c) schematic of boxed area in (b), showing the muscles and nerves visible in a top - down, superior view. (d) schematic showing the muscles and nerves visible in a bottom - up, inferior view. the views in (c) and (d) are those used in figure 3. color coding : cn3 and the muscles it innervates (mr, ir, so, io) are green, cn4 and the muscle it innervates (so) are blue, and cn6 and the muscles it innervates (lr and rb) are pink. beginning at e12.5, differences are noted in development of ocular motor nerves in the presence and absence of eoms (fig. distal cn4 does not form the extensive fan - like terminal branches seen in wild - type embryos. the cn3 inferior division decision region remains large ; its appearance is similar to wild - type at e11.5. a branch of cn3 extends toward where the io muscle should be located, but it appears thinner, straighter, and blunter than in wild - type embryos (fig. 1f, caret). 2b), and the position changes slightly as the embryo grows, so further anatomic descriptions are in reference to the eye. by e13.5, orbit growth requires that orbital dissections be performed from either the superior (looking down from above) or inferior view (looking up from below ; see schematic figs. 2c, 2d). in e13.5 wild - type embryos, the eoms continue to grow and separate. the distal aspect of cn4 widens considerably, and terminal branches emanate into the so muscle from one - half of the decision region, while branches from the other half are oriented away from the muscle (figs. multiple branches of the cn3 superior division extend from the main trunk and travel along and into the sr muscle (fig. the cn3 inferior division decision region contains multiple nerve branches extending directly into the ir and mr muscles, while a long fasciculated branch extends to and sends terminal branches into the io muscle. cn6 crosses inferiorly to cn3, extends along the lr muscle, and sends terminal branches into it (figs. orbits are imaged from a superior (a, a, b, e, e, f, i, i, j) or inferior (c, c, d, g, g, h, k, k, l) view, as per figures 2b through 2d schematics. (a d) at e13.5, myf5 eoms share a common origin at the annulus of zinn (a, asterisk ; c). from the superior view (a, a) the so muscle begins to curve at its insertion onto the globe, and the rectus muscles have further differentiated from each other. cn3 superior division has multiple branches extending to the sr muscle (a, triple - headed arrow). cn6 has crossed inferiorly to cn3 and extends terminal branches into the lr muscle. from the inferior view (c, c), cn3 inferior division sends terminal branches to the ir and mr muscles (c, long arrow), and a well - formed branch to the io muscle (c, caret). at e13.5, the myf5 orbit is devoid of eoms (b, d). from the superior view (b), cn4 has fasciculated but lacks an extensive distal decision region and terminal branches (arrowhead), and cn3 superior division is not visible. from the inferior view (d), cn3 distal decision region (d, long arrow) and the branch to the io muscle (d, caret) are hypoplastic and misoriented, and cn6 is stunted and barely crosses over cn3 (d, double - headed arrow). (e h) at e14.5, the myf5 eoms continue to grow and change in orientation (e, g) : the sr muscle is now a broad band inserting on the top of the globe, the lps muscle has appeared between the sr and so muscles, and the so muscle has obtained its stereotypic curved shape. there is extensive terminal branching of cn3 superior division into the sr muscle (e, e), and cn4 has developed a blunt - ended branch that is not in association with an eom (e, e). cn3 inferior division branch has continued extending terminal branches to the mr and ir muscles, and the branch to the io muscle extends into the muscle (g, g). in the absence of eom, the myf5 cn4 appears thin and has developed multiple aberrant branches (f), cn3 main trunk widens (h) where the absent superior branch should have formed (h, triple arrowhead), the inferior division decision region is small, with no visible branches to the ir and mr muscles, and the branch to the io muscle is now hypoplastic and aberrant (h). cn6 trajectory is aberrantly parallel to cn3 and its growth cones pathologically wander (h, double arrowhead). (i l) at e15.5, myf5 orbits reveal the near - final ocular motor innervation patterns, and each nerve sends terminal and intramuscular branches into the appropriate eom. cn3 superior division branches have coalesced into a single trunk, and the blunted branch of cn4 is retracting (i, i, k, k). by contrast, in myf5 orbits the nerves have failed to develop further since e14.5 and appear to be regressing, while the abducens nerve wanders (double arrowhead). although more of the oculomotor nerve appears visible in figure 2j than in figure 2f, this is a staining and imaging artifact. in both images, the oculomotor nerve can be seen traveling inferiorly to the trochlear nerve and descending out of view near the stained artery ; the embryo in (j) was imaged slightly deeper. cn4 terminates in a single blunt end adjacent to where the so muscle should be (fig. the cn3 superior division has not formed, and the nerve trunk is significantly wider where it should have formed, averaging 62.05 8.17 m in myf5 embryos and 35.92 8.43 m in wild type (p = 0.0052) (figs. 4a, 4b). the cn3 inferior division decision region is small and no terminal branches extend toward the region where the ir and mr should be. the branch extending toward where the io muscle should be is short, thin, and straight rather than arching as seen in wild - type embryos (figs. cn6 appears thinner and shorter than in wild type and ends bluntly just after passing inferiorly to cn3 (fig. the myf5 cn3 widens at the origin of the absent superior division and has a shorter branch to the io muscle as compared to the myf5 orbit. (a) orbit of an e13.5 myf5 from the inferior view as pictured in figure 2c with measurements illustrated. the diameter of cn3 was measured at the location where the superior division normally forms in control embryos. the length of the inferior division branch to the io muscle was defined as the distance from the termination of the distal decision region to the distal tip of the nerve and was measured in three dimensions by using two to three straight lines that approximated the curvature of the nerve. (b) cn3 superior division branch point is significantly wider in myf5 embryos from e13.5 to e15.5 (average from 62.05 8.17 m at e13.5 to 63.96 2.08 m at e15.5) than in the wild - type embryos (average 35.92 8.43 m at e13.5 to 38.26 2.89 m at e15.5). this difference in width was significant at each age measured (e13.5 : p = 0.0052 ; e14.5 : p < 0.0001 ; e15.5 : p < 0.0001). (c) at e13.5, cn3 inferior division branch to the io muscle is significantly longer in myf5 (373.8 138.7 m) than in myf5 embryos (138.7 56.6 m ; p = 0.0017). while this branch continues to lengthen in wt orbits, reaching an average length of 412.8 25.5 m at e14.5 and 477.6 57.9 m at e15.5, it fails to lengthen substantially in mutant orbits (155.3 87.7 m at e14.5 and 106.7 44.0 m at e15.5). the branch to the io muscle is significantly shorter in the mutant at each age (e14.5 : p = 0.0007 ; e15.5 : p < 0.0001)., cn4 provides terminal innervation to the so muscle. a second branch oriented away from the cn3 superior division branches have grown in length and are running along the body of the sr muscle (figs. the cn3 inferior division continues to send terminal branches into the mr and ir muscles, and the branch to the io muscle extends further into the muscle (figs. cn6 crosses inferiorly to cn3 and extends terminal branches into the lr muscle (figs. 3 g, 3g). in e14.5 myf5 embryos, eoms remain absent (figs. 3f, 3h). cn4 is thin and forms multiple aberrant distal nerve branches that appear incorrectly oriented relative to the artery visible within the orbit (fig. the cn3 superior division has not formed, and the nerve trunk widens where the superior division normally forms (figs. 3h, 4b). the cn3 inferior division does not extend branches to the area of the ir or mr muscles. the branch to the io muscle is only a thin wisp extending toward where the muscle would normally be located (figs. cn6, which had crossed inferiorly to the oculomotor nerve at e13.5, now has an aberrant trajectory parallel to the oculomotor nerve. at e15.5, the ocular motor nerves in wild - type embryos are close to their mature configuration (figs. so muscle has defined terminal branches associated with the muscle, while the second blunted branch appears thinner and less developed than at e14.5 (figs. the cn3 superior division has formed a single fasciculated main trunk and developed more extensive intramuscular nerve branches into the sr (figs. the cn3 inferior division sends longer terminal branches to the ir and mr muscles, and the io nerve branch has continued to elongate with its terminal branches in close association with the io muscle (figs. 3k, 3k, 4c). cn6 continues to expand into the lr muscle, and branches to the rb muscle are now visible (figs. cn4 maintains a few wandering branches that are mislocalized relative to the normal location of the so muscle (fig. the cn3 trunk where the cn3 superior division should have formed remains significantly wider in myf5 embryos (63.96 2.08 m) than in wild - type embryos (38.26 2.89 m, p < 0.001) (figs. cn3 inferior division branches to the mr and ir muscles are not visible and the branch to the io muscle has failed to elongate (155.3 87.7 m at e14.5, 106.7 44.0 m at e15.5 ; figs. this branch is significantly shorter than that in the wild type at e15.5 (477.6 57.9 m ; p < 0.001). the cn6 trajectory remains aberrantly parallel to cn3, and its terminal axons are defasciculated (fig. to determine if eoms are required for motor neuron survival, we examined the number of motor neurons in oculomotor and trochlear nuclei at e11.5 and e18.5. at e11.5, myf5 and control embryos had a similar number of isl1-positive motor neurons in the oculomotor and trochlear nuclei (4285 214 control, 4477 182 myf5, p = 0.84 ; figs. c), indicating that there is normal initial proliferation of motor neurons. by e18.5, there is substantial normal developmental cell death in the control embryos, as reported previously. myf5 embryos had greater levels of cell death, however, and have significantly fewer oculomotor and trochlear neurons than control embryos (2465 271 vs. 868.2 81.7, p = 0.00049, figs. myf5 oculomotor and trochlear neuron number and position are similar to control at e11.5, but are significantly reduced in number by e18.5. (a, b) oculomotor nucleus motor neurons were labeled with anti - isl1 in control (a) or myf5 (b) brainstems at e11.5. (c) the number of motor neurons in the oculomotor and trochlear nuclei is equal in myf5 and control embryos at e11.5 (4285 214, n = 3, vs. 4477 182, n = 3, p = 0.84), indicating that motor neurons are formed in equal numbers. (d, e) oculomotor nucleus motor neurons were labeled with anti - isl1 in control (d) or myf5 (e) brainstems at e18.5. (f) the number of motor neurons is reduced by 2/3 in myf5 oculomotor nucleus (2465 271, n = 3, vs. 868.2 81.7, n = 3, p = 0.00049), indicating that extraocular muscles are necessary for oculomotor neuron survival. most prior studies of ocular cranial nerve development in mouse have relied on dii labeling or neurofilament staining, highlighting the initial nerve trajectories but not permitting visualization of their terminal processes in the orbit. by dissecting and visualizing the mouse orbit from timed embryos with genetically labeled motor axons, we characterized in greater detail the development of mouse oculomotor, trochlear, and abducens nerves, and showed that formation of the initial nerve trajectory does not require signals from the eoms, but formation of terminal branching does. ocular motor nerves arrive to the orbit and form distal decision regions in a spatially and temporally similar fashion in myf5 and wild - type embryos, demonstrating that axon growth and guidance from the brainstem to the proper position within the orbit is independent of eoms and likely occurs through cues from the mesenchyme and/or through cell autonomous processes. subsequent full nerve extension and terminal branching into the eom targets, maintenance of correct nerve position as the orbit expands, and survival of axons and their motor neurons, however, are dependent on the presence of eoms. the role of eoms in ocular motor nerve development is similar to the findings from previous reports of the contribution of skeletal muscle to spinal nerve development and survival. silver stains and histologic sectioning following surgical ablation or irradiation of developing chick limb muscles reveal normal development of the main motor nerve trunks and distal nerve branches adjacent to the missing muscles, but failure of the formation of terminal branches that normally would innervate the missing individual muscles. similarly, myod::myf5 mouse embryos lack all skeletal muscle and have normal initial growth and guidance of spinal motor nerves but fail to develop terminal nerve branches, followed by motor neuron apoptosis. together, these data support the influence of locally acting mesenchymal guidance cues on proximal nerve patterning and locally acting eom cues on terminal nerve branching into target muscles. we found minor differences in the relative timing of ocular motor nerve development in mouse compared to chick and zebrafish. in mouse we found that cn6 and cn3 reach the orbit simultaneously and before cn4, which is similar to zebrafish but different from chick, in which the arrival of cn6 is followed by cn3 and then cn4. in both mouse and chick, cn3 projects an initial nerve branch to the io muscle, followed by branches to the mr, ir, and sr muscles. by contrast, in zebrafish a branch is sent to the sr muscle many hours after branches are sent to inferior division muscles, correlating with the temporal development of the cn3 nucleus, in which zebrafish sr motor neurons are born after mr and ir motor neurons. first, we identified a trochlear branch in developing wild - type embryos that is not associated with a muscle, forms a blunt end, and subsequently retracts. consistent with its apparent lack of muscle target, the growth and regression of this wild - type trochlear branch resembles the growth and regression of ocular nerves in myf5 embryos. second, we found that the cn3 superior division, which in the mature state forms a single branch that then divides distally to innervate both the sr and lps muscles, fails to form in myf5 embryos. this suggests that, while axons within the main trunk of cn3 are guided in a cell - autonomous manner, by axon axon interactions, or by mesenchymal cues, the superior division axons exit from the main cn3 trunk in response to muscle cues. we have long been puzzled by the observation that the cn3 superior division first appears as multiple small branches extending away from the main cn3 trunk, which subsequently coalesce and elongate. our current imaging approaches now reveal that, at e11.5 to e12.5, the sr / lps muscle anlage lies in close juxtaposition to the main trunk of cn3, and these multiple small branches extend directly along and into the muscle anlage. as the orbital size expands over subsequent days, the distance between the origin and eom targets of these small branches increases and, by e15.5, the branches have fasciculated into the superior division. myf5 embryos do not form these multiple small branches, and, at the position where they normally form, the cn3 main trunk is wider than in wild - type embryos. this is reminiscent of the pathology of kif21a mice. in these mutant mice that recapitulate a human ccdd, the superior division also fails to form, and a widened bulge is visible along cn3 that, by electron microscopy, contains convoluted axon trajectories and stalled growth cones. the kif21a bulge is located far proximally to the normal superior division branch - point and forms secondarily to attenuated kif21a autoinhibition. by contrast, we found the myf5 widening to be located at the position of the normal distal superior division branch - point. the parallels, however, suggest that the myf5 nerve widening may result from paused growth cones that fail to exit the main cn3 trunk secondary to the absence of eoms. ocular motor neurons, like other motor neurons, receive trophic support from their target muscles, and are responsive to neurotrophic factors, particularly bdnf and gdnf (see ref. it is therefore not surprising that survival of ocular motor neurons depends on the presence of eoms. it is remarkable, however, that in the absence of target muscles, more than one - third of the oculomotor and trochlear neurons remain at e18.5. by contrast, in myod::myf5 embryos, which lack all skeletal muscles, all somatic motor neurons in the spinal cord die by e17.5, and most motor neurons in the facial nucleus are dead or dying. the ocular motor nerves are selectively spared until late in the course of motor neuron degenerative disorders such as amyotrophic lateral sclerosis and spinal motor atrophy. this selective sparing is theorized to result from increased resistance of the ocular motor neurons to environmental stressors or excitotoxicity. the retention of some motor neurons in cn3 and cn4 as late as e18.5 without trophic support from their target muscles may be another example of the intrinsic relative resistance of oculomotor neurons to pathologic conditions. using genetic tools, orbital dissection, and a developmental framework in timed mouse embryos, our findings catalogued the development of the ocular motor nerves and they implicate the eoms in the control of their terminal branching. the identities of the signals provided by eoms, whether they differ among different eoms, and whether they are mediated through short - range diffusible cues, direct contact, or both, remain to be determined. | purposeto spatially and temporally define ocular motor nerve development in the presence and absence of extraocular muscles (eoms).methodsmyf5cre mice, which in the homozygous state lack eoms, were crossed to an islmn : gfp reporter line to fluorescently label motor neuron cell bodies and axons. embryonic day (e) 11.5 to e15.5 wild - type and myf5cre / cre : islmn : gfp whole mount embryos and dissected orbits were imaged by confocal microscopy to visualize the developing oculomotor, trochlear, and abducens nerves in the presence and absence of eoms. e11.5 and e18.5 brainstems were serially sectioned and stained for islet1 to determine the fate of ocular motor neurons.resultsat e11.5, all three ocular motor nerves in mutant embryos approached the orbit with a trajectory similar to that of wild - type. subsequently, while wild - type nerves send terminal branches that contact target eoms in a stereotypical pattern, the myf5cre / cre ocular motor nerves failed to form terminal branches, regressed, and by e18.5 two - thirds of their corresponding motor neurons died. comparisons between mutant and wild - type embryos revealed novel aspects of trochlear and oculomotor nerve development.conclusionswe delineated mouse ocular motor nerve spatial and temporal development in unprecedented detail. moreover, we found that eoms are not necessary for initial outgrowth and guidance of ocular motor axons from the brainstem to the orbit but are required for their terminal branching and survival. these data suggest that intermediate targets in the mesenchyme provide cues necessary for appropriate targeting of ocular motor axons to the orbit, while eom cues are responsible for terminal branching and motor neuron survival. |
obsessive - compulsive disorder (ocd) has a lifelong prevalence rate of approximately 2.5% in the general population.1 sobin have suggested that about half of ocd patients exhibit moderate to severe levels of schizotypy, and the authors have also identified a subtype of ocd with specifically high schizotypy. (ie, schizotype).3 meehl4 extended the concept of schizotypy, proposing that a genetically determined defect in neural function invariably results in the development of a set of schizotypal features. schizotypal features were later considered to be common features in the general population and were considered related to character traits and cognitive style. recently, it has been considered that schizotypy results from an interaction between a schizophrenic genotype and environmental factors.5,6 the concept of schizotypy has been considered as multidimensionally similar to schizophrenia.7 the schizotypal personality questionnaire (spq) rating of schizotypy was developed in accordance with the criteria for schizotypal personality disorder (spd) in the diagnostic and statistical manual of mental disorders (third edition, revised [dsm - iii - r]).8 spd, as defined by the dsm - iii - r, is similar to schizophrenia, but it does not meet the diagnostic criteria for schizophrenia. the validity of the spq as a screening tool for spd has been confirmed, and some patients with a high spq score present symptoms consistent with the diagnostic criteria for spd.9 the spq is made up of three factors : (1) the cognitive - perceptual factor includes the items concerning ideas of reference, odd beliefs or magical thinking, and unusual perceptual experiences ; (2) the interpersonal factor includes the items concerning excessive social anxiety and no close friends ; and (3) the disorganized factor includes the items concerning constricted affect, odd or eccentric behavior, and odd speech. ocd patients have a high score on cognitive - perceptual and disorganized factors of schizotypy in comparison with other anxiety disorder patients.10 moreover, the correlation between schizotypy and the severity of obsessive - compulsive symptoms (ocss) has been consistently found in university students and ocd patients.1114 enright and beech15 have explored the links between schizotypy and ocd by utilizing experimental paradigms drawn from cognitive science. the authors found a task designed to investigate inhibitory mechanisms in selective attention showed that ocd patients were different from other anxiety disorder patients. ocd patients exhibited a non - inhibition response pattern very similar to that previously observed in schizotypy and schizophrenia patients.16 relationships between schizotypy and specific ocss have also been reported. tallis and shafran14 showed that schizotypy was correlated with impulse, precision, and rumination ocss rather than washing and checking ocss. the authors suggested that the reduced cognitive inhibition observed in both schizotypy and ocd may result in the frequent entry into awareness of unacceptable urges and intrusive thoughts. therefore, the present authors hypothesized that schizotypy was correlated with the severity of obsession rather than the severity of compulsion. in the present study, the authors examined the relationship between the severity of obsession or compulsion and schizotypy to verify this hypothesis. furthermore, the authors investigated the relationship between the severity of obsession or compulsion and schizotypal factors of the spq. a total of 61 ocd outpatients in the university hospital at the kyoto prefectural university of medicine (kyoto, japan) during the period 20082010 were recruited as subjects for the study. the patients were primarily diagnosed with ocd in accordance with the criteria of the dsm - iv - r.17 all subjects were diagnosed using the structured clinical interview for dsm - iv - r axis i disorders. the final number of ocd outpatients enrolled in the study was 60 (25 males and 35 females). two patients had comorbidities that included major depressive disorders, two had specific phobias, one had social anxiety disorder, and 55 had ocd as their sole diagnosis. with regard to medication, 13 patients were not taking any drugs and 47 subjects were taking selective serotonin reuptake inhibitors. all procedures were approved by the kyoto prefectural university of medicine research ethics committee (approval number c-379) before the study was undertaken. after a description of the study was given to all subjects, the authors obtained consent in writing from each patient. the authors used the japanese version of the yale brown obsessive compulsive scale (y - bocs), which comprises ten items (five items for obsession, five items for compulsion) used to measure the severity of ocss.18 each item was rated on a five - point scale (ranging from 0 to 4), and a total score could range from 0 to 40 points. the japanese version of the spq is a self - completed questionnaire comprising 74 items.19 as mentioned earlier, the spq is made up of three factors : (1) the cognitive - perceptual (33 items) ; (2) the interpersonal (25 items) ; and (3) the disorganized factors (16 items). is rated 1 point, and a total score could range from 0 to 74 points. the japanese version of the hamilton depression rating scale (ham - d) is a structured interview comprising 21 items used to evaluate depression.20 a total score could range from 0 to 63 points. the japanese version of the hamilton anxiety rating scale (ham - a) is a structured interview comprising 14 items used to evaluate anxiety.21 each item was rated on a five - point scale (ranging from 0 to 4), and a total score could range from 0 to 56 points. the pearson correlation coefficients between the y - bocs and spq scores were estimated so as to evaluate the relationship between ocs severity and three schizotypal factors. hierarchical multiple linear regression analyses were then conducted to assess whether schizotypy predicted the severity of obsession and compulsion. in these models, the total score of spq was entered as an independent variable and the scores of obsession and compulsion on the y - bocs were entered as dependent variables. therefore, the total ham - d and ham - a scores were entered to control for the effect of depression and anxiety in the first step of the analysis, and the total spq score was entered in the second step. all analyses were conducted using spss software (v 17.0 for windows ; spss inc, chicago, il). a total of 61 ocd outpatients in the university hospital at the kyoto prefectural university of medicine (kyoto, japan) during the period 20082010 were recruited as subjects for the study. the patients were primarily diagnosed with ocd in accordance with the criteria of the dsm - iv - r.17 all subjects were diagnosed using the structured clinical interview for dsm - iv - r axis i disorders. the final number of ocd outpatients enrolled in the study was 60 (25 males and 35 females). two patients had comorbidities that included major depressive disorders, two had specific phobias, one had social anxiety disorder, and 55 had ocd as their sole diagnosis. with regard to medication, 13 patients were not taking any drugs and 47 subjects were taking selective serotonin reuptake inhibitors. all procedures were approved by the kyoto prefectural university of medicine research ethics committee (approval number c-379) before the study was undertaken. after a description of the study was given to all subjects, the authors obtained consent in writing from each patient. the authors used the japanese version of the yale brown obsessive compulsive scale (y - bocs), which comprises ten items (five items for obsession, five items for compulsion) used to measure the severity of ocss.18 each item was rated on a five - point scale (ranging from 0 to 4), and a total score could range from 0 to 40 points. the japanese version of the spq is a self - completed questionnaire comprising 74 items.19 as mentioned earlier, the spq is made up of three factors : (1) the cognitive - perceptual (33 items) ; (2) the interpersonal (25 items) ; and (3) the disorganized factors (16 items). is rated 1 point, and a total score could range from 0 to 74 points. the japanese version of the hamilton depression rating scale (ham - d) is a structured interview comprising 21 items used to evaluate depression.20 a total score could range from 0 to 63 points. the japanese version of the hamilton anxiety rating scale (ham - a) is a structured interview comprising 14 items used to evaluate anxiety.21 each item was rated on a five - point scale (ranging from 0 to 4), and a total score could range from 0 to 56 points. the pearson correlation coefficients between the y - bocs and spq scores were estimated so as to evaluate the relationship between ocs severity and three schizotypal factors. hierarchical multiple linear regression analyses were then conducted to assess whether schizotypy predicted the severity of obsession and compulsion. in these models, the total score of spq was entered as an independent variable and the scores of obsession and compulsion on the y - bocs were entered as dependent variables. therefore, the total ham - d and ham - a scores were entered to control for the effect of depression and anxiety in the first step of the analysis, and the total spq score was entered in the second step. all analyses were conducted using spss software (v 17.0 for windows ; spss inc, chicago, il). the mean ocs severity was 24.2, as measured by the y - bocs total score. the mean depression (measured by the ham - d) and anxiety scores (measured by the ham - a) were 7.8 and 8.0, respectively, and both depression and anxiety were mild. table 2 shows the pearson correlation coefficients between the subindicators of the y - bocs and the spq. the y - bocs total score was correlated with the spq cognitive - perceptual (r = 0.34, p = 0.008) and disorganized factors (r = 0.28, p = 0.02). the y - bocs obsession score also was correlated with the spq cognitive - perceptual (r = 0.36, p = 0.005) and disorganized factors (r = 0.31, p = 0.01). by contrast, the y - bocs compulsion score had no correlation with either spq subscales. moreover, the authors then performed the hierarchical multiple linear regression analyses to evaluate whether schizotypy predicted the severity of obsession and compulsion. table 3 shows the result of linear regression analysis predicting the y - bocs obsession score. the spq total score was a significant predictor of the y - bocs obsession score after accounting for control variables (= 0.26, adjusted r = 0.09, f = 2.84, p = 0.04). on the other hand, the spq total score did not predict the y - bocs compulsion score after accounting for control variables (f [3, 55 ] = 0.18 ; not significant). in addition, table 4 indicates pearson correlation coefficients between dependent variables for linear regression analyses. the results of this study showed the correlation between the severity of ocss and schizotypy in ocd patients. this result is consistent with findings from a previous study of ocd patients.14 although these previous studies used ocs rating scales other than the y - bocs (eg, the padua inventory, the maudsley obsessive - compulsive inventory [moci ], and the obsessive - compulsive inventory), these studies showed the same results the correlation between the severity of ocss and schizotypy in ocd patients.1114 it is worth noting here that the clinician - rated y - bocs is more objective than self - completed questionnaires. the results of this study showed the correlation between the severity of ocss and schizotypy in ocd patients. this result is consistent with findings from a previous study of ocd patients.14 although these previous studies used ocs rating scales other than the y - bocs (eg, the padua inventory, the maudsley obsessive - compulsive inventory [moci ], and the obsessive - compulsive inventory), these studies showed the same results the correlation between the severity of ocss and schizotypy in ocd patients.1114 it is worth noting here that the clinician - rated y - bocs is more objective than self - completed questionnaires. in the present study, the authors report that the y - bocs obsession score, not the y - bocs compulsion score, was correlated with the spq total score. to the best of the authors knowledge, this is the first study investigating the relationship between schizotypy and ocs severity in ocd patients by separating ocs severity into subcategories of obsession and compulsion. y - bocs obsession score was the significant predictor of spq total score. since ham - d and ham - a did nt predict spq total score, it was the authors belief that this result was not affected by depression and anxiety. as the authors hypothesized, the results of this study show that schizotypy was correlated with the severity of obsession rather than the severity of compulsion. this finding suggests that ocd patients with an elevated spq total score experience a reduction of cognitive inhibition, resulting in the frequent entry into obsession. in the correlations between subscales of y - bocs and spq, the y - bocs total score was correlated with the spq cognitive - perceptual and disorganized factors. this result is consistent with the findings of roth and baribeau,11 which indicate that the moci total score is correlated with the spq cognitive - perceptual and disorganized factors. furthermore, the results of the present study show the detailed relationships by classifying ocss into obsession and compulsion. to be specific, the y - bocs obsession score had correlations with the spq cognitive - perceptual and disorganized factors but no correlation with the interpersonal factor. this suggests that obsession is not indefinitely correlated with schizotypy but, rather, that it is correlated with specific schizotypal factors (eg, cognitive - perceptual and disorganized factors). regarding the correlation between the severity of obsession and the spq cognitive - perceptual factor, since the items of the cognitive - perceptual factor include the idea of reference and magical thinking, a patient with a severe obsession may be likely to have a bizarre and incorrect confidence (ie, a delusion). additionally, regarding the correlation between the y - bocs obsession score and the spq disorganized factor, since the items of the disorganized factor include bizarre appearance and talking, a patient with severe obsession may be likely to be disturbed and frequently filled with obsession, resulting in bizarre talking. by contrast, the spq interpersonal factor was interestingly not correlated with either y - bocs subscales. this finding was consistent with the results of roth and baribeau s study,11 which also did not indicate a correlation between moci total score and the spq interpersonal factor. some ocd patients force or persuade their family and/or others to confirm or ensure their ocss (ie, involvement). patients who involve others are generally highly dependent and poor at suppressing emotion (ie, immature type), which may have led to the results in this study. since this was a cross - sectional study, it was impossible to explain the effect of schizotypy on the clinical course of ocd, for example. therefore, future longitudinal studies are recommended to clarify this by examining the changes of schizotypy along the clinical course of ocd patients. since this was a cross - sectional study, it was impossible to explain the effect of schizotypy on the clinical course of ocd, for example. therefore, future longitudinal studies are recommended to clarify this by examining the changes of schizotypy along the clinical course of ocd patients. | purposeobsessive - compulsive disorder (ocd) patients exhibit a noninhibition response pattern very similar to that observed in schizotypy patients in cognitive tasks. it has been suggested that the reduced cognitive inhibition observed in both schizotypy and ocd may result in the frequent entry into awareness of unacceptable urges and intrusive thoughts. the aim of this study was to investigate the relationship between the severity of obsession or compulsion and schizotypy in ocd.patients and methodssixty subjects (25 males and 35 females) who were ocd outpatients in the university hospital at the kyoto prefectural university of medicine during the period 20082010 were enrolled in the study. assessments of these patients were made using the yale brown obsessive compulsive scale (y - bocs), the schizotypal personality questionnaire (spq), the hamilton depression rating scale (ham - d), and the hamilton anxiety rating scale (ham - a). the pearson correlation coefficients between y - bocs and spq scores were calculated. furthermore, hierarchical multiple linear regression analyses were conducted to assess whether schizotypy predicted the severity of obsession and compulsion.resultsby calculating the pearson correlation coefficient, it was found that the y - bocs obsession score, not the y - bocs compulsion score, was correlated with the spq total score. results of the hierarchical multiple linear regression analysis showed that spq total score was a significant predictor of the y - bocs obsession score, after accounting for control variables (ie, ham - d and ham - a).conclusionresults of this study showed that the y - bocs obsession score, not the y - bocs compulsion score, was correlated with the spq total score. this finding suggests that ocd patients with an elevated spq total score experience a reduction of cognitive inhibition, resulting in the frequent entry into obsession. future longitudinal studies are recommended to clarify the effect of schizotypy on the clinical course of ocd. |
although cdte - based czt (cd1-xznxte) crystals are the leading semiconductor compounds for radiation - detection applications, the widespread deployment of czt detectors has been limited by the high cost (due to a low manufacturing yield) of detector - grade materials. property nonuniformity has been the major cause for both poor performance and a low yield of usable portions of ingots. it has long been established that micrometer - scale defects such as tellurium inclusions / precipitates affect carrier transport and uniformity. however, extensive previous efforts to reduce te particles have not resulted in an anticipated improvement in czt property uniformity. one special structural feature of czt crystals is their extreme softness and their propensity to always develop a high density of dislocations during synthesis. experiments have shown that when these dislocations are organized into a network of subgrains, they can directly affect charge carriers to cause electric field perturbations. clearly, eliminating or at least controlling dislocation subgrain structures holds great potential to further improve czt crystals. many types of dislocations may be present in czt crystals, including shuffle dislocations, glide dislocations, and dislocations, partial and perfect dislocations, edge, screw, mixed dislocations, and misfit dislocations. the understanding of the behavior of the various combinations of these dislocations in conjunction with developing methods for improving the corresponding dislocation network structures are therefore challenging. the use of predictive, high - fidelity atomistic simulations can help screen out these structure - limiting dislocation types. here we use a recently developed czt bond order potential (bop) in lammps (large - scale atomic / molecular massively parallel simulator) to perform selected molecular dynamic (md) simulations to determine configurations, energies, and mobilities of 29 different types of dislocations in cdte crystals. [the czt ternary bop is an extension of the cdte binary bop, so they are equivalent if used only for cdte. ] an efficient method to derive activation free energy and activation volume of thermally activated dislocation motion will be explored. our focus is to identify and validate important dislocations and dislocation phenomena in the material, which can reduce future experimental efforts to improve the dislocation network structures. the key to high - fidelity md simulations of dislocations is an interatomic potential that is transferrable to the dislocated regions where the local atomic environment is significantly different from that of the perfect bulk crystal. details of the czt bond order potential applied here are omitted as they are very complex and have been fully described previously. it is demonstrated that the czt bop captures well the property trends of a variety of elemental and compound structures / phases (with local environment varying from coordination 2 to 12), simulates correctly the crystalline growth of stoichiometric compounds during both vapor deposition (under nonstoichiometric vapor fluxes) and melt - growth processes, and predicts precisely the configuration and density of misfit dislocations in vapor deposited multilayers. note that the correct simulation of growth, especially under a nonstoichiometric environment, is a very strong validation of the transferability of a potential as the process involves chemical reactions and samples a variety of configurations and chemistries statistically formed on a growth surface. for this reason, the czt bop is expected to produce reasonable results regarding dislocations. different types of dislocations can be constructed on various planes in a zinc - blende crystal. for instance, mobile dislocations usually lie on the { 111 } slip planes, and misfit dislocations in multilayered films are often seen to lie on interfacial planes, which are not necessarily parallel to { 111}. here we only study dislocations that are on the { 111 } slip planes, with burgers vector b being either partial a/6 or perfect a/2. (a) front projection of (112) planes, (b) top projection of two (111) planes a and b showing glide perfect dislocations, (c) top projection of two (111) planes a and b showing glide partial dislocations, and (d) top projection of two (111) planes b and c showing shuffle dislocations. (a) front projection of (110) planes, (b) top projection of two (111) planes a and b showing glide perfect dislocations, (c) top projection of two (111) planes a and b showing glide partial dislocations, and (d) top projection of two (111) planes b and c showing shuffle dislocations. assuming that the dislocation line direction aligns with the z direction and the slip occurs on the x z plane, our definition of different types of dislocations is shown in figures 1 and 2, where dark and light circles distinguish metal (cd or zn) and te atoms, respectively. dislocations defined in figure 1 are based upon an edge dislocation coordinate system shown in figure 1a where the dislocation line is perpendicular to one of the burgers vectors a/2 (x axis). dislocations defined in figure 2 are based upon a screw dislocation coordinate system shown in figure 2a where is parallel to one of the perfect burgers vectors a/2 (z axis). figures 1a and 2a indicate that a dislocation slip can occur at two different locations, one between two closely separated planes a and b (where the interplane bonds are not perpendicular to the planes), and the other between the more widely separated planes b and c (where the interplane bonds are perpendicular to the planes). according to convention, dislocations between a and b are called glide dislocations, and dislocations between planes b and c are called shuffle dislocations. burgers vectors of glide dislocations can be examined from a top projection of two isolated planes a and b. figures 1b and 2b use such a projection to explore perfect dislocation burgers vectors (a/2). it can be seen from figure 1b that in the edge coordinate system, there are three nonequivalent perfect burgers vectors that can be represented by the angle between b and, i.e., = 30, 90, and 150. there are other perfect burgers vectors, but they are equivalent to the ones listed due to crystal symmetry. for instance, the = 330 dislocation is equivalent to the = 30 dislocation. similarly, it can be seen from figure 2b that in the screw coordinate system there are three more nonequivalent perfect burgers vectors = 0, 60, and 300. figures 1c and 2c use the same top projection of two isolated planes a and b to explore partial dislocation burgers vectors (a/6). it can be seen from figure 1c that in the edge coordinate system, there are two nonequivalent partial burgers vectors = 0 and 120. similarly, it can be seen from figure 2c that in the screw coordinate system there are two more nonequivalent partial burgers vectors = 90 and 330. burgers vectors of shuffle dislocations can be examined from the top projection of two isolated planes b and c. figures 1d and 2d use such a projection to explore shuffle dislocation burgers vectors. here the light and dark atoms in the b and c planes are aligned in the projection (i.e., only light atoms are seen), and hence, there are no partial dislocation burgers vectors for shuffle dislocations. as indicated in figure 1d for the edge coordinate system, we explore three shuffle dislocation burgers vectors = 30, 90, and 150, which are the same as those in the perfect glide dislocation case shown in figure 1b. as indicated in figure 2d for the screw coordinate system, we explore three more shuffle dislocation burgers vectors = 0, 60, and 300, which are the same as those in the perfect glide dislocation case shown in figure 2b. dislocations shown in figures 1 and 2 always occur between a metal (cd or zn) and te { 111 } planes. if a dislocation has an edge component, then the dislocation core can have extra metal atoms or extra te atoms. according to convention, a dislocation with more metal atoms at its core is called an dislocation, and a dislocation with more te atoms at its core is called a dislocation. according to the discussion above, we will consider a combination of glide and shuffle dislocations, partial and perfect dislocations, various burgers vectors, and and dislocations. a cdte crystal system with 336 (112) planes in the x direction, 60 (111) planes in the y direction, and 28 (110) planes in the z direction is used to calculate the (111) stacking fault energy sf. the stacking fault is created by shifting the upper part of a perfect crystal by a partial dislocation vector with respect to the lower part (at a glide dislocation slip plane between planes a and b, see figure 1 or 2). molecular statics (ms) energy minimization simulations are used to calculate relaxed energies of both the perfect and the stacking faulted crystals under the periodic boundary conditions in both x and z directions and a free boundary condition in y direction. the stacking fault energy is then calculated as sf = (esf e0)/(lxlz), where esf and e0 are respectively the relaxed energies for stacking faulted and perfect crystals and lx and lz are respectively the system dimensions in x and z directions. note that the surface energy in the y direction does not affect the calculation as it exists for both stacking faulted and perfect crystals and therefore cancels out. cdte is known to have a low stacking fault energy around 10 mj / m. this in turn allows dislocation energies, i.e., the energy difference between a dislocated crystal and a perfect cdte crystal containing the same number of atoms, to be calculated as a function of dislocation core radius. the key for the calculations is to use the initial dislocation configurations that can be fully relaxed during md and ms energy minimization simulations. we find that an initial dislocation created by simply positioning atoms according to the displacement field of the linear elastic continuum solution does not always fully relax at the core. instead, a dislocation created by shearing the relevant portions of the crystals using md simulations is found to normally relax to lower energies than the other methods. in particular, as will be described below, an md scheme illustrated in figure 3 is effective in creating partial, undissociated perfect, and shuffle dislocations, and an md scheme illustrated in figure 4 is effective in creating dissociated perfect dislocations. these schemes enable atom positions to be strictly determined from atom interactions and also best reflects realistic scenario that dislocations are created under local shear stresses. line energy model for partial, undissociated perfect, and shuffle dislocations. (a) md simulations to create dislocations and (b) md and ms simulations to relax dislocations. the computational system used is periodic in the x and z axes with free surfaces along the y axis. for the undissociated dislocations (including partial and shuffle dislocations) shown in figure 3, the system is divided into different regions as indicated by blue (middle upper), orange (middle lower), black (left and right), and white (the rest) colors. with the black regions held fixed and the white regions free to relax, an md simulation is first performed to uniformly move the blue region by half of the burgers vector 0.5b and the orange region by negative half of the burgers vector 0.5b. for partial dislocations, the movement of the blue and orange regions is completed directly over a total simulated time of 0.1 ns. for perfect dislocations, the movement is through two stages over a total simulated time of 0.2 ns to follow the lowest energy path of two partials, i.e., 0.5(b1 + b2) (= 0.5b) for the blue region and 0.5(b1 + b2) (= 0.5b) for the orange region. in addition, the system temperature is uniformly decreased from 300 to 10 k during simulation. this md procedure creates two dislocations with opposite burgers vectors (also note that if the dislocations have an edge component, then one is an and the other one is a dislocation) as shown in figure 3a. for the dissociated perfect dislocations shown in figure 4, the system is divided into different regions as indicated by light and dark blue (middle upper), light and dark orange (middle lower), black (left and right), and white (the rest) colors. with the black regions held fixed and the white regions free to relax, an md simulation is first performed to uniformly move the light and dark blue region by half of a shockley partial burgers vector 0.5b1 and the light and dark orange region by negative half of the same shockley partial burgers vector 0.5b1 over a total simulated time of 0.1 ns. a second md simulation is then performed to move the dark blue region by half of the other shockley partial burgers vector 0.5b2 and the dark orange region by negative half of the same shockley partial burgers vector 0.5b2 over another total simulated time of 0.1 ns. in addition, the system temperature is uniformly decreased from 300 to 10 k during these two simulations. this md procedure creates the same two perfect dislocation burgers vectors as shown in figure 3a except that each perfect dislocation is dissociated into two shockley partials bounding a stacking fault band as shown in figure 4a. (a) md simulations to create dislocations and (b) md and ms simulations to relax dislocations. once dislocations are created in the md simulations, the blue and orange regions are released. a narrow region in the middle of the system is allocated as shown by the black color in figures 3b and 4b. for perfect dislocations, this narrow region remains a perfect crystal because the blue region is moved by a perfect burgers vector with respect to the orange region. for partial dislocations, the narrow middle region contains a stacking fault. with left, right, and middle black regions fixed, an ms simulation is then conducted to relax the dislocations. in this work, we use an edge dislocation geometry containing 97 (110) planes in the x direction, 60 (111) planes in the y direction, and 24 (112) planes in the z direction and a screw dislocation geometry containing 168 (112) planes in the x direction, 60 (111) planes in the y direction, and 14 (110) planes in the z direction to create various perfect and dislocated computational crystals. relaxed energies of both dislocated and perfect crystals are calculated. to separate and dislocation effects, the total energy is split to the left and right parts respectively by summing up the atomic energies of the left and right halves of the systems. the line energies of the left and the right dislocation, i, are then calculated as i = (ed, i wilzsf)/lz, where ed and e0 are the relaxed energies of the dislocated and the perfect crystals, respectively, lz is the dislocation length (= system dimension in the z axis), sf is the stacking fault energy, w is the stacking fault bandwidth, and the subscript i (= l or r) indicates the left and the right dislocations. we find that of all the simulations using the initial perfect dislocations, the maximum splitting distance is only 25 after energy minimization. table 1 indicates that the screw (= 0) dislocations have lower energies than other dislocations, being 0.38, 0.69, 0.79, and 0.81 ev / for partial, dissociated perfect, undissociated perfect, and shuffle dislocations, respectively. interestingly, the acute angle of the second lowest energy (shuffle, glide partial, glide perfect) dislocations all equal 30 (e.g., = 30, 150, and 330). in addition, the energies of dissociated perfect dislocations are about 8092% of the energies of undissociated dislocations, suggesting that the dissociated dislocations are more stable. however, the observation that the perfect dislocations remain approximately undissociated during md and energy minimization simulations suggests an energy barrier of dissociation. the configuration of perfect dislocations will be examined further below in md simulations of dislocation motion. (a) glide partial dislocations, (b) glide perfect dislocations, and (c) shuffle dislocations. the dislocation line energy is not a constant material property, but rather increases with the material volume within a radius r from the dislocation core. the dislocation line energy at a given radius r can be calculated using the same approach described above except that the energies of the perfect and dislocated crystals are taken as the sum of the atomic energies for all the atoms within the radius. the dislocation line energies as a function of r thus obtained are shown in figure 5a figure 5a c indicates that dislocation line energies calculated with the atomistic models linearly increase with dislocation radius r when r is larger than 40. this is consistent with the boundary conditions used in figures 3 and 4 where the black regions are held fixed during simulations. the dislocation core energies at small radii, say r < 30, come from the relaxed dislocation core structures. in the present work, the system sizes have been chosen to be sufficiently large so that the predicted dislocation core structures are independent of the boundary conditions. as a result, our simulations can give accurate dislocation core energies at r < 30 that can not be predicted from linear elastic theories. it can be seen from figure 5a c that dislocation energies sharply decrease when the radius is decreased. in addition, and dislocations have similar energy vs r curves for partial and shuffle dislocations. while and dislocation energies are also similar for the perfect dislocations at large radii, they become different at the core. in particular, the perfect dislocation core energies are lower than the perfect dislocation core energies. one effective approach to simultaneously study the motion of multiple dislocations is to examine the evolution of a dislocation loop under a shear stress. the computational system consists of a cdte crystal with 120 (112) planes in the x direction, 18 (111) planes in the y direction, and 68 (110) planes in the z direction. periodic boundary conditions are used in the x and z directions and a free boundary condition is used in y direction during the simulation. to create a dislocation loop, the system is divided into different regions as shown in figure 6(a) : the black (top and bottom) regions, the blue (middle upper) hexagonal region, the orange (middle lower) hexagonal region, and the white (remaining) region. as plastic deformation usually proceeds through a glide of dislocations hence, we assume that the boundary between the upper and lower hexagons is between planes a and b as defined in figures 1a and 2a. to create a realistic dislocation loop with a burgers vector of b, an md simulation is performed to uniformly move the top black and blue regions by 0.5b and the lower black and orange regions by 0.5b over a simulated time of 0.1 ns while allowing the white region to relax according to newton s equation of motion and a constant temperature of 10 k. as mentioned above, for a partial dislocation loop, the md simulation is completed directly, and for a perfect dislocation loop, the md simulation is completed in two stages to follow the low energy path of two partial dislocations. this md simulation procedure creates a dislocation loop at a shear strain of approximately = b / t, where b is the magnitude of the dislocation burgers vector and t is the thickness of the sample. the system is then redivided into three regions as shown in figure 6b : the black (top and bottom) regions and the white (remaining) region. the evolution of dislocation loops is examined in subsequent md simulations at a high temperature of 900 k where the top and bottom surface black layers are further moved in positive and negative burgers vector directions to increase the shear strain. (a) md simulation to create dislocation loop and (b) md simulation to evolve dislocation loop. a partial dislocation burgers vector of [112]a/6 is used to create an initial dislocation loop using the procedure shown in figure 6(a). further shear strain is applied by moving the top and bottom black regions in the + x and x directions by 1.394, respectively, using the procedure shown in figure 6b. the equilibrium dislocation loop configurations obtained at 900 k temperature are examined in figure 7 by showing the top view of four consecutive planes above and below the slip plane, where blue and gray circles indicate cd and te atoms respectively, and the yellow hexagon indicates the initial partial dislocation loop at xy = 0.039. panels a and b in figure 7 are obtained using exactly the same conditions except that the cd and te atoms are switched between the two figures, so that in figure 7a, the right side of the loop is an dislocation and the left side of the loop is a dislocation, whereas in figure 7b, the left side is and the right side is. because the movement of a partial dislocation leaves behind a stacking fault, the relaxed partial dislocation loop can be identified by the boundary of the two stacking patterns demonstrated in the figure. it can be seen from figure 7a that the hexagonal dislocation loop becomes asymmetric under the shear and in particular, the dislocation moves by a far longer distance than the dislocation. this is further verified in figure 7b where the asymmetry of the dislocation is flipped as a consequence of the flip of and dislocations. this simulation strongly indicates that dislocations are much more mobile than dislocations (despite their similar line energies in table 1), in good agreement with experiments conducted for many semiconductor compounds. (a) configurations at strains xy = 0.039 and xy = 0.078 and (b) the same as in panel a except that cd and te atoms are switched t = 0.0025 (a), 0.0100 (b), 0.0250 (c), and 0.0350 ns (d). one interesting observation in figure 7 is that to accommodate the motion of the segment (= [110 ]) of the dislocation loop, two segments (= [112 ]) are created. this means that the motion of the mobile partial dislocations can cause elongated screw partials. as a result, cdte crystals may have a high density of such type of screw partials, in agreement with its low energy shown in table 1. on the other hand, this corresponds well to the high energy of the edge partial in table 1. a perfect dislocation loop with a burgers vector of [110]a/2 is created using the procedure described in figure 6a. this procedure produces an initial shear strain of yz,0 = 0.068. by moving the top and bottom black regions in the + z and z directions at a constant speed of 1.2 nm / ns during an md simulation at 900 k, as described in figure 6b, the system is further deformed at a strain rate of dyz / dt = 0.338/ns. the configuration of the dislocation loop during the further strain is shown in figures 8a d as a function of time, where the color scheme represents the magnitude of the slip vector developed previously to show dislocations. figure 8a shows that shortly after further strain at t = 0.0025 ns (yz = 0.069), the dislocation loop remains hexagonal but some portion of the dislocation (the lower part of the [101 ] section) bows out. figure 8b shows that at t = 0.0100 ns (yz = 0.071), the dislocation bows out more. in figure 8c at 0.0250 ns (yz = 0.076), the dislocation is seen to have moved a significant distance that causes it to pass through the low boundary and enter into the image from the top boundary under the periodic boundary condition. clearly, perfect dislocations are also much more mobile than perfect dislocations (despite their similar line energies shown in table 1), in good agreement with experiments. interestingly, figure 8c indicates that, although screw dislocations also have higher mobility than edge dislocations, they are much less mobile than edge dislocations. when the dislocation meets the dislocation at the top of the hexagon, they are annihilated, and the dislocation loop becomes two vertical screw dislocations running across the periodic dimension in the z direction, as can be seen in figure 8d at t = 0.035 ns (yz = 0.080). further strain will cause the two dislocations to continuously move in the x directions until they completely sweep the entire area and are annihilated. interestingly, we find that the dislocation motion is thermally activated and does not occur when the simulation is performed at a low temperature, say, 10 k. in addition, we find that the dislocations do not move as straight lines, but rather through the formation and rapid motion of kinks along the z direction as shown in figure 8c. this is consistent with the traditional kink model of thermally activated motion of dislocations. the observation that screw dislocations are much less mobile than edge dislocations indicates that when the sector of a dislocation loop moves, the screw dislocation sectors (= [110 ]) will elongate. as a result this is consistent with the low energies of screw dislocations shown in table 1. in addition, no dislocation dissociation is observed in figure 8, indicating that the perfect dislocations do not necessarily dissociate under nonequilibrium conditions. the details of dislocation mobility are important to understand because they directly control dislocation network structures. for thermally activated motion of dislocations, the dislocation velocity can be expressed as1where q and are respectively the activation energy and activation volume of the dislocation motion, is the shear stress, k is boltzmann s constant, t is the temperature, and v0 is a constant representing saturation dislocation velocity for barrierless (very high temperature or shear stress) dislocation motion. here q and are the primary parameters quantifying dislocation mobility. the discussion presented above already indicates several distinctive dislocation types that may determine dislocation structures. first, and partial and perfect dislocations have a strong edge component and can contribute to the climb motion responsible for the formation of small angle grain boundaries. dislocations are the most mobile, resulting in special structural features of dislocation networks. screw partial and perfect dislocations can also be important. these screw dislocations are expected to have a high density as they have low energies and can be created by the motion of dislocations. the objective of the present work is not to exhaustively tabulate the mobilities of a variety of dislocations. hence, we focus on calculating the activation energy barrier q and activation volume for and edge type of perfect dislocations. such studies will allow us to fully explore the method for calculating q and and to validate the results using known experimental relative mobilities of and dislocations. in addition, edge dislocations play an important role in dislocation network formation due to their climb motion. the extension of the method to other dislocations that can provide complete inputs for simulations of dislocation network structure evolution using larger scale (e.g., dislocation dynamics) models will be performed in future work. the activation energy barriers of dislocation motion are often directly calculated from energy profiles using the nudged elastic band method. however, the stop - action observation of the dislocation migration case shown in figure 8 indicates that the thermally activated motion of dislocations proceeds through a variety of different paths. without requiring an explicit treatment of migration paths, we perform md simulations of dislocation motion at a variety of temperatures and shear stresses to fit an apparent activation energy barrier and activation volume directly from (1). the activation energy determined this way pertains to a free energy barrier that incorporates the multiple path (entropy) effects. in addition, this approach provides a direct verification of (1) and, hence, the thermally activated dislocation mechanism. the initial crystal contains 144 (110) planes in the x direction, 36 (111) planes in the y direction, and 24 (112) planes in the z direction. the system employs periodic boundary conditions in x and z directions and a free boundary condition in y direction. first, the initial crystal is divided into different regions as shown in figure 9a : the black (left and right) regions, the blue (middle upper) region, the orange (middle lower) region, and the white (remaining) region. since we only explore glide dislocations, we assume that the boundary between the blue and orange regions is between a and b planes as defined in figures 1a and 2a. to create dislocations with a perfect burgers vector of b = b1 + b2, an md simulation is performed where the black regions are fixed, the white regions are relaxed, and the blue and orange regions are first uniformly moved by 0.5b1 and 0.5b1, respectively, over a simulated time of 0.001 ns and then moved by 0.5b2 and 0.5b2, respectively, over another simulated time of 0.001 ns. the crystal is then cut in half in the x direction as shown in figure 9b with a corresponding adjustment of the periodic length so that the two halves remain periodic in the x direction. in particular, we use b = [110]a/2, b1 = [121]a/6, and b2 = [211]a/6 to create two crystals, one containing an perfect dislocation, and the other one containing a perfect dislocation. edge dislocation mobility model. (a) md simulation to create and dislocations, (b) isolate out and dislocations, and (c) md simulation to evolve dislocation under shear stress xy. for dislocation motion simulations, the (half) system is divided into three regions : the black (top and bottom) regions and the white (remaining) region. by adding corresponding forces to the atoms in the top and bottom regions in + x and x directions, respectively, molecular dynamics simulations under an npt (constant atom number, pressure, and temperature) condition are then performed to study migration of dislocations at a variety of stresses and temperatures. we find that in all of these simulations the dislocation splitting is very small, further verifying the discussions presented above that perfect dislocations do not necessarily dissociate. assuming that the slip vector reaches a maximum at the dislocation core, the dislocation position is calculated as a function of time. results obtained for and dislocations at selected stresses and temperatures are shown in figure 10. it can be seen from figure 10 that all the dislocation vs time curves approximately fall on straight lines. this shows that the data can provide reliable information about the steady - state dislocation velocity. the slope of these straight lines changes with stress and temperature, indicating that the simulated conditions capture the thermally activated mechanisms of dislocation motion. figure 10a shows the effect of temperature on dislocation motion at a constant shear stress of 0.5 gpa. increasing the temperature is seen to increase the dynamics, consistent with the thermal activation mechanism. at the same temperature, the dislocation is seen to move faster than the dislocation, further verifying the results seen in figures 7 and 8. figure 10b shows the effect of shear stress at a constant temperature of 600 k. here increasing the stress also correctly increases the dislocation velocity, and the dislocation is again validated to move faster than the dislocation. (a) effect of temperature at a given stress and (b) effect of stress at a given temperature. the steady state velocity can be easily calculated from the slope of the curves shown in figure 10. the results obtained at a constant shear stress of xy = 0.5 gpa are plotted as ln(v) vs (kt) curves in figure 11. here the unfilled and filled circles represent md data for and dislocations respectively, and the lines are fitted to the data using equation (1). figure 11 indicates that the md data falls fairly well on straight ln(v) vs (kt) lines, validating that the method indeed captures well the thermally activated dislocation migration. through the fitting, we find that the activation energy and activation volume are q = 0.14 ev and = 17 for the dislocation and q = 0.27 ev and = 38 for the dislocation. this again verifies that in the thermally activated regime (e.g., xy 1 gpa), the dislocation is more mobile than the dislocation. the thermally activated plastic deformation of cdte has been experimentally analyzed below room temperature. the activation energy and activation volume are assumed to decrease with increasing stress in the experiments. under this assumption, the fitted experimental activation energy and activation volume are approximately 0.8 ev and 5500, respectively, at a small stress of 0.005 gpa, and fall to < 0.5 ev and < 2250, respectively, at a stress of 0.010 gpa. here we assume that q and are constant and are fitted using md data at high stresses (e.g., 0.5 gpa). in addition, simulations target only the peierls potential barrier mechanism of specific (and perfect edge) dislocations, whereas, experiments may include point defect obstacle mechanisms and the contributions from other dislocations. as a result, it is expected that the simulated activation energy and activation volume are smaller than their experimental counterparts. our bop - based md simulations of dislocations in cdte crystals lead to the following conclusions:(1) dislocations move much faster than dislocations, in good agreement with well - known experiments for semiconductor compounds. this, along with previous demonstrations that our bop captures properties of many phases and predicts crystalline growth during md simulations of chemical vapor deposition and melt - growth, provides strong validation that the bop correctly captures the physics of dislocation motion in cdte. the bop - based md simulations, therefore, can provide high - fidelity data to construct large scale (e.g., dislocation dynamics) models that can simulate the evolution of dislocation cell structures.(2)for partial, perfect, and shuffle sets, screw dislocations always have the lowest energies. dissociated glide dislocations always have lower energies than perfect glide dislocations. however, perfect glide dislocations do not necessarily dissociate due to the energy barrier of dissociation. in particular, the splitting distance of perfect glide dislocations is seen to remain very small in our dynamic md simulations.(3)the acute angle of the second lowest energy (shuffle, glide partial, glide perfect) dislocations all equal 30 (e.g., = 30, 150, 330).(4)screw dislocations are much less mobile than edge dislocations. as a result, the screw dislocation sectors perpendicular to the sector are elongated when an sector of a dislocation loop moves. this suggests that screw dislocations are likely to have a higher density than edge dislocations, in good agreement with the low screw dislocation energies.(5)without calculating the energy profile explicitly, we show that md simulations of dislocation motion at a variety of stresses and temperatures can be used to effectively deduce the activation free energy barrier and activation volume of thermally activated glide of dislocations. in particular, we found activation free energy barriers of 0.14 and 0.27 ev and activation volumes of 17 and 36, respectively, for and edge types of glide dislocations. dislocations move much faster than dislocations, in good agreement with well - known experiments for semiconductor compounds. this, along with previous demonstrations that our bop captures properties of many phases and predicts crystalline growth during md simulations of chemical vapor deposition and melt - growth, provides strong validation that the bop correctly captures the physics of dislocation motion in cdte. the bop - based md simulations, therefore, can provide high - fidelity data to construct large scale (e.g., dislocation dynamics) models that can simulate the evolution of dislocation cell structures. for partial, perfect, and shuffle sets, screw dislocations always have the lowest energies. dissociated glide dislocations always have lower energies than perfect glide dislocations. however, perfect glide dislocations do not necessarily dissociate due to the energy barrier of dissociation. in particular, the splitting distance of perfect glide dislocations is seen to remain very small in our dynamic md simulations. the acute angle of the second lowest energy (shuffle, glide partial, glide perfect) dislocations all equal 30 (e.g., = 30, 150, 330). screw dislocations are much less mobile than edge dislocations. as a result, the screw dislocation sectors perpendicular to the sector are elongated when an sector of a dislocation loop moves. this suggests that screw dislocations are likely to have a higher density than edge dislocations, in good agreement with the low screw dislocation energies. without calculating the energy profile explicitly, we show that md simulations of dislocation motion at a variety of stresses and temperatures can be used to effectively deduce the activation free energy barrier and activation volume of thermally activated glide of dislocations. in particular, we found activation free energy barriers of 0.14 and 0.27 ev and activation volumes of 17 and 36, respectively, for and edge types of glide dislocations. | cd1-xznxte (czt) crystals are the leading semiconductors for radiation detection, but their application is limited by the high cost of detector - grade materials. high crystal costs primarily result from property nonuniformity that causes low manufacturing yield. although tremendous efforts have been made in the past to reduce te inclusions / precipitates in czt, this has not resulted in an anticipated improvement in material property uniformity. moreover, it is recognized that in addition to te particles, dislocation cells can also cause electric field perturbations and the associated property nonuniformities. further improvement of the material, therefore, requires that dislocations in czt crystals be understood and controlled. here, we use a recently developed czt bond order potential to perform representative molecular dynamics simulations to study configurations, energies, and mobilities of 29 different types of possible dislocations in cdte (i.e., x = 1) crystals. an efficient method to derive activation free energies and activation volumes of thermally activated dislocation motion will be explored. our focus gives insight into understanding important dislocations in the material and gives guidance toward experimental efforts for improving dislocation network structures in czt crystals. |
integrated care is a relatively new field, which scope and definition is intensively debated. in a contribution to this debate, spreeuwenberg and kodner noticed that some authors are inclined to define integration predominantly as a hierarchical or top - down process driven by generalised organisational exigencies for perfection and optimisation, whereas other authors promote a patient - centred or bottom - up view in which the characteristics and needs of specific patient groups, and their fit with existing systems of care and cure more or less determine the what, how and where of integration. representing the latter group of authors, spreeuwenberg and kodner propose to define the term integrated as a coherent set of methods and models on the funding, and the administrative-, organisational-, service delivery- and clinical levels designed to create connectivity, alignment and collaboration within and between the cure and care sectors. while limiting the focus to patients with complex, long term problems cutting across multiple services, the results of such multi - pronged efforts to promote integration for the benefit of these patients would be called integrated care. in a similar vein, but more comprehensive, hardy. state that integrated care refers to a coherent and co - ordinated set of services, which are planned, managed and delivered to individual service users across a range of organisations and by a range of co - operating professionals and informal carers. in their view, integrated care covers the full spectrum of health and health care - related social care, while it is also inextricably linked to other services that enable people to be properly cared for in their own homes and in their own communities. the essence of integrated care would be to provide individuals with care services they are in need of, when and where they need them. integrated care would appear seamless to the service recipients and would be devoid of overlap or gaps to service commissioners and providers. at the same time, these authors acknowledge that, in practice, integrated care appears in a variety of forms : integral care, comprehensive care, continuing care, intermediate care and so on, partly reflecting different countries of origin and differences in scope and approach. for example, transmural care encompasses many different forms of care directed towards bridging the gap between generalised and specialised care for both acute and chronic patients, with origins in the dutch health care system. according to rosendal the concept of integrated care is more comprehensive than the concept of transmural care since transmural care generally does not include the whole care process of patients but instead tends to focus on one or two crucial transition - steps between different types of health care providers. also, in rosendal 's view, transmural care is similar to shared care and hospital at home care in the uk, and (rather) different from the author states that the latter two approaches more closely match the comprehensive definition of integrated care presented earlier. integrating these views, it is perhaps fair to state that in the past decade a large variety of new care arrangements has emerged which, despite clear differences, are more and more frequently referred to as (alternative forms of) integrated care. integrated care thus may become rather an umbrella term, uniting a field, than an exact definition for all its variations in practice. for reasons of clarity and understanding, in this article integrated care is referred to as a comprehensive concept, while distinguishing between different arrangements on a more detailed level where appropriate. for example, the development of shared care relates to the policy of shifting the balance from secondary to primary care whereas in the netherlands, transmural care was conceptualised in particular as a means to integrate primary health services and hospital services. in these and other countries, the ageing of the population and the concomitant increase of citizens suffering from non - communicable disease in general and chronic disease in particular, are regarded as major drivers of the development of integrated care. related to this, the importance of integrated care arrangements is expected to increase, exemplified by the establishment of this journal, the (forthcoming) creation of a society for integrated care and a concomitant increase in research activities in the field, perhaps in particular in the eu. it is generally assumed that integrated care results in increased effectiveness and quality of care, while being cost - effective or even cost - saving at the same time [13, 6 ]. although many authors agree that integrated care holds a great promise, they warn against expectations that may be unrealistic, while supporting an evaluative approach [1, 35 ]. for example, in a phd thesis on economic evaluation of end - stage renal disease treatment in the netherlands, the author states that : in the context of an efficient use of resources for research, economic evaluation of health care programmes should be oriented increasingly to interventions at the level of the organisation of care or the organisation of the health care system in general, rather than to the plethora of interventions at the micro level of care and prevention (translation hv). analysts in the field agree, indicating that in the dutch context the effectiveness and efficiency of transmural care has hardly been addressed. likewise, in the uk the concern has been expressed that we know too little about the relative cost - effectiveness of providing care in different settings and by professionals with different types of training. in addition, a recent review of the development of integrated care in 6 eu countries indicates that, despite many initiatives, most of these programmes may not yet have achieved their full potential. the combination of these findings indicate a need for evaluation in general and economic evaluation in particular as, due to the relative immaturity of the field, many integrated care programmes are short - lived after initial funding by temporary subsidies and grants at either local or national levels runs out. a positive decision on long - term financing or reimbursement of services can be facilitated by a timely and high - quality economic evaluation demonstrating value for money of the programme in question. likewise, an economic evaluation combined with a budget impact analysis could support decision making on permanent reallocation of some share of e.g. existing hospital and/or home care budgets on behalf of an integrated care arrangement, thus contributing to its long - term survival. in this context this paper intends to stimulate research efforts in this field by providing a brief overview of economic evaluation methods and by discussing a number of issues that seem particularly relevant for economic evaluation of integrated care. in doing so, it has been taken into account that there are quite many different forms of integrated care. as a consequence, concepts are illustrated on the basis of studies evaluating both transmural care-, hospital at home care-, and managed care programmes. the choice of issues has been determined on the basis of general requirements for any economic evaluation, as formulated in the excellent textbook by drummond and colleagues, geared towards integrated care as a result of a plenary discussion following a presentation of a draft version of this paper at the who / ijic international conference on new research and developments in integrated care, held in barcelona, spain, 2122 february 2003. rapid technological change, the ageing of the population, and increased expectations of the public constitute the three major pressures on health services in most industrialised nations, challenging the financial sustainability of health care systems and widely held values of equal access to high quality care. in other words, resources people, time, facilities and knowledge are scarce. choices must and will be made concerning the deployment of resources, in this case to develop and implement integrated care arrangements. in fact, the real costs of committing resources to a particular use is not the number of euros, dollars or other currency appearing on the programme budget, but rather the health outcome achievable in some other programme which have been forgone by committing the resources in question to the first programme. opportunity cost which can be estimated and compared with programme benefits by means of economic analysis. firstly, it deals with both the inputs and outputs, or costs and consequences, of activities. the two characteristics of economic evaluation have led to its definition as the comparative analysis of alternative courses of action in terms of both their costs and consequences. economic evaluation of integrated care thus involves a comparison of its costs and consequences to one or a combination of its most appropriate comparators, e.g. care provided in the this approach is justified where it can be assumed, or has been shown, that the alternative programmes or therapies being compared produce identical outputs. an example of such a study, termed a cost - minimisation study, is the one by shepperd. (1998), who conducted an economic evaluation alongside a randomised clinical trial with 3 months follow - up, comparing hospital at home care with inpatient hospital care for a wide variety of patients, e.g. recovering from hip- or knee replacement. the results of the study were reported in two consecutive articles, the first focusing on health outcomes and the second on the efficiency of the competing care programmes [11, 12 ]. the latter study was reduced to a cost - minimisation analysis, as the analysis of the clinical study demonstrated that there were no major differences in patient 's reported health outcomes between the two arrangements. to date, this form of analysis may have been used most frequently in evaluation of integrated care. the other forms of economic evaluation differ mainly in the method of measuring the outputs. in a cost - effectiveness analysis, outputs are expressed in the most convenient natural units or health effects, such as number of cases successfully treated or years of life gained. analyses like these presuppose that the costs of care are related to a single, common effect, which may differ in magnitude between the alternative programmes. when instead it is deemed more appropriate to present an array of output measures, the associated analysis is termed a cost - consequence analysis. this is often related to the fact that much modern medicine is concerned not only with improving quantity, but also quality of life. to assess quality of life, an impressive number of instruments has been developed, usually distinguishing between disease - specific and generic questionnaires. the latter questionnaires allow comparisons of the quality of life between different patients groups, and between patient groups and the general population. for similar reasons as those underlying the development of quality of life questionnaires, there has also been a growth of interest in cost - utility analysis, where the life - years gained from treatment are adjusted by a series of utility weights reflecting the relative values individuals place on different states of health. in creating these adjustments, health economists are basically attempting to ascertain how much better the quality of life in one health situation or an example is dialysis at home with help from a spouse or a friend versus dialysis in the hospital (see). the output measure most frequently used is known as the quality - adjusted life year (qaly), which is particularly useful for comparing the efficiency of alternative programmes with differences in both mortality and quality of life. finally, in a cost - benefit analysis, the outputs of a programme are, just as the costs, expressed in money terms, usually the currency of the country of study, in order to make them commensurate with the costs of the intervention. an example of a study using an analytical technique that is often applied in cost - benefit analysis to assess the value of outcomes, is provided by a study by barner. (2001), assessing asthma patients ' willingness to pay (wtp) for and give time to an asthma self - management programme. in other cases, a wtp approach is applied to both patients and informal caregivers. yet another example, but now exclusively geared towards informal caregivers, is the study by chiu. (1999), assessing the willingness of families caring for victims of stroke to pay for in - home respite care. in contrast with the previous analytic techniques, where results are expressed as a ratio, the results of cost - benefit analyses are expressed as a net benefit (or cost). the different types of economic evaluation are summarised in table 1. of the different types of economic evaluation, cost - benefit and cost - utility analysis enable decision - makers to assess broader choices, since they address the issue of outcome valuation. worth while when compared to other programmes within and outside the health care sector. in principle, this is highly relevant in case of integrated care arrangements, some of which incorporate a combination of health and non - health services. cost - minimisation and cost - effectiveness analysis tacitly assumes that the treatment objective is worth meeting, and generally address more restrictive questions. in general, the choice of analytic methods should fit the decision making process to be supported. to be categorised as a full economic evaluation, a study needs to examine both costs and effects of the alternative programmes to be compared. usual care, or, more specifically, care in the usual setting as comparator, which may be e.g. regular hospital care, nursing home care, or care provided in hospices [17, 18 ]. in some cases, it may be difficult to choose the most relevant comparator(s). in the netherlands, for example, seven categories of transmural care have been distinguished, of which some are aimed at a single patient group, e.g. diabetes patients. after making the correct choice of comparator, the alternatives need to be described comprehensively. this allows the reader the opportunity to assess whether the results can be transferred to a local setting. (1998), patients randomised to hospital at home care received early discharge with home based rehabilitative care between 08.30 and 11 pm provided by a team of two nurses, a physiotherapist, an occupational therapist and three support workers. the services provided were those for health care, with minimal essential domestic tasks performed, with a case load of 12 patients at any one time or less, with discharge (of hospital at home care) when patients could be managed by routinely available community services [19, 20 ]. health economists recommend to state a viewpoint for the analysis and to place the study in a decision - making context. possible viewpoints include those of the provider institution, the individual clinician or professional organisation, the patient, the purchaser of health care (or third party payer) or society as a whole. this is because data can usually be disaggregated and the analysis carried out from a number of viewpoints. also, the additional costs of adopting a broader perspective at the outset of the study is probably less than the cost of attempting to gather additional information later [9, 21 ]. as noticed earlier, this is relevant in case of integrated care. for example, in the economic evaluation carried out alongside a clinical trial by richards. [19, 20 ], it was attempted to include the viewpoints of both the nhs, social services, and patients. the viewpoints fitted with the aim of the study, evaluating the effectiveness and acceptability of an early discharge, hospital at home scheme and acute hospital care for medically stable elderly patients. it can be added that the relatively important role of informal care in a number of integrated care programmes supports the choice of a broad societal perspective. an example is provided by an arrangement in denmark, where an informal caregiver is allowed to stay home from paid work to care for a terminally ill family member. the quality of an economic evaluation is to a large extent determined by the quality of the evidence of effectiveness of health care programmes. the availability (and if available, the quality) of evidence of effectiveness is almost always a problem, and this goes as well for the field of integrated care. of the different designs that can be used to generate evidence on effectiveness, however, in an assessment of research done in the uk it was stated that few randomised controlled clinical trials of hospital at home services have been done, and that most of these have been small, with little attempt at economic evaluation. so when considering economic evaluation of integrated care programmes, perhaps three issues related to the quality of evidence on effectiveness need to be addressed : for which programmes does reliable evidence of effectiveness exist, and does this provide an adequate basis for economic evaluation?for which programmes are clinical trials being planned and is there any scope for undertaking economic analysis alongside these trials ? (see e.g., for more information on this issue)for which major applications of integrated care is there yet no reliable evidence of effectiveness and what efforts should be made to assemble such evidence ? for which programmes does reliable evidence of effectiveness exist, and does this provide an adequate basis for economic evaluation ? for which programmes are clinical trials being planned and is there any scope for undertaking economic analysis alongside these trials ? (see e.g., for more information on this issue) for which major applications of integrated care is there yet no reliable evidence of effectiveness and what efforts should be made to assemble such evidence ? with regard to these latter points, a meta - analysis of interventions used in disease management programmes for patients with chronic illnesses is illustrative. in this particular study, six interventions were distinguished : provider education (materials or instruction given to healthcare providers regarding appropriate care for patients with the condition targeted by the programme) ; provider feedback (information to health care providers regarding the specific care or results of care received or experienced by their patients) ; provider reminders (prompts given to providers to perform specific patient care tasks) ; patient education (materials and instructions issued to patients providing information on their condition and how it could be managed) ; patient reminders (prompts given to patients to perform specific tasks related to care for their condition) ; and patient financial incentives (payments (direct monetary payments, discounts or services) made to patients for achieving specific treatment related goals). it was concluded that, although these different interventions were associated with improvements in provider adherence to guidelines and patient disease control, the studies did not directly compare different interventions and that, therefore, less is known about which interventions produce the greatest relative improvements in care. it was therefore recommended to organise additional studies to determine the effects and costs of individual intervention strategies. ideally, an economic evaluation may be contemplated in direct association with the results from an overview of clinical studies. typically, the economic analyst would take the respective point estimate of effect from the overview as a base case value and use the confidence interval as the relevant range for sensitivity analysis. with increased research efforts, this option may become realistic in the field of integrated care in a few years time. finally, an ad hoc synthesis of effectiveness data from several sources, including expert opinion, is regarded as justifiable when no well - controlled clinical studies have been performed. given the relative lack of evidence on the effectiveness of integrated care, this is a relevant approach when contemplating new economic appraisal studies. to start with, the range of costs to be included in a given evaluation will be closely related to the viewpoint or the perspective of the analysis. three categories of costs can be identified, health care costs, costs borne by the patients and their relatives, and costs in other sectors, such as costs in the social sector or costs associated with production losses, e.g. due to absence of work. table 2 provides examples of each of these types of costs and their relation to the choice of perspective for an analysis. in general, the identification of relevant categories of resource use is followed by measurement of the quantities of the resources used and their valuation in money terms. the health care resources consumed consist of the costs of organising and operating the programme. the identification of these costs often amounts to the listing of the ingredients of the programme both variable costs such as the time of health professionals or supplies, and fixed or overhead costs such as light heat, rent or capital costs. this means that when, for example, evaluating a rehabilitation programme of stroke patients in a hospital at home setting with attendance at a day centre compared to the standard alternative intervention (rehabilitation in a long stay hospital) it is necessary to take into account both the time of the various professionals involved, the time of secretaries and administrators who help run the service, the cost of food and drugs of stroke patients and a fraction of the capital cost of building the day centre and maintaining a transport service to it. but also when comparing transmural care with outpatient care, overhead costs may be important to consider. for example, in a study in the netherlands comparing the effects and costs of initiation of insulin therapy in type 2 diabetes patients in a transmural care setting and a secondary outpatient care setting, representing usual care, it was calculated that overhead costs contribute 24 and 7%, respectively, of total health care costs in the first year after treatment initiation (2000) report that in the uk typically between 25 and 35% of total hospital costs relate to overhead costs, 30% to 40% relate to specialty level costs (for example medical staffing), whilst the direct costs of ward staffing make up only 3040% of an inpatient bed day. common benefits of quite a few integrated care arrangements that may result in savings compared to usual care are a reduction of hospital stay or, perhaps less commonly, avoiding hospitalisation altogether. how should this be valued ? clearly, it is not satisfactory to take the daily cost of hospitalisation as an estimate of these savings, since the vacated beds will either be filled by other patients, or, if they remain empty, still incur a cost. therefore, the impact of integrated care programmes on the use of other resources needs to be assessed in the local setting, in the knowledge of managerial actions taken to redeploy resources. other authors have pointed to the fact that the possibility to redeploy resources may be dependent on the size of the integrated care programme. a hypothetical example to illustrate this issue, in economists ' terms relating to economies of scale of integrated care programmes, a small programme, taking 5 patients per week from the hospital, may lead to a release of resources for consumables ; at 10 patients per week some staff time may be released, and at 20 patients per week it would be possible to close a small ward. it is clear that, according to these authors, a threshold volume must be reached in order to achieve substitution. following this type of reasoning, the problem may be particularly acute where shared or integrated care programmes are drawing patients from more than one hospital. in this case such a programme needs to be of sufficient size to achieve these threshold volumes in each hospital. in other words, in considering to perform an economic evaluation the sheer size of the integrated care programme and its structuring may determine its potential cost - effectiveness, at least from a health care sector perspective. costs borne by the patient and his or her relatives usually include the costs of travel time, waiting time, and the costs of e.g. over - the - counter medication. a more complex issue is the valuation of resources invested by informal caregivers, who usually are more heavily burdened by integrated care programmes than by conventional care schemes, either by having to learn medical techniques or care for relatively sick relatives at home. a comprehensive introduction and a practical approach to these issues is provided by van busschbach and colleagues (1998), in a study providing an outline for a cost - effectiveness analysis of a drug for patients with alzheimer 's disease. the authors suggest a shadow - price method to value resource use of informal caregivers. firstly, all caregiving and supporting activities that an informal caregiver might perform should be listed with those activities that could be performed by formal caregivers or other professional aids. some activities (e.g. making a bed) will be valued at a lower hourly rate than others (specific caregiving). in this way there is debate about whether the costs of production losses should be included in an economic evaluation. some analysts argue that it introduces inequalities between those interventions that are aimed at individuals who could potentially return to productive activity and those that are not. other analysts state that inclusion of these costs follows straightforward from a societal perspective of analysis. it is therefore, recommended, when included in a study, to report these costs separately, allowing readers the opportunity to interpret study findings with and without taking these costs into account. in case of integrated care arrangements, inclusion of the costs of production losses may not be of paramount importance when evaluating arrangements for elderly patients, most of whom may be retired. the same consideration holds for arrangements aimed at children, who are not yet part of the workforce. however, in evaluating arrangements primarily aimed at working - age populations, inclusion of costs of production losses may be highly appropriate. two different methods are available for this purpose, with the human - capital method using lost earnings until retirement as a proxy for lost production, while the friction - cost method uses lost earnings until the time of replacement of the patient as a measure of lost production. which of these methods is most appropriate is still debated in the health economics literature. a bird in the hand is worth two in the bush. in health as well as in money terms, we value a benefit today more highly than we value a promise of the same benefit in 5 years ' time. therefore, when the costs or benefits of an intervention will occur at least after 1 year in the future, a technique called discounting is used to tackle time preferences. in evaluation of integrated care, discounting of costs and health effects is an important issue, as many of these arrangements are aimed at patients with a chronic condition. more formally, when discounting, the value of the costs and consequences that occur in 10 years are reduced, as they are multiplied with the factor 1/(1+r), where r is the chosen discount rate. for example, a cost of 5000 that has to be paid after 3 years, will with a discount rate of 5% have a present value of 4319, likewise, the present value of a health benefit of 250 qaly 's gained after 5 years, will with a 5% discount rate be the same as the gain of 196 qaly 's today. the exact choice of discount rate is the government recommended rate, usually between 3 and 6%, and a common rate found in the literature is 5% per year. it is also helpful to provide the undiscounted data to allow the reader to recalculate the results using any discount rate. without proper consideration of uncertainty in economic evaluations of integrated care arrangements it seems essential to include e.g. uncertainties surrounding the reduction in the length of stay in the sensitivity analysis, as it usually is (one of) the most important source(s) of potential cost - savings. for example, shepperd (1998), showed that for patients recovering from a hysterectomy, total health service costs were significantly higher for those allocated to hospital at home care compared to hospital care. when subjected to a sensitivity analysis, it was demonstrated that a one day reduction in the length of hospital stay of hospital at home care eliminated the cost difference between these settings, while a two day reduction altered the results so that hospital at home care became significantly less costly than hospital care. in other words, the results of this study were highly sensitive to uncertainties with regard to the length of stay, at least for the particular group of patients, which was reflected in the discussion of the findings. in another study, with a 3 month follow up, it was concluded that, from the combined viewpoint of the nhs and social services, the cost of hospital at home care were less than that of hospital care for medically stable elderly patients. the conclusion was only sensitive to uncertainty when assuming hospital costs to be less than 50% of those used in the initial analysis, indicating that the findings were relatively robust. these examples demonstrate the crucial role of performing a sensitivity analysis as part of an economic evaluation. of all other issues that may need to be addressed when undertaking and reporting an economic evaluation, the generalisability of the findings may be one of the most important. as noted earlier, it is essential, when considering the generalisability of a study, that the programmes and interventions have been comprehensively described. however, authors of studies evaluating new care structures have been criticised for not doing so, e.g. sowden. shared care is used as a black box which has a different content in different studies (). the authors continue : until we have clearer definitions of the key features of a programme of shared care, evaluations will be of limited use, not least because those aspects of shared care that might be important in influencing process and outcome will remain unclear. from a health economics perspective, it can be added that one of the essential features of a programme that need to be reported is related to its size (see earlier), as this co - determines its potential cost - effectiveness. to be categorised as a full economic evaluation, a study needs to examine both costs and effects of the alternative programmes to be compared. usual care, or, more specifically, care in the usual setting as comparator, which may be e.g. regular hospital care, nursing home care, or care provided in hospices [17, 18 ]. in some cases, it may be difficult to choose the most relevant comparator(s). in the netherlands, for example, seven categories of transmural care have been distinguished, of which some are aimed at a single patient group, e.g. diabetes patients. after making the correct choice of comparator, the alternatives need to be described comprehensively. this allows the reader the opportunity to assess whether the results can be transferred to a local setting. (1998), patients randomised to hospital at home care received early discharge with home based rehabilitative care between 08.30 and 11 pm provided by a team of two nurses, a physiotherapist, an occupational therapist and three support workers. the services provided were those for health care, with minimal essential domestic tasks performed, with a case load of 12 patients at any one time or less, with discharge (of hospital at home care) when patients could be managed by routinely available community services [19, 20 ]. health economists recommend to state a viewpoint for the analysis and to place the study in a decision - making context. possible viewpoints include those of the provider institution, the individual clinician or professional organisation, the patient, the purchaser of health care (or third party payer) or society as a whole. this is because data can usually be disaggregated and the analysis carried out from a number of viewpoints. also, the additional costs of adopting a broader perspective at the outset of the study is probably less than the cost of attempting to gather additional information later [9, 21 ]. as noticed earlier, this is relevant in case of integrated care. for example, in the economic evaluation carried out alongside a clinical trial by richards. [19, 20 ], it was attempted to include the viewpoints of both the nhs, social services, and patients. the viewpoints fitted with the aim of the study, evaluating the effectiveness and acceptability of an early discharge, hospital at home scheme and acute hospital care for medically stable elderly patients. it can be added that the relatively important role of informal care in a number of integrated care programmes supports the choice of a broad societal perspective. an example is provided by an arrangement in denmark, where an informal caregiver is allowed to stay home from paid work to care for a terminally ill family member. the quality of an economic evaluation is to a large extent determined by the quality of the evidence of effectiveness of health care programmes. the availability (and if available, the quality) of evidence of effectiveness is almost always a problem, and this goes as well for the field of integrated care. of the different designs that can be used to generate evidence on effectiveness, however, in an assessment of research done in the uk it was stated that few randomised controlled clinical trials of hospital at home services have been done, and that most of these have been small, with little attempt at economic evaluation. so when considering economic evaluation of integrated care programmes, perhaps three issues related to the quality of evidence on effectiveness need to be addressed : for which programmes does reliable evidence of effectiveness exist, and does this provide an adequate basis for economic evaluation?for which programmes are clinical trials being planned and is there any scope for undertaking economic analysis alongside these trials ? (see e.g., for more information on this issue)for which major applications of integrated care is there yet no reliable evidence of effectiveness and what efforts should be made to assemble such evidence ? for which programmes does reliable evidence of effectiveness exist, and does this provide an adequate basis for economic evaluation ? for which programmes are clinical trials being planned and is there any scope for undertaking economic analysis alongside these trials ? (see e.g., for more information on this issue) for which major applications of integrated care is there yet no reliable evidence of effectiveness and what efforts should be made to assemble such evidence ? with regard to these latter points, a meta - analysis of interventions used in disease management programmes for patients with chronic illnesses is illustrative. in this particular study, six interventions were distinguished : provider education (materials or instruction given to healthcare providers regarding appropriate care for patients with the condition targeted by the programme) ; provider feedback (information to health care providers regarding the specific care or results of care received or experienced by their patients) ; provider reminders (prompts given to providers to perform specific patient care tasks) ; patient education (materials and instructions issued to patients providing information on their condition and how it could be managed) ; patient reminders (prompts given to patients to perform specific tasks related to care for their condition) ; and patient financial incentives (payments (direct monetary payments, discounts or services) made to patients for achieving specific treatment related goals). it was concluded that, although these different interventions were associated with improvements in provider adherence to guidelines and patient disease control, the studies did not directly compare different interventions and that, therefore, less is known about which interventions produce the greatest relative improvements in care. it was therefore recommended to organise additional studies to determine the effects and costs of individual intervention strategies. ideally, an economic evaluation may be contemplated in direct association with the results from an overview of clinical studies. typically, the economic analyst would take the respective point estimate of effect from the overview as a base case value and use the confidence interval as the relevant range for sensitivity analysis. with increased research efforts, this option may become realistic in the field of integrated care in a few years time. finally, an ad hoc synthesis of effectiveness data from several sources, including expert opinion, is regarded as justifiable when no well - controlled clinical studies have been performed. given the relative lack of evidence on the effectiveness of integrated care, this is a relevant approach when contemplating new economic appraisal studies. to start with, the range of costs to be included in a given evaluation will be closely related to the viewpoint or the perspective of the analysis. three categories of costs can be identified, health care costs, costs borne by the patients and their relatives, and costs in other sectors, such as costs in the social sector or costs associated with production losses, e.g. due to absence of work. table 2 provides examples of each of these types of costs and their relation to the choice of perspective for an analysis. in general, the identification of relevant categories of resource use is followed by measurement of the quantities of the resources used and their valuation in money terms. the health care resources consumed consist of the costs of organising and operating the programme. the identification of these costs often amounts to the listing of the ingredients of the programme both variable costs such as the time of health professionals or supplies, and fixed or overhead costs such as light heat, rent or capital costs. this means that when, for example, evaluating a rehabilitation programme of stroke patients in a hospital at home setting with attendance at a day centre compared to the standard alternative intervention (rehabilitation in a long stay hospital) it is necessary to take into account both the time of the various professionals involved, the time of secretaries and administrators who help run the service, the cost of food and drugs of stroke patients and a fraction of the capital cost of building the day centre and maintaining a transport service to it. but also when comparing transmural care with outpatient care, overhead costs may be important to consider. for example, in a study in the netherlands comparing the effects and costs of initiation of insulin therapy in type 2 diabetes patients in a transmural care setting and a secondary outpatient care setting, representing usual care, it was calculated that overhead costs contribute 24 and 7%, respectively, of total health care costs in the first year after treatment initiation (2000) report that in the uk typically between 25 and 35% of total hospital costs relate to overhead costs, 30% to 40% relate to specialty level costs (for example medical staffing), whilst the direct costs of ward staffing make up only 3040% of an inpatient bed day. common benefits of quite a few integrated care arrangements that may result in savings compared to usual care are a reduction of hospital stay or, perhaps less commonly, avoiding hospitalisation altogether. how should this be valued ? clearly, it is not satisfactory to take the daily cost of hospitalisation as an estimate of these savings, since the vacated beds will either be filled by other patients, or, if they remain empty, still incur a cost. therefore, the impact of integrated care programmes on the use of other resources needs to be assessed in the local setting, in the knowledge of managerial actions taken to redeploy resources. other authors have pointed to the fact that the possibility to redeploy resources may be dependent on the size of the integrated care programme. a hypothetical example to illustrate this issue, in economists ' terms relating to economies of scale of integrated care programmes, is provided by coast.. a small programme, taking 5 patients per week from the hospital, may lead to a release of resources for consumables ; at 10 patients per week some staff time may be released, and at 20 patients per week it would be possible to close a small ward. it is clear that, according to these authors, a threshold volume must be reached in order to achieve substitution. following this type of reasoning, the problem may be particularly acute where shared or integrated care programmes are drawing patients from more than one hospital. in this case such a programme needs to be of sufficient size to achieve these threshold volumes in each hospital. in other words, in considering to perform an economic evaluation the sheer size of the integrated care programme and its structuring may determine its potential cost - effectiveness, at least from a health care sector perspective. costs borne by the patient and his or her relatives usually include the costs of travel time, waiting time, and the costs of e.g. over - the - counter medication. a more complex issue is the valuation of resources invested by informal caregivers, who usually are more heavily burdened by integrated care programmes than by conventional care schemes, either by having to learn medical techniques or care for relatively sick relatives at home. a comprehensive introduction and a practical approach to these issues is provided by van busschbach and colleagues (1998), in a study providing an outline for a cost - effectiveness analysis of a drug for patients with alzheimer 's disease. the authors suggest a shadow - price method to value resource use of informal caregivers. firstly, all caregiving and supporting activities that an informal caregiver might perform should be listed with those activities that could be performed by formal caregivers or other professional aids. the next step is to assign an hourly wage rate to each specific activity. some activities (e.g. making a bed) will be valued at a lower hourly rate than others (specific caregiving). in this way there is debate about whether the costs of production losses should be included in an economic evaluation. some analysts argue that it introduces inequalities between those interventions that are aimed at individuals who could potentially return to productive activity and those that are not. other analysts state that inclusion of these costs follows straightforward from a societal perspective of analysis. it is therefore, recommended, when included in a study, to report these costs separately, allowing readers the opportunity to interpret study findings with and without taking these costs into account. in case of integrated care arrangements, inclusion of the costs of production losses may not be of paramount importance when evaluating arrangements for elderly patients, most of whom may be retired. the same consideration holds for arrangements aimed at children, who are not yet part of the workforce. however, in evaluating arrangements primarily aimed at working - age populations, inclusion of costs of production losses may be highly appropriate. two different methods are available for this purpose, with the human - capital method using lost earnings until retirement as a proxy for lost production, while the friction - cost method uses lost earnings until the time of replacement of the patient as a measure of lost production. which of these methods is most appropriate is still debated in the health economics literature. a bird in the hand is worth two in the bush. in health as well as in money terms, we value a benefit today more highly than we value a promise of the same benefit in 5 years ' time. therefore, when the costs or benefits of an intervention will occur at least after 1 year in the future, a technique called discounting is used to tackle time preferences. in evaluation of integrated care, discounting of costs and health effects is an important issue, as many of these arrangements are aimed at patients with a chronic condition. more formally, when discounting, the value of the costs and consequences that occur in 10 years are reduced, as they are multiplied with the factor 1/(1+r), where r is the chosen discount rate. for example, a cost of 5000 that has to be paid after 3 years, will with a discount rate of 5% have a present value of 4319, likewise, the present value of a health benefit of 250 qaly 's gained after 5 years, will with a 5% discount rate be the same as the gain of 196 qaly 's today. the exact choice of discount rate is the government recommended rate, usually between 3 and 6%, and a common rate found in the literature is 5% per year. it is also helpful to provide the undiscounted data to allow the reader to recalculate the results using any discount rate. without proper consideration of uncertainty the reader may be unable to judge whether conclusions are meaningful and robust. in economic evaluations of integrated care arrangements it seems essential to include e.g. uncertainties surrounding the reduction in the length of stay in the sensitivity analysis, as it usually is (one of) the most important source(s) of potential cost - savings. for example, shepperd (1998), showed that for patients recovering from a hysterectomy, total health service costs were significantly higher for those allocated to hospital at home care compared to hospital care. when subjected to a sensitivity analysis, it was demonstrated that a one day reduction in the length of hospital stay of hospital at home care eliminated the cost difference between these settings, while a two day reduction altered the results so that hospital at home care became significantly less costly than hospital care. in other words, the results of this study were highly sensitive to uncertainties with regard to the length of stay, at least for the particular group of patients, which was reflected in the discussion of the findings. in another study, with a 3 month follow up, it was concluded that, from the combined viewpoint of the nhs and social services, the cost of hospital at home care were less than that of hospital care for medically stable elderly patients. the conclusion was only sensitive to uncertainty when assuming hospital costs to be less than 50% of those used in the initial analysis, indicating that the findings were relatively robust. these examples demonstrate the crucial role of performing a sensitivity analysis as part of an economic evaluation. of all other issues that may need to be addressed when undertaking and reporting an economic evaluation, the generalisability of the findings may be one of the most important. as noted earlier, it is essential, when considering the generalisability of a study, that the programmes and interventions have been comprehensively described. however, authors of studies evaluating new care structures have been criticised for not doing so, e.g. sowden. shared care is used as a black box which has a different content in different studies (). the authors continue : until we have clearer definitions of the key features of a programme of shared care, evaluations will be of limited use, not least because those aspects of shared care that might be important in influencing process and outcome will remain unclear. from a health economics perspective, it can be added that one of the essential features of a programme that need to be reported is related to its size (see earlier), as this co - determines its potential cost - effectiveness. economic evaluation of integrated care arrangements is a challenging and interesting area of research, offering many opportunities. guidance to realise these opportunities is provided by the general principles for economic evaluation, which can be tailored to suit the evaluation of different forms of integrated care. reasons may be the complexity of the intervention, the fact that evaluation of integrated care arrangements may require detailed data collection across the health care system, the necessity (at least in some arrangements) to take into account other sectors than the health sector as well, the inclusion of a substantial percentage of patients with co - morbidity, and the situation that integrated care programmes may cover multiple patient groups. therefore, on the one hand economic analysis in this field may be more complicated than in case of single technologies or interventions, but on the other hand the results of these studies may support decision making processes that affect the organisation of the health care system and the care processes in multiple patient groups at the same time. a major issue in evaluating integrated care is the choice of design of the study, as this co - determines the quality of the evidence of on effectiveness, which impacts on any subsequent analysis of the cost - effectiveness of the arrangement. although this article has provided a number of examples of studies designed as randomised controlled trials, several authors have pointed at inherent difficulties associated with this design in evaluating specific integrated care arrangements, suggesting comparative cohort studies and audit - type approaches instead, comparing (2002) evaluating costs and effects of a conventional discharge policy after hip fracture versus an early discharge policy in which patients were rehabilitated in a specialised nursing home. in explaining the design of the study, the authors state that randomisation of patients was not considered feasible since the change from conventional discharge to early discharge arrangements required such organisational adjustments that both service models could not be offered simultaneously. despite the methodological disadvantages of such studies, the findings may still be useful to support reimbursement decision making in a situation where, according to van der linden. (2001), most of the current transmural projects are experiencing difficulties in obtaining permanent funding after the conclusion of the experimental phase. a general issue of concern, however, is the demand - generation effect of integrated care programmes. since a considerable proportion of such programmes enable the patient to be treated at home while reducing the length of hospital stay, many categories of patients will prefer this option, and it can therefore be expected that the limits will be pushed of indications that are deemed eligible for care in such settings, which may lead to inefficiencies. it is therefore recommended to explore the cost - effectiveness of broadening the indications of selected integrated care programmes as part of the sensitivity analysis of an appraisal. this article has introduced and discussed some of the issues that need to be addressed when contemplating an economic evaluation of integrated care arrangements. from a societal perspective, assessment of potential savings due to reduced length of hospital stay in integrated care arrangements and issues of cost shifting, e.g. from the hospital to other health care institutions, and from health services to both social services and the patient and their relatives, deserve special attention. for readers who wish to be informed in more detail, finally, to guarantee the highest possible level of economic evaluation in this field, a skilled health economist should be consulted in the early phase of the design of the study. ideally, the health economist would be part of the research team from start to finish of the project. | abstractbackgroundintegrated care has emerged in a variety of forms in industrialised countries during the past decade. it is generally assumed that these new arrangements result in increased effectiveness and quality of care, while being cost - effective or even cost - saving at the same time. however, systematic evaluation, including an evaluation of the relative costs and benefits of these arrangements, has largely been lacking.objectivesto stimulate fruitful dialogue and debate about the need for economic evaluation in integrated care, and to outline possibilities for undertaking economic appraisal studies in this relatively new field.theorykey concepts, including e.g. scarcity and opportunity costs, are introduced, followed by a brief overview of the most common methods used in economic evaluation of health care programmes. then a number of issues that seem particularly relevant for economic evaluation of integrated care arrangements are addressed in more detail, illustrated with examples from the literature.conclusion and discussionthere is a need for well - designed economic evaluation studies of integrated care arrangements, in particular in order to support decision making on the long - term financing of these programmes. although relatively few studies have been done to date, the field is challenging from a methodological point of view, offering analysts a wealth of opportunities. guidance to realise these opportunities is provided by the general principles for economic evaluation, which can be tailored to the requirements of this particular field. |
current recommended treatment goals for chronic obstructive pulmonary disease (copd) include reduction of breathlessness, improvement of exercise capacity and health status, and prevention of exacerbations.1 as dyspnea is a predominant symptom of copd, an appreciation of impaired lung function and breathlessness severity may better reflect the impact of treatment.1 while treatment guidelines recommend a relatively simple approach to evaluating dyspnea (ie, modified medical research council scale1), more sophisticated tools exist to assess breathlessness in more detail.2,3 one example is the transition dyspnea index (tdi),4 a validated tool that assesses the impact of a patient s daily activities on breathlessness.5 it has been shown to reliably denote distinct changes in breathing difficulty due to therapeutic intervention.610 the phosphosdiesterase-4 inhibitor roflumilast, approved to reduce the risk of exacerbations in patients with severe copd associated with chronic bronchitis and a history of exacerbations, has been shown to reduce markers of inflammation in copd patient airways.11 as breathlessness is thought to arise primarily from mechanical limitation of inspiratory effort,12 we wondered whether an agent with potential anti - inflammatory activities like roflumilast would impact dyspnea. small but significant improvements in dyspnea, measured by the tdi, have been previously reported with 1 year of roflumilast treatment compared with placebo in two studies in copd patients;13 however, these data have not been examined in detail, nor was the sample size in previously reported studies large enough to explore relevant clinical associations. in addition, significant lung function improvements with roflumilast treatment have also been reported in placebo - controlled studies in patients with copd,1315 but have not yet been evaluated with regard to dyspnea - related outcomes. thus, to more comprehensively evaluate the impact of roflumilast on breathlessness, including any potential relationship with lung function improvement, individual and pooled analyses of four 1-year clinical studies with roflumilast are presented here. results from this analysis may have potential implications in the pharmacological management of copd with regard to the current recommended treatment goals of reduction of copd symptoms and prevention of exacerbations.1 data were pooled from four 1-year placebo - controlled, double - blind, multicenter, phase 3 clinical trials (m2 - 111, m2 - 112, m2 - 124, and m2 - 125) with once - daily roflumilast 500 g, which were designed to enroll patients with forced expiratory volume in 1 second (fev1), 50% predicted. patients in m2 - 124 and m2 - 125, but not m2 - 111 and m2 - 112, were required to have chronic bronchitis and a history of exacerbations. full details of methodology and patient selection have been reported elsewhere.1315 all four studies were approved by local ethical review committees and performed in accordance with the declaration of helsinki and good clinical practice guidelines. dyspnea was measured at baseline using the baseline dyspnea index (bdi) total score and during treatment (weeks 4, 8, 12, 20, 28, 36, 44, and 52) using the tdi focal score, which evaluates changes from the baseline measurement. bdi total score ranges from 0 (no dyspnea) to 12 (severe dyspnea) ; tdi focal score ranges from 9 (major deterioration) to 9 (major improvement). tdi responders were defined as patients who experienced a clinically meaningful improvement (change of 1 unit from baseline) in tdi focal score;5 tdi deteriorators were patients who experienced a 1 unit deterioration from baseline. outcomes assessed in tdi responders / nonresponders were tdi focal score at week 52, change from baseline to week 52 in postbronchodilator fev1, and percentage of patients achieving a clinically meaningful improvement in fev1 (100 ml)16 from baseline to week 52. data were pooled for all randomized patients who took 1 dose of study medication in each study. data analysis was performed post hoc for the overall patient population from the four studies, as well as for the following six subpopulations of patients with : 1) concomitant inhaled corticosteroid (ics) treatment (m2 - 111 and m2 - 112) ; 2) chronic bronchitis (m2 - 111, m2 - 112, m2 - 124, and m2 - 125) ; 3) chronic bronchitis and concomitant short - acting muscarinic antagonist (sama) treatment (m2 - 111, m2 - 112, m2 - 124, and m2 - 125) ; 4) chronic bronchitis and a history of exacerbations (m2 - 111, m2 - 124, and m2 - 125) ; 5) chronic bronchitis, a history of exacerbations, and concomitant long - acting 2-agonists (laba) treatment (m2 - 124 and m2 - 125) ; and 6) chronic bronchitis, a history of exacerbations, and ics pretreatment (m2 - 124 and m2 - 125). patients included in the subpopulation analyses were required to have non - missing baseline bdi and post - baseline tdi assessments. treatment comparisons on continuous variables were performed at week 52 (last observation carried forward [locf ]) using analysis of covariance with terms for treatment group, bdi, age, smoking status, sex, ics pretreatment, country / region, and study. treatment comparisons on dichotomous / categorical variables were performed using a cochran mantel haenszel test (pooled data were stratified by study) at week 52 (locf) and weeks 4, 8, 12, 20, 28, 36, 44, and 52 (observed cases). data were pooled from four 1-year placebo - controlled, double - blind, multicenter, phase 3 clinical trials (m2 - 111, m2 - 112, m2 - 124, and m2 - 125) with once - daily roflumilast 500 g, which were designed to enroll patients with forced expiratory volume in 1 second (fev1), 50% predicted. patients in m2 - 124 and m2 - 125, but not m2 - 111 and m2 - 112, were required to have chronic bronchitis and a history of exacerbations. full details of methodology and patient selection have been reported elsewhere.1315 all four studies were approved by local ethical review committees and performed in accordance with the declaration of helsinki and good clinical practice guidelines. dyspnea was measured at baseline using the baseline dyspnea index (bdi) total score and during treatment (weeks 4, 8, 12, 20, 28, 36, 44, and 52) using the tdi focal score, which evaluates changes from the baseline measurement. bdi total score ranges from 0 (no dyspnea) to 12 (severe dyspnea) ; tdi focal score ranges from 9 (major deterioration) to 9 (major improvement). tdi responders were defined as patients who experienced a clinically meaningful improvement (change of 1 unit from baseline) in tdi focal score;5 tdi deteriorators were patients who experienced a 1 unit deterioration from baseline. outcomes assessed in tdi responders / nonresponders were tdi focal score at week 52, change from baseline to week 52 in postbronchodilator fev1, and percentage of patients achieving a clinically meaningful improvement in fev1 (100 ml)16 from baseline to week 52. data were pooled for all randomized patients who took 1 dose of study medication in each study. data analysis was performed post hoc for the overall patient population from the four studies, as well as for the following six subpopulations of patients with : 1) concomitant inhaled corticosteroid (ics) treatment (m2 - 111 and m2 - 112) ; 2) chronic bronchitis (m2 - 111, m2 - 112, m2 - 124, and m2 - 125) ; 3) chronic bronchitis and concomitant short - acting muscarinic antagonist (sama) treatment (m2 - 111, m2 - 112, m2 - 124, and m2 - 125) ; 4) chronic bronchitis and a history of exacerbations (m2 - 111, m2 - 124, and m2 - 125) ; 5) chronic bronchitis, a history of exacerbations, and concomitant long - acting 2-agonists (laba) treatment (m2 - 124 and m2 - 125) ; and 6) chronic bronchitis, a history of exacerbations, and ics pretreatment (m2 - 124 and m2 - 125). patients included in the subpopulation analyses were required to have non - missing baseline bdi and post - baseline tdi assessments. treatment comparisons on continuous variables were performed at week 52 (last observation carried forward [locf ]) using analysis of covariance with terms for treatment group, bdi, age, smoking status, sex, ics pretreatment, country / region, and study. treatment comparisons on dichotomous / categorical variables were performed using a cochran mantel haenszel test (pooled data were stratified by study) at week 52 (locf) and weeks 4, 8, 12, 20, 28, 36, 44, and 52 (observed cases). in m2 - 124 and m2 - 125, the proportion of patients pretreated with ics was equal in both the roflumilast and placebo groups (42%) ; the proportions of patients who received concomitant ics or laba were also similar in both roflumilast (21% and 49%, respectively) and placebo (23% and 51%, respectively) groups. similarly, in m2 - 111 and m2 - 112, the proportions of patients who received concomitant ics or laba were comparable in both roflumilast (61% and 8%, respectively) and placebo (60% and 9%, respectively) groups. for the overall pooled population, patient demographic and clinical characteristics were similar between treatment arms (table 1). of randomized patients who took 1 dose of study medication, 1,921/2,864 (67.1%) and 2,083/2,913 (71.5%) patients completed the trials in the roflumilast and placebo groups, respectively. at week 52, roflumilast significantly improved mean tdi focal score versus placebo (roflumilast, 0.368 ; placebo, 0.041 ; difference [95% confidence interval { ci } ], 0.327 [0.1660.488 ] ; p<0.0001). between - group differences in tdi focal score for the pooled population were similar to those in the individual studies (figure 1a) and generally favored roflumilast. significantly higher percentages of roflumilast - treated patients achieved clinically meaningful improvements in tdi focal score (tdi responders) versus placebo after 52 weeks of treatment (locf ; 39.0% versus 33.9%, respectively ; p=0.0001) (figure 2a). furthermore, the significantly higher percentage of tdi responders observed at week 52 was apparent by week 8 and maintained until study end (all, p<0.01) (figure 2a). at week 52, roflumilast - treated patients were more likely to demonstrate clinically meaningful improvements in tdi than placebo - treated patients (figure 1b) ; generally similar results were observed in the individual trials. values of i for both analyses in figure 1 were 0, indicating lack of observed statistical heterogeneity.17 overall, 20 patients need to be treated with roflumilast to have one additional patient with clinically meaningful improvements in tdi focal score over 1 year (crude number needed to treat, 19.9 ; 95% ci, 13.340.0). conversely, roflumilast treatment was associated with significantly fewer tdi deteriorators versus placebo after 52 weeks (locf ; 20.4% versus 23.7%, respectively ; p=0.0022) (figure 2b). this significant between - group difference was also apparent by week 8 and maintained for the remainder of the study (all, p<0.05). to explore the potential association of breathlessness with improved lung function, clinically meaningful improvements in tdi focal score and fev1 were evaluated. in the overall population, the proportion of patients who achieved the minimum clinically important difference (mcid) of 100 ml in fev1 (ie, fev1 responders) was significantly greater with roflumilast versus placebo by week 4, which was maintained until study end (all, p<0.0001) (figure 3). among tdi responders, roflumilast led to significantly higher percentages of patients who achieved mcid in fev1 (36%) versus placebo (29% ; p<0.01) at study end. furthermore, the magnitude of fev1 improvement was significantly greater with roflumilast versus placebo in both tdi responders (p<0.05) and nonresponders (p<0.0001) (figure 4). consistent with this observed treatment effect in the magnitude of fev1 improvement with roflumilast in both tdi responders and nonresponders, a greater proportion of patients treated with roflumilast versus placebo demonstrated improvements from baseline in fev1, a treatment effect (ie, shift to the right in favor of roflumilast) also observed in both tdi responders and nonresponders (figure 5). in addition, as the roflumilast curve is generally above that of placebo across the positive x - axis in both tdi responders and nonresponders, there were also a greater percentage of roflumilast - treated patients than placebo - treated patients for most levels of positive fev1 change from baseline. further analysis of the pooled itt population revealed that particular baseline characteristics were associated with a significantly greater percentage of tdi responders (table 2). these characteristics included being a former smoker (versus current), no concomitant use of labas or samas (versus with), and no concomitant or pre - ics use (versus with). to further analyze whether patient characteristics such as chronic bronchitis, history of exacerbations, or concomitant medications had an effect on tdi response, six subpopulations were examined (figure 6). in copd patients with chronic bronchitis, roflumilast significantly improved tdi focal scores versus placebo (p<0.05) regardless of exacerbation history, concomitant treatment with sama or laba, or pretreatment with ics (table 3). in the patient subpopulation treated with concomitant ics, roflumilast was associated with significantly less deterioration in tdi focal score versus placebo (p=0.0114). tdi responders and deteriorators for each subpopulation are presented in figures s1s6. in all subpopulations, treatment with roflumilast was associated with more tdi responders and fewer tdi deteriorators versus placebo at all treatment weeks ; however, differences were not statistically significant at all treatment weeks. as breathlessness in copd patients worsens gradually over time and is related to lung function, physical activity, and quality of life, dyspnea has been considered a surrogate marker for disease progression.18 moderate improvements in breathlessness with roflumilast have previously been reported in patients with severe to very severe copd.13 to further characterize the effect of roflumilast on dyspnea, data from four 1-year roflumilast studies were pooled for those patients with or without clinically meaningful responses in tdi. in addition, evaluation of the corresponding fev1 improvements among tdi responders explored the relationship between breathlessness and lung function. the results from the current analysis confirm previous findings that roflumilast significantly improves breathlessness compared with placebo, as measured by tdi, in patients with severe to very severe copd.13 furthermore, these results show that a significantly greater percentage of roflumilast - treated patients (40%) experienced clinically meaningful improvements in tdi focal score versus placebo (36%) starting at week 8 (p0.001) ; this 4% between - group difference was maintained to 1 year (locf ; week 52 ; roflumilast, 39% ; placebo, 34% ; p<0.01 for all). conversely, significantly fewer roflumilast - treated patients experienced clinically meaningful deteriorations in dyspnea compared with placebo - treated patients. in patients with copd, improvements in lung function have been reported to occur with decreased breathlessness.1921 patients with copd in several 1-year studies of various inhaled bronchodilators exhibited improvements in trough fev1 over placebo (range, 108160 ml) at study endpoint, which were accompanied by improvements in tdi focal scores (range, 0.61.1 units).2224 as roflumilast is an anti - inflammatory agent, and its effects on lung function in the individual studies pooled here (ie, between - treatment change in prebronchodilator fev1 range, 3658 ml)1315 are lower than those seen for bronchodilators, it is not surprising that the effect of roflumilast on dyspnea reported in this pooled analysis (0.327 units) is lower in magnitude versus those of bronchodilators. however, consistent with studies demonstrating correlations between dyspnea and lung function improvements,19,21,25 the present analysis demonstrates that roflumilast - treated patients with clinically meaningful improvements in dyspnea (tdi responders) had a significantly greater magnitude of improvement in fev1 and were more likely to have a clinically meaningful improvement in lung function compared with placebo - treated patients. furthermore, the present analysis is consistent with a previous study that reported significant lung function improvements occurring earlier than significant reductions in breathlessness.26 in the individual 1-year roflumilast studies, small but significant treatment - associated improvements in lung function were demonstrated as early as week 4.14,27 in the current analysis, significantly more patients achieved the fev1 mcid with roflumilast treatment versus placebo as early as week 4 and at every study visit thereafter. in comparison, more roflumilast - treated patients achieved the tdi focal score mcid at week 4 compared with placebo ; however, this difference did not reach significance until week 8, at which point it was maintained until study end. together, these time courses suggest that significant lung function improvements may be observed earlier than significant dyspnea improvements. the mechanisms by which roflumilast may lead to improved airflow are not fully defined, but an anti - inflammatory effect has been suggested.11,28 similar to results in the overall population, improvements in tdi focal scores at study end were small but consistently significant over placebo in patients with chronic bronchitis, regardless of exacerbation history, concomitant sama or laba treatment, or ics pretreatment. although greater proportions of tdi responders were observed with roflumilast versus placebo in the subgroups of patients receiving concomitant ics, laba, or sama treatment throughout the treatment period, these differences were not significant at every study visit. in contrast, significantly greater percentages of tdi responders were consistently observed with roflumilast versus placebo from treatment weeks 8 through 52 in patients with chronic bronchitis, a history of exacerbations, and ics pretreatment. consistent with the results presented here, a 6-month placebo - controlled study examining the effects of roflumilast in patients with moderate to severe copd and chronic bronchitis who were already being treated with tiotropium demonstrated that significantly more roflumilast+tiotropium patients achieved clinically meaningful improvements in tdi focal score compared with placebo+tiotropium patients,29 with a between - group difference (0.4 units) that was generally similar to that reported in the current analysis. in addition, compared with placebo, significantly more patients receiving roflumilast+tiotropium in the 6-month study29 achieved clinically meaningful differences in an instrument that assessed dyspnea associated with specific activities of daily living, the university of california, san diego shortness of breath questionnaire (sobq),30 further demonstrating the positive impact of roflumilast treatment on breathlessness. numerical improvements in tdi focal scores were also observed with the addition of roflumilast to salmeterol in a separate 6-month study in patients with moderate to severe copd ; however, these differences were not significantly different from patients treated with salmeterol and placebo (treatment difference of 0.1 unit).31 it is of note that the inclusion criteria for the roflumilast+tiotropium trial (ie, chronic cough and sputum production and frequent use of short - acting 2-agonists during the run - in period while being treated with tiotropium 3 months prior to study enrollment) led to the recruitment of more symptomatic patients compared with patients recruited in the roflumilast+salmeterol trial. as such, the variation in the magnitude of treatment effect of roflumilast on dyspnea in tiotropium - treated patients versus salmeterol - treated patients may have been due to these differences in patients baseline characteristics between the two studies. inclusion of both of these 6-month roflumilast trials in a recent meta - analysis by pan demonstrated a moderate but significant increase in tdi focal scores with roflumilast (weighted mean difference of 0.30 units ; 95% ci, 0.140.46), similar to what was reported in the current analysis that focused on 1-year studies alone. increased dyspnea is strongly associated with reduced quality of life in copd patients.33,34 in m2 - 111, the st george s respiratory questionnaire (sgrq),35 a disease - specific quality of life instrument that assesses causes and impacts of dyspnea, was used to evaluate health status. compared with placebo at week 52, roflumilast provided moderate but significant improvements in sgrq total score, and a significantly greater percentage of patients achieved a clinically meaningful improvement in sgrq with roflumilast (p<0.05 for both).36 furthermore, pooled data in patients with chronic bronchitis and/or emphysema (m2 - 111 and m2 - 112) demonstrated a significant difference in sgrq total score improvement versus placebo after 52 weeks of treatment.15 this suggests that roflumilast may similarly provide clinically meaningful improvements in quality of life in some copd patients, possibly due to a reduction of breathlessness. were performed post hoc, and the included component studies were not designed to detect clinically meaningful improvements in dyspnea, these comparisons should be interpreted with caution and used primarily to generate hypotheses for future studies. findings may not be generalizable to all patients with copd, as inclusion criteria for each individual study contributing to this pooled analysis may have excluded patients typically seen in clinical practice. the use of locf in the current analysis may not have taken into account missing data that were nonrandom (ie, treatment - related discontinuations). the average placebo - adjusted improvements in dyspnea observed in the analyses presented here were small in magnitude ; however, patients who improved their tdi focal score by 1 unit were observed with roflumilast treatment throughout the studies, suggesting that some patients treated with roflumilast may experience clinically meaningful improvements in breathlessness. since worsened breathlessness has been associated with an increased risk for copd exacerbations,37 and the effects of roflumilast on dyspnea may be related to a reduction in exacerbation rates, it is possible that longer studies in which more exacerbations would be prevented with roflumilast may enable observation of greater magnitudes of dyspnea improvement. as two of the four studies included in this pooled analysis had a population enriched with patients with a history of exacerbations, it is unclear whether copd patients with chronic bronchitis who are frequent exacerbators would experience greater improvements in breathlessness compared with non - exacerbators. finally, this analysis was based on monotherapy trials that limited concomitant use of medications commonly used to treat copd, such as fixed - dose ics / laba, long - acting muscarinic antagonist, or theophylline. while data derived from subgroups of patients who were pretreated with ics or who received concomitant sama, laba, or ics treatment during the studies can be suggestive of potential treatment combinations, this may not be directly applicable to patients who are simultaneously prescribed multiple medications to manage their copd.38 in conclusion, these results further explore the effects of roflumilast on breathlessness, demonstrating small but generally consistent improvements in dyspnea with roflumilast treatment over placebo. patients being treated with roflumilast to reduce their risk of exacerbations may also experience reduced dyspnea, regardless of patient characteristics such as chronic bronchitis, history of exacerbations, or previous (ics) or concomitant (sama, laba, or ics) treatment. the added benefit of reducing breathlessness with roflumilast treatment, although small in magnitude, may provide an added value to consider in the use of this phosphodiesterase-4 inhibitor in the management of copd. tdi responders (a) and deteriorators (b) over time for the subpopulation of patients with chronic bronchitis. notes : p<0.05 ; p0.01 ; p0.001 versus placebo. abbreviations : locf, last observation carried forward ; n, number of responders ; n, number of patients analyzed ; tdi, transition dyspnea index. tdi responders (a) and deteriorators (b) over time for the subpopulation of patients with chronic bronchitis and a history of exacerbations. notes : p<0.05 ; p0.01 ; p0.001 versus placebo. abbreviations : locf, last observation carried forward ; n, number of responders ; n, number of patients analyzed ; tdi, transition dyspnea index. tdi responders (a) and deteriorators (b) over time for the subpopulation of patients with chronic bronchitis, a history of exacerbations, and pretreatment with inhaled corticosteroids abbreviations : locf, last observation carried forward ; n, number of responders ; n, number of patients analyzed ; tdi, transition dyspnea index. tdi responders (a) and deteriorators (b) over time for the subpopulation of patients with chronic bronchitis, a history of exacerbations, and concomitant long - acting 2-agonist treatment. notes : p<0.05 ; p0.01 ; p0.001 versus placebo. abbreviations : locf, last observation carried forward ; n, number of responders ; n, number of patients analyzed ; tdi, transition dyspnea index. tdi responders (a) and deteriorators (b) over time for the subpopulation of patients with chronic bronchitis and concomitant short - acting muscarinic antagonist treatment. abbreviations : locf, last observation carried forward ; n, number of responders ; n, number of patients analyzed ; tdi, transition dyspnea index. tdi responders (a) and deteriorators (b) over time for the subpopulation of patients with concomitant inhaled corticosteroid treatment. notes : p<0.05 ; p0.01 ; p0.001 versus placebo. abbreviations : locf, last observation carried forward ; n, number of responders ; n, number of patients analyzed ; tdi, transition dyspnea index. | purposebreathlessness is a predominant symptom of chronic obstructive pulmonary disease (copd), making it a valuable outcome in addition to lung function to assess treatment benefit. the phosphodiesterase-4 inhibitor roflumilast has been shown to provide small but significant improvements in dyspnea, as measured by the transition dyspnea index (tdi), in two 1-year studies in patients with severe to very severe copd.patients and methodsto provide a more comprehensive assessment of the impact of roflumilast on dyspnea, post hoc analyses of four 1-year roflumilast studies (m2 - 111, m2 - 112, m2 - 124, and m2 - 125) in patients with moderate to very severe copd were conducted.resultsin this pooled analysis (n=5,595), roflumilast significantly improved tdi focal scores versus placebo at week 52 (treatment difference, 0.327 ; p<0.0001). roflumilast was associated with significantly greater tdi responders and significantly fewer tdi deteriorators (1-unit increase or decrease from baseline, respectively) versus placebo at week 52 (p<0.01, both) ; these significant differences were apparent by week 8 and maintained until study end (p<0.05, all). at study end, the postbronchodilator forced expiratory volume in 1 second improvement in tdi responders was significantly greater with roflumilast versus placebo (p<0.05). similar to the overall population, improvements in tdi focal scores at week 52 were small but consistently significant over placebo in patients with chronic bronchitis, regardless of exacerbation history, concomitant treatment with short - acting muscarinic antagonists or long - acting 2-agonists, or pretreatment with inhaled corticosteroids.conclusionthis analysis shows that patients treated with roflumilast to reduce exacerbation risk may also experience small but significant improvements in dyspnea, with accompanying improvements in lung function. |
hernia of morgagni was first described by giovanni battista morgagni, an italian anatomist and pathologist in 1769. it is a rare cause of diaphragmatic hernia usually presenting in children. the defect involves failure of fusion of the septum transversus, the diaphragm and the costal arches. even though the resulting space is often congenital, conditions such as pregnancy, trauma, chronic cough, obesity and constipation, all of which may increase intraabdominal pressure predisposes the condition [3, 4 ]. usually asymptomatic patients may have symptoms such as abdominal discomfort, bloating, vomiting and bowel obstruction. morgagni hernia is an uncommon cause of gastric outlet obstruction with very few case reports in the literature. an 80-year - old man presented to our emergency room with multiple episodes of non - bilious vomiting for 4 days and not - passing stool or flatus for 2 days. he also complained of intermittent pain in the upper abdomen, which was not associated with fever, chest pain, melena or diarrhea in the recent past. however, he complained of increased cough and shortness of breath for the last 4 days on the background of a diagnosed chronic obstructive pulmonary disease. his vital parameters were normal, other than an oxygen saturation of 82% for which oxygen had to be given via face mask. laboratory investigations revealed hypokalemia (3.1 meq / l) with other parameters being normal. on the chest x - ray 1). computed tomography (ct) scan of the abdomen performed demonstrated a right anterior diaphragmatic hernia with stomach as content. (figs 2 and 3) the patient was taken to the operating room, and an upper midline laparotomy was performed. findings included an ~4 3 cm defect to the right of the sternum in the anterior diaphragm (fig. 4), with incarcerated pylorus / antrum of stomach as contents and a grossly distended stomach. the contents were reduced, and the defect closed primarily with 2 - 0 non - absorbable interrupted mattress sutures (fig. 5). postoperative period was uneventful, and chest x - ray revealed a corrected defect with reduced contents (fig. the patient was started orally on the first postoperative day and discharged on the sixth postoperative day. morgagni hernia is the rarest of all congenital diaphragmatic hernias with an incidence of ~3%. it occurs through the foramina of morgagni lying between the sternal and the costal margins of the thoracic diaphragm and was first described in 1769. although the hernias are congenital, they rarely present in children and usually are incidentally found on routine radiological imaging in adults. symptoms if present are often nonspecific, including abdominal discomfort, bloating, vomiting and bowel obstruction. emergency presentation in such patients are commonly acute respiratory distress, gastric outlet obstruction or strangulation / ischemia of entrapped bowel [5, 6 ], with an incidence of 1214%. diagnosis is usually suggested by chest x - rays indicating an air fluid level in the chest with barium studies revealing contrast in the stomach / bowel herniating through the diaphragm. ct scan of the chest and diaphragm are highly accurate and help proceed with surgical management. surgical management via laparotomy is the most common surgical approach with advantages of easier reduction of hernial contents, inspection of the contralateral diaphragm for other hernia defects and easier evaluation and surgical treatment of the acute complications, such as strangulation and ischemia of luminal abdominal organs. the laparoscopic approach has the benefits of less postoperative pain and earlier return to activities and work but is less suited to emergency presentations where surgical resection or treatment of necrotic or ischemic contents may be required [3, 9 ]. morgagni hernia is an exceedingly rare hernia in adults and may present with gastric outlet obstruction in the emergency room. this clinical entity should be kept in mind while evaluating the patient, and early surgical intervention should be initiated. | morgagni hernia is the rarest of all congenital diaphragmatic hernias, first described in 1769. it is rarely symptomatic and found on routine radiological examinations for other conditions. gastric outlet obstruction in adults with morgagni hernia is exceedingly rare. an 80-year - old man was taken to the operating room with a diagnosis of morgagni hernia with gastric outlet obstruction. an upper midline laparotomy was performed, and the incarcerated pylorus and antrum of the stomach reduced with primary closure of the defect. postoperative period was uneventful, and the patient was discharged on the sixth postoperative day. morgagni hernia is exceedingly rare in adults and may present with gastric outlet obstruction in the emergency room. this clinical entity should be kept in mind while evaluating the patient, and early surgical intervention should be initiated. |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.