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many environmental chemicals and pesticides have been found to be estrogenic and have been shown to stimulate the growth of estrogen receptor - positive (er - positive) human breast cancer cells. since it is difficult to avoid human exposure to environmental estrogens, a potentially important area of research is the development of dietary strategies to prevent the stimulated growth of breast tumors by environmental estrogens. in this context, the inhibitory action of curcumin and a combination of curcumin and isoflavonoids were studied in er - positive human breast cancer cells (mcf-7 and t47d) and er - negative mda - mb-231 cells induced by the pesticide o, p'-ddt and the environmental pollutants 4-nonylphenol and 4-octylphenol. the median inhibitory concentration (ic50) for curcumin in t47d cells was 10 microm when measured at either a 48-hr or a 6-day incubation time. the ic50 value for curcumin was within the 8 - 10 microm range for inhibiting the growth of t47d cells induced by a 10- microm concentration each of 4-nonylphenol, 4-octylphenol, and o, p'-ddt. the ic50 for curcumin in mcf-7 cells induced by 10 microm of either o, p'-ddt, 4-octylphenol, or 4-nonylphenol were 9, 39, and > 50 microm, respectively. a combination of curcumin and isoflavonoids was able to inhibit the induced growth of er - positive cells up to 95%. for mda - mb-231 cells, the ic50 for curcumin was 17 microm, which was reduced to 11 microm in the presence of 25 microm genistein. curcumin and genistein induce drastic changes in the morphological shape of both er - positive and er - negative cells. data presented here indicate that a mixture of curcumin and isoflavonoids is the most potent inhibitor against the growth of human breast tumor cells. these data suggest that combinations of natural plant compounds may have preventive and therapeutic applications against the growth of breast tumors induced by environmental estrogens.imagesfigure 1figure 2figure 3figure 4figure 5figure 6 |
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normal tension glaucoma (ntg) is an optic neuropathy associated with glaucomatous optic nerve head damage, progressive retinal nerve fiber layer thinning, characteristic visual field defects, open anterior chamber angles on gonioscopy and maximum intraocular pressure (iop) below 21 mmhg. glaucoma is a major optic neuropathy characterized by significant death of retinal ganglion cells. according to global surveys, the second leading cause of blindness after cataracts is glaucoma. glaucoma affects more than 66 million people and is the second leading cause of visual loss worldwide. ntg accounted for 92% of primary open - angle glaucoma (poag) in a japanese population. patients with ntg tend to be older than those with primary open angle glaucoma (poag)female subjects have a higher prevalence of the disease than male counterpartsntg is more frequent among japanese peoplethe most important risk factor for glaucoma is elevated iop. it has been established that high iop is a part of the pathogenic process of ntg ; however, the pressure theory can not sufficiently explain how ntg causes loss of vision. multicenter clinical trials have confirmed the value of reducing iop both in poag and ntg. there is yet to be a consensus regarding the specific relationship between iop and ntg. however, in many cases the progression of gon has been observed even after lowering iop. patients with ntg have wider diurnal fluctuations of iop as compared to the healthy population. iop spikes may occur at night, and thus iops measured during office hours may miss nocturnal spikes in many patients. associated changes in nocturnal orbital blood pressure and iop may affect optic nerve head blood perfusion differently in glaucomatous eyes as compared to healthy eyes, and this issue may also influence the susceptibility of the optic nerve to damagecentral corneal thickness (cct) in patients with ntg is lower than poag subjectsvascular dysfunction and ischemia have been considered as important factors in the progression of ntg. however, the ischemia in glaucoma is not simply insufficiency of blood flow, but also due to improper vascular autoregulation, and hypothetically episodes of transient ischemia and re - perfusion injury occurcold hands and feet, as an over - reaction to cold or stress, are suggestive of defective vasoregulation. abnormal vasoregulation such as raynaud 's phenomenon and migraine are more associated with ntg than poag. a study of peripheral vascular endothelial function in patients with ntg found impaired acetylcholine - induced peripheral endothelium - mediated vasodilation in comparison to healthy age- and sex - matched control subjects, and polymorphisms of the endothelin receptor type a gene have been associated with ntgpatients with ntg have an increased frequency of headaches with or without migraine featuresnon - iop - related cardiovascular dysregulation factors, such as systemic hypertension, systemic hypotension, nocturnal hypotension, and cardiac arrhythmia are implicated in ntgthere are reports suggesting that signs of glaucoma in certain eyes may be related to an acute ischemic episode (shock - induced neuropathy), or chronic obstructive arterial disease, which may be non - progressivemojon suggested that patients with sleep apnea syndrome are at high risk for glaucoma. the prevalence of obstructive sleep apnea syndrome (osas) was higher in patients with ntg. osas may cause optic nerve head hypoperfusion and glaucomatous optic neuropathy by creating transient hypoxemia and increasing vascular resistanceabnormally low diastolic double product (ddp = diastolic blood pressure heart rate) may represent a state of cardiovascular autonomic dysregulation, resulting in low ocular perfusion in certain ntg patientsntg patients with lower heart - rate variability, which reflects autonomic dysfunction with sympathetic predominance, manifested a faster rate of central visual field progression as compared to patients with higher heart - rate variabilityglaucoma patients have a decrease in central retinal vein (crv) blood velocities. this is particularly important in ntg patientsduplication of the tank - binding kinase 1 (tbk1) gene can be a rare cause of ntglifestyle factors such as smoking and high body mass index can cause progression of glaucomatous visual field defect. in the blue mountain eye study, smokers were found to have higher iop than non - smoker counterpartsout of the metabolic syndrome components, hypertension and impaired glucose tolerance (igt) may contribute to an increased risk of ntg. patients with ntg tend to be older than those with primary open angle glaucoma (poag) female subjects have a higher prevalence of the disease than male counterparts ntg is more frequent among japanese people the most important risk factor for glaucoma is elevated iop. it has been established that high iop is a part of the pathogenic process of ntg ; however, the pressure theory can not sufficiently explain how ntg causes loss of vision. multicenter clinical trials have confirmed the value of reducing iop both in poag and ntg. there is yet to be a consensus regarding the specific relationship between iop and ntg. however, in many cases the progression of gon has been observed even after lowering iop. patients with ntg have wider diurnal fluctuations of iop as compared to the healthy population. iop spikes may occur at night, and thus iops measured during office hours may miss nocturnal spikes in many patients. associated changes in nocturnal orbital blood pressure and iop may affect optic nerve head blood perfusion differently in glaucomatous eyes as compared to healthy eyes, and this issue may also influence the susceptibility of the optic nerve to damage central corneal thickness (cct) in patients with ntg is lower than poag subjects vascular dysfunction and ischemia have been considered as important factors in the progression of ntg. however, the ischemia in glaucoma is not simply insufficiency of blood flow, but also due to improper vascular autoregulation, and hypothetically episodes of transient ischemia and re - perfusion injury occur cold hands and feet, as an over - reaction to cold or stress, are suggestive of defective vasoregulation. abnormal vasoregulation such as raynaud 's phenomenon and migraine are more associated with ntg than poag. a study of peripheral vascular endothelial function in patients with ntg found impaired acetylcholine - induced peripheral endothelium - mediated vasodilation in comparison to healthy age- and sex - matched control subjects, and polymorphisms of the endothelin receptor type a gene have been associated with ntg patients with ntg have an increased frequency of headaches with or without migraine features non - iop - related cardiovascular dysregulation factors, such as systemic hypertension, systemic hypotension, nocturnal hypotension, and cardiac arrhythmia are implicated in ntg there are reports suggesting that signs of glaucoma in certain eyes may be related to an acute ischemic episode (shock - induced neuropathy), or chronic obstructive arterial disease, which may be non - progressive mojon suggested that patients with sleep apnea syndrome are at high risk for glaucoma. the prevalence of obstructive sleep apnea syndrome (osas) was higher in patients with ntg. osas may cause optic nerve head hypoperfusion and glaucomatous optic neuropathy by creating transient hypoxemia and increasing vascular resistance abnormally low diastolic double product (ddp = diastolic blood pressure heart rate) may represent a state of cardiovascular autonomic dysregulation, resulting in low ocular perfusion in certain ntg patients ntg patients with lower heart - rate variability, which reflects autonomic dysfunction with sympathetic predominance, manifested a faster rate of central visual field progression as compared to patients with higher heart - rate variability glaucoma patients have a decrease in central retinal vein (crv) blood velocities. this is particularly important in ntg patients duplication of the tank - binding kinase 1 (tbk1) gene can be a rare cause of ntg lifestyle factors such as smoking and high body mass index can cause progression of glaucomatous visual field defect. in the blue mountain eye study, smokers were found to have higher iop than non - smoker counterparts out of the metabolic syndrome components, hypertension and impaired glucose tolerance (igt) may contribute to an increased risk of ntg. history has to be taken regarding migraine, raynaud 's phenomenon, episodes of shock, head injury, headache and other neurological symptoms. use of medications including systemic steroids and antihypertensive agents such as beta blockers should also be taken into account. goldmann applanation tonometry, gonioscopy, stereoscopic biomicroscopy of the optic nerve head, optical coherence tomography and humphrey field analyser are the main tools of investigation for diagnosis of ntg. increased cup to disc ratio or asymmetry of cupping between the two eyes (difference more than 0.2) is significant [figure 1 ]. a region of absent retinal pigment epithelium (rpe) is more often seen as a crescent or halo at the disc border in ntg. the cupping is often worse in the region of absent rpe, with field loss more marked in the corresponding region. occasionally, there is a notch due to thin or absent neuro - retinal rim, referred as a this is associated with a highly localized dense arcuate field defect or even a dense hemifield defect. other discs have diffuse shallow cupping and pallor, leading to the designation senile sclerotic disc. patients with high myopia may be particularly susceptible to ntg. with a frequent temporal crescent, scotomas tend to be closer to fixation than the paracentral scotomas of non - myope cases. splinter hemorrhages are seen more commonly in ntg, but may also be found in poag. hemorrhages may simply indicate poor control. although the optic nerve head may be larger in ntg than in poag, glaucomatous cupping is comparable. normal - tension glaucoma eyes can have greater optic nerve head (onh) torsion as compared to poag eyes with matched axial length. the direction of the onh tilt and torsion can be related to the location of the visual field defect only in ntg eyes. as compared to normal subjects, peripapillary choroidal thickness was significantly thinner in ntg patients, at least in some locations. another revealed a difference in the progression pattern as compared to poag patients ; in poag eyes, field defects initially increased in area and later in depth, whereas in patients with ntg, the increases in area and depth remained in constant proportion. other investigations include 24-hour blood pressure monitoring to exclude nocturnal systemic hypotension ; blood tests to rule out other causes of glaucomatous optic neuropathy such as vitamin b12 and folate levels, esr / crp and serum ace. cranial mri may be necessary to rule out intracranial space occupying lesions (sols) ; and nail fold capillaroscopy with cold provocation may detect blood flow abnormalities. a number of congenital disorders may be confused with ntg ; these include optic nerve anomalies including coloboma, pits, oblique insertion of the optic nerve, in addition to autosomal dominant optic atrophy (kjer type). acquired disorders which need to be considered in the differential diagnosis of ntg include history of steroid use by any route which may have led to prior elevated iop, prior trauma or surgery which may have caused elevated iop, hemodynamic crisis, methyl alcohol poisoning, optic neuritis, ischemic optic neuropathy (both arteritic and non - arteritic), compressive lesions of the optic nerve and tract (e.g., meningioma, vascular lesion) ; and wide diurnal fluctuation in iop. the early manifest glaucoma trial showed that glaucoma progression was decreased by 10% with reduction of each mmhg of iop. according to the collaborative normal tension glaucoma study group, choices for medical treatment in progressive cases include betaxolol eye drops which have a beneficial effect on optic nerve blood flow in addition to iop reduction. other beta blockers and adrenergic drugs (such as dipivefrine) should better be avoided because of the probability of nocturnal systemic hypotension and optic nerve hypoperfusion.. dorzolamide - timolol fixed combination (dtfc) is a safe and effective iop - lowering agent in patients with ntg. brimonidine significantly improved retinal vascular autoregulation in ntg patients ; however, short - term alterations in visual function could not be demonstrated. in the collaborative normal tension glaucoma study (cntgs), 57% of the patients achieved 30% iop reduction with topical medications, laser trabeculoplasty or both. the remaining 43% required filtering surgery which may prevent progressive damage. a single session of selective laser trabeculoplasty (slt) for ntg achieved iop reduction of 20% from pre - study iop and 30% reduction from baseline iop at 6 months. deep sclerectomy seems to be effective and safe in reducing iop in patients with ntg. intraoperative use of mmc results in lower postoperative iop after 12 months without an increased rate of complications. an additional part of managing ntg is treatment of any cardiovascular abnormality such as anemia, hypotension, congestive heart failure (chf), transient ischemic attacks and cardiac arrhythmias to increase optic nerve head perfusion. if significant nocturnal dips in blood pressure are detected, it may be necessary to reduce the antihypertensive medication especially at bedtime. finally treatment is aimed at neuroprotection to improve the retinal ganglion cell or optic nerve head function. nilvadipine, a calcium channel blocker, increases blood flow to the optic nerve head and fovea. there was a significant reduction in the rate of disc and field damage in ntg patients who received calcium channel blockers. | normal tension glaucoma (ntg) is labelled when typical glaucomatous disc changes, visual field defects and open anterior chamber angles are associated with intraocular pressure (iop) constantly below 21 mmhg. chronic low vascular perfusion, raynaud 's phenomenon, migraine, nocturnal systemic hypotension and over - treated systemic hypertension are the main causes of normal tension glaucoma. goldmann applanation tonometry, gonioscopy, slit lamp biomicroscopy, optical coherence tomography and visual field analysis are the main tools of investigation for the diagnosis of ntg. management follows the same principles of treatment for other chronic glaucomas : to reduce iop by a substantial amount, sufficient to prevent disabling visual loss. treatment is generally aimed to lower iop by 30% from pre - existing levels to 12 - 14 mmhg. betaxolol, brimonidine, prostaglandin analogues, trabeculectomy (in refractory cases), systemic calcium channel blockers (such as nifedipine) and 24-hour monitoring of blood pressure are considered in the management of ntg. the present review summarises risk factors, causes, pathogenesis, diagnosis and management of ntg. |
in the iranian traditional medicine, anbarnesa smoke derived from burning female donkey s dung has long been used for treatment of inflammatory ulcers and infections of the middle and external ear with no significant side effects. we conducted a systematic search in pubmed, medline, google, and google scholar databases to find studies on anbarnesa. the keywords searched were as follows : anbarnesa, traditional medicine, medicinal smoke, donkey, dung, antimicrobial, inflammation, infection, and cytotoxicity. literature review reveals that annas (anbarnesa smoke) enhances wound healing, decreases scar formation, inhibits growth of cancer cells (hela and kb) and has antimicrobial properties. also, annas combined with propylene glycol is nontoxic in 1/64, 1/128, and 1/256 dilutions. the constituents of anbarnesa smoke mainly possess antibacterial, anti - inflammatory, and growth inhibition effects on cancer cells. considering the numerous side effects of synthetic drugs and advances in the alternative medicine in recent years, traditional medicine and herbal medications have gained the spotlight and found their way into modern medicine and dentistry (1, 2). the iranian traditional medicine contains valuable information in this respect (3). in iranian traditional medicine, anbarnesa smoke derived from burning female donkey s dung collected in spring has long been used for the treatment of inflammatory oral ulcers such as aphthous ulcers and other inflammatory conditions such as infections of the middle and external ear with no significant side effects (3 - 8). avicenna used anbarnesa smoke for vaginal infections and decreasing the duration of menstrual period and stanch bleeding (3, 6, 8). evidence shows that using the smoke of medicinal plants for treatment of diseases is common not only in iran, but also in over 50 countries (9). the preliminary research with contemporary standard methodology on the effects of this traditional treatment (anbarnesa smoke) was started in 2010 at shahid beheshti university of medical sciences by preparing anbarnesa solution and mouthwash (in propylene glycol solvent) ; the study is still ongoing (3 - 5). the aim of this study was to introduce anbarnesa and discuss its therapeutic effects through literature review. we conducted a systematic search in pubmed, medline, google, and google scholar databases on anbarnesa smoke. the keywords searched were as follows : anbarnesa, traditional medicine, medicinal smoke, donkey, dung, antimicrobial, inflammation, infection, and cytotoxicity. all the identified abstracts, letters to the editors, review articles, original articles, animal studies, and other documents were reviewed. the publication language was not an exclusion criterion, and studies published from july 1970 to july 2015 were included. reference lists of the retrieved articles were also reviewed to identify citations that complied with our inclusion criteria. eventually, 5 studies (3, 5, 10, 11) were chosen upon complete agreement between the two reviewers. a literature search using the above - mentioned criteria yielded a total of 4 articles on anbarnesa smoke (4, 5, 10, 11). two articles were in english (4, 5) and 2 in farsi (10, 11). also, 3 doctoral (dds) theses had been conducted on anbarnesa smoke under the supervision of dr h. shafiee (3, 12, 13). of them, 2 had been converted to english articles (4, 5, 12, 13) and the remaining one was conducted on wistar rats (3) (table 1). shafiee. (4) analyzed the smoke from burning of anbarnesa using chromatography by gc mass device and isolated its constituents as hexane, acetic acid, aconitane, beta carotene, dimethyl amine. these compounds mainly have antibacterial, antifungal, anti - inflammatory and antioxidant properties and some of them are used for treatment of neuralgia, rheumatism, capillary hemorrhage, and skin disorders (3, 4, 14). an in vitro study showed that a mouthwash (0.2% annas sbmu 1) prepared from anbarnesa smoke had antimicrobial and bacteriostatic properties and yielded a growth inhibition zone diameter (izd) and minimum inhibitory concentration (mic) similar to those of 0.2% chlorhexidine (chx) against streptococcus mutans, streptococcus salivarius, and streptococcus pyogenes (5). chx mouthwash has strong antibacterial activity against different oral microorganisms and is used as the positive control group in many studies. since s. mutans is an acidogenic and aciduric bacterium responsible for caries and also the most important microorganism in bacterial plaque, annas1 can also be used for the prevention of caries and periodontal disease (5). showed that the mic value of chx for s. sanguinis and particularly enterococcus faecalis was significantly higher than that of annas1 (5). the exact mechanism of annas1 mouthwash against bacteria has yet to be understood but it appears to be via the destruction of cell integrity (5). also, analysis of annas with nitro blue tetrazolium (nbt) test showed that constituents of anbarnesa smoke induce the activity of neutrophils in the tested blood and enhance their antimicrobial activity (12). however, it should be noted that chx, despite its potent antimicrobial property, has several side effects and shows cytotoxic effects in bactericidal concentrations (15). in another study, shafiee. demonstrated that annas1 mouthwash in 1/64, 1/128 and 1/256 concentrations had antibacterial properties while being nontoxic for l929 fibroblasts and can be used with fewer side effects for plaque control and prevention of periodontal disease and caries (4). parvin. showed that anbarnesa smoke in direct contact (without solvent) via at least 6 seconds of fumigation created growth inhibition zones on pseudomonas aeruginosa and staphylococcus aureus cultures (11). however, further studies with a more accurate methodology are required to further elucidate this finding. the antibacterial efficacy of anbarnesa is believed to be due to the presence of some antibacterial agents in donkey s dung produced through the process of fermentation and digestion of foods in the digestive system as well as the presence of probiotics (10, 11). also, it seems that the smoke from burning female donkey s dung increases the permeability of cells for its effective agents (10, 11). sadeghi aliabadi. showed the cytotoxic effects of anbarnesa smoke combined with water and n - hexane solvent on hela and kb cancer cells. they reported that this solution was nontoxic for l929 fibroblasts in dilutes with higher than 2 mg / ml concentrations (10). also, based on previous studies, they stated that hydrolysis of dung lignin produces some compounds with bacterial growth inhibition properties (10, 11, 16). based on their findings, further investigations are required to identify the bactericidal and bacteriostatic compounds in anbarnesa smoke. in another study, researchers applied annas1 solution on wounds created on the back of the neck of wistar rats and showed that number of myofibroblasts (which are effective in scar formation) decreased by 3 folds following the application of this solution to superficial and deep areas of wounds after 14 and 21 days, and skin, hair follicles, and sebaceous glands appeared after 21 days in the area as well. this indicates that annas1 solution enhances wound healing and decreases scar formation (3). the process of scar formation is complex and depends not only on the number of myofibroblasts but also on the environment in which their function is regulated. it appears that the effects of annas1 solution on decreasing scar formation relate to regulation of the level of expression of tgf - b1 because scar formation decreases by inhibiting the activity of tgf - b1 (3, 17). however, the exact mechanism of function of annas1 has yet to be clearly understood (3) and further investigations as well as clinical trials are required for its use in medicine and dentistry. | context : in the iranian traditional medicine, anbarnesa smoke derived from burning female donkey s dung has long been used for treatment of inflammatory ulcers and infections of the middle and external ear with no significant side effects. the aim of this study was to introduce anbarnesa and discuss its therapeutic effects.evidence acquisition : we conducted a systematic search in pubmed, medline, google, and google scholar databases to find studies on anbarnesa. the keywords searched were as follows : anbarnesa, traditional medicine, medicinal smoke, donkey, dung, antimicrobial, inflammation, infection, and cytotoxicity.results:literature review reveals that annas (anbarnesa smoke) enhances wound healing, decreases scar formation, inhibits growth of cancer cells (hela and kb) and has antimicrobial properties. also, annas combined with propylene glycol is nontoxic in 1/64, 1/128, and 1/256 dilutions.conclusions:the constituents of anbarnesa smoke mainly possess antibacterial, anti - inflammatory, and growth inhibition effects on cancer cells. |
a coronary artery fistula (caf) is an abnormal communication between one of the coronary arteries and a cardiac chamber or vessels, including the coronary sinus, pulmonary artery, or superior vena cava.1)2)3) caf is involved in 0.002% of general population and accounts for 0.4% of all cardiac malformation.4) caf mostly drains into the venous structures of circulation, such as the right - sided chambers, pulmonary artery, coronary sinus and superior vena cava, but drainage into the left - sided chambers is less frequent.5) we present a case of caf which is originated from left coronary artery and drained into left ventricle. a 48-year - old male presented with abdominal pain that had lasted for 2 months. he had no cardiovascular risk factor, and no cardiovascular symptom such as chest pain or dyspnea. preoperative electrocardiography was within normal limit, and chest x - ray showed no pathologic abnormality. he was consulted to cardiovascular department because surgeon heard a continuous cardiac murmur and ordered echocardiography for a preoperative evaluation. the mitral inflow e / a ratio was 1.63, and e / e ' ratio was 12.0. however, there was abnormal color flow within left ventricle predominantly during diastole (fig. 1, supplementary movie 1,2, 3, 4). the maximal velocity of blood flow draining into left ventricle was approximately 3.0 m / s. in short - axis great arterial view, left coronary artery appeared dilated. multiple tortuous dilated vascular structures with internal blood flows disclosed by color doppler image were also found around left ventricular myocardium. especially, dilated large echo - free vascular structure was detected in the apical area. the connection between those dilated vascular structures and left coronary artery was suspected, but clear visualization of the connection was limited in 2-dimensional echocardiography. coronary artery computed tomography (ct) scan was performed to confirm the pathologic anatomy of these abnormal findings. coronary ct revealed markedly dilated (up to 16 mm) and serpentine whole left coronary arteries (fig. 2). the left anterior descending artery was communicating with left circumflex artery at the apical posterior epicardium, and was directly connected to the basal posterior side of left ventricular cavity (fig. since the patient had no cardiovascular symptom, he underwent laparoscopic cholecystectomy without specific treatment of caf. caf communicating with the cardiac chambers are called coronary cameral fistula, as in our case. however, the incidence of coronary cameral fistula is very low and coronary cameral fistula involving left ventricle has been very rarely reported.4)5) this case is also unique because almost whole left coronary artery was dilated and looked like cystic lesion in the apical portion, which could be clearly detected by transthoracic doppler echocardiography. the majority of coronary cameral fistula (up to 90%) drains into the right - sided chambers of the heart, while only 10% of coronary cameral fistula shows communications with the left - sided chambers or both left and right - sided chambers.6) doppler echocardiography is a good screening imaging tool for the diagnosis of caf, but evaluation of whole anatomy of caf can be best visualized by coronary ct image. chest pain, exertional dyspnea and fatigue may develop, but many patients do not suffer from any symptoms.7) the mechanism of symptoms appeared to be coronary steal phenomenon and diastolic overload.8) the most common complication is myocardial ischemia due to coronary steal phenomenon which occurs in 15% of patients with caf.9) congestive heart failure and arrhythmia could be caused by excessive loading of cardiac chambers. intravascular thrombosis, infective endocarditis, and rarely hemopericardium due to rupture of aneurysmal coronary artery might be complications. surgical closure of caf may have benefit for the patients with large, hemodynamically significant caf.10) there were several reasons for not doing any surgical interventions for this patient. first, this pathology was incidentally found without any cardiovascular symptom, and may carry only mild risk in undergoing laparoscopic surgery. therefore, the first thing we had to do for this patient was to let the surgeon not to postpone this necessary surgery because of the cardiac problem. second, good functional status of this patient indicates low possibility of coronary steal causing myocardial ischemia due to this pathology. lastly, surgical treatment for this coronary cameral fistula was not technically easy, because almost entire portion of the coronary artery was dilated as noticed in coronary ct. the parasternal long axis view revealed abnormal color flow within left ventricle predominantly during diastole. the parasternal short axis view revealed diastoledominant abnormal color flow draining from posterior left ventricular wall. dilated large vascular structure was detected in the apical area. abnormal vascular structure was originated from basal posterior wall and drained into the left ventricular cavity. | we present a case of 48-year - old male who presented with coronary artery fistula draining into left ventricle. transthoracic echocardiography showed abnormal blood flow draining into left ventricle, with enlarged coronary arteries and multiple vascular structures around ventricular myocardium. coronary computed tomography revealed dilatation of entire left coronary artery which was wrapping around left ventricle, and draining into the posterior side of left ventricle. he did not undergo any invasive treatment, because he was not symptomatic. |
pregnancy does not translate to being medically compromised ; therefore dental treatment should not be denied simply because a woman is pregnant. the pregnant woman who presents for dental care may require special considerations.1 therefore, the management of these patients may require adjustments in the timing and type of dental treatment, as well as in the drugs to be prescribed.2 proper risk assessments should be done for the mother and fetus. the following should be considered : risk of teratogenicity in the fetus due to drugs taken by the mother, susceptibility to supine hypotensive syndrome as a result of a decrease in blood pressure and cardiac output while the patient is in a supine position, and the potential danger of disseminated vascular coagulopathy due to increase in clotting factors.1 oral health during pregnancy has, for long, been a focus of concern 3 with obstetricians acknowledging its importance and attendant effect on pregnancy outcomes.45 the physiologic changes in pregnancy include changes in the oral cavity with an attendant increase in susceptibility to oral infections.6 also, increased consumption of carbohydrates, increased acid in the mouth from vomiting and reduced production of saliva and possibly too, increased acidity of saliva have been believed to increase the risk of dental caries in pregnancy.7 a study found that pregnant women were 1.97 times more likely to suffer from dental caries than the nonpregnant women.8 a 74.5% prevalence of dental caries among the pregnant women was observed in a study by mahmud.9 usually, pregnant patients are not immunocompromised ; however, there is suppression of the maternal immune system in response to the fetus 6 subsequently causing a decrease in cell - mediated immunity, as well as natural killer cell activity.10 with the foregoing, odontogenic infections have the potential to progress rapidly to deep - space infections eventually compromising the oropharyngeal airway.10 in addition, pregnant women may also receive a prescription and/or over - the - counter analgesics to control serious pulpal pain. abuse of these drugs rather than receiving the appropriate dental treatment may have deleterious effects on the fetus and the pregnant mother. therefore, it is imperative that odontogenic infections should be treated promptly at any time during pregnancy. one of the possible treatment options is endodontic treatment involving the extirpation of the diseased pulp. endodontic treatment may entail the use of radiographs, local anesthetic agents, root canal irrigants, intra - canal medications, and drugs (analgesics and antibiotics). they are required for proper diagnosis, determination of working length, proper obturation, and posttreatment evaluation. as the x - rays are directed to the mouth and not the abdomen, along with the use of protective measures such as high - speed film, collimation, filtration, lead apron, and a thyroid collar.11 it has been proposed that, concerned pregnant patients be reassured that in all cases requiring such imaging, the as low as reasonably achievable (alara) principle will be practised, and only radiographs necessary for diagnosis and treatment will be obtained.12 x - ray radiation exposure during pregnancy totaling < 5 - 10 cgy, and a full mouth series of dental radiographs of only 8 10 - 4 cgy has been reported to show no increase in congenital anomalies or intrauterine growth retardation.111314 local anesthetics are relatively safe when administered properly and in the correct amount during pregnancy.10 the quantity of anesthetic agent administered could be a probable cause for concern among endodontists. this may be because of the uncertainty of the initial dose administered being ineffective in achieving anesthesia, thus requiring an additional anesthetic agent to make the patient feel more comfortable. pain incurred during treatment may induce stress which could be more damaging to the fetus than the effect (s) of additional quantities of anesthetic agent. this drug has been studied in amount of up to 0.1 mg added to local anesthetics. no unusual side effects or complications were reported following its use for epidural anesthesia during labor.15 it has been reported that the local anesthetic with epinephrine administered as an intravascular injection may, at least supposedly, cause a deficiency of uteroplacental blood flow.10 however, for a healthy pregnant patient, the 1:100,000 epinephrine concentration used in dentistry, administered by proper aspiration technique and limited to the minimal dose required, is safe.16 endodontic treatment in pregnancy is directed towards controlling disease, maintaining a healthy oral environment, and preventing potential problems that could occur later in the pregnancy or during the postpartum period.16 it has been asserted that neither the cleansing irrigant, hypochlorite nor root canal filling materials used in endodontic treatment is detrimental to the fetus.17 the initial 3 months of pregnancy are considered vital to the growth of the fetus. it has been recommended that any avoidable treatment in the first trimester should be moved to the next trimester to prevent any threat of untoward effects of dental treatment.10 by the end of the first trimester, organogenesis is complete, the uterine size is not large enough to make sitting on the dental chair uncomfortable and nausea has generally waned. dentists frequently have to face the anxiety associated with the safety of the dental treatment during pregnancy18, as well as the need to eliminate the odontogenic infection. assessment of the potential risk in rendering endodontic treatment during pregnancy is important while paying attention to the physiologic changes associated with pregnancy. probably due to the wrong suppositions propagated following the lack of proper information, dentists are wary of rendering treatment to pregnant women.1920 reviews on amalgam fillings and periodontal therapy have been reported 21 but there is a paucity of studies reviewing perceptions of endodontic treatment in the pregnant woman. this study to assess the perception of dentists about endodontic treatment during pregnancy thus became imperative as the purpose of this study. this cross - sectional study was carried out among the resident doctors in the different dental specialties in nigeria preparing for the various levels of the fellowship examinations of the west africa college of surgeons and the national postgraduate medical college of nigeria, who attended the revision course in lagos state in september 2012 and april 2013. the 17-itemed questionnaire sought information on respondents ' demography, their considerations while rendering endodontic treatment to the pregnant patients and their perceptions of the safety of endodontic treatment in pregnancy. the data collected were analyzed using the ibm statistical package for social science (spss) for windows version 21.0. the results were presented as tables and cross - tabulations. the nonparametric analysis in the form of chi - square test most (86.9%) of the respondents were junior residents and 42.6% were residents in oral and maxillofacial surgery [table 1 ]. demographic characteristics of the respondents the majority (86.9%) thought that appropriate treatment of dental pain and infection was important in pregnancy. while rendering such treatment, 82% were aware that the pregnant patient should be positioned in a special way [table 2 ] with 36.1% recommending that the pregnant patient 's positioning should allow her head to be higher than her feet. responses regarding dental treatment among pregnant women with regards to the safety of endodontic treatment during pregnancy, 91.8% considered it safe [table 2 ] and this was not statistically significant in relation to the specialty or status of the respondent. however, 52.4% of the respondents recommended that the ideal period to carry out endodontic treatment was the second trimester while 21.3% thought that any time during pregnancy was safe if endodontic treatment was indicated. table 3 displays the responses of respondents concerning agents employed in the course of endodontic treatment. the majority (77.0%) of the respondents felt use of local anesthetic with epinephrine was safe in pregnancy. all the females were certain of the safety of local anesthetic with epinephrine, and this was statistically significant (p = 0.03). all the respondents in oral medicine and orthodontics considered the use of local anesthetic with epinephrine in pregnancy safe while respondents in oral pathology were uncertain, and this was statistically significant (p = 0.0001). responses regarding agents employed during endodontic treatment in pregnancy as regards the safety of the use of irrigants during root canal treatment, 13.1% of the respondents thought that their use posed some danger to the fetus while 42.6% were uncertain of any danger their use posed. this was statistically significant in relation to the specialty of the respondents (p = 0.02) with a majority of respondents who thought that it was not safe being oral and maxillofacial surgery residents. in the same vein, it was statistically significant in relation to the gender of the respondents with no female agreeing that irrigants used during endodontic treatment posed any danger to the fetus (p = 0.006). however, there was no statistically significant association between the status of the respondents and their perception of the safety of irrigants use. more than half (57.4%) of the respondents agreed they would expose dental x - rays for a root canal treatment procedure during pregnancy. the majority of those who were uncertain if they would expose x - rays for a root canal treatment during pregnancy were residents in oral and maxillofacial surgery, and this was statistically significant (p = 0.002). likewise, 59.0% agreed that they would place interappointment medicaments during a root canal treatment procedure in pregnancy. the majority of the respondents in restorative, periodontics, pedodontics, prosthodontics, and orthodontics specialties were willing to use interappointment medicaments during root canal treatment on the pregnant patient while all those in oral pathology were uncertain and this was statistically significant (p = 0.0001). also statistically significant was the association of gender with the use of interappointment medicaments for a root canal treatment during pregnancy with more females agreeing (p = 0.01) but no such association with the status of the respondents. in relation to the use of root sealers for a root canal treatment during pregnancy, majority (80.3%) admitted they would use them, and this was statistically significant in relation to the specialty of the respondents (p = 0.0001). all respondents in prosthodontics, pedodontics, and orthodontics affirmed they would use root sealers while those in oral pathology were not certain. when asked if root canal obturation points could have any adverse effect on the fetus, only 1.6% claimed they could, and these participants were in restorative dentistry and this was statistically significant (p = 0.005). majority (77.0%) agreed they would undertake a root canal treatment on a pregnant patient with all respondents in restorative dentistry, prosthodontics, periodontics, and pedodontics in the affirmative while all in oral pathology would refuse to do such (p = 0.0001, table 4). understanding the physiologic changes associated with pregnancy, as well as the effects of dental procedures including radiography and drug use on the pregnant patients and the developing fetus is requisite in rendering dental treatment to pregnant patients.22 the male preponderance in this study is similar to other nigerian studies 2324 reflecting that there is still a male - domination of the dental profession in nigeria.. this may be due to the fact that it was one of the first specialties of dentistry to be developed in nigeria. the importance of appropriate treatment of dental pain and infection has been well - emphasized, and this was affirmed in this study. it has been advocated that endodontic treatment, when indicated, should not be deferred till after delivery to avoid the inappropriate long - term use of analgesics to relieve pain.16 however, proper positioning of the pregnant patient during dental treatment is necessary, given the likelihood of hypotensive syndrome and the attendant loss of consciousness. in this study, the need for this proper positioning of pregnant women during dental treatment was recognized by a majority but only a few had an idea of which was the best position. this reflects a gap in knowledge among residents who are in active, specialization training and who are expected to be up - to - date with current trends. dental treatment, including endodontic treatment, has been certified safe in pregnancy. nevertheless, it has been recommended that elective procedures be avoided until the end of pregnancy and for only emergency treatment to be given or if possible, be delayed until the second trimester.22 the findings of this study suggest that dental residents have a clue of the timing of dental treatment for the pregnant patient. local anesthetic with vasoconstrictor can be safely administered to the pregnant patients during the dental treatment 25 but aspiration must always be done to minimize the likelihood of intravascular injection.26 dental residents seem to be conversant with this especially as most dental procedures require the use of local anesthetic. the scope of oral medicine specialty consists of the nonsurgical management of oral diseases where applicable and includes the use of pharmacological agents in the management of dental conditions. therefore, it is not surprising that all the residents in oral medicine were conversant with the safety of the use of local anesthetic with epinephrine in pregnancy. not much has been reported on the use of endodontic irrigants for root canal treatment during pregnancy, and this was reflected in the varied opinions regarding the safety of endodontic irrigants use in pregnancy. it is imperative therefore that research into the possible adverse effects of endodontic irrigants in pregnancy be looked into, this will provide informed choices for dentists while selecting which irrigant to use for root canal treatment procedures in pregnant women. a lot has been done to ascertain the safety of dental x - rays during pregnancy. however, a reasonable percentage of dental residents in this study were not confident exposing x - rays for root canal treatment procedures on pregnant women. there is the need for more emphasis on the safety of dental x - rays and the use of protective measures such as the use of modern imaging machines (with low - dose, high - yield radio - diagnosis), high - speed films, collimation, filtration, lead aprons, and a thyroid lead collars. the female residents in this study exhibited a better knowledge of the use of local anesthetics, interappointment medicaments, and endodontic irrigants. this may be because women tend to be interested in or concerned with medication use in pregnancy. endodontics is a subspecialty in restorative dentistry in nigeria, so residents in this specialty tended to display more confidence by their eagerness to render endodontic treatment to pregnant patients. the hierarchical status of the respondents did not play any role in the outcomes of all the questions asked. this shows that increased clinical exposure, experience, and knowledge did not influence the perception of dental residents toward rendering endodontic treatment to the pregnant women. limitations exist in the ability to generalize the findings of this study given the fact that some specialties such as oral medicine and oral pathology were poorly represented in the study. in addition, the authors recognize that specific agents used as irrigants, sealers, and obturating points were not enquired about. this could have thrown more light on the depth of knowledge of the respondents about these materials and their use in endodontic treatment in pregnancy. the findings of the study suggest that dental residents are aware of the safety of endodontic treatment in pregnant women. however, there are gaps in knowledge with regards to the proper positioning of pregnant women, the timing of treatment, the safety of the use of irrigants, and radiographic exposure for pregnant women. it is imperative that the postgraduate training should include pregnancy - specific, confidence - boosting knowledge for better endodontic treatment of the pregnant women. | introduction : in order to control serious pulpal pain following odontogenic infections in pregnant women, endodontic treatment may become necessary. the aim of this study was to assess the perception of dentists about rendering endodontic treatment to pregnant women.materials and methods : this was a cross - sectional study of resident doctors in the different dental specialties in nigeria preparing for the various levels of the fellowship examinations of the west africa college of surgeons and the national postgraduate medical college of nigeria. data were collected by the means of a 17-itemed questionnaire which sought information on respondents ' demography, their considerations while rendering endodontic treatment to the pregnant patients and their perceptions of the safety of endodontic treatment in pregnancy. the data collected were analyzed using the statistical package for social science version 21.0.results:with regards to the safety of endodontic treatment during pregnancy, 91.8% considered it safe, and this was not statistically significant in relation to the specialty or status of the respondent. majority (77.0%) agreed they would undertake a root canal treatment on a pregnant patient with all respondents in restorative dentistry, prosthodontics, periodontics, and pedodontics in the affirmative while all in oral pathology would refuse to do such (p = 0.0001).conclusion : dental residents are aware of the safety of endodontic treatment in pregnant women. however, gaps exist in their knowledge, bringing to the fore, the need for inclusion of pregnancy - specific training in the dental postgraduate curriculum. |
today, a large number of patients are searching for personal satisfaction and increasing the demand for an attractive smile. tooth bleaching has become one of the most popular cosmetic procedures offered in dental practice.1 several products and techniques are available for vital tooth bleaching, and vary in concentration and type of end products released.2,3 the whitening procedures can be performed in the office by the dentist or at home by the patient without dentist supervision. overall, when applied over an enamel surface, the peroxide - containing agents break down into water and oxygen, which diffuse through the enamel, causing oxidation and reduction of organic pigments that are mainly located within dentin, resulting in a reduction or elimination of the discoloration.4 at - home vital tooth bleaching with custom trays is the most common modality used.5,6 the advantages of this treatment are its application, reduced chair time, safety and low incidence of tooth sensitivity.57 supervised at - home tooth bleaching involves the use of custom trays loaded with low concentrations of carbamide (10%22%) or hydrogen peroxide gels (3.4%7.5%) that are used by the patient for a few hours per day.2,5 the side effects frequently associated with tray bleaching systems are gingival irritation and tooth sensitivity, which can either be related to shape of the tray or to concentration of the bleaching agent.8,9 in recent years, an increasing number of over - the - counter bleaching products have appeared in the market, which can be used by the patient at home without dentist supervision. these products have increased in popularity and represent a large percentage of the over - the - counter products sold for oral hygiene. even though such products have low concentrations of peroxide agents, they may produce side effects, including an erosive effect on dental structure, because patients tend to ignore the manufacturers recommendations and use them for a long period of time.10 although the manufacturers have introduced different concentrations of bleaching agents onto the market, 10% carbamide peroxide gel is the only one that has a seal of acceptance from the american dental association assuring its safety and efficacy for at - home tooth bleaching.11 in an attempt to provide an evidence base for dental practitioners, the aim of this review was to evaluate the erosive potential of at - home bleaching agents, which could increase susceptibility to tooth abrasion. a literature search of the electronic database, medline, was performed up to february 28, 2011. the following search format was performed using mesh terms : (tooth bleaching[mesh ]) and (tooth erosion[mesh ] or tooth abrasion[mesh ]). tooth wear can be defined as a progressive loss of hard dental tissues due to the processes of abrasion and erosion.12 dental erosion is an irreversible loss of tooth substance due to a chemical process without the presence of bacteria,13 while abrasion is caused by oral habits and the use of abrasive substances, such as abrasive toothpastes.14 even though these processes can occur individually or together, the effect of erosion is often dominant.1416 it has been suggested that for enamel demineralization to take place, the ph on the enamel surface must fall below 5.5.17 dental erosion is a multifactorial condition that may be idiopathic or caused by a known acid source.18,19 several acids, including some of those regularly found in the human diet, such as acidic food and soft drinks ; or those originating in the stomach, like gastric acid from regurgitation or reflux, and some drugs, are related to the pathogenesis of dental erosion.13,14,18 some bleaching agents have a lower than ideal ph, which can cause changes in the mineral content of the enamel, contributing to the formation of shallow depressions, increasing enamel porosity and promoting slight erosion.20 these changes can be higher when the contact time between bleaching agent and tooth surface is increased.21,22 however, studies have shown that the addition of calcium or fluoride to the composition of a bleaching agent can minimize mineral loss in the enamel.23,24 brushing is a hygiene procedure for maintaining oral health, usually done with abrasive dentifrices, and plays an important role in prevention of formation or removal of extrinsic stains.25,26 nevertheless, the use of more abrasive toothpastes can increase wear on the tooth surface.2729 few in vitro studies have evaluated changes in the bleached enamel and dentin after methods of acid exposure or toothbrushing.3033 the majority of studies have reported that the abrasive or erosive actions of combinations containing 10% carbamide peroxide30,32 or 35% hydrogen peroxide33 have no deleterious effects on enamel or dentin. one study has evaluated the effect on enamel and dentin of the interaction between 10% carbamide peroxide bleaching, erosion with 1% citric acid, and abrasion using low and high abrasive dentifrices. the authors concluded that bleaching did not increase the susceptibility of enamel to erosive and abrasive wear, but that dentin wear was affected by the interaction of bleaching, erosion, and dentifrices.30 another study that evaluated the roughness of human enamel exposed to 10% carbamide peroxide showed that use of this concentration of bleaching agent alone did not alter enamel surface roughness, but when the bleaching treatment was associated with brushing using fluoridated or nonfluoridated abrasive dentifrices, there was a significant impact on enamel surface roughness.31 a number of studies have evaluated the influence of at - home bleaching agents on the properties of enamel and dentin.21,24,3032,3451 most of them have used the 10% carbamide peroxide and different methodologies to investigate the softening effect produced by bleaching agents on mineralized dental tissues, including surface microhardness, surface morphology, surface roughness, and calcium loss. while some studies have found no significant alteration in enamel surface caused by 7.5%22% carbamide peroxide or 6% hydrogen peroxide,21,31,32,3439 others showed that 10%22% carbamide peroxide solutions can cause morphological changes and erosive lesions, decreasing microhardness and increasing enamel surface roughness.24,4049 therefore, the question arises as to whether these morphological changes in the enamel surface are transient, permanent, and/or clinically significant ? in general, findings of damage to the enamel surface after bleaching treatment come from studies carried out in vitro, with the methodological limitations inherent in this type of study. such findings may not be representative of the in vivo condition, in which the oral cavity is continually bathed with saliva containing various minerals, including fluoride, calcium, and phosphate, lipids, carbohydrates, proteins, and other substances.44 evaluation of specimens was usually performed soon after the bleaching protocols, without any period of storage in artificial saliva and consequently with no remineralizing effect.44,4648 storage in artificial saliva was performed only between clinical sessions or from the first to the last session.24,4049 the relevant studies identified in the medline database are summarized in table 1, with information regarding the type of study, measurement used, tissue evaluated, product concentration, ph values, changes observed, and possible reversibility after remineralization. surface evaluations were performed by scanning electron microscopy24,42,43 or enamel microhardness41 after exposure to 10%22% carbamide peroxide for 14,24,41 84,42 and 9043 days. erosive lesions were observed for periods up to 84 days after conclusion of bleaching42 and decreased microhardness even after 14 days.41 basting showed that an increase in enamel microhardness occurred after the end of bleaching treatment, but without reaching baseline microhardness values. this can be explained by the absence in artificial saliva of proteins present in human saliva, which prevents the formation of the acquired pellicle, a protective barrier of dental tissue formed in vivo. even though 10% carbamide peroxide caused alterations in the surface morphology of enamel in one study, these alterations were reversed within 3 months following treatment.43 additionally, it was observed that enamel microhardness decreased after bleaching treatments containing 10% carbamide peroxide with or without fluoride, but hardness values gradually recovered after cessation of bleaching.24 these discrepant results may be attributed to wide variation in methodology, which may limit any comparisons between studies. using scanning electron microscopy to compare in vitro and in situ methodologies to detect effects of 10% carbamide peroxide on enamel topography, calcium loss, and microhardness, while rougher surface, higher mineral loss, and lower microhardness were observed for bleaching treatment performed in vitro, such alterations were not detected in situ, which is very similar to the in vivo condition. in figure 1 the effects of 10% carbamide peroxide treatment on the enamel surface can be seen, and the different patterns of erosion caused by 10% carbamide peroxide using in vitro and in situ methodologies are compared with unbleached enamel. some authors have reported that the erosive effect associated with bleaching treatment may be related to the low ph of the whitening solutions.24,43,45 however, when enamel erosion was detected after bleaching with 10%22% carbamide peroxide, the products used generally had ph values ranging from 6 to 7.8, ie, neutral or very close to neutral.24,40,41,43,47,48 one study used an agent with an acidic ph (4.7),44 but the others did not evaluate the ph of the solutions tested.42,46 such findings demonstrate that morphological changes induced by bleaching procedures could not be exclusively related to ph.40 an investigation was conducted to evaluate the time period required to re - establish enamel surface microhardness after bleaching with fluoridated or nonfluoridated 10% carbamide peroxide gels with neutral or acidic ph that used a daily demineralization and remineralization protocol. after seven days of whitening treatment, a statistically significant loss of hardness ranging from 7%15% was observed in all groups. nevertheless, fluoridated gels provided some protective effect, with less loss of hardness than with the nonfluoridated gels. the ph of acidic gels does not seem to contribute significantly to demineralization of enamel.49 no significant changes in surface roughness of the enamel have been observed after bleaching with 10%31 or 16%21 carbamide peroxide, but susceptibility to abrasion was increased when brushing was performed with abrasive toothpaste.21,31 however, these studies were carried out in vitro, so would have had more pronounced effects than in conditions in vivo. another study39 evaluated enamel microhardness after bleaching with 10% carbamide peroxide, and the authors were unable to find a significant difference before and after bleaching, but their study did show decreased resistance to abrasion. additionally, the authors observed that bleached enamel showed a greater loss of tooth structure when abraded against both a harshly and mildly abrasive substrate than the unbleached enamel did.39 the potentially higher susceptibility of bleached enamel to erosion and demineralization was also evaluated in a study of human incisors. the study was designed to determine if enamel bleached using different carbamide peroxide gel concentrations of 10%, 16%, 22%, or 10% and containing xylitol, fluoride, and potassium, had an increased risk of either acid erosion or demineralization as compared with unbleached enamel. the authors observed that erosion was detected in all samples, and that there was no statistically significant difference between the bleached and nonbleached areas, regardless of the bleaching agent used. when submitted to cariogenic challenges, all samples showed caries - like lesions, in bleached and unbleached specimens. they concluded that tooth bleaching with carbamide peroxide does not increase the susceptibility of enamel to acid erosion or demineralization as occurs during caries formation.32 only one in vivo study evaluated the influence of at - home bleaching agents on enamel microhardness. this clinical trial compared the effects of a neutral fluoridated and a nonfluoridated whitening product, both containing 15% carbamide peroxide, on enamel microhardness after tooth extraction.35 the authors concluded that 15% carbamide peroxide with or without fluoride in the composition does not seem to alter enamel microhardness. this is in accordance with other in vitro studies using artificial saliva as the storage solution, in which investigators reported that 7.5% or 10% carbamide peroxide did not cause any significant alteration in enamel surface topography37 or microhardness.37,38 additionally, another in vitro study that assessed the amount of calcium loss in enamel previously bleached using 10% carbamide peroxide, did not detect an increase in enamel solubility. 36 furthermore, a study comparing the erosive effect of 6% hydrogen peroxide indicated for at - home tooth bleaching and that of orange juice concluded that the erosive effects of 6% hydrogen peroxide on enamel surface were not statistically significant compared with orange juice.34 the authors also reported that daily intake of acidic soft drinks was potentially more harmful to hard dental tissues than periodic application of hydrogen peroxide - based tooth bleaching products.34 due to the highest organic content of dentin when compared with enamel, it has been suggested that dentin is more prone to mineral loss resulting from tooth bleaching. some studies have evaluated the effect of carbamide peroxide on the dentin surface.30,45,50,51 we found two studies, one in situ50 and the other in vitro30, that evaluated enamel and dentin resistance to abrasion after bleaching with 10% carbamide peroxide. neither study found a significant effect of bleaching on enamel surface wear, but a higher wear depth was detected for bleached dentin, regardless of the abrasiveness of the dentifrice used. moreover, even with the formation of an acquired pellicle being shown by the in situ study, there was still abrasion on the dentin surface. however, the analysis was done immediately after the bleaching protocol, without any period of recovery for the dental surface. in summary, most of the studies have shown that at - home tooth bleaching with low concentrations of hydrogen or carbamide peroxide have no significant harmful effects on enamel and dentin surface morphology, microhardness, roughness, or calcium loss. the few studies that showed alterations in enamel or dentin surfaces all had limitations in their in vitro methodology or used highly acidic bleaching agents. in addition, these harmful effects on tooth substrates were generally transitory, and were not significant when remineralization periods were allowed. at - home tooth bleaching with 10% carbamide peroxide placed in a custom - tray is considered the safest and efficacious method of bleaching, having the additional benefits of a lower incidence of tooth sensitivity and gingival irritation.5,11,53,54 although these side effects may occur,55,56 they usually disappear at the end of treatment.57,58 gingival irritation could be attributed either to the design of the tray or to the concentration of the bleaching agent. interruption of treatment for 12 days or adjustment of the tray generally resolves this side effect.59 tooth sensitivity may cause discomfort to the patient, but is a reversible effect that does not last more than 24 hours and rarely leads to cessation of treatment.60,61 most sensitivity occurs within the first two weeks of treatment54 and it may be the result of the glycerine or other vehicle used to carry the active ingredient, which cause tooth dehydration, enabling easier penetration into dental tissues and leading to reversible pulpitis.62 the risk of tooth sensitivity increases if there is gingival recession with exposure of cementum and/or dentin.63 combination of the bleaching agent with potassium nitrate and fluoride can reduce this undesirable effect.58 another method that has been shown to be effective in reducing the intensity of tooth sensitivity is application of fluoride before the bleaching treatment.64 additionally, manufacturers have introduced at - home bleaching gels with fluoride in their composition in order to decrease any post - treatment tooth sensitivity.8,23 studies have reported that the histological modifications to the pulp after vital tooth bleaching with 10% carbamide peroxide might cause initial mild, localized pulp reactions. however, the minor histological changes observed did not affect the overall health of the pulp tissue and were reversible within two weeks post - treatment.6567 thus, at - home bleaching has shown to be a safe and well tolerated method for whitening teeth. the side effects, mainly tooth sensitivity and gingival irritation, are easily controlled when the correct technique is employed, with the use of a well adapted tray, an adequate amount of bleaching gel, and application of fluoride or desensitizing agents before and after treatment. based on the present literature review, the following conclusions could be drawn : the majority of the studies have shown that at - home tooth bleaching agents based on hydrogen or carbamide peroxide have no harmful effects on enamel and dentin properties. in vitro studies have shown that at - home tooth bleaching agents based on hydrogen or carbamide peroxide has no clinically relevant effect on enamel mineral loss caused by erosion or abrasion ; additionally, artificial saliva is an efficient media for reversing possible mineral loss associated with bleaching treatment in vitro. the bleaching agents used in at - home tooth bleaching have shown satisfactory safety and tolerability, and any adverse effects can be easily controlled by application of fluoride or desensitizing agents between sessions, using well adapted trays or lower concentrations of bleaching agents. more randomized clinical trials are needed to have a better understanding of the effects of bleaching products on the predisposition to erosion and abrasion. | this review investigates erosion and abrasion in dental structures undergoing at- home bleaching. dental erosion is a multifactorial condition that may be idiopathic or caused by a known acid source. some bleaching agents have a ph lower than the critical level, which can cause changes in the enamel mineral content. investigations have shown that at - home tooth bleaching with low concentrations of hydrogen or carbamide peroxide have no significant damaging effects on enamel and dentin surface properties. most studies where erosion was observed were in vitro. even though the treatment may cause side effects like sensitivity and gingival irritation, these usually disappear at the end of treatment. considering the literature reviewed, we conclude that tooth bleaching agents based on hydrogen or carbamide peroxide have no clinically significant influence on enamel / dentin mineral loss caused by erosion or abrasion. furthermore, the treatment is tolerable and safe, and any adverse effects can be easily reversed and controlled. |
neuronal migration disorders are diseases that develop due to disruption of normal movement of postmitotic neurons from the ventricular zone to the cortical plate during embryogenesis that result in the total or subtotal absence of cortical sulci and severely disturbed architecture of the cortical plate. however, the location of these neurons along their migratory route and its severity, which also forms the basis of classification of these anomalies, depends on the pathological mechanism of the disorder. these disorders have been discussed classically under umbrella of lissencephaly, which means smooth brain. in agyria, there is absence of deep sulci in more than one lobe and the thickness of the cortex is 1020 mm, whereas in pachygyria sulci are present but are wider than in the normal cortex. in addition, the affected cerebral cortex has only four developed layers instead of the normal six layers. pachygyria is currently considered a subtype of lissencephaly spectrum of disorders which ranges from classical lissencephaly (agyria or pachygyria), in which microscopic examination reveals total disorganization of the cortex and absence of any distinguishable layers to subcortical band heterotopia or double - cortex syndrome. although exact incidence of this disorder is not known, the incidence of all forms of type i lissencephaly is estimated to be 1 in 100,000 births. clinical presentation may be varied including microcephaly or normal head circumference, facial dysmorphism including esotropia and/or exotropia, telecanthus, seizures, mental retardation, and hypotonia with or without pyramidal and cerebellar signs. mutations of several genes such as lis1 and dcx have been reported in neuronal migration disorders. historically, neuronal migration disorders have been classified based on a postmortem examination ; however, mainstay of diagnosis and classification of these disorders is currently based on magnetic resonance imaging (mri) of the brain. thus, knowledge of typical and atypical mri findings in these disorders may be of utmost importance in delineating further plan of management including preparation of treatment paradigms and selection of correct investigation panel, especially for genetic studies. here, we present a case of pachygyria who presented to us with some atypical features including tigroid - like stripes and leopard - like pattern on mri brain which has not been reported in the medical literature previously. a 12-month - old boy belonging to a hindu family born as a first child, and a product of non - consanguineous marriage, a full term normal delivery and without any history of prenatal or perinatal complications presented to our outpatient department with a history of global developmental delay and infantile spasms since the age of 6 months. he was able to sit with support since 10 months of age, developed social smile at the age of 9 months and was not able to utter any sound at the time of presentation. at 6 months of age, his parents noted sudden onset epileptic spasm of flexion and extension (mixture) type which occurred initially just on awakening. however, the frequency of these attacks had increased for last 2 months to around 510 episodes per day. these attacks now occurred in clusters throughout the day, and each episode lasted for 35 min. tone in all the extremities was normal and deep tendon reflexes were normally elicited, however, plantar was bilaterally extensor. mri of brain showed frontal predominant pachygyria, periventricular, and bilateral frontoparietal symmetric hyperintensities along with (t2-weighted sagittal image showing radial stripes) [figures 1, 2, 3a and b ] and leopard - like appearance - scattered dots in form of hyperintensities on a normal background white matter [figure 3c and d ]. fluid - attenuated inversion recovery image [figure 2c ] showed hyperintensities in bilateral subcortical white matter suggesting myelination abnormality and ruling out the enlargement of perivascular space as a cause of this appearance. awake electroencephalogram showed background activity of high amplitude polymorphic delta wave with multifocal spikes [figure 4 ]. urine examination for metachromatic granules was negative, and serum aryl sulfatase was also normal. we also performed mri brain of the parents to look for any malformation which turned out to be normal. 12-month - old boy presented to our outpatient department with a history of global developmental delay and infantile spasms since age of 6 months diagnosed as a case of pachygyria. (a and b) t2-weighted axial images show hyperintensities in periventricular (single arrow) and bilateral frontoparietal region with frontal predominant cortical thickening (double arrow) with paucity of sulci favoring cortical dysplasia with pachygyria. 12-month - old boy presented to our outpatient department with a history of global developmental delay and infantile spasms since age of 6 months diagnosed as a case of pachygyria. (a and b) t2-weighted axial images show tigroid - like stripes(single arrow) along with frontal predominant cortical thickening (double arrow) and (c) fluid - attenuated inversion recovery image shows hyperintensities in bilateral subcortical white matter (thin triple arrow) suggesting myelination abnormality. 12-month - old boy presented to our outpatient department with a history of global developmental delay and infantile spasms since age of 6 months diagnosed as a case of pachygyria. t2-weighted sagittal images (a and b) show radial stripes (single arrow) ; (c and d) : show scattered dots in form of hyperintensities on a normal background white matter (double arrow) - leopard - like appearance along with a normal appearing cerebellum 12-month - old boy presented to our outpatient department with a history of global developmental delay and infantile spasms since age of 6 months diagnosed as a case of pachygyria. awake electroencephalogram of the child shows background high amplitude polymorphic delta wave activity with multifocal spikes. initially, he received 40 iu of acth per day by subcutaneous route for 2 weeks followed by tapering off over another 2 weeks. apart from that, the patient was also maintained on sodium valproate at a dose of 200 mg / day which he was receiving before presentation to our center. there was an overall clinical improvement with respect to frequency of spasms which decreased to 12 per day after about 6 weeks of initiation of therapy. currently, he is in close follow - up and further management paradigm is to be planned according to the clinical progression and evolution of the disease. there are three stages of development of the cerebral cortex : cell proliferation, cell migration, and cortical organization. in the stage of cellular proliferation, both the neuronal and glial precursors are generated ; during migration, these cells travel from the proliferative zone to their final designated destination and during the stage of cortical formation there is formation of the cellular network. lissencephaly is classified into two types - (a) classic (type i) lissencephaly (4-layer lissencephaly), in which there may be a smooth brain surface in the complete form, or more commonly, a smooth surface with some gyral formation along the inferior frontal and temporal lobes in the incomplete form resulting from arrest of the neuronal migration process and (b) cobblestone (type ii) lissencephaly (congenital muscular dystrophy), which is characterized by a nodular brain surface, ocular anomalies, and congenital muscular disorders which results from over migration of the neuroblasts and glial cells beyond the external glial limitations into the subarachnoid space. as lissencephaly is a broad spectrum of disorders, a severity scale has also been proposed to classify various disorders under this heading ; lower grade signifies more severe disease. lissencephaly grades 1 and 2 consist of diffuse agyria ; however, patients with lissencephaly grade 2 generally have a few shallow sulci over the frontal or occipital pole. dieker syndrome (caused by microdeletion of 17p 13.3), in which the child presents with a combination of lissencephaly with dysmorphic facial features, visceral abnormalities, and polydactyly. lissencephaly grade 3 comprises of mixed agyria and pachygyria, and grade 4 comprises only of pachygyria. grade 5 includes mixed pachygyria and subcortical band heterotopia, and grade 6 comprises only subcortical band heterotopia. mutations of many genes have been reported in neuronal migration disorders such as lis1 on chromosome17p13.3 and dcx on xq23 in classical lissencephaly spectrum. reln and vldlr genes have been found to be contributory in lissencephaly with cerebellar hypoplasia. arx has been found to be mutated in x - linked lissencephaly with abnormal genitalia which often also result in lissencephaly grade 3, in addition to the basal ganglia, white matter and callosal abnormalities, and similarly, tuba1a mutation results in micro lissencephaly with agenesis of the corpus callosum. the location of lesions also sometimes depend on the genetic factors such as mutations of lis1, arx, or tuba1a result in a posterior more severe than anterior gradient, while mutations of dcx or reln lead to an anterior more severe than the posterior gradient of abnormalities. although microcephaly can be frequently noted in patients of pachygyria, normal head size as seen in this case and predominant frontotemporal pachygyria kurul., described mri findings of cases with localized pachygyria ; six cases had bilateral frontoparietal agyria - pachygyria, and five of their cases had bilateral frontotemporo - parietal agyria - pachygyria. similarly, developmental delay, focal or generalized seizures, and infantile spasms have been described in the literature. the typical tigroid pattern on axial mri or leopard pattern on the sagittal plane is marked by symmetric diffuse high signal intensity on t2-weighted images in the cerebral white matter with radially oriented stripes of low signal intensity. it has been reported classically in disorders of myelin formation such as metachromatic leukodystrophy (mld) and pelizaeus merzbacher disease (pmd), where it results due to sparing of dysmyelination along the venules. the hallmark of imaging in mld is symmetrical dysmyelination in the periventricular white matter and centrum semiovale with sparing of the subcortical white matter. mri in pmd shows diffuse hyperintensity in the central white matter of the cerebral hemispheres, cerebellum, and brain stem along with reduction in white matter volume. similar pattern has also been reported in globoid cell leukodystrophy and infantile gm1 gangliosidosis in which it is less prominent. ad is another disorder of white matter due to mutations in the gene encoding for the glial fibrillary acidic protein resulting in progressive motor and mental retardation, seizures, and megalencephaly. lowe (oculocerebrorenal) syndrome is an x - linked recessive disorder characterized by cognitive developmental delay, congenital cataract, glaucoma, and fanconi syndrome. the tigroid pattern has also been reported in lissencephaly with cerebellar hypoplasia (lis - ch) by kono., who speculated that the reln gene which is mutated in lis - ch may be involved in white matter formation and that the tigroid pattern of the white matter may be associated with the heterogeneity of myelination as seen in pmd and mld. there is a clear cut evidence that limited axonogenesis begins during neuronal migration, and earliest stages of white matter formation consist primarily of radial glial fibers and the neurons migrating on them. nevertheless, cortical migration and myelination abnormalities are not mutually exclusive disease processes, and it has been clearly pointed by researchers that a gene which is responsible for neuronal migration may also be involved in white matter formation. hence, we postulate that the cause of defective myelination as seen in our case may also be due to some defect in a particular gene and subsequently defective protein formation which possibly has a role in white matter formation apart from playing a role in neuronal migration. furthermore, a defect encompassing radial glial fibers and the neurons migrating on them may also hypothetically affect earliest stages of white matter formation simultaneously leading to both these disorders. although we noted both, tigroid - like stripes or reverse tigroid pattern on axial mri sequence and leopard - like pattern on sagittal sequence, the tigroid - like stripes on axial mri were formed by radial hyperintensities on a normal background white matter and leopard - like pattern was formed by scattered dots in form of hyperintensities on a normal background white matter as opposed to small scattered foci of normal white matter signal intensity within the dysmyelinated white matter, as seen in mld. so, our findings may be referred to as tigroid - like stripes or reverse tigroid pattern. presentation of agyria - pachygyria, a lissencephaly spectrum of disorders can be varied including normal head circumference or microcephaly, developmental delay, facial dysmorphism, seizures, and mental retardation. mri of the brain is thus of immense value in identifying the exact nature and extent of the disease and also helps in delineating further plan of management. the tigroid pattern on axial mri and leopard appearance on sagittal mri is classically reported in disorders of myelin formation such as metachromatic leukodystrophy and pelizaeus merzbacher disease. nevertheless, these are not always sine qua non of disorders of dysmyelination, as tigroid - like stripes or reverse tigroid pattern can be found in disorders of neuronal migration as well. in future, the presence of these signs on mri brain may be helpful in keeping this possibility in mind, especially in cases of mild pachygyria which can be occasionally missed otherwise. | pachygyria is considered a subtype of lissencephaly which, in turn, is a spectrum of disorders caused by abnormal neuronal migration. clinical presentation in this disorder may be varied including microcephaly, developmental delay, facial dysmorphism, seizures, and mental retardation. magnetic resonance imaging (mri) of brain identifies the exact nature and extent of the disease and helps in delineating further plan of management. a tigroid pattern on axial mri scan and leopard pattern on a sagittal plane has been classically reported in disorders of myelin formation such as metachromatic leukodystrophy and pelizaeus merzbacher disease. we present here a case of pachygyria who presented to us with some atypical features including tigroid - like stripes and leopard - like pattern on mri brain which has not been reported in the medical literature previously. |
the 20 century witnessed a myriad of advances in technology, which have found several applications in the field of medicine. video technology was one of the most important factors that enhanced the ability of surgeons to perform complex laparoscopic surgeries. the advantages of laparoscopy were realized in adult surgeries long before its acceptance in pediatric surgery. the reasons for initial non - acceptance included lack of suitable equipment for the pediatric patient, uncommon need for cholecystectomy and perhaps the traditionally smaller incisions in pediatric patients making the cosmetic benefit of laparoscopy less evident. currently, laparoscopy can now be performed in infants weighing less than 1.5 kg without significant mortality or morbidity. the advent of minimal access technique of laparoscopy marked a new dawn in surgical care of patients. video - laparoscopy allows surgical assistants ; anesthesiologists and nurses to view what the surgeon is doing and to actively participate in the procedure in their respective roles. the other benefits of laparoscopy include less post - operative pain, post - operative morbidity, early recovery and return to normal activity with shorter hospital stay. the cosmetic advantage has played a major role in the acceptance of this type of surgery amongst patients. a developing country like nigeria with a lean health budget and an unresolved primary communicable disease challenge has been slow in joining the trend. there is still a palpable skepticism even among surgeons of the feasibility and relevance of laparoscopy in our environment. albeit pioneering efforts are documented from some parts of the country highlighting the feasibility of this minimal access practice despite immense challenges with a clarion emphasis on local adaptations. to the best of our knowledge, this is the first documented study from the niger delta region to evaluate laparoscopy as a useful tool for management of common surgical abdominal conditions in a developing country. this is a prospective outcome study of all consecutive surgical patients who had laparoscopic procedures in general and pediatric surgery units of our institution - a regional tertiary health facility, from august 2011 to december 2012. patients were selected after clinical, laboratory and radiological evaluation to exclude significant co - morbidities. an american society of anesthesiology class not greater than 2 was an inclusion criterion. in cases of adhesive small bowel obstruction, a non - dedicated theatre suite was used with a combination of privately - sourced and hospital equipment. in the absence of a trained endoscopy nurse or technician, resident doctors in the unit were trained by the operating surgeon on equipment handling and disinfection of laparoscopy instruments. there was non - invasive monitoring of temperature, spo2, blood pressure, respiration, pulse rate and urinary output. there was manual ventilation and no functional capnography. based on surgeon 's preference and presence of previous abdominal scar a closed or open technique visibility was achieved with a 30 10 mm laparoscope mounted on a karl storz (germany) or hawk (china) single - chip camera unit connected to a video monitor. the light source was from either a karl storz 175w xenon cold light or hawk halogen light sources. two or three ports were used in the diagnostic laparoscopies and conventional 3-ports (10 mm optical and two 5 mm working ports) were used for laparoscopic appendicectomy. there was random lateralization or centralization of the optical port i.e. 5 mm working ports at either left iliac and supra - pubic or right lumbar and left iliac fossa respectively to compare ergonomic advantage. tissue retrieval was routinely carried out using the cannula method and scarcely with an endo - bag. the age range was from 2 to 65 years (mean : 32.27 17.86 years). the surgeries included : six laparoscopic appendicectomies, five diagnostic laparoscopies biopsy, one laparoscopic - assisted orchidopexy, two laparoscopic adhesiolysis for adhesive small bowel obstruction and one trans - abdominal pre - peritoneal hernia repair for adult bilateral inguinal herniorrhaphy [figure 1 ]. the patients for laparoscopic appendicectomy had an even sex distribution (male : female 1:1). all were successfully completed irrespective of the position of the optical port [table 1 ]. laparoscopic surgery during study period laparoscopic appendicectomies performed a case of chronic abdominal pains had no pathology observed during diagnostic laparoscopy. the positive findings during the other diagnostic laparoscopies performed are as shown in table 2. operating time was from 40 to 300 min and challenges encountered included power outages, equipment failures and technical difficulties. oral sips of water was commenced within 24 h and patients discharged by 2 day post - operation except for a case of conversion for an intra - abdominal tumor (soft - tissue sarcoma) from uncontrollable bleeding. surgical complications encountered were grade i and ii of the clavien - dindo classification : one wound dehiscence of a 5 mm port site ; two post - operative pyrexia ; one neuropraxia of superficial peroneal nerve and leg edema from pressure effect of stirrup in lithotomy position during evaluation of the pelvis prior to an appendicectomy. a two - center study from the south - eastern part of the country also had laparoscopic appendicectomy as the most performed procedure. this is probably due to the fact that acute appendicitis is a common abdominal pathology and laparoscopic appendicectomy requires basic laparoscopic skills to execute. the lateralization or centralization of the optical port was observed not to have a significant effect on the outcome of laparoscopic appendectomy in the early career of a laparoscopic surgeon since all cases were successfully completed. there was no laparoscopic cholecystectomy due to a low incidence of gall stone disease in our center. the open hasson port technique is favored by many surgeons in view of less likelihood of visceral and vascular injury, however, the closed access method using the veress needle is safe, fast and efficacious even in children of all ages. we routinely used the closed access method in the absence of previous abdominal surgery ; however following access the working ports were inserted under direct vision. a two - port laparoscopic and single port laparoscopic appendicectomy are feasible but these have limitations in conditions of extensive adhesion and gangrenous appendix. there was no perforation of viscus, blood vessel injury nor hemodynamic instability resulting from capnoperitoneum peri - operatively. this conversion rate of 6.7% (n = 1) is similar to 4.1% (n = 1) recorded from 24 laparoscopic surgeries performed in a tertiary health facility in nigeria. comparatively, another nigerian study of 145 laparoscopic surgeries recorded a conversion rate of 0.027% (n = 4). some other challenges encountered during were power outages and lack of trained support staff (endoscopy nurses and technicians). the onus is on the laparoscopic surgeon to acquire an in - depth knowledge of all aspects of the practice from a certified oversea or on - site training to effectively train his support team. it is important to note that the operational cost for laparoscopy is not huge after initial set - up i.e., training of personnel, acquisition of equipment and relevant infrastructure. cost reducing measures adopted included : use of reusable instruments, cannula method for tissue retrieval and the choice of extracorporeal knot placement for ligation. these measures though cheap and effective prolong set - up and operating time. in the face of competing theatre usage the practice of laparoscopic surgery in our environment is feasible and safe despite the numerous, but surmountable challenges. an institutional will to achieve a routine laparoscopy in a resource - poor setting is achievable with adequately trained surgeons, support staff and necessary infrastructure. | background : video - laparoscopic surgery has long been practiced in western countries ; however documented practice of this minimal access surgical technique are recently emanating from nigeria. to the best of our knowledge, this is the first documented study on laparoscopic surgery from the niger delta region.aim:to evaluate the feasibility of laparoscopy as a useful tool for management of common surgical abdominal conditions in our environment.patients and methods : this was a prospective outcome study of all consecutive surgical patients who had laparoscopic procedures in general and pediatric surgery units of our institution from august 2011 to december 2012. data on patient 's age, gender, indication for surgery, duration of hospital stay and outcome of surgery were collected and analyzed.results:a total of 15 laparoscopic procedures were performed during this study period with age range of 2 - 65 years ; mean : 32.27 17.86 years. there were 11 males and four females. six laparoscopic appendicectomies, one laparoscopy - assisted orchidopexy, five diagnostic laparoscopy biopsy, one laparoscopic trans - abdominal pre - peritoneal herniorrhaphy for bilateral indirect inguinal hernia and two laparoscopic adhesiolysis for small bowel obstruction were performed. all were successfully completed except one conversion (6.7%) for uncontrollable bleeding in an intra - abdominal tumor.conclusion:the practice of laparoscopic surgery in our environment is feasible and safe despite the numerous, but surmountable challenges. there is the need for adequate training of the support staff and a dedicated theatre suite. |
copd is a common cause of disability, morbidity, and mortality worldwide and a major global health problem with enormous direct and indirect health care costs.1,2 the worst notice, however, is that the mortality rate (number of deaths per 100,000 persons) for copd in the last 50 years (at least in usa) has progressively increased in both men and women older than 60 years in every 5-year period.3 moreover, among the 20 leading causes of deaths in usa, the only one that has substantially increased the age - standardized relative rank of death in the last 20 years is copd.4 in europe, the situation seems better, and in many european countries, the mortality rate (number of deaths for 100,000 persons) for copd is decreasing, particularly in men.5 however, for 100,000 deaths, the percentage of mortality due to copd is increasing, suggesting that among chronic noncommunicable diseases copd is less effectively treated.5 therefore, according to this trend, it is estimated that copd will become the third worldwide cause of mortality in 2030.6 of course, there are many causes for this, but among them the possibility that the recommended diagnostic and therapeutic approaches to copd were less effective than what they could be should be seriously considered. the most disseminated definition says that copd is a disease.1 in fact, d in the copd acronym stands for disease. it must be acknowledged, however, that copd is recognized in the definition as an obstructive functional defect associated with a chronic immune inflammatory process, occurring in the lungs of susceptible individuals with known risk factors, in the absence of other causes. since the 1960s, based on pathological findings, it has been clear that similar functional disorder could arise from different diseases, essentially two, one originating from small airways and the other from lung parenchyma.7 today, we know that chronic bronchiolitis, which is a progressive, proliferative, and fibrosing pathology of the membranous and terminal bronchioli due to persistent inflammatory, immune, and remodeling process, is by far the most common disease leading to copd.8 in contrast, panlobular emphysema, which is a destructive and hyporegenerative pathology of the alveolar walls with some autoimmune features, is a rare disease that may originate among individuals with severe alpha-1-antitrypsin deficiency and heavy smokers and also leads to copd.9,10 moreover, chronic bronchiolitis can be frequently associated with centrilobular emphysema, when the inflammatory process involves the respiratory bronchioli with subsequent destruction of the secondary lobule, from the center to periphery. this may configure the presence of mild (5% of the lung volume with laa < 950 hu, showing polygonal- or irregular - shaped laa with ill - defined border), or confluent (showing irregular - shaped laa with ill - defined border coalescent with each other) centrilobular emphysema.11 actually, centrilobular emphysema does not occur without chronic bronchiolitis, and there is growing evidence that injury of terminal bronchioli would precede the enlargement of centrilobular spaces.12 in a cross - sectional high - resolution computed tomography analysis, it is often impossible to distinguish between panlobular emphysema and confluent centrilobular emphysema, the only distinctive clue being the prevalence of destructive lesions in the lower lobes in the former. at the stage of severe - to - very severe airflow obstruction, fibrosing chronic bronchiolitis with (same degree of) centrilobular emphysema is the pathological condition most frequently encountered. although not definitely proved, isolated fibrosing chronic bronchiolitis should be the most common form of copd at earlier stages of airflow obstruction. in all circumstances, however, a progressive reduction in maximal expiratory and inspiratory flow rates may develop because of an increase in airway resistance and/or a decrease in pulmonary static elastance. diminution of the elastic recoil pressure can augment the expiratory flow resistance by distortion and collapse of the small airways because of reduction in tethering forces. this phenomenon is amplified by the loss of airway parenchyma interdependence due to progressive disappearance of alveolar attachments. with the above - mentioned background, in a patient with copd, the priority should be to define the prevalent underlying disease. generally, clinical, radiological (chest x - ray), and adequate functional assessments are sufficient to achieve this (tables 1 and 2), but sometimes this task requires a more extensive work - up including high - resolution computed tomography lung scan. many lines of evidence suggest yes. based on different underlying disease, namely prevalent chronic bronchiolitis versus prevalent pulmonary emphysema, in patients with copd the effect of anti - inflammatory treatments (inhaled corticosteroids and/or phosphodiesterase 4 inhibitors, for instance) on top of long - acting bronchodilators is different in terms of improvement in function, symptoms, quality of life,13,14 and prevention of exacerbations.1517 in addition, the functional decline is differently affected by treatment,18,19 the all - cause and respiratory mortality is different in long - term follow - up,20 and finally, the clinical phenotypes and some comorbidities tend to cluster differently.21,22 after that it is mandatory to stage the copd severity, which can not rely only on forced expiratory volume in 1 second (fev1) and, as recently suggested, on symptoms, requiring instead a multidimensional approach not only to have a prognostic evaluation but also to establish the level of initial treatment and timing of monitoring. presently, the updated bode (body mass index, airflow obstruction, dyspnea and exercise capacity) index appears the most suitable tool for such purpose,23 taking into account also the distribution of the symptoms during the day and the gas exchange abnormalities. prospectively, determination of some plasma biomarkers, such as c - protein and fibrinogen, might be useful.24 all of this information must be weighted for the age of the patient. subsequently, an annual history of copd exacerbations, as much as is possible to detail, must be performed because a high frequency of copd exacerbations is an undisputable marker of disease severity that needs to be aggressively controlled. it is of limited utility to simply count the previous copd exacerbations, and every effort should be made to recognize the prevalent type, and so choose the best available strategy to prevent them.25 the various comorbidities should finally be investigated and graduated by severity in order to treat adequately those known to have a major impact on mortality in patients with copd.26 the pharmacological baseline treatment of stable copd has been widely based on the severity of airflow obstruction and recently also of chronic symptoms and on the number of exacerbations in the previous year.1 with the interesting but somehow confusing exception of the spanish guidelines,27 these recommendations do not take into account the underlying prevalent disease that should be treated and the future risk. moreover, the exacerbations are just enumerated with no attempt to define their etiology, always suggesting the stereotyped use of inhaled corticosteroids (ics) on top of bronchodilators when they are two or more in the previous year (or one severe, requiring hospitalization).1 in contrast, the therapy must be first tailored on the prevalent disease underlying copd, independent from the degree of fev1 reduction and chronic dyspnea score, and only after that, according to the severity of the disorder (and age of the patient), to establish the level of the treatment in order to freeze, when possible, and not to follow the underlying pathological process, running after it (figure 1). we do not need expensive randomized controlled trials (rcts) anymore where thousands of patients with copd having different underlying diseases and having different severity of airflow obstruction are enrolled all together, trying to understand the effect of a same treatment. on the contrary, more valuable information about any given targeted intervention might be collected, studying small numbers of well - selected patients with copd, with same underlying disease, similar clinical phenotypes, same degree of airflow obstruction and/or bode index, and similar age range, for an adequate span of time. it can be speculated, but it must be proved with well - designed prospective studies that this approach will be more effective in terms of lung function decline and patient - related outcomes, particularly if applied in the initial phases of copd, implying an early diagnosis of chronic airflow obstruction and a better characterization of the underlying disease. perhaps, we would learn that more aggressive treatments have to be implemented in the earlier stages of copd, instead of using them in the more advanced ones as recommended today, to obtain the best possible outcomes. this has been suggested by the post hoc subgroups analysis in the previous interventional large studies on copd where improvement in symptoms, exacerbation frequency, fev1 annual decline, and all - cause mortality was demonstrated only for patients with copd stages ii and iii (global initiative for chronic obstructive lung disease).28,29 the goal should be to have patients dying with copd (when allowed by the underlying disease, essentially chronic bronchiolitis) and not because of copd. the prevention of copd exacerbations is a point of paramount importance in the management of copd that needs a completely different approach because it can not be addressed simply with the baseline pharmacological treatment. we know a lot about copd exacerbations, even if their diagnosis, essentially based on worsening of chronic symptoms reported as relevant by the patients, is presently still based on the exclusion of other diseases. to suffer from two or more copd exacerbations or from one severe copd exacerbation leading to hospitalization in the previous year is without doubt a marker of copd severity, independent from the underlying disease, degree of airflow obstruction, and entity of symptoms or bode index, even if lower fev1 is associated with higher risk of frequent and more severe exacerbations. although the probability of having a new copd exacerbation is greater in those patients with copd who previously experienced copd exacerbations (so - called frequent exacerbators),30 such status, with few exceptions,3133 can not be identified as a distinct phenotype, rather as a condition requiring more strict social and medical attention. in fact, it is quite easy to shift from a frequent exacerbator to a nonfrequent exacerbator and vice versa,34 sometimes without an obvious reason, but often clearly because of a more adequate and comprehensive treatment.35 given the prognostic importance of copd exacerbations,36 however, we can not be limited to simply counting exacerbations ; we must learn how to consistently recognize the prevalent type in a single patient. such an approach is crucial to prevent more effectively the copd exacerbations using more tailored and specific therapies (figure 2). some plasma, blood, or sputum biomarkers have been shown to be associated with high sensitivity and specificity to a prevalent clinical type of copd exacerbation : eosinophilic, infectious either virus or bacteria associated, or pauci - inflammatory, due to several possible causes that have to be identified.37 more interestingly, these specific biomarkers tend to be detectable also in stable conditions, at least in eosinophilia- and bacteria - associated exacerbations, allowing an easier identification of the most likely future pattern of copd exacerbations.37 thus, when possible, each prevalent type of copd exacerbation in a given patient who is a frequent exacerbator should be appropriately prevented by specific treatments that can not be invariably the use of high - dose ics, as presently recommended.1,27 long - acting bronchodilators and mainly ultralong - acting bronchodilators (both beta-2 agonist and antimuscarinic drugs) are able to prevent up to 30% of copd exacerbations when given alone38 and possibly more when given in combination.39 ics are very useful to prevent the eosinophilic exacerbations,1517 but it is difficult to see how ics can prevent infectious exacerbations, or even noneosinophilic and noninfectious exacerbations, apart from strengthening the action of bronchodilators such as long - acting beta-2 agonists (laba) in some circumstances.38 in patients with copd having bronchiectasis and chronic bronchitis, copd exacerbations are often infectious and bacterial in nature and must be prevented with antibiotic prophylaxis, at least in the winter season. there is strong evidence that macrolides may significantly reduce copd exacerbations,4042 suggesting that this kind of exacerbation can be prevented in some clinical phenotypes of patients with copd having high risk of bacterial colonization or chronic infections. presently, only adequate immunization against influenza virus can be offered for viral exacerbations in patients with copd, and we urgently need the same effective tools for rhinovirus infection, the most common cause of virus - associated copd exacerbation. all other types of copd exacerbations (noninfectious and noneosinophilic) deserve accurate work - up and specific preventative treatment should be applied when the cause is recognized (figure 2). many of these exacerbations, in fact, can be largely avoided with targeted approaches.38,4345 for instance, in the presence of patients with copd having presumable high oxidative stress (those who continue smoking, those with chronic bronchitis, and those with high exposure to environment pollution), an adequate antioxidant support might significantly reduce copd exacerbations.46,47 finally, a strict adherence to baseline chronic therapy should be assured throughout a strong relationship between patients with copd and their physicians and caregivers because this aspect is crucial to obtain improved control of copd exacerbations and their consequences.48 in summary, in patients with copd, first treat as soon as possible the underlying disease, aiming to freeze the disease and halt progression, ready to implement treatment (essentially with different bronchodilators, including theophylline) to control symptoms and improve lung function, exercise tolerance, and quality of life, if necessary. second, look at the exacerbations of copd, if they are frequent (more than one in a year), define their prevalent phenotype (eosinophilic, bacterial, or due to other causes) and treat to prevent accordingly. | copd is a common cause of disability, morbidity and mortality worldwide and a major global health problem with enormous direct and indirect health care costs. different reasons can be advanced to explain it, but among them the possibility that the recommended diagnostic and therapeutic approaches to copd were less effective than they could be, should be also considered. the pharmacological baseline treatment of stable copd has been widely based on the severity of airflow obstruction and recently, of chronic symptoms and on the annual number of previous exacerbations. these recommendations do not take into account the underlying prevalent disease that should be treated and the future risk. our suggestion is that the therapy must be firstly tailored on the prevalent disease leading to copd, independently from the degree of fev1 reduction and chronic dyspnea and only after that, according to the severity of the disorder (and age of patient), to establish the level of the treatment in order to freeze, when possible, and not to follow the underlying pathological process, running after it. moreover, given the relevance of exacerbations in the natural history of copd, greater effort should be placed on recognition of their prevalent type in frequent exacerbators and to prevent them using more tailored and specific treatment. |
over the past decade it has been increasingly acknowledged that the understanding of metalorganic interfaces is crucial for further improving organic (opto)electronic and single - molecule devices. in this context, adsorbing organic acceptors or donors onto electrodes provides a convenient tool for modifying metal work functions and consequently for tuning the alignment between the metal fermi level and the organic semiconductor states. a prototypical organic acceptor is 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (f4tcnq) (see right inset in figure 1). f4tcnq is a highly promising candidate for decreasing hole - injection barriers and has been thoroughly investigated in several spectroscopic and theoretical studies. for efficiently decreasing electron - injection barriers, doubly reduced viologens have recently been suggested as particularly potent materials. this is especially true for 1h,1h-[4,4]bipyridinylidene, hv0 (see left inset in figure 1), for which a very strong charge transfer to the au(111) surface has been predicted theoretically.(17) this has later been confirmed experimentally for the more stable doubly methylated derivative 1,1-dimethyl-[4,4]bipyridinylidene. this material has been found to decrease the work function of a au(111) surface by 2.2 ev(12) and to concomitantly decrease also the electron injection barrier into subsequently deposited organic electron transport layers such as c60 and alq3.(12) comparison of the molecular orbital occupation of f4tcnq on ag(111) (circles), and hv0 on au(111) (squares). the filled (open) symbols represent molecular orbitals that are occupied (unoccupied) in the isolated molecules. the right y - axis representing the occupations for f4tcnq orbitals has been shifted by a constant to ease the comparison. the chemical structures of hv0 (left, 1h,1h-[4,4]bipyridinylidene) and f4tcnq (right, 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane) are shown as insets. a more in - depth discussion of these data can be found in refs (16) and (17). for both systems examined throughout this study, f4tcnq on ag(111) and hv0 on au(111), the induced work - function modifications have been found to primarily originate from interfacial charge transfer. for f4tcnq it is dominated by a nearly complete filling of the lumo and for hv0 by a partial emptying of the homo. a more in - depth understanding of the interfacial charge - transfer processes can be obtained from the density of states (dos) projected onto the states of the monolayer without the metal present (i.e., essentially onto the orbitals of the noninteracting molecules). this quantity has been referred to as the molecular orbital dos.(18) integrating the individual molecular orbital projected doss up to the fermi level yields the occupation of the associated molecular states in the interacting system. the respective results for f4tcnq on ag(111) and hv0 on au(111) are shown in figure 1 ; an in - depth discussion of the molecular orbital doss of these systems can be found in refs (16) and (17). the underlying process that allows for fractional occupations is a hybridization of the molecular states with the metal states and the filling of only part of the resulting hybrid orbitals. the reduced occupation of the homo in hv0 on au(111) and the nearly complete filling of the molecular lumo for f4tcnq on ag(111) are clearly visible ; for the latter, also a back - donation of electrons from deeper lying f4tcnq orbitals to the ag(111) surface is visible as a reduced occupation of the homo-12 to the homo-9. these states have been identified as being localized on the terminal cn substituents, which is a first indication for the strong contribution of these groups to the metalmolecule bonding. their important role for the interaction can, furthermore, be deduced from the strong molecular distortions occurring upon adsorption that have been observed by x - ray standing wave experiments for f4tcnq on cu(111) and by density functional theory - based modeling on all coinage metals : the f4tcnq molecule, which is planar in gas phase, bends in a way that the terminal cn groups are closer to the surface by several tenths of an angstrom than the central ring (with the absolute magnitude depending on the substrate metal). a bent adsorption geometry has recently been confirmed by scanning tunneling microscopy experiments on tcnq (the nonfluorinated analogue to f4tcnq) on cu(100).(19) a nonvanishing (albeit much smaller) distortion has been calculated also for hv0 on au(111).(17) in both cases, the bending gives rise to an intramolecular dipole which, in addition to the above - discussed charge transfer, determines the induced change in the substrate work function. the latter, amounting to + 0.85 ev for f4tcnq on ag(111) and 1.21 ev hv0 on au(111), is another indication for a strong metalmolecule interaction. strong chemisorption usually leads to the establishment of (partial) bonds between the adsorbates and the substrate that goes hand in hand with a change of the bonding pattern within the adsorbed molecule. a technique to analyze these processes based on theoretical modeling was first introduced by hoffmann. they defined a crystal orbital overlap population (coop) to interrogate the bonding and antibonding behavior of the atomic orbitals and their overlap with the metal bands resolved on the energy scale. in the present contribution, we describe what can be learned from such atomic orbital - based overlap populations about the covalent contributions to the bonding between organic adsorbates and metal surfaces by performing an in - depth analysis of the f4tcnq / ag(111) and hv0/au(111) interfaces. moreover, we expand the analysis tool to bonding and antibonding contributions arising from the interaction between certain molecular orbitals and the metal. these analyses allow identification of the individual atoms, the metal bands, and the molecular orbitals dominating the bonding process. all calculations presented here are based on optimized adsorption geometries obtained from the plane - wave density functional theory (dft) code vasp, version 4.6. details on the applied methodology can be found in the respective papers dealing with the electronic structure of f4tcnq and hv0 adorbates. here, the same unit cells as in those contributions are used, i.e., for f4tcnq on ag(111) and (3 3 5) for hv0 on au(111) ; the respective structures are included in the supporting information. to ease the projection onto atomic and molecular orbitals, we used the vasp geometries as an input to perform a single self - consistency cycle in the atomic orbital based code siesta(24) version 2.0. there, the pbe exchangecorrelation functional, norm - conserving pseudopotentials based on relativistic calculations using the troulliermartins scheme, as well as the same (3 3 1) monkhorstpack(25)k - point grids (as in the vasp calculations) were used. a double- polarized (dzp) basis set with 15 atomic orbitals (aos) for the metal atoms, 5 aos for the hydrogen atoms, and 13 aos for all other atoms has been used as implemented in the siesta code. test calculations applying a single- polarized (szp) basis as implemented in siesta(24) and a user - generated(28) triple- polarized (tzp) basis with 21 aos for the metal and 17 for the molecule (6 for h) were made to test the influence of the basis - set size on the obtained results. the single- calculations yielded equivalent trends but resulted in some quantitative deviations, as discussed in the supporting information. in this context it should be noted that, naturally, well - known shortcomings of density functional theory will to some extent adversely impact the full quantitative validity of the results in any such calculations. to minimize their role, we have, however, carefully chosen the test systems so that their impact should be comparably small. moreover, they affect in no way the main purpose of the present paper, which is showing the versatility of orbital overlap populations for describing metalmolecule bonding. they can, of course, also be applied in future calculations that contain corrections to the flaws of current (semi)local dft implementations. the above - mentioned shortcomings include the neglect of van der waals interactions by (semi)local functionals, which can lead to an overestimation of the bonding distance. during the past few years, several remedies to that problem have been discussed.(29) here, to minimize its impact, we chose to study strongly bonded systems, where at least for f4tcnq on cu(111) a good agreement between theory and experiment has been observed.(15) also the studied metal substrates were deliberately chosen among the coinage metals, as the charge transfer and interactions between a donor and au (with the largest work function) and an acceptor and ag (with the smallest work function) are particularly strong. other effects including the lack of derivative discontinuity of the functionals,(30) the occurrence of self - interaction errors,(30) and improperly captured correlation - screening at the metalorganic interface affect the relative alignment between the adsorbate and the metal states. in our test systems, this does at least not impact the positions of the frontier orbitals, for which it has been established theoretically and experimentally that they are pinned at the fermi energy. also the positions and widths of the metal bands are known to be reasonably well described using relativistic pseudopotentials as in our calculations. to elucidate the details of the bonding process, three different types of quantities are used : (i) densities of states (doss), (ii) orbital overlap populations (oops), and (iii) total overlap populations (tops). (i) densities of states in various forms are frequently applied to analyze interactions between adsorbate layers and metals. they give the numbers of states per energy interval either of the total system or projected onto a certain region of space (e.g., molecular density of states, metal density of states) or onto a volume element (local density of states). alternatively, the projection can also be onto individual molecular orbitals of the noninteracting adsorbate (molecular - orbital - dos), as discussed in introduction (cf. figure 1). a detailed dos - based analysis of the hv0 and f4tcnqmetal bonding can be found in refs (16) and (17). (ii) orbital overlap populations (oops) for analyzing bonds have originally been introduced by hughbanks and hoffmann(20) in the form of a crystal orbital overlap population (coop). its general definition is given by here, x and y denote two groups of atoms and m and l the corresponding orbitals representing the basis set ; the cimk denote the linear combination of atomic orbital (lcao) coefficients for state ik and the smlk indicate the overlap matrices, where i is the band index. g is a line shape function, which in our case is a normalized gaussian. one can distinguish between different types of oops depending on which orbitals are included in the above summation ; furthermore, one can sum over oops between different pairs of atoms or use different types of consistent basis sets, with respect to which the above expansion coefficients are defined. the latter results in different values for the s and c s in eq 1. first, we will use atomic orbitals in line with the work by hoffmann.,(18) which contains also a number of examples for the application of such oops to instructive test cases. subsequently, we will combine the molecular orbitals of the isolated monolayer and the atomic orbitals of the metal slab to create a new basis(36) that is particularly useful for analyzing the interaction in a chemically intuitive way. a schematic illustration to explain this kind of oop is provided in figure 2, which shows how a molecular orbital interacts with a metal band. among other effects those that are of bonding character are typically located at energies below the original orbital, while antibonding states are located above. hence, the oop of the derived hybrid band is positive at low energies, cuts through zero at the position of the molecular orbital, and is negative at higher energies. this characteristic pattern is observed here for all molecular orbital - related oops (see section 4.3 and the corresponding supporting information), although the detailed shapes of the oops are usually more complex than in the schematic picture in figure 2. note that a very strong interaction with the substrate, such as the formation of covalent bonds, could result in deviations from this picture, if it causes a rehybridization of the states within the molecule. this is because in such a situation, the assumption of a single broadened molecular orbital is no longer appropriate. schematic illustration of the interaction between a molecular orbital and a metal band giving rise to the metalmolecular orbital overlap population (oop). the rightmost panel shows the oop integrated over energy with the value of that curve at ef corresponding to the total overlap population (top) associated with that orbital. (iii) to analyze the total contribution of the interaction between x and y to the bonding in the investigated system, a total overlap population (top) can be introduced. it is defined as the integral over the corresponding oop up to the fermi energy ; i.e., it is the integral over the occupied this quantity has been shown to scale with the bond order(18) and, consequently, is closely related to the bond length and the bonding strength. tops can be calculated for all of the above - described oops and will be shown in the following whenever they provide additional insight. they will simply be denoted by replacing oop with top in the quantities described in table 1. to avoid confusion, we will refrain from an excessive use of acronyms ; i.e., we will write the full names of the above quantities apart from the acronyms oop and top. the focus of the current manuscript is on using various types of overlap populations for analyzing the metalmolecule interactions. therefore, in the main manuscript, we will refrain from discussing changes in the intramolecular oop that occur due to adsorption - induced geometric deformations of the molecules. the latter can, however, be found in the supporting information. as a first step, the metalmolecule oop will be used to investigate the bonding between the molecules and the metal. the metalmolecule oop of hv0 on au(111) is shown as a black, solid line in figure 3. its shape around the fermi energy is reminiscent of the classical situation described by hoffmann for co on ni(100)(18) that is also sketched in figure 2 : the lower energy part of the band adopts a bonding and the upper energy part an antibonding character, as seen in the region marked by an ellipse in figure 3. as the homo - derived band of the hv0 layer is only partially occupied due to the charge - transfer (cf., introduction), it is not surprising that the fermi energy depicted as a vertical dash - dotted line cuts right through the bonding to antibonding wave. at this point, only the significantly larger magnitude of the bonding feature might appear somewhat surprising. we will return to that and also identify the origin of the strongly bonding features at 0.6 ev and 3.6 ev, when discussing the molecular orbitalmetal oop for this system. the dash - dot - dot line represents the metalmoleculepart oop where the part of the molecule consists of only the outer secondary amine groups with their hydrogen atoms. the fermi edge is set to zero, and the horizontal line divides bonding (positive) and antibonding (negative) areas. the ellipse marks the area around ef that is discussed in the main text. to identify the parts of the molecules that most strongly contribute to the metalmolecule interaction, metalmolecule part oops the most relevant is the one associated with the two outer secondary amine groups together with their hydrogen atoms, the metalmoleculeamine oop. it is shown as a dash - dot - dotted line in figure 3 and reproduces the positions of the main features of the metalmolecule oop. this indicates that the secondary amines are to a large extent responsible for the covalent contribution to the bonding between hv0 and au. this can at least to some degree be associated with the calculated slight bending of the these groups toward the surface.(17) as the bending of the f4tcnq molecules adsorbed on ag(111) is much stronger,(16) it is advisable to first study the hypothetical case of a planar adsorbate (see structure in the top of figure 4) and only as a second step investigate the impact of bending. the metalmolecule oop for planar f4tcnq on ag(111) with the central ring at the same adsorption distance as in the fully relaxed structure (i.e., 3.21 above the metal)(16) is shown in figure 4. a side view of the adsorbed molecule with the top metal layer is shown above the graph. the region around ef looks qualitatively similar to hv0, with a bonding to antibonding transition., the features around ef are related to the former lumo which, due to the accepting nature of f4tcnq, becomes slightly occupied. however, the amount of charge transfer is significantly lower than for the fully relaxed, bent adsorption geometry where a nearly complete filling of the lumo has been observed as discussed in the introduction section and in ref (16). metalmolecule oop for the hypothetical case of a planar f4tcnq monolayer adsorbed on ag(111). the fermi edge is set to zero, and the horizontal line divides bonding (positive) and antibonding (negative) areas. on top of the graph, a side view of an adsorbed planar f4tcnq molecule is shown together with the top metal layer. the bending down of the terminal cn groups has dramatic consequences for the metalmolecule oop. the result for the fully relaxed (i.e., strongly bent) adsorption geometry is shown in figure 5 as a solid black line. compared to the planar case in figure 4, the whole region around ef has become antibonding, and there is a strong negative peak at 3.3 ev. these are more than compensated by strongly bonding oop contributions between 4.0 ev and 7.0 ev, which results in an increase of the corresponding total overlap population from 0.09 for the planarized adsorbate layer to 0.26 for the fully optimized case. it should be noted that the y - scales in figures 3 and 4, on the one hand, and figure 5, on the other hand, differ by a factor of 10 (!). metalmolecule oop of f4tcnq on ag(111), black line, and metalmoleculepart oop of only the cn atoms of the molecule with the metal, red - shifted curve. the fermi edge is set to zero and the horizontal line divides bonding (positive) and antibonding (negative) areas. on top of the plot, a side view of an adsorbed fully relaxed f4tcnq molecule is shown together with the top metal layer. considering that the main difference between the planar and the fully optimized geometry is that in the latter the cn groups are bent down by 1.23 (position of the n - atoms), it is reasonable to assume that they must also be responsible for the huge changes in the oop. this can be checked, by calculating the metalmoleculecn oop in which only the interaction between the n and c atoms of the four terminal cn groups with the metal are considered. the corresponding oop curve is shown as a dash, dot - dotted gray line in figure 5. to be visible in the plot, the curve had to be shifted because the result is virtually identical to the full metalmolecule oop. this confirms the leading contribution of the cn groups in the covalent part of the metalmolecule bonding in the fully relaxed geometry. this finding also implies that the prominent features in figure 5 are related to molecular orbitals largely localized on the cn groups. a more in - depth analysis of the origin of the peaks addressing this question will be provided in section 4.3. in the next step, the metals bandmolecule and metald bandmolecule oops for hv0 on au(111) are shown in the top part of figure 6. the corresponding tops are contained in the bottom part of figure 6 for an extended energy range. (top) metalmolecule oop (black, solid line) and metals, d bandmolecule oop of hv0 on au(111) (top graph) and integrated metalmolecule and metals, d bandmolecule oop of hv0 on au(111) (bottom graph). the red, dashed line represents the metals - bandmolecule interaction, the green, dash, dot - dotted line the metald - bandmolecule interaction. the vertical dash, dot - dotted line represents the fermi energy, which is set to zero. in this context it needs to be mentioned that in our calculations, the au(111) d - band starts around 2 ev below ef and has a width of 4.5 ev consistent with the calculations in ref (37). interestingly, all strong d - band contributions to the overlap population have a positive sign below the fermi edge. the metals bandmolecule oop starts contributing much lower in energy (approximately 22 ev below ef, which is attributed to the hybridization of low - lying molecular orbitals with parts of the metal s - band at higher energies). its contributions to the overlap population are essentially bonding below the fermi edge as well. as a result, about 2/3 of the metalmolecule top originates from the bonding to the au s - electrons. qualitatively, a different pictures arises when studying the influence of the metal bands for the bonding process of f4tcnq on ag(111). in ag 3 ev (i.e., 1 ev lower than for au) and the d - bandwidth as ca. 4 ev. here, the interaction with the ag d - band plays a strong role for the bonding oop between 7 and 4 ev and even dominates the antibonding feature between 4 and 2 ev, as shown in figure 7 (top), i.e., the interaction of the bent - down cn groups with the metal has a strong contribution from the ag d - orbitals. the integral over the metaldmolecule oop in figure 7 (bottom) reveals that the bonding and antibonding contributions largely cancel. in fact, in contrast to hv0 on au(111) with its small positive metaldmolecule top, here the antibonding d - band contributions slightly outweigh the bonding ones, resulting in a small negative metaldmolecule top. this is far outweighed by the contribution from the s - band, for which especially the interaction with the cn groups is nearly exclusively bonding (figure 7 (top)). (top) metalmolecule oop (black, solid line) and metals, d bandmolecule oop of f4tcnq on ag(111) and (bottom) integrated metalmolecule (black, solid line) and metals, d bandmolecule oop of f4tcnq on ag(111). the red, dashed line represents the metals - bandmolecule interaction, the green, dash, dot - dotted line the metald - bandmolecule interaction. the vertical dash, dot - dotted line represents the fermi energy, set to zero. finally, the molecular orbitals need to be identified, whose hybridization with the metal states gives rise to the various bonding and antibonding features in the above oops ; i.e., we will discuss the metal molecular orbital as in the present adsorbates, one frequently encounters groups of orbitals with similar character, we will group them in the following discussion for the sake of clarity. the results for the oops associated with the most relevant (groups of) orbitals for hv0 on au(111) are displayed in the left column of figure 8. it is shown that the strongly bonding oop of hv0 on au(111) peaking at 3.6 ev is mostly a superposition of contributions from homo-3 and homo-2 derived states. interestingly, an integration of the metalhomo-2 and metalhomo-3 oops shows that for these orbitals this strongly bonding peak is completely compensated by antibonding contributions closer to ef (see right column of figure 8). also for the homo-1 (shown only in the supporting information), bonding and antibonding features cancel and the top becomes zero. of all occupied orbitals considered in figure 8, only the metalhomo top does not vanish. the corresponding oop, i.e., the metalhomo oop is also responsible for the bonding to antibonding transition at ef. however, it can not explain its asymmetry as well as the magnitude of the peak at 0.6 ev. as shown in figure 8, these features can only be rationalized by the partial occupation of unoccupied orbitals, namely the lumo+3 and lumo+4 (cf. this is surprising, as the molecular orbital dos of these two orbitals in figure 1 shows that they do not bear significant electron density because they are filled to only about 2%(17) and, therefore, have received no attention in previous studies.(17) an orbital representation of these two states reveals the orbitals -character and their large amplitudes around the secondary amine parts of the molecule. exactly these parts of the molecule have been identified above to be of particular importance, when comparing the metalmoleculeamine ooo to the metalmolecule oop. thus, as a net effect, the hybridization between metal states and unoccupied molecular states contributes more than 1/3 of the overall metalmolecule top, which is surprising for the adsorption of a strong electron donor. the rest of the top stems from deeper lying orbitals that are not included in figure 8. left column : metalmolecular orbital oop analysis for hv0 on au(111). the most significant (groups of) molecular orbitals and their oops with the metal are shown. from top to bottom : homo-2 plus homo-3, homo, lumo+3 plus lumo+4, and the total moleculemetal oop ; central column : shapes of the homo-2, the homo, and the lumo+3 as representative examples ; right column : corresponding integrated oops. tops and oops for all individual orbitals can be found in the supporting information. oop for f4tcnq on ag(111) confirms the notion that deep lying orbitals with large amplitudes on the cn groups are crucial for the development of the strong bond. introduction) are responsible for the most strongly bonding feature at about 6.3 ev, as shown in the left column of figure 9. however, they do not completely explain the bonding to antibonding transition at somewhat higher energies, which is mostly due to the contributions from the hybrid states formed by the homo-8 to homo-5 and the metal. albeit the details of the shapes of the homo-12 to homo-9, the homo-8/homo-7, and the homo-6/homo-5 orbitals differ (see central column in figure 9), they are all strongly localized on the terminal cn group. left column : metalmolecular orbital oop analysis for f4tcnq on au(111). the most significant (groups of) molecular orbitals and their oops with the metal are shown. from top to bottom : sum of homo-12 to homo-9, sum of homo-8 to homo-5, lumo and the total molecule - metal oop. note that the scale for the metallumo oop differs from the others by a factor of 10. central column : shapes of the homo-9, the homo-7, the homo-5, and the lumo shown as representative examples ; right column : corresponding integrated oops. the lumo - derived oops display a bonding to antibonding transition at 0.2 ev (similar to the homo - derived feature giving rise to a bonding to antibonding transition at about 1.2 ev ; see supporting information). compared to the oops related to homo-12 to homo-5, the respective contributions are, however, so weak that they are hardly visible in the metalmolecule oop. an analysis of the corresponding tops displayed in the right column of figure 9 shows that, not unexpectedly, the dominant contribution to the covalent part of the metalmolecule bonding comes from the homo-12 to homo-9 contributions. finally, the relation between the metalmolecule oop and the dos projected onto the molecular region need to be discussed. here, one has to keep in mind that the prerequisite for a nonvanishing metalmolecule oop in a certain energy region is that there metalmolecule hybrid orbitals must exist, i.e., the region will typically be close to the respective bands of the isolated molecular layer and metal slab. moreover, the orbitals on the molecule and the metal need to overlap, which finds its mathematical manifestation in the overlap integral in eq 1. keeping that in mind, the similarities and differences between the molecular dos and the metalmolecule oop can be well understood and are shown in figure 10 for the example of f4tcnq on ag(111). normalized metalmolecule oop (black solid line), dos projected onto the molecular region (green dash - dotted line), and dos projected onto the region around the n - atoms (red dashed line) for f4tcnq adsorbed on ag(111). the dominant metalmolecule oop features between 7.0 and 2.5 ev correlate well with an energy range of increased dos in the molecular region, especially in the region around the downward bent n - atoms (green dash - dotted and red - dashed line in figure 10, respectively) and a very large dos in the metal in exactly that energy range due to the d - bands (vide supra). also for the features at higher energies, a correlation with the dos projected on the n - atoms and the metalmolecule oop is well visible. the overall magnitude of the oop is, however, somewhat lower due to the lack of metal - d - band contributions. the bonding to antibonding transitions in the oop are, of course, not visible in the dos. figure 10 also shows that the oop is clearly different from a dos simply separated into bonding to antibonding contributions. this is evidenced, for example, by only very small oop peaks associated with the dos maxima below 6.8 ev and at 1.8 ev that are not associated with the downward - bent n - atoms, which manifests itself in small overlap integrals and, thus, in a reduced oop. we introduce a number of versatile tools for analyzing the covalent contribution to the bonding between organic adsorbates and metal surfaces that are derived from the crystal orbital overlap population introduced by hoffmann.(18) these tools allow the identification of energies at which metalmolecule hybrid states have bonding and antibonding character, respectively, and together with the calculation of a total overlap population they provide a measure for the total covalent bonding strength. overlap populations enable the identification of the atoms and groups of the molecule that most strongly contribute to the bonding and also make it possible to quantify the role of different metal bands as well as (sets of) molecular orbitals in the bonding process. as instructive examples, we apply various overlap populations to explain the bonding between a particularly strong electron donor, hv0, and au(111) and the prototypical acceptor f4tcnq and ag(111). for the former example, they highlight the contribution of otherwise easily overlooked unoccupied orbitals that become only slightly occupied in the course of the bonding but have large amplitudes on those parts of the molecules that are closest to the metal. for f4tcnq on ag(111), the pivotal contribution of the terminal cn groups and the orbitals localized on those groups is identified. | the electronic structure of metalorganic interfaces is of paramount importance for the properties of organic electronic and single - molecule devices. here, we use so - called orbital overlap populations derived from slab - type band - structure calculations to analyze the covalent contribution to the bonding between an adsorbate layer and a metal. using two prototypical molecules, the strong acceptor 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (f4tcnq) on ag(111) and the strong donor 1h,1h-[4,4]bipyridinylidene (hv0) on au(111), we present overlap populations as particularly versatile tools for describing the metalorganic interaction. going beyond traditional approaches, in which overlap populations are represented in an atomic orbital basis, we also explore the use of a molecular orbital basis to gain significant additional insight. on the basis of the derived quantities, it is possible to identify the parts of the molecules responsible for the bonding and to analyze which of the molecular orbitals and metal bands most strongly contribute to the interaction and where on the energy scale they interact in bonding or antibonding fashion. |
the protective action of dietary fiber against colorectal cancer (crc) has been attributed to the fermentation of fiber by anaerobic bacteria in the colon, producing butyrate 1, an inhibitor of histone deacetylases (hdaci), which produces cell cycle arrest, differentiation, and/or apoptosis of crc cells 2 - 4. wnt activity derives from the accumulation of beta - catenin, which becomes associated with dna - binding tcf factors ; the resulting beta - catenin - tcf complexes stimulate transcriptional activity from target genes 8. the constitutive activation of wnt signaling, primarily due to mutations in the apc and beta - catenin genes, promotes tumorigenesis in the colon 8. wnt signaling also plays a role in rna metabolism that may have consequences for tumorigenesis 9,10. for example, the wnt factor beta - catenin has been shown to influence various steps of rna metabolism in crc cells, modulating the levels of gene products relevant to colonic tumorigenesis, including cox-2 and cyclin d1 10. since wnt activity can influence rna metabolism 10 and butyrate can influence wnt signaling 5 - 7, the findings that butyrate can directly modulate rna metabolism is provocative. for example, butyrate inhibits the splicing of rna from the wnt - target gene c - myc, important for many forms of cancer including crc 11, and also alters alternative splicing of the survival motor neuron (smn) gene, resulting in enhanced smn levels in vitro and in vivo 12. in addition, butyrate influences the relative levels of calcitonin and calcitonin gene - related peptide mrnas 13, the ratio of which is controlled by alternative polyadenylation. this latter finding suggests that butyrate modulates 3 ' end processing as well as splicing. these findings indicate that wnt signaling, and the activation of this signaling by butyrate, influences rna metabolism, and that altered rna metabolism modifies patterns of gene expression related to tumorigenesis. further, wnt signaling itself can be differentially modulated by the products of alternative rna processing, suggesting the possibility of bidirectional crosstalk between wnt activity and rna metabolism. as part of our study of differential gene expression affecting colonic tumorigenesis, a full human genome microarray analysis (genus biosystems) was performed on rna derived from hct-116 crc cells, to determine genes modulated by a physiologically relevant concentration (5 mm) of butyrate. the expression of a number of genes involved with rna metabolism was found to be influenced by butyrate. for example, butyrate significantly (p < 0.001) decreased by 2.4-fold the expression of the splicing factor gene srp20 ; this latter action by butyrate may mediate its therapeutic activity, since srp20 has been linked to crc tumorigenicity 14. expression of the intronless gene c - jun, which can promote colonic tumorigenesis (see below), was upregulated by butyrate 9.5-fold (p < 0.001). furthermore, the expression of other genes important in rna metabolism was up- or downregulated by butyrate treatment of hct-116 cells. genes whose expression was upregulated include the nuclear rna export factor nxf3, while a number of genes exhibited downregulation of expression, including genes whose products influence 3 ' end processing (cstf3, several other cpsf and cstf genes, and pabpn1), as well as genes whose products influence rna stability (hnrnp d) and splicing (srsf10 and ptbp1). the modulation, by butyrate, of the expression of cstf3, srsf10, ptbp1, and pabpn1 is also influenced by wnt activity, as the degree of that modulation was decreased in hct-116 cells expressing an inducible form of dominant - negative tcf4 (dn - tcf4), which is a potent inhibitor of canonical wnt signaling 8. to evaluate effects of wnt activity on overall intron splicing efficiency in crc cells, we utilized the luciferase - beta galactosidase double reporter system from the eperon laboratory 15 which is based on a tropomyosin tmp3 intron. two plasmids, ptn23 and ptn24, were used, which differ in the structure of the tmp3 intron sequences. with efficient intron splicing, coding sequences from both the luciferase and beta - galactosidase cassettes are fully included in the final transcript and both reporter activities are detected. however, in the absence of splicing, only beta - galactosidase reporter activity is observed. thus, the ratio of luciferase to beta - galactosidase reporter activity is indicative of splicing efficiency. sw620 crc cells exhibit a relatively high basal level of canonical wnt signaling that is strongly inhibited by dn - tcf4 (5 - 7 and refs. we performed a pilot experiment by cotransfecting the ptn23 or ptn24 vectors with a control vector or with the dn - tcf4 expression vector. inhibition of wnt activity in sw620 cells enhanced splicing efficiency by more than three - fold with the ptn23 vector and more than 1.5-fold with the ptn24 vector (fig. 1). this demonstrates wnt activity - specific effects on intron splicing in a well characterized crc cell line, with the general trend of increased intron splicing with repressed wnt activity. based on these preliminary findings, a review of the literature was conducted to evaluate evidence for physiologically relevant cross - talk between wnt activity and rna processing that is (a) of relevance to crc, and (b) potentially modifiable by butyrate / hdacis, and thus possibly amenable to preventive and/or therapeutic approaches against crc. as part of our study of differential gene expression affecting colonic tumorigenesis, a full human genome microarray analysis (genus biosystems) was performed on rna derived from hct-116 crc cells, to determine genes modulated by a physiologically relevant concentration (5 mm) of butyrate. the expression of a number of genes involved with rna metabolism was found to be influenced by butyrate. for example, butyrate significantly (p < 0.001) decreased by 2.4-fold the expression of the splicing factor gene srp20 ; this latter action by butyrate may mediate its therapeutic activity, since srp20 has been linked to crc tumorigenicity 14. expression of the intronless gene c - jun, which can promote colonic tumorigenesis (see below), was upregulated by butyrate 9.5-fold (p < 0.001). furthermore, the expression of other genes important in rna metabolism was up- or downregulated by butyrate treatment of hct-116 cells. genes whose expression was upregulated include the nuclear rna export factor nxf3, while a number of genes exhibited downregulation of expression, including genes whose products influence 3 ' end processing (cstf3, several other cpsf and cstf genes, and pabpn1), as well as genes whose products influence rna stability (hnrnp d) and splicing (srsf10 and ptbp1). the modulation, by butyrate, of the expression of cstf3, srsf10, ptbp1, and pabpn1 is also influenced by wnt activity, as the degree of that modulation was decreased in hct-116 cells expressing an inducible form of dominant - negative tcf4 (dn - tcf4), which is a potent inhibitor of canonical wnt signaling 8. to evaluate effects of wnt activity on overall intron splicing efficiency in crc cells, we utilized the luciferase - beta galactosidase double reporter system from the eperon laboratory 15 which is based on a tropomyosin tmp3 intron. two plasmids, ptn23 and ptn24, were used, which differ in the structure of the tmp3 intron sequences. with efficient intron splicing, coding sequences from both the luciferase and beta - galactosidase cassettes are fully included in the final transcript and both reporter activities are detected. however, in the absence of splicing, only beta - galactosidase reporter activity is observed. thus, the ratio of luciferase to beta - galactosidase reporter activity is indicative of splicing efficiency. sw620 crc cells exhibit a relatively high basal level of canonical wnt signaling that is strongly inhibited by dn - tcf4 (5 - 7 and refs. therein). we performed a pilot experiment by cotransfecting the ptn23 or ptn24 vectors with a control vector or with the dn - tcf4 expression vector. inhibition of wnt activity in sw620 cells enhanced splicing efficiency by more than three - fold with the ptn23 vector and more than 1.5-fold with the ptn24 vector (fig. this demonstrates wnt activity - specific effects on intron splicing in a well characterized crc cell line, with the general trend of increased intron splicing with repressed wnt activity. based on these preliminary findings, a review of the literature was conducted to evaluate evidence for physiologically relevant cross - talk between wnt activity and rna processing that is (a) of relevance to crc, and (b) potentially modifiable by butyrate / hdacis, and thus possibly amenable to preventive and/or therapeutic approaches against crc. at least 60% of human genes exhibit alternative splicing, and microarray methodologies have been developed to analyze the patterns of pre - mrna splicing in different cells and under varied conditions 16 - 18. tumor - specific splicing variants have been identified and some of these variants influence wnt activity 19 - 22. for example, the splicing factor srp20 is a wnt target gene ; srp20 upregulates crc cell proliferation and has been linked to tumorigenesis 14. it is known that variants of tcf / lef factors such as tcf1 and tcf4, which bind to beta - catenin and drive wnt transcriptional activity, can be generated by alternative splicing 23. further, lee. 10 demonstrated that an rna aptamer that interferes with the interaction between beta - catenin and tcf4 in crc cells not only repressed wnt activity but also regulated the alternative splicing of rna from an adenovirus e1a minigene. conversely, alternative splicing influences wnt activity ; thus, while the full length form of sulfatase 1 (sulf1a) stimulates wnt signaling, a shorter sulf isoform (sulf1b) produced by alternative splicing inhibits wnt activity 19. thus, cross - talk between rna splicing and wnt signaling activity is bidirectional, and is associated with colonic neoplasia. importantly, wnt activity is influenced not only by the expression of the splicing factor srp20 14,22 but also by that of asf / sf2 22,24. thus, changes in the relative levels of srp20 and asf / sf2 results in altered rna splicing and differential expression of rac1b, a rac1 variant overexpressed in certain colon tumors that contributes to cell survival 14,22, and which can enhance wnt signaling 25. srp20 enhances skipping of the rac1 alternative exon 3b in crc cells, while asf / sf2 promotes exon 3b inclusion ; thus, asf / sf2 increases and srp20 decreases rac1b expression. increased levels of wnt activity enhance expression of srp20, which in turn results in repressed expression of the rac1b variant 22. rac1b is more highly expressed in crc cells with lower levels of wnt activity, promoting survival of these low - wnt activity cells 20. conversely, the expression of asf / sf2 is blocked by phosphatidylinositol 3-kinase (pi3-k) signaling ; therefore, formation of rac1b is ultimately controlled by levels of wnt and pi3-k signaling 22,24. crc cells characterized by low levels of wnt activity and pi3-k signaling utilize rac1b to promote survival ; however, crc cell types with higher levels of wnt and pi3-k signaling exhibit low or no expression of rac1b and utilize alternative signaling pathways to promote cell survival 20. these findings are consistent with our observations that very high levels of wnt activity induced by hdacis promote apoptosis of crc cells, since lower levels of wnt signaling, associated with increased expression of rac1b, promote cell survival 5 - 7. further, rac1b itself upregulates wnt activity when overexpressed in hct-116 cells, a cell line that normally expresses relatively low levels of rac1b 25. this upregulation of wnt activity by rac1b occurs upstream of beta - catenin 24,25 ; this is similar to the activation of wnt activity by hdacis at the plasma membrane, resulting in enhanced levels of active beta - catenin 5 - 7. therefore, one intriguing possibility is that upregulation of wnt activity by hdacis may be partially mediated by the action of rac1b. remarkably, rac1b - negative sw480 crc cells can be made to endogenously express levels of rac1b similar to that of the rac1b - positive ht-29 crc cell line, through a combination of asf / sf2 overexpression and sirna knockdown of srp20 22. this suggests plasticity of rac1b phenotypes, perhaps extending to downstream effects of rac1b expression. manipulation of rac1b levels likely can induce rac1b - mediated cell survival pathways, including induction of moderate levels of wnt signaling activity. however, since increased wnt activity downregulates rac1b expression via srp20, a negative feedback loop would likely occur. thus, low levels of wnt activity promotes increased expression of rac1b, which simulates wnt activity ; this increase in wnt signaling would in turn downregulate levels of rac1b through alternative rna splicing. our previous findings 5 - 7 are consistent with the hypothesis that moderate levels of wnt activity drive crc cell proliferation and survival, while wnt hyperactivation promotes crc cell death. thus, the negative wnt - rac1b feedback loop may serve to keep levels of wnt activity in the pro - proliferative range and below levels that would promote crc cell death. the overall relationship between wnt signaling, rac1b, and crc, with respect to alternative rna splicing, is outlined in fig. the development of other forms of cancer is also influenced by splicing factors whose expression is controlled by cancer - related cell signaling pathways. for example, the levels of asf / sf2 as well as of hnrnp a1 are increased in lung neoplasia ; this altered expression is associated with cancer - related changes in the alternative splicing of cd44, which is involved in cell - cell and cell - matrix interactions 26. thus, the effects of srp20 and asf / sf2 on rac1b may be part of a broader modulation of rna processing in tumorigenesis, responsible for altering the levels of protein variants in the neoplastic cell 22,26. consistent with a significant role for rna metabolism in mediating the effects of cell signaling pathways in normal and neoplastic cells, analysis of beta - catenin - containing nuclear complexes showed the presence of a variety of proteins that bind rna and/or influence rna metabolism, such as hnrnp a2/b1, hnrnp m, hnrnp g, hnrnp a1, and hnrnp k, among others 27. in addition, beta - catenin interacts with fusion (fus)/translocated in liposarcoma (fus / tls), a rna binding protein that influences mrna transcription and splicing 27. overexpression of fus / tls repressed wnt activity in crc cells and repressed crc cell clonogenic growth ; in addition, fus / tls levels are increased in crc and may serve as negative feedback, limiting the effects of deregulated wnt activity 27. this interaction further links rna metabolism to wnt activity, which in turn influences crc cell growth. thus, fus / tls and similar factors may integrate wnt - mediated rna transcription with effects on rna processing, to exert more fine - grained control of the expression of genes associated with tumorigenesis. statistical analyses demonstrated that naturally intronless genes are over - represented among genes coding for proteins with binding or signal transduction / receptor activities and under - represented among genes coding for proteins with catalytic functions 33. signal transduction / receptor proteins are highly relevant to the signaling pathways involved in the development of cancer ; therefore, these data suggest the possibility that intronless rna metabolism is associated with tumorigenesis. rapid - response genes, which include c - jun, are often intronless ; rna metabolism of these genes reflects the requirement to bypass splicing processes for more efficient expression and function 34. the protein product of the intronless human c - jun gene associates with c - fos, forming the transcription factor ap-1, which participates in jnk signaling 35,36. ap-1/jnk signaling plays an important role in the development of crc 37, and a dominant negative mutant of c - jun has been proposed as a gene therapy approach for crc, through disruption of ap-1 activity 38. supporting this view, c - jun is overexpressed in crc, and jnk signaling activates intestinal tumorigenesis in mouse models of crc (39 and refs. therein). jnk signaling stimulates progenitor cell proliferation in intestinal crypts ; importantly, expression of the wnt target genes axin 2 and lgr5 is upregulated by jnk activity 39. further, expression of tcf4, a key component of the wnt signaling complex, is upregulated by jnk activity, and chromatin immunoprecipitation analyses demonstrated that tcf4 is a direct c - jun target gene 39. c - jun itself is a wnt target gene ; therefore, these findings suggest that positive feedback between wnt and jnk activity, mediated through c - jun, contributes to intestinal tumorigenesis 39. in addition, c - jun, together with the wnt signaling factors beta - catenin and tcf, associate with a 3 ' enhancer region of the wnt - target gene c - myc, a proto - oncogene necessary for colorectal neoplasia 40,41. with respect to rna metabolism, while intron - containing genes often require the presence of introns for proper mrna accumulation ; naturally intronless genes contain sequence elements which can substitute for introns to allow for efficient gene expression (42 and references therein). an example of such an element is the human c - jun gene enhancer (cje) element 42. the rna metabolism of intronless c - jun, by influencing levels of c - jun protein, may alter signaling cross - talk (e.g., wnt - jnk) and therefore contribute to the development of crc 41. in addition, preliminary findings from our laboratory (unpublished data) indicate that wnt activity can enhance the activity of sequences that confer efficient intron - independent gene expression ; this suggests that wnt activity influences c - jun expression both at the level of transcription as well as at the level of post - transcriptional modulation of intronless rna metabolism (e.g., rna stability). further research into the effects of wnt activity on intronless rna metabolism is required to evaluate this possibility. importantly for crc, c - jun expression is upregulated by butyrate both in vitro and in vivo 43,44, and our microarray data (above) show a marked upregulation of c - jun expression in butyrate - treated hct-116 cells. thus, while hdaci treatment is generally pro - apoptotic and hence therapeutic against cancer, enhanced wnt activity resulting from exposure to hdacis can also increase the expression of pro - tumorigenic genes. therefore, it is important to understand the various mechanisms whereby basal and hdaci - induced wnt activity influences the expression of c - jun, including effects on intronless rna metabolism. induction of c - jun expression by hdacis can be rapid ; for example, exposure of leukemic cells to the butyric acid prodrug pivaloyloxymethyl butyrate (a-9) induces significant c - jun expression in only 15 minutes 45. genes that contain introns, the interplay between rna processing and gene expression, including the kinetics of gene expression, likely differs in a gene specific manner. for example, the relative importance of the intronless condition for rapid gene expression may be influenced by factors such as promoter strength, chromosome location and surrounding chromatin structure, and/or the presence or absence of other regulatory sequences. certain intronless genes, such as c - jun, may require the intronless state for efficient and rapid expression ; this dependence on the intronless state influences the levels of the relevant gene products, with consequent effects on cell physiology, including neoplasia. therefore, differences of rna metabolism in intron - dependent vs. intron - independent pathways of expression may in part determine the degree to which induction of gene expression by wnt signaling contributes to tumorigenesis. further, a complete understanding of how cell signaling pathways influence intronless gene expression in tumorigenesis may lead to approaches to suppress expression of tumorigenic factors such as c - jun, which would enhance the efficacy of, e.g., hdaci - based anti - cancer therapeutics. 3 ' end processing is also of relevance to cancer ; for example, alternative 3 ' end processing can enhance oncogene expression in cancer cells, and differential 3 ' end formation is associated with clinically relevant cancer subtypes 46. altered gene expression in cancer, caused by alternative 3 ' end processing, often results from enhanced rna stability, in some cases involving shortened 3 ' untranslated regions (3 ' utrs) and the consequent loss of sites mediating repression by mirnas 47. several genes linked to neoplasia exhibit regulation of expression through control of 3 ' end processing. the wnt target cyclin d2, involved in cell cycle regulation, is regulated through modulation of 3 ' end processing, and other cancer related genes (e.g., n - acetyltransferase 1) also exhibit regulation of expression through alternative polyadenylation (48,49 and refs. therein). further, alternative polyadenylation produces a version of the wnt factor tcf4 that inhibits wnt activity 50, suggesting a direct link between 3 ' end processing, wnt activity, and colonic neoplasia. a classic example of a crc - related gene whose rna stability 51 and expression is controlled by alternative polyadenylation is the wnt target cyclooxygenase-2 (cox-2) (52 and refs. ht-29 crc cells primarily use the cox-2 distal polyadenylation site, while hepg2 liver cancer cells use both the distal and proximal cox-2 polyadenylation sites 52. this tissue - specific difference in polyadenylation usage has practical consequences, as the longer cox-2 mrna (distal site usage) tends to be more stable than the shorter form (proximal site usage), resulting in higher steady - state levels of the cox-2 protein that may promote colonic neoplasia. thus, differential rna stability, such as that resulting from alternative 3 ' end processing of cox-2 rna, can influence neoplasia. further, since our preliminary microarray data (see above) demonstrated that wnt activity and butyrate influence the expression of factors involved in 3 ' end processing, levels of wnt signaling may influence alternative polyadenylation (e.g., of cox-2 rna). of particular relevance for crc, beta - catenin influences cox-2 rna stability 10,51 ; beta - catenin stabilizes cox-2 transcripts through interactions with au - rich elements in the 3 ' utr, again demonstrating cross - talk between wnt signaling and rna metabolism. the rna binding protein hur likely mediates these effects of beta - catenin ; the hur protein stabilizes transcripts correlated to the number of binding sites and level of association of hur with the targeted rna 50, 53, 54. rnas containing hur binding sites in both intronic as well as 3 ' utr sequences exhibited greater stability than transcripts containing either intronic or 3 ' utr sites only ; these findings suggest that hur links pre - mrna processing with mature mrna stability 55. therefore, wnt activity not only influences the levels of cox-2 at the transcriptional level, but also post - transcriptionally, at the level of rna stability. in addition, the association of hur with thrombospondin-1 mrna modulates the translation of the angiogenesis inhibitor tp1, affecting neoplastic progression via altered tumor vascularity 56. therefore, rna metabolism involving the 3 ' ends of transcripts can influence multiple points along the neoplastic continuum. at least 60% of human genes exhibit alternative splicing, and microarray methodologies have been developed to analyze the patterns of pre - mrna splicing in different cells and under varied conditions 16 - 18. tumor - specific splicing variants have been identified and some of these variants influence wnt activity 19 - 22. for example, the splicing factor srp20 is a wnt target gene ; srp20 upregulates crc cell proliferation and has been linked to tumorigenesis 14. it is known that variants of tcf / lef factors such as tcf1 and tcf4, which bind to beta - catenin and drive wnt transcriptional activity, can be generated by alternative splicing 23. further, lee. 10 demonstrated that an rna aptamer that interferes with the interaction between beta - catenin and tcf4 in crc cells not only repressed wnt activity but also regulated the alternative splicing of rna from an adenovirus e1a minigene. conversely, alternative splicing influences wnt activity ; thus, while the full length form of sulfatase 1 (sulf1a) stimulates wnt signaling, a shorter sulf isoform (sulf1b) produced by alternative splicing inhibits wnt activity 19. thus, cross - talk between rna splicing and wnt signaling activity is bidirectional, and is associated with colonic neoplasia. importantly, wnt activity is influenced not only by the expression of the splicing factor srp20 14,22 but also by that of asf / sf2 22,24. thus, changes in the relative levels of srp20 and asf / sf2 results in altered rna splicing and differential expression of rac1b, a rac1 variant overexpressed in certain colon tumors that contributes to cell survival 14,22, and which can enhance wnt signaling 25. srp20 enhances skipping of the rac1 alternative exon 3b in crc cells, while asf / sf2 promotes exon 3b inclusion ; thus, asf / sf2 increases and srp20 decreases rac1b expression. increased levels of wnt activity enhance expression of srp20, which in turn results in repressed expression of the rac1b variant 22. rac1b is more highly expressed in crc cells with lower levels of wnt activity, promoting survival of these low - wnt activity cells 20. conversely, the expression of asf / sf2 is blocked by phosphatidylinositol 3-kinase (pi3-k) signaling ; therefore, formation of rac1b is ultimately controlled by levels of wnt and pi3-k signaling 22,24. crc cells characterized by low levels of wnt activity and pi3-k signaling utilize rac1b to promote survival ; however, crc cell types with higher levels of wnt and pi3-k signaling exhibit low or no expression of rac1b and utilize alternative signaling pathways to promote cell survival 20. these findings are consistent with our observations that very high levels of wnt activity induced by hdacis promote apoptosis of crc cells, since lower levels of wnt signaling, associated with increased expression of rac1b, promote cell survival 5 - 7. further, rac1b itself upregulates wnt activity when overexpressed in hct-116 cells, a cell line that normally expresses relatively low levels of rac1b 25. this upregulation of wnt activity by rac1b occurs upstream of beta - catenin 24,25 ; this is similar to the activation of wnt activity by hdacis at the plasma membrane, resulting in enhanced levels of active beta - catenin 5 - 7. therefore, one intriguing possibility is that upregulation of wnt activity by hdacis may be partially mediated by the action of rac1b. remarkably, rac1b - negative sw480 crc cells can be made to endogenously express levels of rac1b similar to that of the rac1b - positive ht-29 crc cell line, through a combination of asf / sf2 overexpression and sirna knockdown of srp20 22. this suggests plasticity of rac1b phenotypes, perhaps extending to downstream effects of rac1b expression. manipulation of rac1b levels likely can induce rac1b - mediated cell survival pathways, including induction of moderate levels of wnt signaling activity. however, since increased wnt activity downregulates rac1b expression via srp20, a negative feedback loop would likely occur. thus, low levels of wnt activity promotes increased expression of rac1b, which simulates wnt activity ; this increase in wnt signaling would in turn downregulate levels of rac1b through alternative rna splicing. our previous findings 5 - 7 are consistent with the hypothesis that moderate levels of wnt activity drive crc cell proliferation and survival, while wnt hyperactivation promotes crc cell death. thus, the negative wnt - rac1b feedback loop may serve to keep levels of wnt activity in the pro - proliferative range and below levels that would promote crc cell death. the overall relationship between wnt signaling, rac1b, and crc, with respect to alternative rna splicing, is outlined in fig. the development of other forms of cancer is also influenced by splicing factors whose expression is controlled by cancer - related cell signaling pathways. for example, the levels of asf / sf2 as well as of hnrnp a1 are increased in lung neoplasia ; this altered expression is associated with cancer - related changes in the alternative splicing of cd44, which is involved in cell - cell and cell - matrix interactions 26. thus, the effects of srp20 and asf / sf2 on rac1b may be part of a broader modulation of rna processing in tumorigenesis, responsible for altering the levels of protein variants in the neoplastic cell 22,26. consistent with a significant role for rna metabolism in mediating the effects of cell signaling pathways in normal and neoplastic cells, analysis of beta - catenin - containing nuclear complexes showed the presence of a variety of proteins that bind rna and/or influence rna metabolism, such as hnrnp a2/b1, hnrnp m, hnrnp g, hnrnp a1, and hnrnp k, among others 27. in addition, beta - catenin interacts with fusion (fus)/translocated in liposarcoma (fus / tls), a rna binding protein that influences mrna transcription and splicing 27. overexpression of fus / tls repressed wnt activity in crc cells and repressed crc cell clonogenic growth ; in addition, fus / tls levels are increased in crc and may serve as negative feedback, limiting the effects of deregulated wnt activity 27. this interaction further links rna metabolism to wnt activity, which in turn influences crc cell growth. thus, fus / tls and similar factors may integrate wnt - mediated rna transcription with effects on rna processing, to exert more fine - grained control of the expression of genes associated with tumorigenesis. statistical analyses demonstrated that naturally intronless genes are over - represented among genes coding for proteins with binding or signal transduction / receptor activities and under - represented among genes coding for proteins with catalytic functions 33. signal transduction / receptor proteins are highly relevant to the signaling pathways involved in the development of cancer ; therefore, these data suggest the possibility that intronless rna metabolism is associated with tumorigenesis. rapid - response genes, which include c - jun, are often intronless ; rna metabolism of these genes reflects the requirement to bypass splicing processes for more efficient expression and function 34. the protein product of the intronless human c - jun gene associates with c - fos, forming the transcription factor ap-1, which participates in jnk signaling 35,36. ap-1/jnk signaling plays an important role in the development of crc 37, and a dominant negative mutant of c - jun has been proposed as a gene therapy approach for crc, through disruption of ap-1 activity 38. supporting this view, c - jun is overexpressed in crc, and jnk signaling activates intestinal tumorigenesis in mouse models of crc (39 and refs. therein). jnk signaling stimulates progenitor cell proliferation in intestinal crypts ; importantly, expression of the wnt target genes axin 2 and lgr5 is upregulated by jnk activity 39. further, expression of tcf4, a key component of the wnt signaling complex, is upregulated by jnk activity, and chromatin immunoprecipitation analyses demonstrated that tcf4 is a direct c - jun target gene 39. c - jun itself is a wnt target gene ; therefore, these findings suggest that positive feedback between wnt and jnk activity, mediated through c - jun, contributes to intestinal tumorigenesis 39. in addition, c - jun, together with the wnt signaling factors beta - catenin and tcf, associate with a 3 ' enhancer region of the wnt - target gene c - myc, a proto - oncogene necessary for colorectal neoplasia 40,41. with respect to rna metabolism, while intron - containing genes often require the presence of introns for proper mrna accumulation ; naturally intronless genes contain sequence elements which can substitute for introns to allow for efficient gene expression (42 and references therein). an example of such an element is the human c - jun gene enhancer (cje) element 42. the rna metabolism of intronless c - jun, by influencing levels of c - jun protein, may alter signaling cross - talk (e.g., wnt - jnk) and therefore contribute to the development of crc 41. in addition, preliminary findings from our laboratory (unpublished data) indicate that wnt activity can enhance the activity of sequences that confer efficient intron - independent gene expression ; this suggests that wnt activity influences c - jun expression both at the level of transcription as well as at the level of post - transcriptional modulation of intronless rna metabolism (e.g., rna stability). further research into the effects of wnt activity on intronless rna metabolism is required to evaluate this possibility. importantly for crc, c - jun expression is upregulated by butyrate both in vitro and in vivo 43,44, and our microarray data (above) show a marked upregulation of c - jun expression in butyrate - treated hct-116 cells. thus, while hdaci treatment is generally pro - apoptotic and hence therapeutic against cancer, enhanced wnt activity resulting from exposure to hdacis can also increase the expression of pro - tumorigenic genes. therefore, it is important to understand the various mechanisms whereby basal and hdaci - induced wnt activity influences the expression of c - jun, including effects on intronless rna metabolism. induction of c - jun expression by hdacis can be rapid ; for example, exposure of leukemic cells to the butyric acid prodrug pivaloyloxymethyl butyrate (a-9) induces significant c - jun expression in only 15 minutes 45. genes that contain introns, the interplay between rna processing and gene expression, including the kinetics of gene expression, likely differs in a gene specific manner. for example, the relative importance of the intronless condition for rapid gene expression may be influenced by factors such as promoter strength, chromosome location and surrounding chromatin structure, and/or the presence or absence of other regulatory sequences. certain intronless genes, such as c - jun, may require the intronless state for efficient and rapid expression ; this dependence on the intronless state influences the levels of the relevant gene products, with consequent effects on cell physiology, including neoplasia. therefore, differences of rna metabolism in intron - dependent vs. intron - independent pathways of expression may in part determine the degree to which induction of gene expression by wnt signaling contributes to tumorigenesis. further, a complete understanding of how cell signaling pathways influence intronless gene expression in tumorigenesis may lead to approaches to suppress expression of tumorigenic factors such as c - jun, which would enhance the efficacy of, e.g., hdaci - based anti - cancer therapeutics. 3 ' end processing is also of relevance to cancer ; for example, alternative 3 ' end processing can enhance oncogene expression in cancer cells, and differential 3 ' end formation is associated with clinically relevant cancer subtypes 46. altered gene expression in cancer, caused by alternative 3 ' end processing, often results from enhanced rna stability, in some cases involving shortened 3 ' untranslated regions (3 ' utrs) and the consequent loss of sites mediating repression by mirnas 47. several genes linked to neoplasia exhibit regulation of expression through control of 3 ' end processing. the wnt target cyclin d2, involved in cell cycle regulation, is regulated through modulation of 3 ' end processing, and other cancer related genes (e.g., n - acetyltransferase 1) also exhibit regulation of expression through alternative polyadenylation (48,49 and refs. therein). further, alternative polyadenylation produces a version of the wnt factor tcf4 that inhibits wnt activity 50, suggesting a direct link between 3 ' end processing, wnt activity, and colonic neoplasia. a classic example of a crc - related gene whose rna stability 51 and expression is controlled by alternative polyadenylation is the wnt target cyclooxygenase-2 (cox-2) (52 and refs. ht-29 crc cells primarily use the cox-2 distal polyadenylation site, while hepg2 liver cancer cells use both the distal and proximal cox-2 polyadenylation sites 52. this tissue - specific difference in polyadenylation usage has practical consequences, as the longer cox-2 mrna (distal site usage) tends to be more stable than the shorter form (proximal site usage), resulting in higher steady - state levels of the cox-2 protein that may promote colonic neoplasia. thus, differential rna stability, such as that resulting from alternative 3 ' end processing of cox-2 rna, can influence neoplasia. further, since our preliminary microarray data (see above) demonstrated that wnt activity and butyrate influence the expression of factors involved in 3 ' end processing, levels of wnt signaling may influence alternative polyadenylation (e.g., of cox-2 rna). of particular relevance for crc, beta - catenin influences cox-2 rna stability 10,51 ; beta - catenin stabilizes cox-2 transcripts through interactions with au - rich elements in the 3 ' utr, again demonstrating cross - talk between wnt signaling and rna metabolism. the rna binding protein hur likely mediates these effects of beta - catenin ; the hur protein stabilizes transcripts correlated to the number of binding sites and level of association of hur with the targeted rna 50, 53, 54. rnas containing hur binding sites in both intronic as well as 3 ' utr sequences exhibited greater stability than transcripts containing either intronic or 3 ' utr sites only ; these findings suggest that hur links pre - mrna processing with mature mrna stability 55. therefore, wnt activity not only influences the levels of cox-2 at the transcriptional level, but also post - transcriptionally, at the level of rna stability. in addition, the association of hur with thrombospondin-1 mrna modulates the translation of the angiogenesis inhibitor tp1, affecting neoplastic progression via altered tumor vascularity 56. therefore, rna metabolism involving the 3 ' ends of transcripts can influence multiple points along the neoplastic continuum. we summarize the interactions between wnt activity and aspects of rna processing discussed in this paper in fig. wnt signaling interacts with various points of rna metabolism, and the gene products so influenced themselves feed back to affect wnt activity and, hence, influence neoplasia. analysis of cancer - specific rna metabolism is a relatively unexplored area of research, with potentially significant implications for the prevention and treatment of crc. rna metabolism can be altered by wnt signaling as well as by butyrate and other hdacis. rna metabolism is likely a major control point mediating the changes in gene expression that result from deregulated wnt signaling. in particular, intron - dependent expression of genes such as c - jun, and cross - talk between wnt signaling, rac1b expression, and mrna processing likely play fundamental roles in colonic neoplasia. determining how rna processing and intron - independent gene expression influences wnt - initiated crc may lead to novel interventions aimed at restoring normal gene expression for therapeutic benefit. | rna processing involves a variety of processes affecting gene expression, including the removal of introns through rna splicing, as well as 3 ' end processing (cleavage and polyadenylation). alternative rna processing is fundamentally important for gene regulation, and aberrant processing is associated with the initiation and progression of cancer. deregulated wnt signaling, which is the initiating event in the development of most cases of human colorectal cancer (crc), has been linked to modified rna processing, which may contribute to wnt - mediated colonic carcinogenesis. crosstalk between wnt signaling and alternative rna splicing with relevance to crc includes effects on the expression of rac1b, an alternatively spliced gene associated with tumorigenesis, which exhibits alternative rna splicing that is influenced by wnt activity. in addition, tcf4, a crucial component of wnt signaling, also exhibits alternative splicing, which is likely involved in colonic tumorigenesis. modulation of 3 ' end formation, including of the wnt target gene cox-2, also can influence the neoplastic process, with implications for crc. while many human genes are dependent on introns and splicing for normal levels of gene expression, naturally intronless genes exist with a unique metabolism that allows for intron - independent gene expression. effects of wnt activity on the rna metabolism of the intronless wnt - target gene c - jun is a likely contributor to cancer development. further, butyrate, a breakdown product of dietary fiber and a histone deacetylase inhibitor, upregulates wnt activity in crc cells, and also modulates rna processing ; therefore, the interplay between wnt activity, the modulation of this activity by butyrate, and differential rna metabolism in colonic cells can significantly influence tumorigenesis. determining the role played by altered rna processing in wnt - mediated neoplasia may lead to novel interventions aimed at restoring normal rna metabolism for therapeutic benefit. therefore, this minireview presents a brief overview of several aspects of rna processing of relevance to cancer, which potentially influence, or are influenced by, wnt signaling activity. |
an ectopic thyroid gland is defined as thyroid tissue that is not located anterolaterally to the second to fourth tracheal cartilages. anatomically, an ectopic thyroid can be lingual (at the base of the tongue), sublingual (below the tongue), prelaryngeal (in front of the larynx), or can be found at other rare sites. mediastinal ectopic thyroids are very rare, accounting for less than 1% of all cases, but rare mediastinal ectopic thyroid is also important to consider in the differential diagnosis of mediastinal masses. here, we review a rare case of mediastinal ectopic thyroid presenting with a paratracheal mass compressing the superior vena cava but without symptoms, a condition that has not been reported previously in korea. a 65-year - old male presented with a right paratracheal mass, incidentally detected on a computed tomography (ct) scan performed during a health examination after a traffic accident (fig. he had no history of exposure to radiation, and there was no family history of thyroid disease. the results of his laboratory tests were all within normal limits and included a thyroid stimulating hormone level of 0.96 mu / l (normal range, 0.40 to 4.00), t3 of 1,090 pmol / l (normal range, 600 to 1,950), and free t4 of 11.2 the titers of serum thyroid autoantibodies were also within normal ranges ; antithyroid peroxidase antibody was < 25 u / ml (normal range, < 100) and antithyroglobulin antibody was < 25 u / ml (normal range, < 100). a ct scan of the neck (fig. 1) revealed a 4.5 2.9 cm heterogeneously enhanced mass in the right paratracheal area. the mass was located at the intersection of the caudal margin of the left brachiocephalic vein and the trachea, and it compressed the superior vena cava. the thyroid gland was located in the normal position and showed slightly heterogeneous enhancement and no cervical adenopathy. 2) and ebus - transbronchial needle aspiration were performed to obtain a tissue sample of the paratracheal mass. during ebus, the large mass was observed as a right upper paratracheal lesion (2r lesion). the radiologic diagnosis of the mass by ebus was metastatic adenopathy, castleman 's disease, or tuberculous lymphadenitis. recently, he had undergone a coil embolization for an aneurysm in his left posterior communicating artery ; he is now waiting for the ectopic thyroid tissue to be surgically excised after his general condition has stabilized. ectopic thyroid tissue is the result of abnormal gland migration from the foramen caecum to its normal pretracheal position. lingual thyroid tissue accounts for 90% of these abnormalities, and sublingual ectopic tissues are much less frequent. dual ectopic thyroid has been described, with thyroid gland tissue also present in the normal location. the wall of a thyroglossal duct cyst is a common site for ectopic thyroid tissue, and the presence of a solid mass along a thyroglossal duct cyst should raise a suspicion of ectopic thyroid tissue. furthermore, other rare sites, such as the mediastinum, gall bladder, porta hepatis, and duodenum have also been described. biallelic mutations in foxe1 have been shown to result in thyroid ectopy in mice ; however, to date, no mutations in known genes have been associated with human ectopic thyroid tissues. ectopic thyroid tissue in the thorax with no connection to the cervical thyroid gland is very rare. we could find only a small number of cases of mediastinal ectopic thyroid tissues in the literature, and there were no reported cases in korea. most korean cases reported have been lingual or sublingual (infrahyoid) ectopic thyroids, although several cases of dual ectopic thyroids, one lateral ectopic thyroid, and two cases of intratracheal thyroids have been reported [4 - 6 ]. to our knowledge, this is the first case of mediastinal ectopic thyroid in korea. the mediastinum is a unique anatomic area containing several structures and pluripotent cells that allow for the development of a range of tumors. mediastinal tumors include primary thymic carcinomas, neuroendocrine carcinomas, germ - cell tumors, and lymphomas, as well as neurogenic, endocrine, and mesenchymal tumors. more rarely, primary thyroid tumors (adenomas or carcinomas) may occur in the mediastinum without cervical disease. ct or magnetic resonance imaging is mandatory for evaluating the site and size of the lesion. thyroid scintigraphy with i or technetium-99 m is highly sensitive and specific for detecting normal and ectopic thyroid tissues. ebus has been shown to be of increasing importance in the diagnosis of mediastinal masses. most mediastinal ectopic thyroid cases showed normal thyroid follicles, and one case revealed a papillary thyroid cancer. based on the location, ct - guided fine needle aspiration, ebus - transbronchial needle aspiration or surgical excision is chosen to obtain tissues. because of their invasiveness, mediastinal thyroid carcinomas usually present with dyspnea, wheezing, and chest pain, whereas benign lesions are usually discovered incidentally. mediastinal goiter can remain asymptomatic until the structures located in the thoracic inlet are compressed. the most common symptoms are dyspnea, dysphagia, cough, and hoarseness, and, occasionally, some patients present with superior vena cava syndrome. the chief complaints in reported mediastinal ectopic thyroid cases are painful or pulsating retrosternal mass, dyspnea, and cough. our case was found incidentally and was asymptomatic, although the mass compressed the superior vena cava. if this mass had remained undiscovered, the patient might have suffered superior vena cava syndrome. in mediastinal ectopic thyroid cases, both euthyroidism and hypothyroidism are found, regardless of the presence of a normal thyroid gland. in previous korean studies, hypothyroidism has been found in up to two - thirds of patients with ectopic thyroid, and these patients did not have a normal thyroid gland. however, in one case of hypothyroidism there was a normally located thyroid ; thus, a thyroid function test is necessary, irrespective of the existence of a normal thyroid. in the present case, the thyroid gland was located in the normal position, and the thyroid function test showed euthyroidism. surgery for mediastinal goiters should always be considered, even in elderly patients, because of the high risk of tracheal compression and the low morbidity of the surgery. although there is no real consensus regarding the proper management of mediastinal ectopic thyroids, surgical excision must be considered because they can be malignant, and can have mass effects on the surrounding structures. in summary, ectopic thyroid is a rare condition, and its location in the mediastinum is even rarer. although entirely intrathoracic ectopic thyroids are rare, they must be considered in the differential diagnosis of all mediastinal masses. because they have the potential to become malignant and to compress mediastinal structures, surgical excision of mediastinal ectopic thyroids | mediastinal ectopic thyroid is a very rare condition, with few reported cases in the literature and no reported cases in korea. this report describes an asymptomatic 65-year - old man with a right paratracheal mass compressing the superior vena. additionally, the epidemiology, clinical manifestation, diagnosis, and management of mediastinal ectopic thyroids are discussed. a mediastinal ectopic thyroid should be considered in the differential diagnosis of all mediastinal masses. surgical excision is recommended for both the diagnosis and treatment of this condition, because of its potential for malignancy and compression of mediastinal structures. this case demonstrates the clinical importance of mediastinal etopic thyroid. |
drug abuse or dependence is derived from a set of factors including social and family issues, availability, and individual s disposition (1, 2). personality is another factor that may play an important role in preparation, acceleration, or maintenance of drug abuse or dependence behaviors. personality disorders are highly observed among drug abusers both in clinical population (3, 4) and non - clinical population (5 - 7). moreover, personality is a significant clinical feature that also affects smoking patterns and may influence the nicotine withdrawal syndrome expressions (8). the influential role of a number of personality traits in drug abuse and dependence is proved. transferring from regular smoking stage to dependence stage numerous studies recognized that in addition to personality traits, onset age of smoking is also one of the important predictor variables in future dependence to other illegal substances. many studies concluded that smoking is a risk factor among teenagers to be addicted to other drugs. kandel. reported a significant relation between smoking cigarettes and use of other illegal drugs among high school students ; the use of illegal drugs in smokers who smoked daily was much higher than non - smokers (10). findings of several studies indicate that 98% of hashish users, before starting to use hashish, had used to smoke tobacco, and onset age of using hashish in these individuals was lower than the starting age of hashish consumption (11). individual traits such as genetic preparedness and personality traits, or environmental factors such as drug availability or peer influence, and lower age of smoking onset are among the strong predicting factors in future dependence that can explain the sequence of starting from one drug to another (12). the current research aimed to investigate the personality traits and onset age of smoking cigarettes as predicting variables in future use of illegal drugs. the present research was a causal - comparative study performed on 45 male nicotine addicts and 45 male opiate dependent individuals. using random sampling, the researchers attended a smoking cession center in qazvin, iran, and then, individuals who had been recognized to be nicotine dependent, according to the fagstrome questionnaire and diagnosis of the physician in that center, were selected. these individuals had passed six months of their treatments in this center and did not display smoking withdrawal symptoms. the inclusion criteria were lack of severe psychiatric disorders and non - dependence on different types of drugs. additionally, the researchers randomly attended four treatment centers for drug abuse and dependence in qazvin and individuals recognized to be drug dependents, according to the maudsley addiction profile and physicians diagnosis, were selected. these individuals had passed six months of their treatments and they did not display any drug withdrawal symptoms. the inclusion criteria were smoking, lack of severe psychiatric disorders and non - dependence on different types of drugs. moreover, the onset age of smoking cigarettes in these individuals was recorded. at the beginning of the sampling process, in both groups, individuals who were qualified to participate in the groups data were analyzed by spss software version 16, using anova and multivariate regression tests. temperament includes novelty seeking, harm avoidance, reward dependence, and persistence ; and character dimension includes self - directedness, cooperativeness, and self - transcendence. the internal reliability of the revised version, translated by ayati, chemikar, and pourshahbaz is 0.82 (14). this 60-item questionnaire was applied to find the therapeutic results including four areas : 1) drug use, 2) physical health, 3) mental health, and 4) high - risk behaviors. the internal correlation coefficient of this questionnaire is higher than 0.75 and its reliability is reported 0.65 - 0.74 (15). temperament includes novelty seeking, harm avoidance, reward dependence, and persistence ; and character dimension includes self - directedness, cooperativeness, and self - transcendence. the internal reliability of the revised version, translated by ayati, chemikar, and pourshahbaz is 0.82 (14). this 60-item questionnaire was applied to find the therapeutic results including four areas : 1) drug use, 2) physical health, 3) mental health, and 4) high - risk behaviors. the internal correlation coefficient of this questionnaire is higher than 0.75 and its reliability is reported 0.65 - 0.74 (15). both groups were homogeneous in terms of their variables. in the education variable, a significant inter - group difference was observed. table 2 indicates the statistics related to the comparison of seven personality dimensions between the two groups. as indicated, there was a significant difference between the two groups. it means that novelty seeking in dependents of opiate drugs was higher than those of dependents of nicotine (p = 0.000). cooperativeness of opiate addicts was lower than that of nicotine addicts (p = 0.000). the obtained results indicated that the mean of smoking onset in dependents of opiate drugs was lower than that of the dependents of nicotine. table 3 demonstrates the t - test results for the variable of onset age of smoking. the obtained results indicated the significance of logistic regression in variables of onset age of smoking cigarettes, education, and personality traits in predicting the dependence on opiate substances (table 4). according to table 4, low onset age of smoking cigarettes, lower education level, along with lower scores in self - directedness and cooperativeness traits, predict future dependence on opiate substances. the obtained results indicated a significance difference between the two groups in the dimensions of novelty seeking and cooperativeness. novelty seeking reflected a hereditary orientation in onset or activation of novelty seeking response, approach to reward cues, active avoidance of conditioned punishment cues, and unconditioned punishment escape. this dimension is observed as an exploratory activity in responding to novelty, impulsivity, exaggeration in approaching to reward cues, indulgence and disorder, fatigue and quick fed up, and active avoidance of failure. behavioral responses sent after novelty seeking includes exploratory pursuit, voracity, active avoidance, and escape. therefore, opiate addicts have traits such as indulgence in approach to reward cues, impulsivity, novelty seeking and pursuit of reward cues that distinguish them from the other groups. many of these traits cause an individual to go towards activities in which the person looks for reward cues, seeks novelty, and acts impulsively. also, novelty seeking dimension in drug addicts was higher than that of the ordinary population, and in some studies, it was higher than that of the alcohol addicts (16, 17). thus, findings of the current research can be explained as follows : opiate dependent individuals have traits such as indulgence in approach to reward cues, impulsivity, novelty seeking, and pursuit of reward cues that distinguishes them from the other groups. drug, itself, is a very strong incentive reward and affects the reward system of the brain. drug dependent individuals use drugs to supply the dopamine for their limbic system which is the reward system of brain, (18). also, these people display behaviors such as exploratory pursuits of drugs, active avoidance of withdrawal cues (including pain, and unpleasant physical and mental states) and voracity. voracity is one of the important factors in drug abuse, staying in withdrawal, and failure to return. voracity is high in drug addicts and high voracity has a negative correlation with successful withdrawal. this dimension of personality traits in the current research was compatible with many of the previous studies in this area such as results gained by ball. (22). also, cooperativeness dimension in opiate dependent inpatients was significantly lower than those of the other groups. individuals with higher degrees of this dimension consider themselves as an inseparable member of the human society and are, generally, sympathetic, patient, compassionate, merciful, and supportive, and adhere to the principles and rules. people with low cooperativeness are self - attracted, impatient and intolerant, critical, avenger, and opportunistic. these individuals do not usually assist others, and in every situation, first think about themselves. also, they intend to be inattentive about other people s emotions and rights. these people are intolerant, do not adhere to the principles and rules, and are inattentive to others emotions and rights. the difference of nicotine addicts and ordinary population with opiate dependent individuals in this dimension relates to supportiveness, adherence to the rules and principles, participation in group works, compassion, kindness, and patience. this finding is in agreement with the findings of many previous studies such as those of evren. evren. (21) argued that high novelty seeking is one of the distinguished features in category b of personality disorders, namely, the same category in which anti - social personality disorder is placed ; therefore, drug dependent individuals with higher novelty seeking may have symptoms of anti - social personality disorder. le bon. (8) found that heroin abusers (an opiate substance) personality profile indicate more anti - social personality traits compared to that of alcohol addicts. evren. (21) also pointed out that reward dependence is one of the characteristics of category a, high reward novelty seeking is one of the characteristics of category b, and high harm avoidance is one of the characteristics related to category c of personality disorders (21). moreover, results showed that low onset age of smoking cigarettes, lower education, and low cooperativeness and self - directedness dimensions predicted future use of opiate drugs after onset of smoking cigarettes. in a research performed on the onset age of drug use, chen. (23) identified that low onset age of smoking is one of the predisposing factors for future drug use. this implies that the lower the onset age of smoking, the more probable that the person refers to using other illegal drugs (23). (12) reported that the average onset age of smoking in their research samples was 13.4 (13.3 for boys, 13.5 for girls), and the mean (average) onset age of using alcohol was 15.1 (15.0 for boys, 15.2 for girls). it implies the lower age mean in smokers. experiencing tobacco before starting to use hashish occurred in 98% of hashish users (12). guxens. (11) indicated that tobacco and alcohol use preceded the use of hashish and the performed meta - analyses showed that it is 1.7 to 2.6 times more likely that tobacco and alcohol consumers use hashish. therefore, the current research results are in accordance with those of other studies, indicating that lower onset age of smoking is a predisposing factor for future use of other drugs. also, it is natural that when self - directedness traits are cases such as responsibility, purposefulness, trouble - shooting, self - acceptance, and adapting to society s norms, then this dimension can play an effective role to predict drug dependence. self - directedness is, to some extent, a component of a coherent ego that enables individuals to delay satisfying their needs, be self - accepted, purposeful, and responsible. low cooperativeness dimension in the present research is another predicting factor of drug dependence. as mentioned in the previous sections, cases such as empathy, sympathy, usefulness, and having a clean heart therefore, cooperativeness can be another component of a coherent ego, it means that individuals can consider norms of the society, be socially useful, and have empathy and a clean heart. in general, three dimensions of self - directedness, cooperativeness, and self - transcendence can, to some extent, reflect an individual s coherent ego and degree of sophistication. then, it can be explained that low character dimensions in opiate addicts, that were low in all three dimensions in this research, can indicate ego incoherence and non - sophistication. 21) in their research concluded that high novelty seeking and low cooperativeness along with low onset age of smoking cigarettes predict drug dependence. in their study, subscales predicting drug dependence were more reasonable than low age, lower scores in subscales of sympathy and compassion vs. malice and usefulness, and higher scores in subscale of spiritual acceptance vs. materialism. therefore, low self - directedness and cooperativeness, low onset age of smoking cigarettes and low education can predict drug dependence. according to the current and prior studies, it is clarified that many of the people using illegal substances have some personality characteristics such as novelty seeking, and antisocial traits that cause to start smoking in lower ages, and then approach to use any kind of substances. | background : according to drug gateway theory, smoking cigarettes, especially, low onset age of smoking, is one of the risk factors for future use.objectives:the present study aimed to compare nicotine and opiate addicts to identify the differences in personality traits and onset age of smoking in the two groups that cause some individuals to appeal to other substances after starting to use cigarettes.patients and methods : two groups of opiate and nicotine addicts were randomly selected. revised version of the cloninger temperament inventory questionnaire, the fagrastrom nicotine dependence and the maudsley addiction profile were used. anova and logistic regression were applied for data analysis.results:opiate addicts had higher scores in novelty seeking dimension and lower scores in cooperativeness compared to nicotine addicts. the onset age of smoking cigarette in opiate addicts was lower than nicotine addicts.conclusions:low onset age of smoking cigarettes, high novelty seeking and low cooperativeness in opiate dependents are among the important personality traits in future use of drugs that can predict the subsequent onset of using opiate drugs. |
type 1 diabetes mellitus (dm) and graves disease are autoimmune diseases, and a number of genetic and environmental factors are thought to be involved in their etiology. we have demonstrated an association between both hla class ii genotype and ctla-4 (cytotoxic t lymphocyte antigen-4) gene polymorphism and childhood - onset type 1 dm (1, 2). the incidences of hla - drb1 0405, 0901, and dqb1 0303, 0401 were found to be significantly higher in children with type 1 dm than in controls, whereas the incidences of drb1 0803, 1501, 1502 and dqb1 0301, 0601, 0602 were significantly lower (1). the g allele in the ctla-4 a / g polymorphism at position 49 was more frequent in type1 dm children than in controls (2). we have also found an association between both hla class ii genotype and ctla-4 gene polymorphism and childhood - onset graves disease in japanese patients (submitted for publication). in 1994 aaltonen. assigned the disease locus in finnish families with apeced (autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy) to chromosome 21q22.3 based on the results of a linkage analysis between two markers, d21s49 and d21s171 (3), and in 1997 a novel gene was isolated from this region and named the aire-1 (autoimmune regulator-1) gene (4, 5). the aire-1 gene is composed of 14 exons. aire-1 protein is expressed in the thymic medulla, where t - cell immune tolerance is established, as well as in the lymph nodes, spleen, and fetal liver, but it is not expressed in the affected organs of apeced patients. kogawa. recently found that aire-1 protein is also restrictively expressed in peripheral cd14-positive monocytes and in differentiated dendritic cells (6). apeced is an autosomal recessive disease that is especially frequent in finnish and iranian jews, and is characterized by the simultaneous presence of at least two of three major diseases in the same individual : hypoparathyroidism, addison s disease, and chronic mucocutaneous candidiasis. it may also be associated with clinical manifestations of autoimmune thyroid disease, type 1 dm, and gonadal dysfunction. apeced causes multiple organ dysfunction in a wide variety of endocrine and extra - endocrine organs, and production of autoantibodies against the affected organs and lymphocyte invasion of the affected organs have frequently been demonstrated. a high percentage of patients with type 1 dm produce autoantibody against the thyroid gland as well as against the cells of the pancreas and have autoimmune thyroid disease, including graves disease and hashimoto thyroiditis (7, 8). the patients with graves disease may not only have autoantibodies against thyroid epithelial cells or the tsh receptor, but antinuclear antibodies and antineutrophil cytoplasmic antibodies (anca) that are not specific for endocrine organs (9). these findings suggest that patients with type 1 dm and graves disease tend to have autoimmune reactions not only against specific target organs but against several other organs and that they may possess heterozygous aire-1 mutations. in this study we examined japanese children with isolated type 1 dm or graves disease for the presence of heterozygous aire-1 mutations. as a first step we analyzed the r257x heterozygous mutation in exon 6 and the k83e heterozygous mutation in exon 2 which can be easily analyzed. the r257x mutation is the most common mutation in apeced which has been reported in many ethnic groups and in exon 2 many kinds of missense mutation have been reported. forty - six unrelated children with type 1 dm (17 males and 29 females ; age at the time of diagnosis, 0.516 yr) and 44 unrelated children with graves disease (10 males and 34 females ; age at the time of diagnosis, 316 yr) were the subjects of this study. type 1 dm was diagnosed according to the criteria of the who study group (10). the age of the subjects with type 1 dm at the time of diagnosis ranged from six mo to 16 yr. the interval between the time of diagnosis and examination for mutations ranged widely, from 0 to 18 yr. twenty - one of the 39 dm patients tested were positive for anti - gad antibody, and 20 of the 34 dm patients tested were positive for ia-2 antibody. graves disease was diagnosed on the basis of the presence of biochemical hyperthyroidism, the presence of tsh receptor antibodies (trab), and the presence of clinical evidence, such as palpable diffuse goiter, exophthalmos, or tachycardia, plus the absence of other causes of hyperthyroidism. the age of the graves disease patients at the time of diagnosis ranged from 3 to 16 yr. the microsome test (mchc) was positive in 37 of the 44 graves disease patients, and the thyroid test (tgha) was positive in 10 of the 44. this study was carried out in accordance with the principles of the declaration of helsinki. in this study we analyzed aire-1 genes for an a > g transition in exon 2 (k83e mutation) and a c > t transition in exon 6 (r257x mutation). dna was extracted from peripheral blood leukocytes with dna quick ii (dainihonseiyaku, japan). the aire-1 gene mutations were typed by pcr of genomic dna and restriction fragment - length polymorphism analysis (pcr - rflp) (4). the primers used for the k83e mutation were 5-tccaccacaagccgaggagat-3 and 5-acgggctcctcaaacaccact-3. the pcr was performed in a thermocycler at 94c for 5 min followed by 35 cycles of 94c for 30 sec, 60c for 30 sec and 68c for 30 sec, and a final step at 68c for 10 min. the amplified products were dissolved with restriction enzyme taqi (roche, germany) and run on a 2% agarose gel. the a allele corresponds to the 424-base pair (bp) uncleaved fragment with no taqi site and the g allele corresponds to the presence of 261-bp and 163-bp cleaved fragments generated by taqi dissolution. the primers used for the r257x mutation were 5-gcggctccaagaagtgcatccagg-3 and 5-ctccaccctgcaaggaagaggggc-3. the pcr was performed in a thermocycler at 94c for 5 min followed by 35 cycles of 94c for 30 sec, 65c for 30 sec and 72c for 30 sec, and a final step at 72c for 10 min. the amplified products were dissolved with restriction enzyme taqi and run on polyacrylamide gel. the c allele corresponds to the presence of three cleaved fragments (222 bp, 61 bp, and 55 bp), and the t allele corresponds to the presence of two cleaved fragments (283 bp and 55 bp) generated by taqi dissolution (4). 1 electrophetograms showing the results of digestion with taq1. (a) analysis for the a > g transition (k83e mutation) in exon 2. figure (a) shows the 424-bp uncleaved fragment in two representative samples of type 1 dm and graves disease (gd) subjects. (b) analysis for the c > t transition (r256x mutation) in exon 6. figure (b) shows three cleaved fragments (222 bp, 61 bp, and 55 bp) in two representative samples of type 1 dm and graves disease (gd) subjects.. analysis of the a > g transition in exon 2 (k83e mutation) revealed the 424-bp uncleaved fragment in all of the type 1 dm and graves disease subjects, and analysis of the c > t transition in exon 6 (r257x mutation) revealed three cleaved fragments (222 bp, 61 bp, and 55 bp) in all of the subjects. none of the childhood - onset type 1 dm or graves disease subjects had the k83e or r257x heterozygous mutation in the aire-1 gene. (a) analysis for the a > g transition (k83e mutation) in exon 2. figure (a) shows the 424-bp uncleaved fragment in two representative samples of type 1 dm and graves disease (gd) subjects. (b) analysis for the c > t transition (r256x mutation) in exon 6. figure (b) shows three cleaved fragments (222 bp, 61 bp, and 55 bp) in two representative samples of type 1 dm and graves disease (gd) subjects. meyer. examined 139 patients with graves disease, 224 with type 1 dm, 83 with addison s disease, and 75 with hashimoto s thyroiditis, in germany, for an association between heterozygous aire-1 mutations and isolated autoimmune endocrinopathy (11). the r257x mutation was found in one patient with hashimoto s thyroiditis in its heterozygous form, but not in any patients with type 1 dm, graves disease, or addison s disease. it was noteworthy that the patient who had the r257x mutation in its heterozygous form later developed other endocrine diseases. in the present study in japanese patients we did not find any k83e or r257x heterozygous mutations of the aire-1 gene in 46 children with type 1 dm and 44 children with graves disease. these results suggest the absence of any association between k83e or r257x heterozygous mutations and isolated type 1 dm and graves disease in japanese children. although apeced is very rare in japan, the following mutations have been discovered in japanese patients : l28p in exon 1 and ivs9 - 1g > c (12), missense mutation r15c in exon 1 (13), insertion of a cytosine at nucleotide 29635 in exon 10, and a deletion of guanine at nucleotide 33031 in exon 13 (14). however, there have been no reports of japanese apeced patients with r257x or k83e mutations. an analysis of the heterozygous mutations that have been discovered in japanese apeced patients should be included in future studies of genetic factors in childhood - onset type 1 dm and graves disease in japanese patients. in conclusion, no k83e and r257x heterozygous mutations in the aire-1 gene were found in japanese children with isolated type 1 dm or graves disease. further study is needed to conclude whether there is an association between heterozygous aire-1 gene mutations or polymorphisms and isolated type 1 dm or graves disease in japanese patients. | type 1 diabetes mellitus (dm) and graves disease are autoimmune diseases, and a number of genetic factors, including hla and ctla-4 genes, have been reported to contribute to their etiology. the gene responsible for autoimmune polyendocrinopathy- candidiasis - ectodermal dystrophy (apeced) has been cloned and named the autoimmune regulator-1 (aire-1) gene. aire-1 protein is thought to be a transcription regulatory protein and to have a role in the maintenance of immunological tolerance. the aim of this study was to determine whether heterozygous aire-1 gene mutations are associated with childhood - onset type 1 diabetes and graves disease in the japanese population. we investigated 46 children with type 1 dm (29 females and 17 males ; age at the time of diagnosis, 0.516 yr) and 44 children with graves disease (34 females and 10 males ; age at the time of diagnosis, 316 yr) for the presence of the k83e mutation in exon 2 and the r257x mutation in exon 6 of the aire-1 gene. the alleles were identified by polymerase chain reaction of genomic dna and restriction fragment - length polymorphism analysis (pcr - rflp) with endonuclease taqi. since no patients with type 1 dm or graves disease were found to carry the k83e or the r257x heterozygous mutation, we concluded that neither the k83e nor the r257x heterozygous mutation in the aire-1 gene seem to be the cause of the more common isolated endocrinopathies, i.e., type 1 diabetes mellitus and graves disease, in japanese children. |
there have been numerous investigations of the effects of noise in neurobiological dynamical systems, for both single neurons and neural networks. a large number of interesting noise - related phenomena have been analyzed, including synchronization, stochastic resonance, and coherence resonance (reviewed in lindner. most often, noise leads to increased responses (stein. 2005) and sometimes to the phenomenon of stochastic resonance in which a maximum occurs in a response variable at a particular noise strength (wiesenfeld and moss 1995 ; gammaitoni. 1998 ; badzey and mohanty 2005). this phenomenon has been demonstrated in neuronal systems, particularly, but not exclusively, in the case of periodic inputs (gang. 1993 ; collins. 1996 ; longtin 1993 ; nozaki. 1999). 2006 ; sim and forger 2007) and in neuronal models (gutkin. 2008) that noise can subdue or turn off repetitive neuronal activity. in this article, we report further investigations of such properties in hodgkin huxley single neurons. we find that there is a tuning effect of noise that has the opposite character to stochastic resonance, and thus, it might be called inverse stochastic resonance. we argue that these phenomena will occur generically in neural and other dynamical systems that exhibit a specific kind of bistability between a stable limit cycle and a stable focus. in the central nervous system, the responses of many types of neurons to input currents, whether injected or synaptic, have been much investigated experimentally and theoretically (mccormick. they receive input signals from many other neurons through thousands of excitatory and inhibitory synapses at unpredictable or random times (destexhe. 2001). in order for a neuron to send out a signal, called an action potential (bean 2007), it must receive sufficient net excitation (over inhibition) in a small enough time interval so that the current or voltage distribution in the cell passes through some threshold condition. once the threshold is reached, self - exciting processes lead to the emission of an action potential. huxley model (hodgkin and huxley 1952), which is capable of reproducing spiking behavior similar to those of many real neurons and has often been employed in investigations of the effects of noise on neuronal dynamics (brown. 1999 ; schmid., 2004 ; tuckwell 2005 ; torcini. we employ two commonly used different models for the input to the cell, which are described as current - driven and conductance - driven (destexhe. 2001 ; tiesinga. 2000). in the first of these, the input current is additive, being of the form i(t) = + w(t), where t is time, and are constant, and w(t) is standard gaussian white noise. such an input may represent somatic current injection by microelectrode in the laboratory. in the second model, which is closer to the nature of synaptic stimulation as it occurs in the nervous system, the driving current is of the form i(t) = g(t)(ve v(t)), where v(t) is the membrane depolarization, ve > 0 is the reversal potential for synaptic excitation (tuckwell 1988), and g(t) satisfies a first - order differential equation containing a noise amplitude e and a conductance parameter ge, along with a time constant (destexhe. if there is inhibition, there is a similar term involving the inhibitory reversal potential, but here, we consider excitation only. all the model equations for both kinds of input current and the relevant parameter values are given in the appendix. we first demonstrate the unusual inhibitory effect of noise in the case of a hodgkin huxley neuron with a current - based input when the mean current is just greater than the critical value for repetitive firing. figure 1a shows the voltage responses of the model neuron in such a case with various noise levels. initially, the neuron is taken to be at rest, which, by definition (see hodgkin and huxley 1952), implies that the depolarization is zero. the applied signal has a mean of strength and a noisy component of amplitude. without noise (top left record), there is, for this value of the steady input, = 6.6, a repetitive stream of output spikes, there being eight in the time period duration of 150 ms shown. adding noise makes the output sequence of spikes irregular. extremely weak noise naturally has little effect, but slightly larger amounts can have a significant effect on the neuron s spiking activity. moreover, moderate amounts of noise can actually arrest the spiking for a long time. in the examples shown, a noise level of = 0.2 can halt the firing of action potentials after three spikes, and a somewhat larger noise level of = 0.5 here stops the spiking after just one spike. when the noise level is turned up to = 2, more spikes are emitted, there being 9 in the trial shown. in fig. n (hodgkin and huxley 1952), are shown. these phase - space diagrams are useful in understanding the effects of noise of various levels, as discussed below. the mean input current density is 6.6 a / cm and the effects of noise of various magnitudes,, are shown. b orbits of voltage vs potassium activation variable corresponding to the plots of a. the limit cycle is clearly seen in the noise - free case and the manner in which small noise, = 0.2, and intermediate noise, = 0.5 may switch the orbit away from the limit cycle. when = 2, the orbits are close to the noise - free case a showing voltage trajectories with spikes for a current - driven the mean input current density is 6.6 a / cm and the effects of noise of various magnitudes,, are shown. b orbits of voltage vs potassium activation variable corresponding to the plots of a. the limit cycle is clearly seen in the noise - free case and the manner in which small noise, = 0.2, and intermediate noise, = 0.5 may switch the orbit away from the limit cycle. when = 2, the orbits are close to the noise - free case we further explored the effects of noise on the spiking activity of varying the mean of the input and its noise level in both the current - driven and conductance - driven cases. in fig. 2a, results are shown for the current - driven neuron. the mean number of action potentials, n, emitted over a 1,000-ms period, is plotted for various values of the mean current against the noise level. it should be noted that, in characterizing most central nervous system activity, except for example some brain stem neurons, 1,000 ms is a very long time, as the natural scale is milliseconds. for each data point with noise, 200 trials were performed. the mean number of spikes n over a 1,000 ms period is shown as the noise intensity increases for three values of the mean input current. a minimum in the output is clearly seen as increases when is near the critical value c. the mean number of spikes n over a 1,000-ms period is shown as the noise intensity increases for three values of the mean input signal strength ge (ms / cm). a minimum in the output is clearly seen as e increases when ge is near the critical value at 0.112 and also when ge = 0.1318 ms / cm a inverse stochastic resonance in the current - driven the mean number of spikes n over a 1,000 ms period is shown as the noise intensity increases for three values of the mean input current. a minimum in the output is clearly seen as increases when is near the critical value c. b inverse stochastic resonance in the conductance - driven hodgkin huxley model. the mean number of spikes n over a 1,000-ms period is shown as the noise intensity increases for three values of the mean input signal strength ge (ms / cm). a minimum in the output is clearly seen as e increases when ge is near the critical value at 0.112 and also when ge = 0.1318 ms / cm without noise (= 0), there is a critical value of = c, which is about 6.44, at which a stable and unstable limit cycle appear and co - exist with a stable focus. for values of > c up to a second critical value \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{\text{c}}^ { } $ $ \end{document } with approximate value 9.8 (hassard 1978), at which a subcritical hopf bifurcation occurs, the system is bistable, and repetitive periodic firing may persist without noise. for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu > \mu_{\text{c}}^ { } $ $ \end{document }, the focus becomes unstable, and only the stable limit cycle exists (until a further critical value is reached). for values of between c and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{\text{c}}^ { } $ $ \end{document }, perturbations, which may be stochastic or deterministic, can drive the system from near the stable limit cycle or spiking state to near the rest point, or vice - versa. in this article, we are mainly concerned with the effects of noise for values of between c and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{\text{c}}^ { } $ $ \end{document}. when = 5.5, below the critical value c, there is only one spike without noise. as the noise level increases beyond = 0.2, so does the mean number of spikes. however, when = 6.8, not far above the critical value and when there are 57 spikes without noise, increasing the noise level makes the mean number n of spikes at first drop dramatically. there is a pronounced minimum of six spikes at about = 0.5, representing a drop in mean spike count of 89%, and thereafter, the mean number of spikes increases at first quite sharply and then more slowly as the noise level increases. for the larger value of the mean input current, = 8, there are 62 spikes without noise and a noticeable, yet less pronounced, minimum value of 48 in the mean number of spikes when is just less than one. in order to examine what role the initial conditions might have played in these results, the values of v, n, m, and h at t = 0 were all chosen randomly. for v, the initial value was uniformly distributed from the minimum to the maximum voltage obtained in rhythmic spiking without noise. for n, m, and h, their values at t = 0 were uniformly distributed on (0,1). the effects of additive noise were qualitatively the same as for the case of initial resting conditions. for example, with = 6.8 at a time interval of 500 ms, the mean number of spikes dropped from 20.3 without noise to 3.08 as increased to 0.5. thereafter, as increased, the mean number of spikes increased monotonically to about 31 at = 4. with = 8, there is a small decline in mean spike count from 29.9 to 24.9 as increases from 0 to 0.7, and then a slow increase to n = 32.4 as increased further to four. the results for = 5.5 also paralleled those for resting initial conditions. it is pointed out that, with some of the randomly chosen initial conditions, some of the solutions would, in the absence of noise, evolve to the rest point, whereas others would tend to the limit cycle. as a further test of the robustness of the results we have described, we performed simulations in which the initial conditions were fixed at the resting values given in the appendix, but the noise was switched on at a random time tr. the latter was uniformly distributed over the period of rhythmic spiking, which is about 20 ms. thus, the neuron is set spiking with a given value of > c, and then at tr = 100 + 20u, where u is a uniform (0,1) random variable, the noise is switched on. we recorded the mean number n of spikes for tr < t < 500 ms, that is, after the noise is switched on until t = 500. the dependence of n on for the two values of previously employed (6.8 and 8) again paralleled those shown in fig. for example, with = 6.8, the mean spike count n declined sharply from 21.5 with no noise to a minimum of 4.2 with = 0.5. it may be concluded that the observed inhibitory effect of noise for certain values of the mean input current and the occurrence of a minimum as the noise level increases are robust phenomena for the hodgkin huxley system. in further support of this latter claim, 2b shows corresponding results obtained for the case of a conductance - driven neuron, for which the equations are also given in the appendix. when there is no noise, there is a critical conductance value just less than ge = 0.112 ms / cm, below which there is no sustained firing. the bottom curve shows the effects of increasing noise when ge = 0.0906 ms / cm, which is below the critical value. with increasing noise levels when ge = 0.112 ms / cm and there is no noise, there are 55 spikes in a 1,000-ms period. a small amount of noise causes a very large decrease in spike rate, and as the noise level increases, there is a well - defined minimum at about e = 0.005. for a stronger mean stimulus level, ge = 0.1318 ms / cm, there are 63 spikes without noise. as the noise level increases, a distinct minimum occurs at about e = 0.0075. thus, for both kinds of input current, current - driven and conductance - driven, a noise - induced decrease in firing rate occurs for inputs with means near the critical value for periodic spiking, and this inhibitory effect occurs even if the neuron is driven by a purely excitatory synaptic input. in this article, we reported new and interesting findings on the effects of noise on the behavior of certain dynamical systems relevant to neuronal activity. these arose from our investigations of the properties of pairs of coupled type 1 (gutkin and ermentrout 1998) neurons with noisy inputs (gutkin. huxley neurons, which are of type 2 excitability, came from our studies of pairs of such coupled neurons, to be reported elsewhere. our main observation is that, contrary to what is generally accepted, noise does not always have an excitatory or positive effect (cope and tuckwell 1979 ; yu and lewis 1989 ; stein. 2005), but it can lead to inhibitory or negative effects, which are also amenable to tuning. the occurrence of a minimum in the response as the noise level increases through a certain value might be called inverse stochastic resonance, a term which derives from its having the opposite character to stochastic resonance, in which a maximum in a response variable occurs as the noise level increases (collins. huxley variables were chosen randomly or when the noise was switched on at a random time, thus lending support to the robustness of the findings. preliminary simulations of more complex nerve models and larger networks have yielded the same kind of behavior, both in regard to silencing and the occurrence of a minimum in the firing rate, indicating that these phenomenona are quite general in neural systems. the effects of noise reported above are explainable in terms of the behavior of the voltage and other variables on what are called stable limit cycles (murray 1993), which occur when, for example, a neuron fires repetitively at the same frequency (see fig. such a stable limit cycle in a dynamical system often appears by a bifurcation mechanism when a parameter, like the mean input current strength in the hodgkin huxley model, varies continuously and crosses some critical value. just above that critical value, the basin of attraction of the limit cycle, the stable limit cycle then coexists with one or more other attractors, which is the key to the occurrence of the phenomena we have reported. in the hodgkin huxley model, for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \mu_{\text{c } } < \mu < \mu_{\text{c}}^ { } $ $ \end{document }, the only other attractor is a stable quiescent or resting state. noise can make the solutions leave the basin of attraction of the limit cycle for that of the quiescent state so that spiking ceases. a minimum in the spike count as noise increases is likely to occur for values of not far above c. the transitions to and from the attractors can be explored technically using classical stochastic process theory, as we will report with mathematical detail elsewhere. when the noise is small, the solution will then typically stay near the rest state for a very long time, but for larger noise, there is a considerable probability that the solutions will get kicked back up to threshold so that spiking may resume. 1b, where the voltage variable v is plotted against the potassium conductance variable n for = 6.6, with no noise, = 0, (top left), and values of the noise parameter = 0.2, 0.5, and 2. it is seen that weak noise can act to switch off the activity and induce a long period without spikes, whereas stronger noise tends to make the system switch back and forth between spiking and non - spiking states. the rate at which transitions then occur between spiking and non - spiking can be so rapid that the activity seems almost uninterrupted. the switching behavior for small noise has been observed in recent experiments (paydarfar. thus, the functional significance of these effects of noise on rhythmic activity is that a very small disturbance can lead to a drastic change in behavior. in the brain, electrical activity is often broadly rhythmic, involving limit - cycles in both normal and epileptic activity (steriade 2000 ; buzski and draguhn 2004). if such oscillations arise near a bifurcation point, then a small noisy signal could lead to the cessation of, or a sharp modification of, rhythmic activity. it is feasible that this could be the basis of a therapeutic approach to alleviate symptoms in the case of pathological oscillatory activity. also, in a population of cells, for example, with functionally tuned receptive fields, those which are weakly responding and operating near a bifurcation point akin to c could be silenced easily by noisy inputs, whereas cells firing at higher levels would have their activity augmented by noise, giving noise - induced tuning to the population responses. this may represent a new noise - induced mechanism for the sharpening of neural responses. note that the perturbing inputs (noise) do not have to be smooth but could be just as effective if they had an impulsive nature. since dynamical systems in diverse fields exhibit stable limit cycles coexisting with a stable focus, we expect to find that the phenomena of inhibition of cyclic, repetitive, or rhythmic activity by noise and inverse stochastic resonance will have widespread occurrence. examples of fields or systems where a stable limit cycle coexists with a stable rest state occur in circadian rhythms (jewett and kronauer 1998), cardiology (panfilov and holden 1997), cell kinetics and tumor growth (lahav. 2004 ; goldbeter 1991), and oscillating neural networks (steriade 2000 ; buzski and draguhn 2004), as well as in climatology, ecology, and astrophysics. the underlying mathematical bifurcation pattern suggests that the phenomena we have detected are of a general nature and not restricted to the hodgkin although the phenomena we have described are of interest in themselves, as indeed is stochastic resonance, their functional significance in neurobiological and other dynamical systems remains to be fully explored. a related finding was reported in a heuristic nonlinear stochastic model of affective disorders (huber., 2004). the original hodgkin huxley system (hodgkin and huxley 1952) is employed as follows, in which v(t) is the depolarization from resting potential in millivolts at time t (ms) and n(t), m(t), and h(t) are the (dimensionless) auxiliary variables for potassium activation, sodium activation, and sodium inactivation, respectively : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ dv = \left [{ \mu + g_{\text{k}}^ { } n^4 \left ({ v_{\text{k } } - v } \right) + g_{\text{na}}^ { } m^3 h\left ({ v_{\text{na } } - v } \right) + g_{\text{l } } \left ({ v_{\text{l } } - v } \right) } \right]dt / c + \sigma dw / c $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ dn = \left [{ \alpha_n \left ({ 1 - n } \right) - \beta_n n } \right]dt $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ dm = \left [{ \alpha_m \left ({ 1 - m } \right) - \beta_m m } \right]dt $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ dh = \left [{ \alpha_h \left ({ 1 - h } \right) - \beta_h h } \right]dt. $ $ \end{document}here, c is the membrane capacitance per unit area ;, which may depend on t, is the mean input current density ; and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g_{\text{k}}^ { } $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g_{\text{na}}^ { } $ $ \end{document }, and gl are the maximal (constant) potassium, sodium, and leak conductances per unit area with corresponding equilibrium potentials vk, vna, and vl, respectively. the input current has a stochastic component, w being a standard wiener process and being the noise amplitude. the voltage - dependent coefficients in the auxiliary equations are \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \alpha_n (v) = \left ({ 10 - v } \right)/\left [{ 100\left ({ e^{{\left ({ 10 - v } \right)/10 } } - 1 } \right) } \right ] $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \beta_n (v) = e^{- v/80 } /8 $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \alpha_m (v) = \left ({ 25 - v } \right)/\left [{ 10\left ({ e^{{\left ({ 25 - v } \right)/10 } } - 1 } \right) } \right ] $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \beta_m (v) = 4e^{- v/18 } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \alpha_h (v) = 7e^{- v/20 } /100 $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \beta_h (v) = 1/\left [{ e^{{\left ({ 30 - v } \right)/10 } } + 1 } \right ]. $ $ \end{document } the standard parameter set is c = 1, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g_{\text{k}}^ { } = 36 $ $ \end{document }, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g_{\text{na}}^ { } = 120 $ $ \end{document }, gl = 0.3, vk = 12, vna = 115, and vl = 10, and the standard initial conditions are taken as resting values v(0) = 0, n(0) = 0.35, m(0) = 0.06, and h(0) = 0.6. for conductance - based noise, the current terms dt + dw in the voltage equation are replaced by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left [{ g_{\text{e } } (t)\left ({ v_{\text{e } } - v } \right) + g_{\text{i } } (t)\left ({ v_{\text{i } } - v } \right) } \right]dt $ $ \end{document }, where ve and vi are the excitatory and inhibitory synaptic reversal potentials, and the excitatory and inhibitory conductances ge and gi satisfy the stochastic differential equations \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ dg_{\text{e } } = - \tau_{\text{e}}^{- 1 } \left ({ g_{\text{e } } - g_{\text{e}}^ { } } \right)dt + \sigma_{\text{e } } dw_{\text{e } } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ dg_{\text{i } } = - \tau_{\text{i}}^{- 1 } \left ({ g_{\text{i } } - g_{\text{i}}^ { } } \right)dt + \sigma_{\text{i } } dw_{\text{i } }. $ $ \end{document}here, \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g_{\text{e}}^ { } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g_{\text{i}}^ { } $ $ \end{document } are equilibrium values, e and i are time constants, we and wi are independent standard wiener processes, and e and e are constant noise amplitudes. the parameters employed in the simulations, with excitation only, were ve = 80 mv, e = 2 ms. | the effects of noise on neuronal dynamical systems are of much current interest. here, we investigate noise - induced changes in the rhythmic firing activity of single hodgkin huxley neurons. with additive input current, there is, in the absence of noise, a critical mean value = c above which sustained periodic firing occurs. with initial conditions as resting values, for a range of values of the mean near the critical value, we have found that the firing rate is greatly reduced by noise, even of quite small amplitudes. furthermore, the firing rate may undergo a pronounced minimum as the noise increases. this behavior has the opposite character to stochastic resonance and coherence resonance. we found that these phenomena occurred even when the initial conditions were chosen randomly or when the noise was switched on at a random time, indicating the robustness of the results. we also examined the effects of conductance - based noise on hodgkin huxley neurons and obtained similar results, leading to the conclusion that the phenomena occur across a wide range of neuronal dynamical systems. further, these phenomena will occur in diverse applications where a stable limit cycle coexists with a stable focus. |
the patient data analyzed in this study were obtained from the united kingdom glaucoma treatment study (ukgts). the ukgts was a randomized, double - masked, placebo - controlled, multicenter treatment trial for open - angle glaucoma. the study recruited newly diagnosed (untreated) glaucoma cases with glaucomatous visual field defects consistent with optic nerve head changes and with open angles on gonioscopy. subjects with moderately advanced visual field loss (mean deviation worse than 10 db in the better eye or worse than 16 db in the other eye) were excluded from the study and subjects with iop greater than 35 mm hg on two consecutive visits were also excluded from the study. subjects with lens opacity greater than p1 (on the lens opacity classification system iii grading) and those with other ocular comorbidities such as diabetic retinopathy were also excluded from the study. further details on the inclusion and exclusion and other types of tests included in the ukgts study can be found elsewhere. we analyzed test results for perimetry, neuroretinal rim area, and circumpapillary rnfl thickness. the visual field sensitivity measures were obtained using the goldmann size iii stimulus on the humphrey field analyzer (hfa) ii or ii - i, (carl zeiss meditec, dublin, ca, usa) with the sita 24 - 2 program. humphrey field analyzer test results with fixation losses greater than 20% or false - positive rate greater than 15% were excluded. the exclusion criteria did not exempt any subjects in the original ukgts dataset from our study. the rim area measurements were obtained using the hrt-3 (software version 3.0.60 ; heidelberg engineering, heidelberg, germany). when the mean pixel sd of the hrt image was greater than 40 m, the hrt data were regarded as unreliable and were excluded. rim area data were collected on 482 right eyes and 481 left eyes. after applying the exclusion criteria we were left with hrt data for 482 and 480 right and left eyes, respectively. the rnfl thicknesses were acquired using the stratus oct (software version 5.0 ; carl zeiss meditec). the fast rnfl thickness scanning protocol was used (3.4-mm diameter circle centered on the disc). the oct data were screened to exclude scans with signal strength less than 7. for our analysis we also excluded oct scans with errors : with an error message, a pixel lower than 10 m, or the range across repeated scans greater than 15 m. there were oct data on 289 right eyes and 277 left eyes in the original ukgts data set. after applying our inclusion and exclusion criteria, we were left with rnfl thickness measures for 284 and 261 right and left eyes, respectively. we matched the hrt, oct, and hfa data by date and eye for the last visit of each patient. we required that all three tests were acquired on the same day, in order to avoid artifacts from progression. we were able to successfully match the three tests for 245 right eyes and 223 left eyes. the 55 controls were from a pool of 62 whose oct and hfa data had already been published in swanson. only subjects who also had reliable data on the hrt the criteria for excluding unreliable data and matching reliable data were the same for the controls as for the patients. the instruments used in acquiring the control data were comparable with those used in the ukgts study. in the original studies it was reported that the methods used in gathering the data were in accordance with the declaration of helsinki. informed consent was obtained from subjects after explanation of the nature and goals of the study, before testing began. in our primary analysis, we analyzed data from the right eyes in an effort to replicate the finding that between - subject variability in controls is the primary source of structural functional (and structural structural) discordance in patients with glaucoma. we analyzed data from the left eyes in a secondary confirming analysis, in the same way as for the right eye. global measures from hfa, oct, and hrt were used to reduce the impact of between - subject variations in nerve fiber projection to optic disc sectors. for the hfa, global visual field sensitivity was calculated by first converting the sensitivity at the various test locations to a linear scale before averaging. two visual field points, at and just above the blind spot, were excluded from this index. in order to overcome the impact of the differences in the metric of measurement of the various clinical measuring techniques on our analysis, we computed depth of defect (difference from mean normal) for data from each device. depth of defect was used as a measure of the severity of the glaucomatous damage. our analysis was centered around the limits of agreement between two measures as described by bland and altman ; the difference in depth of defect for two tests was plotted against their average depth of defect. the difference in defect depth may vary with the severity of damage, so we calculated the residuals from linear regression of difference versus mean. the absolute values of the residuals were plotted against the average of the two measures and then fitted with a regression to estimate whether the limits of agreement varied with severity of damage. in our analysis, the limits of agreement did not vary with the average depth of defect and so we did not proceed with the other methods described in section 3.3 of the bland and altman article (that describe computing limits of agreement that vary with the average depth of defect). functional comparisons (perimetry versus rnfl thickness, perimetry versus rim area) and structural structural comparisons (rnfl thickness versus rim area). it has been found, that relative to the hfa, depth of defect estimated from structural measures is limited by a floor (due to the nonneural component contained in the structural measures). this floor introduces a potential artifact to the bland - altman analysis when two measures (containing different amounts of nonneural components) are being compared. a floor of 0.5 was used in swanson. in the bland - altman plot comparing hfa and oct (and oct and hrt) we also used a floor of 0.5. for lack of a study that established a floor for hrt measures and for the sake of simplicity we also used a floor of 0.5 in the hfa - hrt comparison. in the primary analysis, statistical significance was set to a p value of 0.017 (a bonferroni adjustment) because three f values (two structural functional comparisons and one structural structural comparison) were assessed. for a p value of 0.017 and the sample sizes in the patient and control groups, for these sample sizes, the critical value will be f(244, 54) = 1.63, p less than 0.017. to compare our findings with those of hood., we applied the hood - kardon model (which predicts the relationship between rnfl thickness and perimetry) to our data. the hood - kardon model assumes that the nonneural component of the rnfl thickness contributes approximately 33% of the measured thickness before the onset of the glaucomatous damage. it also assumes that rnfl thickness does not increase beyond mean normal even when visual field sensitivity is above mean normal. to allow for a direct comparison with our analyses we made similar assumptions as hood. (although our analysis was focused on global indexes and that for hood. was for superior temporal and inferior temporal disc sectors). the model predicts that approximately 95% of the patient data points should be within the prediction interval based on variability in the control data. the patient data analyzed in this study were obtained from the united kingdom glaucoma treatment study (ukgts). the ukgts was a randomized, double - masked, placebo - controlled, multicenter treatment trial for open - angle glaucoma. the study recruited newly diagnosed (untreated) glaucoma cases with glaucomatous visual field defects consistent with optic nerve head changes and with open angles on gonioscopy. subjects with moderately advanced visual field loss (mean deviation worse than 10 db in the better eye or worse than 16 db in the other eye) were excluded from the study and subjects with iop greater than 35 mm hg on two consecutive visits were also excluded from the study. subjects with lens opacity greater than p1 (on the lens opacity classification system iii grading) and those with other ocular comorbidities such as diabetic retinopathy were also excluded from the study. further details on the inclusion and exclusion and other types of tests included in the ukgts study can be found elsewhere. we analyzed test results for perimetry, neuroretinal rim area, and circumpapillary rnfl thickness. the visual field sensitivity measures were obtained using the goldmann size iii stimulus on the humphrey field analyzer (hfa) ii or ii - i, (carl zeiss meditec, dublin, ca, usa) with the sita 24 - 2 program. humphrey field analyzer test results with fixation losses greater than 20% or false - positive rate greater than 15% were excluded. the exclusion criteria did not exempt any subjects in the original ukgts dataset from our study. the rim area measurements were obtained using the hrt-3 (software version 3.0.60 ; heidelberg engineering, heidelberg, germany). when the mean pixel sd of the hrt image was greater than 40 m, the hrt data were regarded as unreliable and were excluded. rim area data were collected on 482 right eyes and 481 left eyes. after applying the exclusion criteria we were left with hrt data for 482 and 480 right and left eyes, respectively. the rnfl thicknesses were acquired using the stratus oct (software version 5.0 ; carl zeiss meditec). the fast rnfl thickness scanning protocol was used (3.4-mm diameter circle centered on the disc). the oct data were screened to exclude scans with signal strength less than 7. for our analysis we also excluded oct scans with errors : with an error message, a pixel lower than 10 m, or the range across repeated scans greater than 15 m. there were oct data on 289 right eyes and 277 left eyes in the original ukgts data set. after applying our inclusion and exclusion criteria, we were left with rnfl thickness measures for 284 and 261 right and left eyes, respectively. we matched the hrt, oct, and hfa data by date and eye for the last visit of each patient. we required that all three tests were acquired on the same day, in order to avoid artifacts from progression. we were able to successfully match the three tests for 245 right eyes and 223 left eyes. the 55 controls were from a pool of 62 whose oct and hfa data had already been published in swanson. only subjects who also had reliable data on the hrt the criteria for excluding unreliable data and matching reliable data were the same for the controls as for the patients. the instruments used in acquiring the control data were comparable with those used in the ukgts study. in the original studies it was reported that the methods used in gathering the data were in accordance with the declaration of helsinki. informed consent was obtained from subjects after explanation of the nature and goals of the study, before testing began. in our primary analysis, we analyzed data from the right eyes in an effort to replicate the finding that between - subject variability in controls is the primary source of structural functional (and structural structural) discordance in patients with glaucoma. we analyzed data from the left eyes in a secondary confirming analysis, in the same way as for the right eye. global measures from hfa, oct, and hrt were used to reduce the impact of between - subject variations in nerve fiber projection to optic disc sectors. for the hfa, global visual field sensitivity was calculated by first converting the sensitivity at the various test locations to a linear scale before averaging. two visual field points, at and just above the blind spot, were excluded from this index. in order to overcome the impact of the differences in the metric of measurement of the various clinical measuring techniques on our analysis, we computed depth of defect (difference from mean normal) for data from each device. depth of defect was used as a measure of the severity of the glaucomatous damage. our analysis was centered around the limits of agreement between two measures as described by bland and altman ; the difference in depth of defect for two tests was plotted against their average depth of defect. the difference in defect depth may vary with the severity of damage, so we calculated the residuals from linear regression of difference versus mean. the absolute values of the residuals were plotted against the average of the two measures and then fitted with a regression to estimate whether the limits of agreement varied with severity of damage. in our analysis, the limits of agreement did not vary with the average depth of defect and so we did not proceed with the other methods described in section 3.3 of the bland and altman article (that describe computing limits of agreement that vary with the average depth of defect). functional comparisons (perimetry versus rnfl thickness, perimetry versus rim area) and structural structural comparisons (rnfl thickness versus rim area). it has been found, that relative to the hfa, depth of defect estimated from structural measures is limited by a floor (due to the nonneural component contained in the structural measures). this floor introduces a potential artifact to the bland - altman analysis when two measures (containing different amounts of nonneural components) are being compared. a floor of 0.5 was used in swanson. in the bland - altman plot comparing hfa and oct (and oct and hrt) we also used a floor of 0.5. for lack of a study that established a floor for hrt measures and for the sake of simplicity we also used a floor of 0.5 in the hfa - hrt comparison. in the primary analysis, statistical significance was set to a p value of 0.017 (a bonferroni adjustment) because three f values (two structural functional comparisons and one structural structural comparison) were assessed. for a p value of 0.017 and the sample sizes in the patient and control groups, for these sample sizes, the critical value will be f(244, 54) = 1.63, p less than 0.017. to compare our findings with those of hood., we applied the hood - kardon model (which predicts the relationship between rnfl thickness and perimetry) to our data. the hood - kardon model assumes that the nonneural component of the rnfl thickness contributes approximately 33% of the measured thickness before the onset of the glaucomatous damage. it also assumes that rnfl thickness does not increase beyond mean normal even when visual field sensitivity is above mean normal. to allow for a direct comparison with our analyses we made similar assumptions as hood. (although our analysis was focused on global indexes and that for hood. was for superior temporal and inferior temporal disc sectors). the model predicts that approximately 95% of the patient data points should be within the prediction interval based on variability in the control data. for 245 right eyes and 223 left eyes, the patients had mean (sd) age of 65.5 (11.1) and 65.6 (11.2) years, respectively. the 55 controls had mean (sd) age of 63.0 (9.9) years. for the controls, mean (sd) perimetric sensitivity was 0.49 (0.14) log contrast sensitivity, mean rnfl thickness was 1.99 (0.04) log m and mean rim area 0.18 (0.04) log mm. the mean control hrt disc area was 1.93 (0.41) mm and that for the patients was 2.02 (0.40) mm and 2.00 (0.47) mm for od and os, respectively. the table shows the sds of the residuals when depths of defect measured by two devices were compared on a bland - altman plot. the sd in log units of the residuals around the regression line on a bland - altman plot comparing two clinical device outputs in patients the left panels in figure 1 show plots of discordance against average depth of defect comparing perimetry and rnfl thickness. it also features the hood - kardon model modified for the plot. in the right and left eyes, 4.7% and 5.4%, respectively, of the patient data fell outside the 95% limits based on the dispersion in the control population. the upper plots show data for left eyes and the lower plots show data for right eyes. the left panels show limits comparable to the hood - kardon model, and the right panels show the 95% limits of agreement comparable to the limits of swanson. the red ellipses on the left show the 95% prediction interval for the controls ; the black broken lines show the 95% prediction interval for patients. the slope of the regression line on the bland - altman plot was 0.87 (se = 0.07) and 0.88 (se = 0.06) for right and left eyes, respectively ; the intercepts of the regression lines were 0.07 (se = 0.01) and 0.06 (se 0.01) for left and right eyes respectively. a second structural functional comparison is shown in figure 2, perimetry versus rim area. the slope of the regression line was 0.44 (se = 0.10) for right eyes and 0.59 (se = 0.10) for left eyes ; the intercepts were 0.13 (se = 0.02) and 0.10 (se = 0.02) for the right and left eyes, respectively. the upper plot shows data for left eyes and the lower plot shows data for right eyes. a structural structural comparison is shown in figure 3, rim area versus rnfl thickness. the slope of the regression line on the bland - altman plot was 0.56 (se = 0.07) for right eyes and 0.47 (se = 0.08) for left eyes ; the intercept was 0.00 (se = 0.01) for both right and left eyes. a bland - altman plot comparing the rnfl thickness and rim area. as in figures 1 and 2, the upper plot shows data for left eyes and the lower plot shows data for right eyes. in this study, we replicate the finding that normal between - subject variability is the primary source of structural functional discordance in patients with glaucoma. we compared the variability of the discordance in the patients with the variability of the discordance in the controls and found them to be similar. we extended the analysis to include a structural structural comparison and found a similar result ; variability in the control group was similar to variability in the patient group. we also analyzed the perimetry versus rnfl thickness data using an extension of the hood - kardon model, and found that approximately 95% of the patient data fell within the 95% prediction interval computed from the control population. in the hfa - hrt comparison however, we noticed a relatively larger variance as compared with the hfa - oct and oct - hrt comparisons. we were therefore interested in investigating the impact of disc area on the hfa - hrt comparison using the moorfields regression analysis. compared with our original analysis, adjusting for the rim area (using disc area and age) in the hfa - hrt comparison increased the variance in the right of patients by 1% and decreased the variability in the control group by 3%. overall, adjusting for the rim area (using the disc size and age) did not result in a significant improvement in the hfa - hrt comparison. this is not however surprising because we did not see any consistent distribution in depth of defect based on disc size on our plots. this increased variability may be due to some image acquisition factors like the operator tracing the area of the disc and the limit imposed on the rim area by the disc size. in the comparison of perimetry and rnfl thickness, the regression on the bland - altman plot yielded a positive slope, meaning that as the average defect depth increased, perimetry tended to give increasingly deeper defects than did rnfl thickness. the comparison of perimetry and rim area also found positive slopes, but they were not as steep as for perimetry versus rnfl thickness. this is consistent with the proposal by shafi. of a greater nonneural component for rnfl thickness than for rim area. the direct comparison of rim area and rnfl thickness in figure 3 also supports a greater nonneural component for rnfl thickness than for rim area. we recomputed the discordance between the rim area and rnfl thickness measures using a series of other floors to investigate whether the finding on the relative amount of nonneural component contained in the rim area and rnfl thickness measures was impacted by our choice of floor (0.5 log unit). we found that although the magnitude of slope of the bland - altman plot changed depending on the floor, its (negative) direction and implication did not change. the intercept of the bland - altman regression line was nonzero for perimetry versus rnfl thickness and for perimetry versus rim area, but not for rim area versus rnfl thickness. this is an indication that at the early onset of glaucomatous damage, structural measures estimate deeper defects than perimetry. a recent study from our research group found a similar result when comparing sap with new forms of perimetry resistant to peripheral defocus : nonzero intercepts for bland - altman regression line indicating that sap defects were not as deep in mild defects and became deeper in more severe defects. hood. pointed out that when two tests are being compared, the test with a smaller sd in the control population would require less damage to reach statistical significance and be flagged as glaucomatous than the test with a larger sd. drawing from the statistics of identifying which loss constitutes glaucomatous damage and which loss does not, the oct is likely to be more sensitive than the hfa because it had a smaller sd in the control group than the hfa. in an exploratory analysis, we calculated the fifth percentile for the rnfl thickness and visual field measures from the control population. we set the value computed as threshold and identified subjects as showing glaucomatous damage when their rnfl thickness (or visual field sensitivity) fell below the threshold. the oct identified more subjects than the hfa as showing glaucomatous damage consistent with the finding of hood., this may explain why the oct appears to be more sensitive to early damage and suggests a different reasoning (as to why the oct may flag mild defects earlier than the hfa) than preperimetric glaucoma concept (which suggests that a large percentage ofganglion cells are lost before the loss is detected on perimetric testing). from the comparisons, we also noticed that the limits of agreement of oct - hfa comparison (a structural functional comparison) in the patient group were similar to the limits of agreement of the oct - hrt comparison (a structural structural comparison). this provides further evidence that the discordance seen in comparing structural measures to functional measures may not be necessarily due to the fact that one method assesses glaucomatous damage through structural loss while the other uses functional loss. we investigated the characteristics of disc parameters (such as cup - to - disc volume, cup volume, cup disc ratio, etc.) reported by the hrt in the control subjects with the most extreme discordance for each comparison to determine whether there are any specific structural patterns reported by the hrt that may explain the variability of the discordance in controls. for the hfa - oct comparison we did not find any significant difference between subjects with the most extreme positive discordance (greater hfa depth of defect than oct) and those with the most extreme negative discordance (greater oct depth of defect than hfa). for example, the mean disc area of controls with the highest 10% positive discordance (greatest hfa depth of defect than oct) and those with the highest 10% negative discordance (greatest oct depth of defect than hfa) were 1.99 mm (sd = 0.15) and 1.82 mm (sd = 0.30). however, in the hrt - oct comparison we noticed that the maximum cup depth, mean cup depth, horizontal and vertical cup - to - disc area ratio were significantly higher (at p < 0.05) in subjects with the most extreme positive discordance (greater hrt depth of defect than oct) than those with the most extreme negative discordance (greater oct depth of defect than hrt). in a consistent manner, the rim disc area ratio and rim volume were significantly smaller in subjects with the most extreme positive discordance than subjects with the most extreme negative discordance. thus, adjusting for maximum cup depth, mean cup depth, or horizontal and vertical cup - to - disc ratio may reduce the discordance in the healthy population for oct - hrt comparison. we also noticed that the majority of subjects with the most extreme discordance in the hfa - oct comparison were not the same as the subjects with the most extreme discordance in the hfa - hrt comparison. thus, the few subjects who have extreme discordance on both the hfa - oct and hrt - oct comparison may have some underlying structural pattern common to both the hrt and oct that causes the structural depth of defect to differ from the functional depth of defect in a consistent manner. however, it is not clear whether the subjects who have higher depth of defect on the hfa - oct comparison but not on the hfa - hrt measure or vice versa have any specific structural pattern. the trend in this group is consistent with the hrt - oct discordance and suggests a multidimensional source of structural functional discordance. thus, the discordance observed in a subject is not only a property of the structural and functional ganglion cell integrity but is also influenced by the region on the retina where the structural integrity is being assessed. in conclusion, we confirmed the finding of prior studies that between - subject variability in healthy eyes is the primary source of structural functional discordance in patients, and extended this to structural structural comparisons. developing techniques with reduced between - subject variability in the healthy population may be helpful in improving the discordance in comparing two techniques. there have been proposals on how to reduce between - subject variability in controls. patel. proposed using rnfl volume instead of rnfl thickness in structural measures ; elsewhere, there are proposals on how to reduce the impact of prereceptoral factors on perimetric sensitivity. these proposals are geared toward reducing the normal between - subject variability and may consequently yield more sensitive measures with reduced structural functional (or structural structural) discordance when two measures are compared. improved techniques for structural and functional measures yielding improved structural functional discordance may not only be necessary to complement a clinician 's diagnosis but will also open discussions for the use of perimetry for testing ganglion cell dysfunction. to address the challenge of structural functional discordance holistically, other factors that need to be addressed include the spatial sampling of perimetric measures and the difficulty in spatially mapping structural abnormality to functional abnormality using the structural functional maps. while the structural measures (acquired in the region of the disc) assess all the ganglion cell axons present in the retina, perimetric testing with the 24 - 2 protocol samples 54 testing locations on the retina. this predisposes the perimetric tests to miss localized wedge defects that may fall between perimetric testing locations while being identified by the structural measures. due to the fact that quite a number of the structural measures concentrated on determining glaucomatous structural abnormality at the region of the disc, most studies are faced with the challenge of choosing a structural functional map to be able to relate structural abnormality to functional abnormality. while our study concentrated on using global measures to overcome this challenge, it is clear that the wide range of variability in the trajectory of the retinal nerve fiber bundles to the disc is a potential source of structural functional discordance. ballae ganeshrao. have demonstrated that accurately mapping structural measures to functional measures spatially accounting for the anatomical differences in these trajectories yield improved structural the developments in imaging techniques that allow the visualization of the nerve fiber bundles en face are an attractive approach to overcoming the spatial challenges implicated in the structural functional discordance and may provide more insight into the true nature of the structural - functional relationship. | purposeto test with an independent data set the finding that between - subject variability in healthy eyes is the primary source of structural functional discordance in patients with glaucoma.methodsneuroretinal rim area, retinal nerve fiber layer thickness, and perimetric data were analyzed for one eye in each of 55 control subjects and for 245 right eyes of patients in the united kingdom glaucoma treatment study. data were gathered with the heidelberg retina tomograph (hrt), stratus optical coherence tomograph (oct), and humphrey field analyzer (hfa). discordance was quantified as width of the limits of agreement from a bland - altman analysis of depth of defect. the ratio of variances (f test) for the patient and control groups was computed for comparisons of hfa - oct, hfa - hrt, and oct - hrt. bonferroni adjustment required p less than 0.017 for statistical significance. the discordance in the patients was also quantified as the 95% prediction interval computed from the discordance in controls using the hood - kardon model for the hfa - oct comparison.resultsthe f ratio comparing discordance in patients and controls was 0.77, 1.43, and 1.32 for the hfa - oct, hfa - hrt, and oct - hrt comparisons with p values 0.88, 0.06, and 0.11, respectively. for the hood - kardon model, 4.7% of the patients had discordance outside the 95% prediction interval computed from the discordance in controls. similar results were obtained when all comparisons were repeated for left eyes of patients.conclusionsthese results confirm previous findings that between - subject variability in healthy eyes is the primary source of structural functional discordance in patients with glaucoma, and extends this finding to a structural structural comparison. |
hospitals are complex organizations which face a unique challenge in this ever changing economic environment : they need to increase the quality of care offered to their patients, while still reducing the specific associated costs. in this respect, many countries have set multiple forms of future funding policies to pay for healthcare, whereby hospitals receive a certain amount for treating patients according to their number and type, and are unable to impact this price. this system creates clear financial incentives to contain expenses, as hospitals that stay within the threshold will make a profit, while those delivering high - priced care will make a loss. for example, a drg (diagnosis - related group) system used to distinguish between patients with a certain diagnosis does not account for cost variability across disease progression. thus, the main cause for cost variability even between hospital departments is attributed to the case mix index. specialist or teaching hospitals (secondary hospitals, respectively tertiary hospitals) might make an argument that they treat relatively more severe patients due to their reputation for providing high quality care. multi - hospital health systems have become the most popular administrative structure in the hospital industry, leading to both opportunities and challenges for hospital administrators. in government funded healthcare systems, a balance between costs and health services exists. thus, pooling demand for specialized services can help contain healthcare expenses, but it can burden the patients as there will be increased travel distance. one might be restraining operations to become more efficient, or to distinguish them from the competition, by offering enhanced products and services. other possible reasons include increasing spending on public relations, or reinforcing their bargaining power with health plans. specialized care units have the potential to increase the patient volume, reduce expenses, and increase quality. thus, aggregative common tasks can help at reducing healthcare associated costs, while still improving patient outcomes. research showed that quality increases when a high patient - volume for a specialized procedure allows medical personnel to develop their accumulative experience in managing the service and avoid a loss of learning between occasional procedures. in terms of multi - system hospitals, containing expenses for medical capacity can be transmitted to the patients in forms of smaller hospital costs, or used by the network to offer additional services. while some have found that multi - hospital systems have higher costs per case than freestanding hospitals, others have found the contrary. for example, one accounting office might be in charge of all units with little appreciable increase in costs. moreover, hospital systems can scope economies on the production side by reducing identical equipment and efficiently managing employment and supply inventories. research also shows that hospital systems help increase quality of care through better coordination and better tailoring of local needs. they can also have an advantage with bargaining health plans, which can lead to higher prices and lower patient volumes. multi - system hospitals are characterized by the consolidation of information processing and procuring functions, but also by increased access to capital, management expertise, and increased physician recruiting. moreover, they also have improved market power and better capacity to participate in managed care contracting., merged with performance feedback are associated with enhanced acceptance of certain nonsurgical treatments. apart from these, hospital affiliations can impact on the treatment of patients through two channels. firstly, multi - system hospitals may offer a wider array of health services, through service proliferation, by providing management expertise and funds to build new facilities and recruit medical personnel. system affiliation also affects patient treatment patterns, as patients may need to be transferred from one hospital to another in order to receive a certain service. secondly, multi - system hospitals have improved coordination and information transfer among facilities, which also impacts on patient treatment. therefore, system hospitals face lower transaction expenses in developing and implementing procedures and protocols. having protocols in place means that hospitals may have quicker transfer times between facilities. to conclude unfortunately, most of these data are business - oriented reports, ignoring one of the most vulnerable issues in such circumstances healthcare - associated infections, with their tremendous burden (medical, epidemiological, economical etc.). thus, very few details were identified regarding expenses and expenditure control of multi - hospital systems. the aim of the present study is to assess the efficiency in terms of costs of a multi - pavilion hospital from cluj county, romania. hospitals are complex organizations which face a unique challenge in this ever changing economic environment : they need to increase the quality of care offered to their patients, while still reducing the specific associated costs. in this respect, many countries have set multiple forms of future funding policies to pay for healthcare, whereby hospitals receive a certain amount for treating patients according to their number and type, and are unable to impact this price. this system creates clear financial incentives to contain expenses, as hospitals that stay within the threshold will make a profit, while those delivering high - priced care will make a loss. for example, a drg (diagnosis - related group) system used to distinguish between patients with a certain diagnosis does not account for cost variability across disease progression. thus, the main cause for cost variability even between hospital departments is attributed to the case mix index. specialist or teaching hospitals (secondary hospitals, respectively tertiary hospitals) might make an argument that they treat relatively more severe patients due to their reputation for providing high quality care. multi - hospital health systems have become the most popular administrative structure in the hospital industry, leading to both opportunities and challenges for hospital administrators. in government funded healthcare systems, a balance between costs and health services exists. thus, pooling demand for specialized services can help contain healthcare expenses, but it can burden the patients as there will be increased travel distance. one might be restraining operations to become more efficient, or to distinguish them from the competition, by offering enhanced products and services. other possible reasons include increasing spending on public relations, or reinforcing their bargaining power with health plans. specialized care units have the potential to increase the patient volume, reduce expenses, and increase quality. thus, aggregative common tasks can help at reducing healthcare associated costs, while still improving patient outcomes. research showed that quality increases when a high patient - volume for a specialized procedure allows medical personnel to develop their accumulative experience in managing the service and avoid a loss of learning between occasional procedures. in terms of multi - system hospitals, containing expenses for medical capacity can be transmitted to the patients in forms of smaller hospital costs, or used by the network to offer additional services. while some have found that multi - hospital systems have higher costs per case than freestanding hospitals, others have found the contrary. for example, one accounting office might be in charge of all units with little appreciable increase in costs. moreover, hospital systems can scope economies on the production side by reducing identical equipment and efficiently managing employment and supply inventories. research also shows that hospital systems help increase quality of care through better coordination and better tailoring of local needs. they can also have an advantage with bargaining health plans, which can lead to higher prices and lower patient volumes. multi - system hospitals are characterized by the consolidation of information processing and procuring functions, but also by increased access to capital, management expertise, and increased physician recruiting. moreover, they also have improved market power and better capacity to participate in managed care contracting., merged with performance feedback are associated with enhanced acceptance of certain nonsurgical treatments. apart from these, hospital affiliations can impact on the treatment of patients through two channels. firstly, multi - system hospitals may offer a wider array of health services, through service proliferation, by providing management expertise and funds to build new facilities and recruit medical personnel. system affiliation also affects patient treatment patterns, as patients may need to be transferred from one hospital to another in order to receive a certain service. secondly, multi - system hospitals have improved coordination and information transfer among facilities, which also impacts on patient treatment. therefore, system hospitals face lower transaction expenses in developing and implementing procedures and protocols. having protocols in place means that hospitals may have quicker transfer times between facilities. to conclude unfortunately, most of these data are business - oriented reports, ignoring one of the most vulnerable issues in such circumstances healthcare - associated infections, with their tremendous burden (medical, epidemiological, economical etc.). thus, very few details were identified regarding expenses and expenditure control of multi - hospital systems. the aim of the present study is to assess the efficiency in terms of costs of a multi - pavilion hospital from cluj county, romania. the institution analyzed in this article is the adults clinical hospital in cluj - napoca (which, in the meantime, has been re - organized and is currently named professor octavian fodor institute of gastroenterology and hepatology cluj - napoca). the hospital was comprised, in the studied period of 20042010, of four pavilions, offering medical services pertaining to psychiatry, obstetrics - gynecology, orthopaedics - traumatology, and internal medicine, general surgery and gastroenterology. the addressability of the hospital is high, over 30,000 patients from nearby counties benefitting annually from medical services. the flowchart of the medical structure of the investigated multi - pavilion hospital during 2004 2010 is depicted in figure 1. it should be noted, however, that, starting with 2011, the adults clinical hospital cluj - napoca is only comprised of the general surgery and internal medicine compartments (m3-c3). a descriptive retrospective study was conducted from january 2004 to december 2010 to reach the aim of this study. a set of indicators were compiled, divided into three main categories : personnel, statistics, and financial. data related to these indicators was collected at a hospital level, from all four compartments pertaining to the adults clinical hospital cluj - napoca. the main indicators assessed in this study are presented in table i. descriptive statistics were performed on the variables, such as frequencies, means and standard deviations, medians (q1q3). inferential statistics were performed the check for significant differences between the means of the four hospitals, such as an anova test. the institution analyzed in this article is the adults clinical hospital in cluj - napoca (which, in the meantime, has been re - organized and is currently named professor octavian fodor institute of gastroenterology and hepatology cluj - napoca). the hospital was comprised, in the studied period of 20042010, of four pavilions, offering medical services pertaining to psychiatry, obstetrics - gynecology, orthopaedics - traumatology, and internal medicine, general surgery and gastroenterology. the addressability of the hospital is high, over 30,000 patients from nearby counties benefitting annually from medical services. the flowchart of the medical structure of the investigated multi - pavilion hospital during 2004 2010 is depicted in figure 1. it should be noted, however, that, starting with 2011, the adults clinical hospital cluj - napoca is only comprised of the general surgery and internal medicine compartments (m3-c3). a descriptive retrospective study was conducted from january 2004 to december 2010 to reach the aim of this study. a set of indicators were compiled, divided into three main categories : personnel, statistics, and financial. data related to these indicators was collected at a hospital level, from all four compartments pertaining to the adults clinical hospital cluj - napoca. descriptive statistics were performed on the variables, such as frequencies, means and standard deviations, medians (q1q3). inferential statistics were performed the check for significant differences between the means of the four hospitals, such as an anova test. variability in the personnel indicators was observed for several indicators, such as number of filled md positions (figure 2), number of beds per filled md positions (figure 3), and number of beds per filled nurses positions (figure 4). thus, even though the ergo pavilion has the lowest number of filled md positions overall for the investigated period, in 2010 its values for the number of beds per filled md positions were higher than for the m3-c3 pavilion. the values were consistent for the m3-c3 pavilion. however, the lowest number of beds per filled nurses positions pertained to the orto pavilion. even though from 2009 to 2010 the number of physician positions has increased from 34 to 53, the number of doctors per number of beds has decreased, for the same time frame, from 62.05 to 38.63, due to an increase in the number of beds. m3-c3 had the highest values for the number of discharges per medical doctor, compared to the other pavilions. moreover, while the other pavilions had steady values for this indicator for the investigated period, we can observe the 2004 values dropped to almost half in 2010. the case mix index differs for each pavilion, with the highest values identified for m3-c3, compared to the lowest for ergo. moreover, while ergo has had consistent values for the investigated period, the values for m3-c3 have almost doubled in 2010, compared to 2004. an interesting pattern was observed in terms of mortality, as the numbers inconsistent for the ergo, orto and stanca pavilions. by comparison, however, the m3-c3 pavilion had higher mortality indicators, being 13.76 in 2010 when compared to the other pavilions, which had values between 0.08 and 1.05 in 2010. while the m3-c3, orto and stanca pavilions have had relatively consistent values for the average hospitalization indicator within the observed timeframe, a drastic increase was observed from 2006 to 2007 for the ergo pavilion, from 24.52 to 47.40. heterogeneity between different years was observed for the continuous hospitalization indicator (figure 10) and the wage budget indicator (figure 11). the highest variability was observed between the budget and expenses indicators (figure 12), while a smaller variability was observed at the average costs per patient (figure 13). the continuous hospitalization budget for m3-c3 was almost triple for 2010 (29,244,529 ron) compared to 2004 (11,726,246 ron). similarly, the continuous hospitalization budget for stanca for 2010 was almost four times higher than the 2004 budget (2,906,982 ron compared 12,400,112 ron). while the same budget increase was observed for orto pavilion as for stanca until 2008, this budget dropped until 2010, being similar to the ergo budget (4,978,232 ron for orto and 4,459,000 ron for ergo). the budget - expenses rapport for m3-c3 has shown increased variability in the observed timeframe, varying between positive and negative from one year to another. the ergo budget - expenses rapport showed a dropping tendency, from 3,201,208 ron in 2004 to minus 723,420 ron in 2010. the orto budget - expenses rapport has also shown a dramatic drop tendency, being minus 962,754 ron in 2004, culminating with minus 4,640,203 ron in 2010. the stanca pavilion is the only one showing a positive rapport between budget and expenses, being minus 617,377 ron, increasing until 3,971,397 ron in 2010. the costs per patient have increased at all pavilions in the observed time frame, the higher costs being at m3-c3 (10203 ron in 2010). however, the most dramatic increase was for the ergo pavilion, from 2006 (2368 ron) to 2007 (6838 ron). variability in the personnel indicators was observed for several indicators, such as number of filled md positions (figure 2), number of beds per filled md positions (figure 3), and number of beds per filled nurses positions (figure 4). thus, even though the ergo pavilion has the lowest number of filled md positions overall for the investigated period, in 2010 its values for the number of beds per filled md positions were higher than for the m3-c3 pavilion. the values were consistent for the m3-c3 pavilion. however, the lowest number of beds per filled nurses positions pertained to the orto pavilion. even though from 2009 to 2010 the number of physician positions has increased from 34 to 53, the number of doctors per number of beds has decreased, for the same time frame, from 62.05 to 38.63, due to an increase in the number of beds. m3-c3 had the highest values for the number of discharges per medical doctor, compared to the other pavilions. moreover, while the other pavilions had steady values for this indicator for the investigated period, we can observe the 2004 values dropped to almost half in 2010. the case mix index differs for each pavilion, with the highest values identified for m3-c3, compared to the lowest for ergo. moreover, while ergo has had consistent values for the investigated period, the values for m3-c3 have almost doubled in 2010, compared to 2004. an interesting pattern was observed in terms of mortality, as the numbers inconsistent for the ergo, orto and stanca pavilions. by comparison, however, the m3-c3 pavilion had higher mortality indicators, being 13.76 in 2010 when compared to the other pavilions, which had values between 0.08 and 1.05 in 2010. while the m3-c3, orto and stanca pavilions have had relatively consistent values for the average hospitalization indicator within the observed timeframe, a drastic increase was observed from 2006 to 2007 for the ergo pavilion, from 24.52 to 47.40. heterogeneity between different years was observed for the continuous hospitalization indicator (figure 10) and the wage budget indicator (figure 11). the highest variability was observed between the budget and expenses indicators (figure 12), while a smaller variability was observed at the average costs per patient (figure 13). the continuous hospitalization budget for m3-c3 was almost triple for 2010 (29,244,529 ron) compared to 2004 (11,726,246 ron). similarly, the continuous hospitalization budget for stanca for 2010 was almost four times higher than the 2004 budget (2,906,982 ron compared 12,400,112 ron). while the same budget increase was observed for orto pavilion as for stanca until 2008, this budget dropped until 2010, being similar to the ergo budget (4,978,232 ron for orto and 4,459,000 ron for ergo). the budget - expenses rapport for m3-c3 has shown increased variability in the observed timeframe, varying between positive and negative from one year to another. the ergo budget - expenses rapport showed a dropping tendency, from 3,201,208 ron in 2004 to minus 723,420 ron in 2010. the orto budget - expenses rapport has also shown a dramatic drop tendency, being minus 962,754 ron in 2004, culminating with minus 4,640,203 ron in 2010. the stanca pavilion is the only one showing a positive rapport between budget and expenses, being minus 617,377 ron, increasing until 3,971,397 ron in 2010. the costs per patient have increased at all pavilions in the observed time frame, the higher costs being at m3-c3 (10203 ron in 2010). however, the most dramatic increase was for the ergo pavilion, from 2006 (2368 ron) to 2007 (6838 ron). efficient cost containment has been an important issue ; hospitals and healthcare facilities in general have been trying to resolve, without impacting the quality of services offered to their patients. the aim of this study was to assess the efficiency in terms of costs of a multi - pavilion hospital from cluj county, romania. the adults clinical hospital cluj - napoca was comprised of four pavilions, each having a different addressability. after comparing the pavilions in terms of personnel, financial and statistical indicators, our study identified several differences across the pavilions. regarding personnel indicators, it was identified that the m3-c3 pavilion had the highest values in terms of number of filled md positions, number of beds per filled md positions and number of beds per filled nurses positions. we believe this difference can be attributed to the higher number of beds the pavilion has, being thus able to capacitate a higher number of patients. namely, m3-c3 pavilion had 341 beds in 2010, compared to 120, 126, and 133 beds for ergo, orto and stanca respectively. moreover, this is also a sign of the hospital s prestige in treating illnesses pertaining to internal medicine. the higher case mix index for the m3-c3, compared to the other three pavilions, can be attributed to the profile of each pavilion. while psychiatry, obstetrics - gynecology, orthopaedics - traumatology, and gastroenterology are more disease - specific, internal medicine and general surgery imply a wider range of diseases treated and of procedures performed. moreover, the hospital is renowned at a regional level for its performance in diagnosing and treating gastroenterological diseases. the mortality indicator showed a higher mortality for the m3-c3 pavilion, compared to the other three. we can speculate that this increased mortality rate can be associated with the higher case mix index, as a higher case mix index also implies more severe cases as well. on the other hand, a study found that a lower patient outcome can be correlated with lower staffing levels, especially for nurses, compared to the patient volume. it was interesting to observe that while the continuous hospitalization budget for ergo, orto and stanca had had a relatively slow or steady increase or even decrease during the observed period, the continuous hospitalization budget for m3-c3 had increased significantly from 2004 to 2010. again, we can attribute this increased hospitalization to the more severe cases treated at the m3-c3, which required prolonged hospital stay post - procedures. moreover, more complex cases also require more complex procedures pre- and post - surgery, having an impact on expenses as well. thus, one hospitalization day at m3-c3 cost on average 1728 ron, compared to 593 ron for stanca, 520 for orto, and only 281 ron for ergo. to further sustain this idea, the data also showed increased costs per patient for m3-c3 pavilion (10,203 ron), compared to 6308 ron for ergo, 5841 ron for orto, and 2792 ron for stanca. research shows that gastrointestinal diseases impose a great burden in the united states in terms of mortality and expenses ; 32.4 billion dollars being spent in 2009 on endoscopy examinations. moreover, at a global level, world health organization data place digestive diseases as a third cause of death for middle - income countries. the budget vs expenses indicator provided a good insight into the financial situation of each pavilion. according to the data gathered from 2004 until 2010, an interesting phenomenon occurred. out of all pavilions, stanca was the only one whose budget was greater than its expenses. contrarily, the orto pavilion was the only one whose expenses increased relatively high compared to its available budget. few studies approached the issue of cost efficiency in healthcare, especially in the context of a multi - system hospital. in romania, the cost efficiency was approached from the perspective of resource allocation to hospitals based on diagnosis - related groups. a study identified new models for researching costs based on the diagnosis - related group system in the romanian health sector. another study approached the issue of efficiency in terms of analyzing hospital s utility costs. cost comparisons between hospitals, or, in this case, hospital pavilions, results in the identification of the most efficient hospitals, and their classification. a precise definition of each service delivered and the identification every cost unit for delivering the service have been highlighted as essential in making comparisons across hospitals. the institute of medicine has identified six dimensions important for a identifying a performing hospital : safety, effectiveness, patient - centeredness, timeliness, efficiency, and equity. these dimensions are essential for determining if a hospital is providing quality care to its patients, and how efficient it is in cost - containment without jeopardizing care quality. hospital systems are associated with increases in hospital market concentration, as their services respond to a wide array of procedure demand. moreover, if these hospitals are located in the same area, they can rationalize care delivery more efficiently, as they can coordinate the procedures effectively. however, the link between quality increase and expense containment is difficult to assess, as one might have to discern whether they are complementary or in competition one with the other. to the best of our knowledge, this study is the first to approach the issue of cost - efficiency in the context of a multi - pavilion hospital in romania. the pavilions included in the adults clinical hospital cluj - napoca have different expenses patterns, as each pavilion is focused on different specialties. this in turn results in different expense patterns across each pavilion. we must study this trend more in depth, in order to gain a more comprehensive understanding of the phenomenon, so that effective expenditure containment strategies could be implemented. future research can also focus on comparing the 20042010 timeframe with present times, so that we could identify other expenditure patterns that might have occurred. | background and aimmulti - hospital health systems have become the most popular administrative structure in healthcare, leading to both opportunities and challenges for hospital administrators. in government - funded healthcare systems, there is a balance between costs and the provision of health services.the aim of the present study is to assess the efficiency in terms of costs of a multi - pavilion hospital from cluj county, romania.methodsthe institution analyzed in this article is the adults clinical hospital in cluj - napoca. a descriptive retrospective study collected data from january 2004 to december 2010. a set of indicators were compiled, divided into three main categories : personnel, statistics, and financial.resultstwenty-one financial indicators were investigated. heterogeneity between different years was observed for the continuous hospitalization indicator and the wage budget indicator. the highest variability was observed between the budget and expenses indicators, while a smaller variability was observed at the average costs per patient. the costs per patient have increased at all pavilions in the studied time frame, the higher costs being at the internal medicine and surgery pavilions : 10,203 ron in 2010 (1 euro ~ 4.4 ron)conclusionthe pavilions included in the adults clinical hospital cluj - napoca have different expenses patterns, as each pavilion is focused on different specialties. each pavilion serves different target groups, requiring different procedures. this in turn results in different expense patterns across each pavilion. |
sepsis is a major healthcare problem. despite advances in supportive care of critically ill patients, sepsis remains an important cause of death worldwide in adults and children [13 ]. the surviving sepsis campaign (ssc), which standardized the approach to sepsis, was recently updated. several efforts have been made to improve adherence to ssc guidelines [5, 6 ]. nevertheless, mortality and costs are still high [2, 7, 8 ]. first, the presence of microorganisms in the bloodstream causes an innate immune response characterized by the stimulation of monocytes and release of proinflammatory cytokines and the activation of a medley of different immune pathways. toll - like receptors (tlrs) play a key role in this initial immune activation, acting as innate immune system sensors through the recognition of highly conserved components of a variety of microorganisms. the activation of tlrs induces an inflammatory response to control the infection, which results in local vasodilatation, release of various cytotoxic chemicals, and, hopefully, destruction of the invading pathogen. many of these same components of inflammation that are beneficial in host defenses against infection can, under some circumstances, be deleterious, causing cell and tissue damage and hence multiple organ failure. endotoxin, also known as lipopolysaccharide, is a component of gram - negative bacteria and a strong activator of tlr4. the recognition of endotoxin by immune cells is important in the pathogenesis of septic shock [10, 11 ]. conventional therapy such as antibiotics and surgical procedures to remove the source of infection is crucial for treating sepsis, but these approaches can not reverse the effects of the bacterial toxins already released into blood or of the endogenous mediators produced by the host in response to bacteria. over recent years, numerous attempts have aimed to intervene in the inflammatory cascade. attempts to stop the inflammatory cascade using antiendotoxin strategies such as monoclonal antibodies or vaccines have failed [12, 13 ]. a recent phase 2 trial found a nonsignificant trend toward better survival in patients with severe sepsis treated with eritoran tetrasodium, a tlr-4 antagonist. blood purification techniques including hemoperfusion, plasma exchange, and hemofiltration with hemoperfusion are associated with lower mortality in patients with sepsis as it has been demonstrated in a recent meta - analysis. devices to remove endotoxin or inflammatory cytokines have been designed as a strategy to reduce the morbidity and mortality associated with sepsis, especially with sepsis due to gram - negative bacteria. these devices have also been successfully used in patients with sepsis due to gram - positive microorganisms and in patients with acute respiratory distress syndrome (ards), suggesting that they could have an immunomodulating action in addition to endotoxin elimination [1721 ]. indeed, this review aims to summarize the immune modulation actions of polymyxin b - immobilized cartridge to understand the potential usefulness of this device beyond endotoxin elimination. over recent years, devices to eliminate endotoxin, inflammatory molecules such as cytokines and immune cells, have been designed to mitigate the deleterious effects of the inflammatory cascade. most of these devices are designed to combine the effect of molecules removal with renal replacement therapies (hemofiltration, dialysis, or hemodiafiltration). the biocompatibility of these devices is the main limitation for its use, thrombocytopenia and bleeding risk are the potential side effects. polymyxin b is a cationic polypeptide antibiotic with activity against gram - negative bacteria and a high affinity to endotoxin, but its intravenous use has been limited due to nephrotoxicity and neurotoxicity. since 1994, polymyxin b has been fixed and immobilized with polystyrene fiber in a hemoperfusion column polymyxin b - immobilized cartridge (pmx) that allows endotoxin removal without the toxic effects of this antibiotic. this treatment has been widely used in japan for septic shock due to gram - negative bacteria, and its use was authorized in europe in 1998. recent studies support the safety and efficacy of this treatment [16, 2426 ]. this medical device designed for extracorporeal use contains a series of porous polyethylene plates coated with a peptide specific to endotoxin and has a high adsorption capacity. compared lps adsorber and pmx hemoperfusion in a small sample of patients with gram - negative sepsis. however, due to limitations of the study, the authors concluded that further studies were necessary to clarify the efficacy of lps adsorber. this an-69 (polysulfone and polyacrylonitrile) based membrane adsorbs a large spectrum of plasma inflammatory mediators such as endotoxin and cytokines [30, 31 ]. to date, clinical experience with this device is limited, but two trials are underway in septic patients. the results of these two trials are crucial to determine its usefulness compared with the current standard of care. based on the endotoxin - binding abilities of human albumin, this adsorber contains human serum albumin immobilized on polymethacrylate beads. this extracorporeal treatment is based on nonspecific adsorption of cytokines and other proinflammatory mediators onto a specially designed resin cartridge, coupled with hemofiltration. this is a promising therapy, although further studies are necessary to determine its usefulness in septic patients. one clinical trial, compact 2, is underway to clarify whether adding high doses of cpfa to current clinical practice can reduce hospital mortality in septic shock patients (clinicaltrials.gov number nct01639664). this extracorporeal device removes cytokines through adsorption to a high - surface - area biocompatible porous polymer sorbent. this device does not target endotoxin, but it does rapidly eliminate several key cytokines by adsorption in both in vitro and in vivo experiments [38, 39 ]. this device is very promising, but more studies in septic patients are needed. due to the broad information existing about safety and efficacy of polymyxin b - immobilized cartridge, we will review the immunological mechanisms described in this treatment. whereas some of these mechanisms are derived from endotoxin elimination, others result from direct action on other inflammatory molecules and cells or from a combination of endotoxin elimination and direct action on these mediators. immune cells recognize endotoxin and other bacterial compounds through the tlr, a group of transmembrane proteins that play crucial roles in the host defense against invading pathogens. during a gram - negative infection, tlr-4 recognizes endotoxin and originates a systemic inflammatory response in sepsis with potentially fatal effects in hosts. as a consequence, proinflammatory molecules such as interleukin-1 (il-1) and tumor necrosis factor alpha (tnf) are released and generate other cell responses in the inflammatory cascade (figure 1). this increase in cytokines is followed by a major expression of tissue factor, which activates coagulation, and by an increase in nitric oxide synthesis, which induces vasodilation. endotoxin levels are high in septic patients [43, 44 ], but they are also high in critical patients without sepsis, such as patients undergoing cardiopulmonary bypass and those with chronic heart failure, chronic kidney disease, and other medical conditions [4447 ]. in critical patients without gram - negative infection, elevated endotoxin levels are related to translocation of gut bacterial antigens and endotoxin into the bloodstream due to gut barrier dysfunction [4850 ]. polymyxin b 's hydrophobic amino acids (phe, leu) form hydrophobic bonds with lipid a fatty acid in endotoxin, and the amino groups of polymyxin b form ionic bonds with the negatively charged phosphate groups of lipid a. this binding results in an antibiotic - endotoxin complex that is highly effective in neutralizing the deleterious effects of endotoxin. many studies have reported diverse benefits of pmx hemoperfusion in septic patients, including improved hemodynamics [24, 25, 5260 ], increased ratio of partial pressure arterial oxygen and fraction of inspired oxygen (pao2/fio2) [24, 54, 55, 57, 58, 60, 61 ], decreased 28-day mortality [24, 52, 53, 59 ], and decreased endotoxin levels [52, 53, 55, 58, 59, 6264 ]. in a multicenter study, vincent. found that pmx hemoperfusion was safe and improved cardiac and renal function due to sepsis or septic shock ; however, they could not demonstrate a reduction in mortality or in endotoxin levels from baseline to the end of treatment. in a systematic review of 28 studies, cruz. concluded that pmx hemoperfusion was associated with lower mortality (rr 0.53, 95% ci : 0.430.65) and improvements in mean arterial pressure (map), use of inotropes, and pao2/fio2. in 17 of these studies in which endotoxin levels were measured, endotoxin levels decreased by 33% to 80% after pmx hemoperfusion. more recently, the same authors published the euphas study, a prospective multicenter randomized controlled trial that enrolled 64 patients with severe sepsis or septic shock who underwent emergency surgery for intra - abdominal infection. patients were randomized to conventional therapy or to conventional therapy plus two sessions of pmx hemoperfusion. after the results of the scheduled interim, analysis revealed that pmx hemoperfusion significantly improved hemodynamics and organ dysfunction and reduced 28-day mortality ; the study was discontinued because it was considered unethical to deprive high risk patients of a potentially beneficial therapy ; however, early discontinuation resulted in a modest sample size. two adequately powered prospective trials are underway, and the results of these trials should elucidate the benefit of endotoxin removal (clinicaltrials.gov numbers nct01046669 and nct01222663) [66, 67 ]. several studies report a reduction in cytokines and inflammatory molecules in patients ' plasma after pmx hemoperfusion [54, 56, 61, 6870 ]. in patients with severe sepsis, tani. found reductions in endotoxin, tnf, il-6, il-10, and plasminogen activator inhibitor-1 (pai-1) activities after pmx hemoperfusion. in patients with ards, kushi. found a reduction in blood levels of pai-1, neutrophil elastase (ne), and il-8 after pmx hemoperfusion. ne is a protease that hydrolyzes lung elastin. in these patients, pao2/fio2 increased significantly after the treatment, and the authors related this increase to the elimination of il-8 and ne. in another study, the same group reported a decrease in ne in 20 septic patients treated with pmx hemoperfusion. in 12 patients with septic shock receiving conventional treatment plus two sessions of pmx hemoperfusion, zagli. found a decrease in il6, il10, and tnf in patients ' serum after the treatment, especially in survivors. most authors attribute the decrease in cytokines and inflammatory molecules to the removal of endotoxin and to the effect of this removal on the inflammatory cascade. patients with sepsis have increased high mobility group box-1 protein (hmgb1), a cytokine secreted by immune cells that triggers inflammatory mediators. the receptor for advanced glycation end - products (rage) is involved in hmgb1 signaling. the inhibition of the hmgb-1-rage axis could be an effective therapeutic strategy for septic shock. nakamura. compared il-6, hmgb1, and rage in serum between 15 patients with septic shock treated with pmx hemoperfusion and healthy volunteers. the levels of the three molecules decreased after pmx hemoperfusion and correlated with a decrease in endotoxin. abe. studied the effects of pmx hemoperfusion on hmgb1 in patients with acute exacerbation of idiopathic pulmonary fibrosis. moreover, hmgb-1 was detected in washing medium from the pmx column, suggesting that the decrease in this molecule was not only secondary to endotoxin removal but also to direct removal by the device. a recent study exploring the meaning of hmgb-1 levels in 60 patients with septic shock treated with pmx hemoperfusion found a significant positive correlation between the sequential organ failure assessment (sofa) score and hmgb-1 level (p < 0.05). the authors concluded that hmgb-1 is a useful prognostic biomarker in sepsis - induced organ failure in patients undergoing pmx hemoperfusion, but formal establishment of the utility of hmgb-1 as a prognostic biomarker still remains to be performed. pai-1, a marker of vascular endothelial cell activation elevated by endotoxin and cytokines, is one of the fibrinolysis inhibitory factors. pai-1 levels decrease after pmx hemoperfusion, decreasing the stimulation of vascular endothelial cells [54, 55, 61 ]. pmx hemoperfusion may have a role in modulating fibrinolysis and inhibiting the development of ischemic organ dysfunction in sepsis. -2 play a contributory role in the pathogenesis of acute lung injury (ali) in septic patients. vascular endothelial growth factor (vegf) is a pluripotent growth and permeability factor that has a broad impact on endothelial cell function. recently studied nine patients with acute exacerbation of idiopathic pulmonary fibrosis treated with conventional therapy and pmx hemoperfusion 6 hours / day on two successive days. they found a high concentration of cytokines and vegf in the eluate from used pmx cartridge fibers, and the clinical improvement in these patients correlated with the amount of vegf in the eluate. this is the first study to demonstrate that cytokines and vegf can be directly adsorbed by pmx hemoperfusion independently from endotoxin removal. the authors suggest that cytokines can bind to pmx hemoperfusion fibers directly through ionic / hydrophobic interactions like endotoxin or indirectly via heparin coated in the fibers. anandamide is an intrinsic cannabinoid that has been related with hypotension in septic shock, although currently its direct link to sepsis is only established in a small patient population. one study in 24 patients with septic shock treated with pmx hemoperfusion found that anandamide levels decreased after pmx hemoperfusion in the nine patients who survived ; the authors conclude that removal of anandamide by pmx hemoperfusion, whether directly or as a result of endotoxin elimination, could be key to successful septic shock treatment. further studies are necessary to elucidate the effect of pmx hemoperfusion on anandamide, in order to establish it as a useful treatment for hypotension. elevation of nitric oxide (no) plays an important role in septic patients, producing vasodilatation and hypotension. nakamura. compared no breakdown products in urine in 20 patients with pmx hemoperfusion, 15 patients with conventional therapy, and 20 healthy controls. they found that septic patients increased no production and that pmx hemoperfusion reduced no levels and thus increased blood pressure. troponin is a biomarker that may be elevated in septic patients as a result of subclinical myocardial cell damage. nakamura. found increased troponin t in septic patients compared to nonseptic patients and age - matched healthy controls ; interestingly, troponin t decreased after pmx hemoperfusion p < 0.05. erythropoietin levels may be higher in patients with sepsis ; erythropoietin levels decrease after pmx hemoperfusion and could be a prognostic indicator in patients with septic shock. during sepsis, different populations of leukocytes are activated and change their adhesive phenotype. the capture of leukocytes through extracorporeal blood purification could alter the immune response to sepsis. after ex vivo perfusion of heparinized blood from patients with sepsis and septic shock through pmx hemoperfusion in a laboratory circuit, kumagai. found significant decreases in neutrophils (78%), monocytes (70%), and lymphocytes (10%). this marked reduction in white blood cells should be attributed mostly to the reduction in the circulation of proinflammatory cytokines that induce cell activation and proliferation, as opposed to a direct effect of removal of these cells by the cartridge. examined the pmx hemoperfusion filters after treating 4 patients with sepsis ; pmx hemoperfusion bound monocytes from the peripheral blood leucocytes. pmx hemoperfusion could produce a beneficial effect by reducing the interaction between monocytes and functionally associated cells, including endothelial cells. nakamura. studied the effect of pmx hemoperfusion on platelet activation, comparing 30 patients treated with conventional therapy plus pmx hemoperfusion and 20 patients with conventional therapy alone. survival was 60% in the group that received pmx hemoperfusion and 30% in the group that received only conventional treatment. septic patients had increased paf (p - selectin, platelet factor 4, and -thromboglobulin), and pmx hemoperfusion reduced the levels of paf. the human body undergoes a biphasic immunological reaction in sepsis. a proinflammatory reaction takes place, marked by the release of proinflammatory cytokines like tnf, as a reaction to the bacterial toxins. on the other hand, the persistence of a marked compensatory anti - inflammatory response is called immunoparalysis (figure 1). this pronounced immunosuppressive state adversely affects immune function, making the patient vulnerable to opportunistic infections. these two phases of sepsis may occur simultaneously with a lasting anti - inflammatory response in later phases. most septic patients survive the initial proinflammatory phase, but they die during this second stage. strategies to stimulate this immunoparalysis phase of sepsis as ifn- and granulocyte - macrophage colony - stimulating factor (gm - csf) have been developed in animals, but extensive clinical studies are needed to test their safety and efficacy. recently, ono. showed that the expression of the surface antigens, hla - dr on monocytes and cd16 on granulocytes, is extremely decreased in patients with septic shock and that pmx hemoperfusion beneficially increases this expression on these leukocytes. the regulatory t cells (treg) that express cd4, cd25, and foxp3 comprise a small percentage of the t - lymphocyte population in the immune system, but they are central to the maintenance of immunological homeostasis and tolerance. in septic patients, the percentage of treg is increased, and this presumably contributes to sepsis - induced immunosuppression. polymyxin - b induces treg cell death in mice through the modulation of the purinergic p2x7 receptor. studied the effect of pmx hemoperfusion on the recovery from the immunosuppression owing to septic shock. treg, il-6, and il-10 were higher in patients with septic shock than in patients with sepsis. after pmx hemoperfusion, treg cells, il-6, and il-10 decreased. in survivors, the decrease in treg cells was accompanied by an increase in cd4 + cells. although further studies are necessary to confirm a causative relationship between treg depletion and pmx hemoperfusion in septic patients, this mechanism could explain why pmx hemoperfusion can be useful in patients without endotoxemia or in those with gram - positive sepsis [89, 90 ]. the authors also suggest that the second pmx hemoperfusion treatment might provide additional benefits for recovery from immunoparalysis. this study sheds new light on the benefits of treating septic patients with pmx hemoperfusion beyond endotoxin removal. endotoxin may cause an inappropriate activation of proapoptotic pathways in immune cells during sepsis, and this may contribute to the impaired immune response that characterizes sepsis. endotoxin can also cause apoptosis of renal tubular cells through fas - mediated and caspase - mediated pathways. tested the hypothesis that pmx hemoperfusion might prevent gram - negative sepsis - induced acute renal failure by reducing the activity of proapoptotic circulating factors. they randomized 16 patients with gram - negative sepsis to receive standard care or standard care plus pmx hemoperfusion. proapoptotic activity was significantly reduced in the plasma of the pmx hemoperfusion group, with decreases in fas upregulation and caspase activity, and these patients also had improved renal function. several studies have found that pmx hemoperfusion has beneficial effects on oxygenation in patients with sepsis [24, 61, 65 ]. moreover, pmx hemoperfusion has been successful in patients with influenza a infection [89, 95 ], ards in drug - induced injury [96, 97 ], interstitial pneumonia [19, 20, 98 ], and idiopathic fibrosis [18, 69, 74, 99 ]. mechanisms other than endotoxin removal could explain the beneficial effects of pmx hemoperfusion in patients with respiratory failure. chemical mediators have an important role in the pathogenesis of ards, and decreasing them through direct or indirect removal could be beneficial in ards patients.. found decreases in pai1, neutrophil elastase (ne), and il-8 in ards after pmx hemoperfusion. studied the role of decreases in hmgb1 after pmx hemoperfusion in patients with acute exacerbation of idiopathic fibrosis. when the same authors investigated the effects of pmx hemoperfusion in a retrospective multicenter study of 160 patients with acute exacerbation of idiopathic pulmonary fibrosis or interstitial pneumonia, they found that the pao2/fio2 ratio significantly increased after pmx hemoperfusion. they concluded that pmx hemoperfusion might be an effective adjunctive therapy for these patients, although the mechanisms underlying the benefits of the treatment are uncertain. likewise, hara. reported that pmx hemoperfusion resulted in improved pao2/fio2 ratio 72 hours and 1 week after treatment in 33 patients with acute exacerbation of interstitial pneumonia. tsushima. treated 20 patients with ards with pmx hemoperfusion and compared the outcomes with a historical control group. they found improved pao2/fio2 ratio and survival ; however, the methodology of the study limits its power to draw conclusions. collectively, the matrix metalloproteinases (mmp) are capable of degrading all kinds of extracellular matrix proteins. mmp-9 is a protease involved in the degradation of the basement membrane, a part of the extracellular matrix associated with the alveolar epithelium and vascular endothelium. mmp-9 is essential for the remodeling of basement membranes in various inflammatory lung diseases, including ards. increased amounts of mmp-9 in the vasculature are likely to enhance vascular permeability and to facilitate cell homing and inflammatory remodeling. nakamura. studied the effect of pmx hemoperfusion on mmp levels in ards patients by treating 12 ards patients with two sessions of pmx hemoperfusion and comparing their laboratory data with those of healthy controls. after pmx hemoperfusion, mmp-9 decreased and chest x - ray findings improved. however, the precise mechanism is still unclear. the authors suggest the need for more studies to elucidate the beneficial effect of pmx hemoperfusion in ards. in a pilot study of 16 patients, abe. studied the effects of pmx hemoperfusion for acute exacerbation of interstitial pneumonia and demonstrated neutrophil adsorption and a decrease in mmp-9. in recent years takahashi. studied the changes in serum s100a12 and rage after pmx hemoperfusion in postoperative septic shock. they found a significant decrease in s100a12 in serum after pmx hemoperfusion and an improvement in pao2/fio2 ratio but no decrease in rage. as mentioned above, oishi. found cytokines and vegf in the eluate from pmx hemoperfusion cartridges used to treat patients with acute exacerbation of pulmonary fibrosis, suggesting a new explanation for the improvement in oxygenation in nonseptic patients treated with pmx hemoperfusion. in recent years, many studies have shown that pmx hemoperfusion is a promising strategy for immunomodulation in septic shock, and two ongoing clinical trials will be key in determining its usefulness. although most studies have focused on the removal of endotoxin as the principal mechanism through which pmx hemoperfusion improves outcome in sepsis, other studies have revealed mechanisms involving diverse immunological pathways through which pmx hemoperfusion could improve outcome not only in sepsis but also in non - septic respiratory failure. however, these studies are limited by their small samples, their observational and in some cases retrospective design, and the lack of control groups in many cases. it is interesting to note that the elimination of endotoxin brings about a reduction in many inflammatory molecules and cells involved in the inflammatory cascade. endotoxin removal devices act at the onset of this complex cascade, and their benefits in terms of immunomodulation are encouraging. only a part of the consequences of endotoxin elimination has been studied and summarized in this review. future studies might reveal other mediators and cells involved in sepsis that might be altered after endotoxin removal. some of the studies reviewed here found mediators and cells in the eluate from pmx hemoperfusion cartridges or by direct examination of the filter. further studies are necessary to elucidate how pmx hemoperfusion eliminates these molecules, whether through ionic / hydrophobic interactions like in endotoxin removal or indirectly via heparin that coats the fibers. additional mechanisms could potentially explain why pmx hemoperfusion can be beneficial in gram - positive sepsis or non - septic respiratory failure. endotoxin can be elevated in other medical conditions apart from gram - negative infections, and its removal could also partially explain this benefit. the immunomodulating effects of pmx hemoperfusion in patients with interstitial pneumonia and acute exacerbations of pulmonary fibrosis are especially interesting, given the high mortality associated with these conditions. however, well - designed clinical trials are needed to assess the efficacy of pmx hemoperfusion in these medical conditions. future potential directions such as combination of different hemoperfusion devices to treat septic patients, in order to alter the host inflammatory response in more than one step, are currently speculative. technically, it could be viable, but no experience has been reported to date. in summary, antimicrobial therapy, surgical treatment of the focus of infection, and hemodynamic stabilization are crucial in the treatment of severe sepsis. it seems that pmx hemoperfusion might have other beneficial immunological mechanisms in addition to endotoxin removal ; however, the limited evidence suggests that we must be cautious with other indications for pmx hemoperfusion, and future studies are necessary. | severe sepsis results in high morbidity and mortality. immunomodulation strategies could be an adjunctive therapy to treat sepsis. endotoxin is a component of gram - negative bacteria and plays an important role in the pathogenesis of septic shock when it is recognized by immune cells. removal of endotoxin could be an effective adjunctive approach to the management of sepsis. devices to adsorb endotoxin or inflammatory cytokines have been designed as a strategy to treat severe sepsis, especially sepsis caused by gram - negative bacteria. polymyxin b - immobilized cartridge has been successfully used to treat patients with sepsis of abdominal origin. although this cartridge was conceived to adsorb endotoxin, several other immunological mechanisms have been elucidated, and this device has also yielded promising results in patients with nonseptic respiratory failure. in this paper, we summarize the immune modulation actions of polymyxin b - immobilized cartridge to explore its potential usefulness beyond endotoxin elimination. |
laparoscopy is widely used as a tool in many clinical situations allowing for diagnosis and/or surgical management in a minimally invasive fashion. nonetheless, attention to surgical technique is paramount to avoid both short and long term complications. a 32-year - old woman had a laparoscopy and a reported left salpingoophorectomy for benign disease of the ovary in september, 1994. shortly thereafter, in january, 1995, she was diagnosed with an intrauterine pregnancy and delivered in october of 1995 by spontaneous vaginal delivery. however, at the time of laparotomy, the patient was found to have a retained foreign body from her prior laparoscopy in the right lower quadrant with a pelvic abscess and evidence of prior right salpingoophorectomy. although laparoscopy is usually done on an outpatient basis, complications can manifest several weeks or months later. this case illustrates and reminds us of the importance of adherence to surgical laparoscopic principles. these include direct visualization when removing equipment and a complete count of surgical instrumentation to confirm the integrity of such at the end of each procedure. operative time is acceptable and recovery periods are significantly less than in open procedures with no significant difference in morbidity and/or mortality, even in pregnancy. a 32-year - old white woman presented to the gynecologic emergency room on november 21, 1995, complaining of tenderness and swelling of her right lower quadrant for three days. at that time she was four weeks postpartum from a spontaneous vaginal delivery. her surgical history was significant for a laparoscopy done in september of 1994 in vienna, austria, where a left salpingoophorectomy reportedly was performed for an ovarian cyst. pelvic examination revealed a normal uterus with a right adnexal mass approximately 8 cm in size, and exquisitely tender to palpation. abdominopelvic ct scan revealed a 5.5 cm 4 cm 5.3 cm right adnexal mass, inflammatory in nature. in addition to this, a 4.3 cm 6 cm 3.4 cm mass was superior to the first mass, in the right lower quadrant, and also inflammatory in nature. the patient was taken to the operating room by the gynecologic service for an exploratory laparotomy. intraoperatively, a normal left adnexae was found, not consistent with the patient 's history of left salpingoophorectomy. on the right side there was a large, 10 cm 12 cm inflammatory mass with the appearance of an abscess. clearly above this mass, a 3 cm 2 cm portion of synthetic tubing was found enveloped by omentum (figure 1). a partial omentectomy and removal of foreign body was performed, along with an excision of the right pelvic abscess and appendectomy. antibiotics were continued until the patient was afebrile for 48 hours. by postoperative day number five the patient was tolerating a regular diet and ambulating without assistance. the patient was seen in the postoperative clinic at four weeks and was doing well with no specific complaints. pathology reports revealed the foreign body to be synthetic tubing and the pelvic mass to be soft tissue with extensive scarring and acute suppurative inflammation with focal abscess formation. a 32-year - old white woman presented to the gynecologic emergency room on november 21, 1995, complaining of tenderness and swelling of her right lower quadrant for three days. at that time she was four weeks postpartum from a spontaneous vaginal delivery. her surgical history was significant for a laparoscopy done in september of 1994 in vienna, austria, where a left salpingoophorectomy reportedly was performed for an ovarian cyst. pelvic examination revealed a normal uterus with a right adnexal mass approximately 8 cm in size, and exquisitely tender to palpation. abdominopelvic ct scan revealed a 5.5 cm 4 cm 5.3 cm right adnexal mass, inflammatory in nature. in addition to this, a 4.3 cm 6 cm 3.4 cm mass was superior to the first mass, in the right lower quadrant, and also inflammatory in nature. the patient was taken to the operating room by the gynecologic service for an exploratory laparotomy. intraoperatively, a normal left adnexae was found, not consistent with the patient 's history of left salpingoophorectomy. on the right side there was a large, 10 cm 12 cm inflammatory mass with the appearance of an abscess. clearly above this mass, a 3 cm 2 cm portion of synthetic tubing was found enveloped by omentum (figure 1). a partial omentectomy and removal of foreign body was performed, along with an excision of the right pelvic abscess and appendectomy. antibiotics were continued until the patient was afebrile for 48 hours. by postoperative day number five the patient was tolerating a regular diet and ambulating without assistance. the patient was seen in the postoperative clinic at four weeks and was doing well with no specific complaints. pathology reports revealed the foreign body to be synthetic tubing and the pelvic mass to be soft tissue with extensive scarring and acute suppurative inflammation with focal abscess formation. complications from laparoscopy such as bleeding or structural injury can usually be recognized immediately intraoperatively. what makes this case unusual is the time interval (14 months) and the fact that the patient had an intervening pregnancy. it is theorized that the device must have been defective and when removing the sleeve thread anchor, probably with the trocar as a whole unit, a portion of it was sheared off and remained intraabdominally. the presentation of the patient was also atypical in that clinical evidence pointed to appendicitis. it is unclear whether the pelvic abscess was directly related to the retained foreign body or if there was another etiology. the authors favor the foreign body as the source since laparotomy revealed normal bowel and absence of the right adnexae in this patient. it is strongly suspected that the presence of a foreign body associated with the physiologic changes in the postpartum period combined to provide a favorable environment for abscess formation. in the literature, both shortand long - term complications exist and precautions to avoid these should be paramount. although most complications are related to hemorrhage, bowel or genitourinary injury, the rare case of a retained foreign body from laparoscopic equipment must be considered. the trend to move to one - piece disposable units where the trocar is self - retaining is one step in the direction of both simplification of laparoscopic procedures and prevention of postoperative complications. | background : laparoscopy is widely used as a tool in many clinical situations allowing for diagnosis and/or surgical management in a minimally invasive fashion. most laparoscopic cases are ambulatory and allow patients to recover quickly. nonetheless, attention to surgical technique is paramount to avoid both short and long term complications.case:a 32-year - old woman had a laparoscopy and a reported left salpingoophorectomy for benign disease of the ovary in september, 1994. shortly thereafter, in january, 1995, she was diagnosed with an intrauterine pregnancy and delivered in october of 1995 by spontaneous vaginal delivery. the pregnancy and delivery were both uncomplicated. the patient presented four weeks postpartum with clinical suspicion of appendicitis. however, at the time of laparotomy, the patient was found to have a retained foreign body from her prior laparoscopy in the right lower quadrant with a pelvic abscess and evidence of prior right salpingoophorectomy. the appendix appeared grossly normal.conclusion:laparoscopy is a safe, effective modality for various surgical and gynecologic conditions. although laparoscopy is usually done on an outpatient basis, complications can manifest several weeks or months later. this case illustrates and reminds us of the importance of adherence to surgical laparoscopic principles. these include direct visualization when removing equipment and a complete count of surgical instrumentation to confirm the integrity of such at the end of each procedure. |
in 2009, the world health organization (who) published the surgical safety checklist (ssc) as part of their safe surgery saves lives campaign. the checklist was adapted from the field of aviation, where checklist use is standard practice. in aviation, checklists were developed in response to a crash involving an experienced pilot operating a new airplane with features that were significantly different from previous models. shortly after takeoff, an investigation revealed that the pilot had forgotten to perform one of the steps necessary for takeoff. in response, the checklist was created to prevent future avoidable disasters.(1) with more than 200 million operations performed annually, the who recognized the importance of addressing surgical safety when the checklist was introduced. the purpose of the checklist was to help operating room (or) teams remember important details that may be missed during an operation. in addition, it served as a tool to encourage teamwork and communication.(2) in a sense, the who came to the same conclusion that the plane crash investigation team had : even highly skilled or teams need tools to help them achieve optimal results. the initial who ssc was piloted at eight diverse hospitals around the world and contained 19 items that were to be addressed at defined time points during the operation (figure 1).(3) the items included in the ssc are aimed at preventing uncommon but serious errors by reminding the team to confirm patient identity, surgical site, and other important characteristics such as comorbid conditions or anticipated complications. results from the initial prospective, sequential, time - series observational study showed significant reductions in complications, in - hospital mortality, rates of unplanned reoperation, and surgical site infection (ssi) compared to pre - checklist rates. (4) since then, the who ssc has been implemented in more than 4,000 hospitals worldwide.(5) hospitals are encouraged to customize the checklist to their needs, but the general format remains the same. studies validating these various checklists have continued to show, for the most part, a benefit when the ssc or similar checklist is used, (611) but the mechanism by which this occurs is unclear. recent high - profile reports have highlighted the pitfalls of sscs, such as inconsistent implementation and compliance.(12) in an era of increasing complexity of care, it appears that the checklist is serving as a conduit for improved teamwork and communication through which the improved outcomes result. the aim of this paper is to review the literature related to ssc use as a communication tool, with a focus on how the checklist is associated with team behaviors and attitudes in the or. in addition, we describe scenarios where use of the ssc is associated with changes in patient outcomes. we reviewed studies that have been collated by the senior author, who has extensively studied the fields of or safety, communication and checklist use for the past 10 years. we included studies that addressed the use of the checklist as a tool for improved communication in the or, with an emphasis on changes in both team behaviors and clinical outcomes after implementation. additional studies were selected that described compliance with the ssc and how it may be affected by variations in implementation strategy. safety within the or is an important public health concern. it is estimated that of the complications that occur within the hospital setting, more than half are associated with surgical procedures.(13) every operation has a series of steps that must be performed correctly every time : surgeons must use the correct equipment, the equipment must be available and in proper working order, and drugs need to be administered in a timely and appropriate fashion. as their roles in an operation are interdependent, it is incumbent on the anesthesia team, the nursing staff, and surgeons to communicate effectively to prevent avoidable complications such as wrong site surgery and inappropriate antibiotic administration. despite this, research has shown that surgeons, anesthesiologists, and nurses have rather different concepts of what constitutes teamwork and communication in the or.(14, 15) one study used the safety attitudes questionnaire (saq) to assess perception of patient safety in the or. the saq is a standardized survey that uses a five - point likert scale to measure items such as teamwork and safety.(16) this particular study found that women reported significantly lower aggregated scores than men on the domain teamwork climate (69 vs 76, p<0.05). (17) a separate study investigated specific aspects of teamwork and found that nurses reported significantly lower scores than surgeons regarding reception of nursing input (3.8 vs 4.3, p<0.001), ability to voice concern (3.5 vs 3.7, p=0.03), and whether physicians and nurses work well as a team (3.3 vs 3.7, p<0.001). (14) the consequences of this disparity can be serious. in one study investigating reports of wrong site surgery, or pooled results predicted that in cases with the potential for wrong - site surgery, concerns would be raised and addressed only 41% of the time.(18) while wrong site surgery is an uncommon event, communication failures are common, occurring every 78 minutes and affecting up to 30% of interactions in the or.(19, 20) for a routine case lasting 23 hours, this means that up to 25 attempts at communication may be unsuccessful. use of a checklist may prevent more than half of communication failures from occurring (21) by orienting the team to the individual patient, alerting each member to potential complications, and encouraging team members to voice concern when they notice an error occurring. one of the primary arguments in favor of checklists is that they help to decrease surgically associated morbidity and mortality, and can be implemented in most settings. use of system - wide checklists can improve compliance with other metrics, such as increased timely antibiotic administration, decreased unexpected delays in the schedule, and reduced time spent outside of the or gathering supplies during an operation.(2123) timely antibiotic administration has been linked to a decrease in surgical site infection. in one study, pre - incision antibiotics were not administered 12.1% of the time ; after introduction of a checklist, this number decreased to 7.1% (p=0.015).(23) while introducing the checklist can initially be viewed as disruptive, staff members typically have a favorable attitude after it has been initiated.(24) substantial work has been undertaken to understand if the use of checklists actually improves communication in the or. in a pilot study investigating the utility of pre - procedural briefing in cardiac surgery (similar to the who ssc), the number of miscommunication events declined by 50% in the briefing group compared to the group that did not use the briefing tool.(21) other studies have found that communication failures declined by two thirds after initiation of a surgical briefing.(24) in a study investigating pre- and post - implementation scores using the saq, respondents were more likely agree that checklists are important for safety (4.58 vs 4.79, p=0.0058), and they were more likely to report a culture that encouraged team members to voice concern (4.02 vs 4.21, p=0.0225). additionally, 93.4% of the clinicians who responded to the survey stated that if they were undergoing an operation, they would want the checklist used.(25) critics of the ssc have noted that while use of the checklist may identify problems, the person conducting the checklist is ultimately responsible for resolving the problem and redirecting the team.(26) for example, if the checklist demonstrates that the patient did not receive appropriate antibiotics in a timely fashion, the surgeon, anesthesiologist, and circulating nurse must rectify this mistake prior to proceeding with the operation. this begins to address an important concern : while the checklist itself might be improving patient safety, there may be something different about teams who routinely use the checklist. checklists are rarely comprehensive enough to catch every possible error. instead, proper use of the checklist may be a marker for teamwork and cooperation within the or. regardless of checklist use, the link between team behaviors and patient safety is well recognized. infrequent use of team behaviors (defined in one study as briefing, information sharing, inquiry, vigilance and awareness, assertion, and contingency management) is associated with increased risk of death and other complications,(27) while high levels of communication and collaboration are associated with overall lower rates of risk - adjusted morbidity.(28) other evidence shows a correlation between increased teamwork and a lower frequency of errors during an operation.(29) wiegmann, in examining when errors in the or are discovered and by whom, concluded that while poor teamwork can lead to errors, good teamwork leads to the detection and correction of mistakes.(30) investigators have attempted to describe the link between checklist use and improved patient outcomes. one explanation is that use of the checklist improves the safety culture within an institution by facilitating communication. makary and colleagues administered an or based version of the saq to assess changes after implementation of an or briefing protocol. respondents reported increased scores on items such as awareness of surgical site brought about by the briefing (3.74 vs 3.18, p<0.001), coordinated efforts by surgical staff and anesthesia staff (4.54 vs 3.68, p<0.000), and on the importance of the briefing to patient safety (3.24 vs 2.75, p<0.001).(31) however, checklist implementation may introduce new challenges that had not previously been considered. in a viewpoint discussing checklist use, rydenfalt contends that merely introducing a checklist without monitoring compliance may actually make the or less safe because previous safety checks are dropped.(32) or staff have reported in interviews that use of the checklist can interrupt the performance of other safety tasks that are simultaneously being performed by individuals. additionally, without a firm sense of commitment to the checklist it may become a routine activity of checking off boxes without actually driving behavior change or improvement. (33) running through the list in such fashion may give or staff a false sense of security that issues have truly been resolved when in fact they have not. (34) without providing team members proper instruction regarding the use and value of the checklist, it may actually become a nuisance to the or staff. while there is a significant amount of data showing that checklist use leads to improvements in patient outcomes, investigators have also performed checklist audits to evaluate how the or team uses the ssc in everyday practice. levy and colleagues examined the efficacy of the checklist for ensuring performance in the or and found that administrative records confirmed 100% performance while auditing by observers in the or recorded less than 50% completion for most elements, and in some cases less than 10% of the checklist elements were completed.(35) subsequently, the same group organized safety workshops as well as a stakeholder engagement group to customize the checklist for local concern. with these two interventions, overall adherence improved from 30% to 96% (p<0.001).(36) a recent report raised serious questions about the utility and effectiveness of surgical checklists. in 2010, the canadian province of ontario mandated that each hospital use the who ssc and that they report their compliance. in this real - world observational study, hospitals were evaluated before and after implementation of the ssc. change in surgical mortality was the primary outcome, but the investigators also looked at other outcomes such as morbidity and readmission. the results of the study showed that despite widespread adoption of the who ssc, there was no significant difference in mortality (0.71% vs 0.65%, p=0.13) or surgical complications (3.86% vs 3.83%, p=0.29). (12) it is unclear why the results of the ontario study were so different from the original who study. the findings sparked a debate about what the surgical community should expect from the ssc, and whether its use was directly associated with a change in outcome. one of the criticisms of the ontario study was related to implementation strategy, as it seemed that individual hospitals were responsible for implementation without being given administrative support. in the who ssc study, the task of implementation required considerable resources and support in order to be effective. additionally, there was concern that compliance with the ssc was likely lower than what it had been in previous studies so the expected effects were not realized.(37) despite operational flaws, many say that the findings from ontario should be seriously considered, as the observational nature of this study is likely to be characteristic of typical use of the checklist.(38, 39) the results found in the rigorously controlled environment of a randomized controlled trial do not always approximate the effects that are seen in real world conditions, which may explain why there was no difference in morbidity or mortality rates in ontario. additionally, simply telling people to change their behavior without providing any guidance or support on how to do so may not be the most effective strategy. the modern surgical environment is complex, and communication errors are relatively common. as described, used of the ssc has become common throughout the world. while checklists show promise in the reduction of surgical morbidity and mortality, there is also evidence that these improvements are not realized without careful attention to implementation strategy. when deciding to implement checklists in the or, administrators should assess the climate of their hospital in order to make the checklist relevant to those who will be using it rather than an additional hurdle to jump over. providing feedback to teams regarding patient outcomes and or performance may be a valuable strategy to promote buy - in at the provider level.(33) in addition, encouraging customization of the checklist to fit the needs of the team may promote a feeling of ownership over the checklist, increasing compliance along the way. (33, 36) without the support of staff members, it is unlikely that the checklist will lead to any changes in patient outcomes. for now, the surgical community should view the checklist as a tool for improving communication and safety culture, and be realistic about its direct impact on patient safety. | existing evidence suggests that communication failures are common in the operating room, and that they lead to increased complications, including infections. use of a surgical safety checklist may prevent communication failures and reduce complications. initial data from the world health organization surgical safety checklist (who ssc) demonstrated significant reductions in both morbidity and mortality with checklist implementation. a growing body of literature points out that while the physical act of checking the box may not necessarily prevent all adverse events, the checklist is a scaffold on which attitudes towards teamwork and communication can be encouraged and improved. recent evidence reinforces the fact the compliance with the checklist is critical for the effects on patient safety to be realized. |
mitochondrial cardiomyopathy can manifest with various cardiac phenotypes, such as left ventricle (lv) hypertrophy, dilatation, wall motion abnormality, and lethal ventricular arrhythmia ; these symptoms range from subclinical to critical conditions (1). the severity of myocardial dysfunction also predicts the prognosis of patients with mitochondrial disease, which highlights the importance of noninvasive assessment for myocardial dysfunction. myocardial perfusion / metabolism mismatch (the decreased uptake of technetium-99 m methoxyisobutylisonitrile [tc - mibi]/the increased uptake of 123-labeled 15 - 4-iodophenyl-3-(r, s)-methyl - pentadecanoic acid [i - bmipp ]) has been reported in patients with mitochondrial cardiomyopathy (2,3). however, the previous studies did not compare the morphological images of patients with this condition. in the present case, we compared images of myocardial perfusion (tc - mibi scintigraphy), fatty acid metabolism (i - bmipp scintigraphy), and morphology (cardiac magnetic resonance [cmr ] and histology), and found that the images of our patient, who had a mismatch between perfusion and metabolism, were unique from those in the previous reports. a 42-year - old man (157 cm, 48.4 kg) was referred to our hospital with left chest pain, diabetes mellitus, and sensorineural hearing loss. his mother had diabetes mellitus ; however, she did not have hearing loss or cardiovascular disease. at admission, his blood pressure was 121/79 mmhg, his heart rate was 82 beats / min with a regular pulse, and his percutaneous oxygen saturation was 98% in room air. although systolic heart murmurs were audible at the base (4lsb), there was no jugular venous distention, and his breathing sounds were normal. hepatomegaly and splenomegaly were not present, and there was no edema in his extremities. chest radiography revealed an increase in the cardiothoracic ratio (59%) and mild pulmonary congestion (fig. twelve - lead electrocardiography detected a normal sinus rhythm and a high amplitude r wave with an inverted t wave in the left lateral precordial leads (fig. transthoracic echocardiography detected thickening of the lv walls (septum, 16 mm ; posterior wall, 16 mm), as well as thickening of the right ventricle (rv) free wall, the slight dilatation of the lv (lv end - diastolic diameter, 54 mm) and diffuse lv systolic dysfunction (ejection fraction, 40% ; using simpson 's method) (fig. (a) chest radiography reveals cardiomegaly (cardiothoracic ratio, 59%) and mild pulmonary congestion. (b) an electrocardiogram reveals a prominent r wave progression in v3 - 6 and a negative t wave in v5 - 6. (c, d) transthoracic echocardiograms reveal the thickening of the left and right ventricle walls. his levels of troponin t and n - terminal pro - brain natriuretic peptide were elevated to 0.107 ng / ml and 1,616 pg / ml, respectively. his fasting glucose level was 172 mg / dl and his glycated hemoglobin was 7.0%. the resting serum and cerebrospinal fluid lactate concentrations had increased to 27.0 mg / dl (normal range, 3 - 17 mg / dl) and 31.8 mg / dl (normal range, 14 - 21 mg / dl), respectively. brain computed tomography and magnetic resonance imaging revealed prominent calcification in the bilateral basal ganglia and cerebellar atrophy. mitochondrial cdna sequencing detected a c.a3243 g mutation, and the patient was diagnosed with mitochondrial disease. to investigate the cause of his cardiac dysfunction, cardiac catheterization was performed after medical treatment. his pulmonary artery pressure was normal (31/11 mmhg, mean 18 mmhg) with an increase in the lv end - diastolic pressure (20 mmhg). his cardiac index was preserved (3.08 l / min / m), and the coronary angiography findings were normal. the microscopic findings revealed the rupture and degeneration of the myocardial fibers, which were surrounded by fibrosis(fig. electron microscopy detected an increase in the number of mitochondria, which varied in size and morphology (fig. (a) a light microscopy image reveals interstitial fibrosis and hypertrophic myocytes with vacuoles (elastica - masson staining, 100). (b) an electron microscopy image reveals an increased number of mitochondria with variable sizes and shapes. the patient subsequently underwent resting tc - mibi cardiac scintigraphy (600 mbq), and single - photon emission computed tomography (spect) images were obtained at 60 minutes (the early phase) and 240 minutes (the delayed phase) after the injection (4). in the early phase, the uptake of tc - mibi did not decrease (fig. the heart - to - mediastinum ratio was 2.89 in the early phase and 2.79 in the delayed phase, respectively. the global washout rate in the delayed phase increased by 25% (normal range : 11 5%). the uptake of tc - mibi in the rv free wall appeared to be enhanced in the early phase (fig. 3a).i - bmipp (111 mbq) was administered intravenously, and spect images were obtained at 35 minutes after the injection (5). the uptake of i - bmipp was decreased in the anterior, septal, and inferior walls (fig. cmr was performed to estimate late gadolinium enhancement (lge), which was observed from the midmyocardium to the epicardium of the anterior wall, as well as in the midmyocardium of the septal and inferior walls (fig. the tc - mibi / i - bmipp mismatch pattern (non - decreased tc - mibi uptake / decreased i - bmipp uptake) was confirmed via bull 's eye mapping (fig. (a) the uptake of tc - mibi is not decreased in the early phase. (b) the uptake of i - bmipp is decreased in the anterior, septal, and inferior walls. (c) late gadolinium enhancement (lge) is observed from the midmyocardium to the epicardium of the anterior wall, as well as in the midmyocardium of the septal and inferior walls. (d) bull s eye mapping shows the tc - mibi / i - bmipp mismatch pattern (non - decreased tc - mibi uptake / decreased i - bmipp uptake). i - bmipp : 123-labeled 15 - 4-iodophenyl-3-(r, s)-methyl - pentadecanoic acid. during the patient 's hospitalization we prescribed enalapril (2.5 mg / day) and carvedilol (1.25 mg / day) to expect preventive effect for the progression of cardiomyopathy (6,7). this is the first report to describe the relationship between myocardial perfusion, metabolism, and morphology, and to reveal a unique tc - mibi / i - bmipp mismatch pattern in a patient with mitochondrial cardiomyopathy. our imaging findings revealed a decrease in the uptake of i - bmipp, using lge in cmr as an anatomical reference. in contrast, previous studies have reported an increase in the uptake of i - bmipp in mitochondrial cardiomyopathy (without providing anatomical reference images). the mechanism underlying this observation may be related mitochondrial respiratory chain failure, which leads to the excessive production of nicotinamide adenine dinucleotide (nadh), which then causes an increase in the concentration of glycerol-3-phospate (to oxidize the excess nadh) and ultimately leads to increased levels of triglycerides. according to this mechanism, i - bmipp (a fatty acid analogue) is incorporated into the cardiomyocytes ' pool of triglycerides (3,8). in our patient, the distribution of the areas that showed a low uptake of i - bmipp was consistent with the distribution of lge, which suggests that the low uptake of i - bmipp might be correlated with the low number of viable myocytes or fibrosis, rather than an energy production from fatty acid metabolism to the glycolytic pathway (as in other cardiomyopathies). thus, the extent of the i - bmipp uptake may depend on the balance between the mitochondrial respiratory chain failure and the viable myocyte mass, whereby the loss of a certain number of myocytes might outweigh the increased uptake of i - bmipp. the uptake of tc - mibi in the early phase was not decreased in this case. one of the interpretations of the preserved uptake of tc - mibi may be the presence of preserved blood flow and viable myocytes. in this case, the unique lge pattern (obscure margins and inhomogeneous intensity) may reflect the scattered viable myocytes that were surrounded by fibrosis, which were also detected in our histological examination. this pattern is distinct from the homogeneous lge pattern that is observed in cases of myocardial infarction. it is possible that the preserved myocytes in the lge - positive area affect the uptake of tc - mibi. the volume effect of the thickening lv wall might also explain the non - decreased uptake of tc - mibi (similar to the effect in patients with hypertrophic cardiomyopathy) and the enhanced uptake of tc - mibi in the rv free wall. furthermore, the washout rate for tc - mibi increased in this case, which is consistent with the findings of previous studies (2,3,8). in this context, mitochondrial dysfunction impairs the retention of the tc - mibi tracer in myocytes, which leads to an increased washout rate for tc - mibi. nevertheless, our unique tc - mibi / i - bmipp mismatch pattern (non - decreased tc - mibi uptake / decreased i - bmipp uptake) is distinct from the patterns in previous reports (decreased tc - mibi uptake / increased i - bmipp uptake) (2,3). the tc - mibi / i - bmipp mismatch pattern in the present case can be considered to be a nonspecific phenomenon in patients with dilated cardiomyopathy (9). this discrepancy may be related to the stages or diverse phenotypes of mitochondrial cardiomyopathy. in conclusion, multimodality imaging allows us to evaluate the relationship between myocardial perfusion, metabolism, and morphology. further studies are needed to elucidate the complex processes of myocardial damage in patients with mitochondrial cardiomyopathy. | a 42-year - old man was referred to our hospital due to chest pain, diabetes mellitus, and sensorineural hearing loss. transthoracic echocardiography revealed diffuse left ventricular hypokinesis. he was diagnosed with mitochondrial disease and a c.a3243 g mutation was identified in his mitochondrial dna. this case of mitochondrial cardiomyopathy demonstrated a low uptake of 123i - bmipp, while the uptake of 99mtc - mibi was preserved. in contrast, previous reports have noted the increased uptake of123i - bmipp and the decreased uptake of 99mtc - mibi. this is the first study to show this unique 99mtc - mibi/123i - bmipp mismatch pattern. we also discuss the relationships among the cardiac scintigraphy, cardiac magnetic resonance imaging, and histopathology findings. |
micrornas (mirnas) are nonprotein coding rnas of between 20 and 22 nucleotides that attenuate protein production by cleavage, translational inhibition, or sequestering of mrna in p bodies. they are implicated in several different biological pathways, including plant and animal development, and cancer [24 ]. to better understand the role that mirnas play in these pathways, large datasets containing rna - seq, expressed sequence tags (ests), and genomic sequences are being investigated for new mirnas [5, 6 ]. as these datasets grow in an ever increasing rate, the precursor folds back to base pair with itself to form a characteristic stem - loop structure. the dicer protein cuts a short, double - stranded rna (mirna : mirna duplex) from the precursor. this double - stranded rna associates with the risc complex, where the mature mirna is retained while the mirna is assumed to degrade. the mirna - loaded risc complex is responsible for mrna cleavage, translational inhibition, or sequestering. many methods for predicting mirnas from sequence data have been reported [6, 816 ]. some of these methods are directed specifically at ests while others are specific for deep - sequencing data [10, 11, 17 ]. several methods use machine learning approaches such as support vector machines (svms) [6, 9, 14 ] or decision trees [13, 15 ]. these methods typically incorporate information based on the predicted minimum free energy (mfe) of the precursor secondary structure. xue. used a sliding triplet method to determine characteristic attributes within the triplets of mirna and non - mirna stem - loop structures for training an svm. the authors reported prediction accuracy for mirna precursors of between 66.7% and 100% on a variety of plant and animal species, as well as viruses after training on known human mirna precursors. it was trained using the same triplet element technique mentioned above, as well as other characteristics including dinucleotide shuffling. the relationship between the mfe of the original sequence and its shuffled counterparts (z statistics) proved to be an important attribute for training this predictor. the mipred method produced a sensitivity of 95.09% and specificity of 98.21%. both machine learning techniques described above agree on the high predictive value of specific triplet elements. these are (i) three unpaired bases with a c in the middle position, (ii) three paired bases with a u in the middle, and (iii) three paired bases with an a in the middle. approaches designed for deep sequencing data, such as mirtools, miranalyzer, and mirdeep2 [12, 17 ] require the read count value to be known for predicting mirnas from rna - seq data. although the read count may have good discriminative power, it prevents its application to genomic or est sequences. the presence of the mirna can be used by mirdeep2. however, the mirna is not always sequenced due to low expression level and degradation. of the three de novo predictors of mirna precursors novomir has the highest reported sensitivity and specificity of 83% and 99%, respectively. hhmmir and triplet - svm report a sensitivity of 64% and 50%, respectively, for mirna precursors only. novomir was trained using arabidopsis thaliana mirna for positive controls and uses trna, rrna, noncoding rna, mrna, and genomic sequences from a. thaliana as negative controls. the program reports the predicted precursor along with the start and end positions of any predicted mature mirna. one can ask, what data are necessary to correctly predict mirnas from sequences ? and, specifically, for deep sequencing data, can an accurate predictor be developed that does not require read count or rely on the presence of the mirna ? the predictive model described here is one of the most specific predictors (98.53% specificity), yet has the least requirements as it only requires that the candidate sequence occurs in a sequence region large enough to encompass the putative precursor. fulfilling this single requirement allows the set of quantitative sequence properties (attributes) for the candidate sequence to be calculated and passed to the predictor. here, positive and negative controls from 18 plant species were used for training and model evaluation. positive controls were taken from mirbase while negative controls were taken from est sequences of each species. the negative controls were collected based on the number and length of the known mirnas in the positive controls for corresponding species. we chose randomly picked ests over specific subsets, such as mrna or various classes of noncoding rnas, to ensure that the predictor is not accidentally trained to detect the characteristics of the negative controls. although this would increase sensitivity and specificity of the predictor, it would also introduce bias. ests broadly represent protein - coding and nonprotein - coding genes including sequences resembling degraded mrna fragments that could exist in deep sequencing data [17, 19, 20 ]. wet - lab validation is costly and time consuming ; therefore, accurately predicting that the candidate rna is truly a mirna is important. our aim was to accurately predict when a sequence is not a mirna at the expense of missing a few true mirnas, which limits the number of false positives. to demonstrate the quality of any predictor the positive controls are known mirnas and non - mirna sequences form the negative controls. the negative controls were picked from ests of the respective species downloaded from tigr plant transcript assemblies. table 1 lists the 18 species by taxonomic group, and figure 1 is a flow chart of data collection and statistical validation. as stated above, the formation of a hair - pin or stem - loop structure by the precursor is critical for mirna biogenesis. to obtain attributes for positive controls, the location of the known mirnas within the precursor stem loop is required. the known precursors from mirbase are only used to confirm two things about the mirnas used as positive controls : (i) the mirna location on the chromosome is inside a known precursor and not just a random match and (ii) the sequence is upstream or downstream of the mirna (in the 5 half or the 3 half of the precursor illustrated in figures 2(a) and 2(b)). only attributes of mirnas for these correct chromosome locations and positions in the precursor are included as positive controls. novoalign was used to align mirna and precursor sequences to the respective genomes downloaded from plantgdb. at this point, the known precursors are no longer useful because all sequences will be treated equally from this point on regardless of whether they are positive controls, negative controls, or the assay sequences yet to be classified. for any short sequence from the above three types, the precursor candidate was defined by first locating the mirna candidate with the strongest duplex binding. next, the region between and including the sequence and the mirna candidate, along with 15 nt on both sides, was extracted. this region defines the operative precursor region (opr), which in real mirnas should form a stem - loop structure. the search for the mirna : mirna candidate duplex with the strongest binding was limited to a 300 nt window. the 300 nt window was used because 95.80% of known plant mirna precursors are less than 300 nt (table 2). for positive controls we have already defined that the correct window is upstream or downstream of the known mirna. for negative controls, both orientations are valid. after training, to use the model for prediction on sequences of unknown class, attributes from both upstream and downstream need to be collected and evaluated. this is essentially the same as for negative controls but without prior knowledge of prediction outcome. we use the opr instead of the precursor region as reported by mirbase to ensure equal treatment of all controls, and later for sequences of unknown class. simply, as unknowns and negative controls do not come with precursors, we must define the opr equally for all, including the positive controls for training. figure 3 illustrates the relationship of the known precursor from mirbase to the opr defined using the method described above. the computationally estimated mfe (in kcal / mol) for both the mirna : mirna duplex and the opr structure are required attributes for training. the rnaduplex function from the vienna rna package was used to find the location of the mirna and the binding energy of a mirna : mirna duplex within the longer genomic sequences. with this information rnafold from the vienna rna package was used to calculate the mfe for the opr, which is represented by deltag in table 3. rnafold also returns the secondary structure in dot - bracket format, representing the mismatch and base - pair matching, respectively, for the opr secondary structure. the dot - bracket structure allows the longest run of uninterrupted matches and mismatches to be calculated, as well as the number of unpaired nucleotides that form the head of the loop. all other attributes are based on characteristics of the mirna sequence, for example, the mirna base composition and the mirna : mirna duplex measurements. the rnaduplex function returns the number of matches and mismatches along with the duplex mfe. it has previously been reported that the number of matching and mismatching base pairs within the precursor stem loop are important attributes for training the precursor predictors [13, 14 ]. the mirnas : mirnas duplex information that includes base - pair matching and mismatching are also valuable attributes for predicting mature mirnas. the attribute duplexenergynorm in table 3 is the mfe of the mirnas : mirnas duplex normalized to the duplex length. the minimum number of consecutive matches for each duplex is stored in the attribute minmatchpercent and is based on both sides of the duplex depending on the shortest side. attributes for negative controls were collected in a similar way as those for positive controls. the randomly picked negative controls only qualified if they contained no ambiguity codes and had a length - normalized shannon entropy consistent with that of the positive controls. the latter is used to avoid low complexity regions and to ensure that strong negative controls are collected. the attribute shannonentropynorm in table 3 is the shannon entropy normalized by the sequence length. not all sequences could be aligned (e.g., they may fall across an intron - exon boundary) sufficient quantities were collected to make up for this expected loss. for negative controls, it does not matter whether the operational mirna is upstream or downstream of the mirna as both orientations are equally valid and either the upstream or downstream attribute set is chosen randomly. in this way, the sequence is represented only once in that control set. the final training set used for validation contains 2073 positive controls and 5306 negative controls. more negative controls than positive were used so that the broadest set of non - mirnas is used to train against the known mirnas. this increases the model 's ability to accurately recognize non - mirnas and to minimize false positive predictions. once attributes have been collected for both positive and negative controls, validation sets can be produced. the model was validated by calculating sensitivity and specificity based on leave - one - out cross - validation. sensitivity is the ability of the classifier to identify positive results (true mirnas), while specificity is the ability to distinguish negative sequences. leave - one - out cross - validation can be described as putting all controls in a stack, taking the first one out and training with the rest. only one of two possibilities will occur : the predictor is correct on the previously unseen control, or not. the one previously removed is returned, and the next one is removed, and again training and testing are done until each control has been excluded and tested. when this is completed, the results are used to calculate sensitivity and specificity based on the following formulas : (1)sensitivity = number of true positivesnumber of true positives+number of false negatives100,specificity = number of true negativesnumber of true negatives+number of false positives100.only sequences that are not highly similar (based on a cutoff value, see below) to the test sequence will be allowed into the training set. it is important that sequences similar to the ones left out are also excluded to ensure the rigour of the leave - one - out approach. a study on precursor prediction used the blastclust program to identify sequences of high similarity for exclusion from the training set. however, we found that blastclust does not always ensure that sequences in two different clusters are sufficiently different, as any given sequence can only belong to one cluster. we aligned each sequence to every other sequence and used their similarity to determine inclusion or exclusion from training. in this way, each control sequence is excluded from training together with similar sequences. all nonsimilar sequences are used to train, and this trained model is tested for its ability to accurately predict the class for the one excluded test case. pools of positive and negative controls have each been pairwise aligned using the needleall program of the emboss package. the results from needleall were used to determine the level of similarity between sequences in the positive controls set, then separately for negative controls. needleall returns the similarity between two sequences, as well as other values. in this case, two sequences are considered similar if the similarity calculated by needleall is greater than 70%, based on the default setting. the c5.0 program from rulequest incorporates a decision - tree machine learning method that we have used to train a mirna predictor using controls of known outcome. c5.0 and the windows version see5 are improved versions of c4.5 that in turn descended from a program called id3. the training data used by c5.0 can be any combination of nominal attributes (e.g., the letter of the first nucleotide in the sequence) and numeric attributes (e.g., mfe). the output is a model in the form of if - then rules or decision trees for classifying cases of unknown outcome.. producing models that are easy to understand can be useful when the goal is to discover the biological relationships between the attributes and class, rather than to classify unknown cases. in some investigations the goal is to determine the attributes and cutoff values critical for separating the classes. c5.0 can be applied to training data containing many thousands of cases with hundreds of attributes each. the typical size of our training set is ~5294 cases, each with only 29 attributes. if, after training, the model had a higher than acceptable false positive rate, a misclassification cost would be applied when retraining. if the opposite was true (i.e., validation cheap and mirna rare), the misclassification cost could be adjusted accordingly. for example, a misclassification cost would be applied to avoid missing true mirnas during training. c5.0 supports a technique called boosting that is used to improve classification results. boosting combines decision trees to produce and test new trees that may improve classification. for example, the branches from one tree are swapped with those from another tree, and the two new classifiers are tested for increased predictive power. different boosting values were tested to determine the level of improvement to the basic predictor. figure s1 (see supplementary material available online at doi : 10.1155/2012/652979) is a graph of roc space for 28 runs from 3 to 30 boosting trials. this shows that, for this type of data, lower boosting values already produce good sensitivity and specificity, while higher boosting provides little improvement. rulequest also provides an evaluator program that uses c5.0-trained classifiers to predict the outcome for data of unknown class. we used this to record the predicted class for each case (e.g., mirna candidate) along with the confidence value. after training, the attribute usage information demonstrates the discriminative importance of each attribute. table 5 shows the attribute usage for one of many possible training runs of the classifier ; other training runs show similar usage. several attributes, such as duplexenergy, minmatchpercent, and gc content, are required for all sequences to be classified. genomic sequences and ests from cdna contain the base t, whereas mirnas from mirbase contain u. for this work, u was converted to t for processing attributes. the attribute t% relates directly to the potential for g - u and a - u base pairing in the rna. not surprisingly, the attribute named duplexenergy is important for training because it relates directly to the stability of the base pairing between the mirna and mirna duplex. this is evident by the 100% usage of duplexenergy and deltagnorm which represents the energy of the opr stem - loop structure normalized by its length. although this is an imperfect method, collecting data from all leave - one - out training runs can provide an overall view of an attribute 's discriminative power. c5.0 has a built - in function called winnowing that, when applied during training, returns the percentage of increase in error if an attribute is removed from training. table 6 shows attributes with the largest average percentage of decline and therefore the level of importance for training the predictor. sensitivity and specificity are critical values for assessing classifier accuracy ; values as high as 84.08% for sensitivity and 98.53% for specificity have been obtained. the question remains whether this high specificity and sensitivity can also be achieved when the predictor is trained with different species than are used for prediction. if all mirnas in each taxonomic category listed in table 7 are systematically excluded from training while including all others, how well does the predictor do when tested on the excluded category ? results from these tests reveal how well the model predicts mirnas in plant species not included in the training set. the ability of the classifier to correctly identify known mirnas ranged from 78% for the dicotyledon salicaceae to 100% for 7 of the 18 groups in table 7. all misclassified mirnas in the salicaceae are unique to the single species of poplar tree. in table 8, four taxonomic groups are formed : monocotyledon, dicotyledon, lycopodiophyta, and embryophyta. when embryophyta is excluded from training, in contrast, when lycopodiophyta, embryophyta, and one of monocotyledon or dicotyledon are combined, the excluded set of monocotyledon or dicotyledon is recognized at 100%. when lycopodiophyta and embryophyta are combined into one group named primitive, training with the combination of any two groups produced a model that correctly classifies all mirnas in the excluded group, as shown in table 9. for all exclusion experiments this demonstrates that an accurate universal predictor of plant mirnas was produced when all groups, and therefore all mirnas, were combined for training. identical mirna sequences are found in multiple plant species, often on multiple chromosomes where they are part of identical or different precursors. although the mirna sequences can be identical, some of their attributes are not. at a minimum leave - one - out sets are required for statistical validation by modelling the predictive accuracy of a control sequence that is unseen during training. for validation, excluding identical or near - identical mirnas is critical. when creating a predictor for unknown sequences, the classifier can be trained using all plant mirnas to ensure the best predictor possible. the taxonomic exclusion tests demonstrate that training with the maximum number of known mirnas produces a classifier with the best cross - species recognition of mirnas. in one training example, the set included 2278 positive controls, some of which are the same mirnas sequence but in different precursors and species. the contrasting negative controls have 5680 short sequences randomly picked from ests. for this training set, from 3 to 300 boosting trials were run. at boosting trials near 300, only five negative controls were misclassified as mirnas during training. although only wet - lab validation can confirm that a sequence is a mirna, two of the five are found in what look like very obvious mirna precursors. the possible mirna precursors for these two est segments are shown in figures s3 and s4 of the supplementary data. these two short sequences taken from the middle of randomly picked ests have yet to be validated as true mirnas. one method of demonstrating that the predictor can be used for de novo prediction is to see how many known medicago truncatula (mt) mirnas can be accurately predicted from the precursor source. this shows the quality of the predictor when applied to short segments taken from ests or chromosomes. for mt, there are 342 unique mirnas from mirbase that occur in one or more of the 581 unique mt precursors. therefore, short sequence segments were taken from the mirna precursors for only these lengths. the segments were extracted by a sliding window method starting at the beginning of the sequence, taking 20 nt, moving one base over and again taking 20 nt. this continues until the end where it becomes too short for a full 20 nt segment. attributes are calculated for this set of short sequences, and then predictions are made by the trained classifier. of the 342 unique mirnas, 309 are correctly classified as mirnas by the predictor for a score of 90.35%. this demonstrates that the predictor can be applied to short segments taken from ests or genomic data as well as to short reads from rna - seq data. we are currently working to achieve the speed increases required to analyze entire chromosomes. in some cases of the 18 plant species used for training, only 17 known mirnas from seven species did not align to the respective genome. when these mirnas can be found in an est, the est sequence can be used in place of a genomic sequence to collect attributes for prediction. there is no genome listed for lja - mir167 in mirbase, and it is not found on any chromosomes tested for that species. it is, however, found in an est [genbank : bw598483 ] at position 43. a comparison between the stem loop from mirbase and our predicted opr for the mirna is shown in figure 3. this demonstrates that when a short test sequence is not found in a genome prediction can still occur if it is found in an est. during development these negative controls were later pooled and the predictor applied to determine the number of potential mirnas in random samples of ests. out of 58,443 sequences, 633 (1.08%) were classified as mirna. as described in the methods section, only one mirna location (either upstream or downstream of the mirna) was kept. it is conceivable that if both were kept, the rate of mirna detection in randomly picked ests could double. if some of these negative controls are true mirna, the specificity would be higher if they were removed from training. while specificity is already high, this suggests that the predictor is more accurate than the value reported here. the true sensitivity may also be higher than the reported value. despite using reported mirnas from mirbase for training fifty mirnas across 10 species that were not classified as mirna were collected in a set. twelve mirnas across four species out of the original 50 were classified as mirna when attributes from both upstream and downstream were tested. closer inspection of the differences between attributes for the same mirna between the two sets revealed that in some cases the relative position of mirna and mirna was different. in these cases the precursor in mirbase was asymmetrical, and the automated location picking script returned the wrong location within the precursor. when the correct location and attributes were tested by the predictor, it was classified as a mirna. an example is gma - mir5034 mi0017906, where the mirna is clearly located in the 3 end. however, it has 69 bases on its downstream side and 60 bases on the upstream side, putting the mirna predominantly in the upstream half of the precursor. although some small set of erroneous attributes based on the wrong locations were included in the training dataset, the classifier correctly classified these as mirna when given both upstream and downstream attributes. including attributes from incorrect locations for training produced a lower sensitivity value than if correct locations were used but did not diminish the predictor 's ability to correctly classify true mirna. we have shown that a highly accurate universal plant mirna predictor can be produced by machine learning using c5.0. this predictor can be applied to any short sequence that aligns to a precursor candidate in a genome or transcriptome. the source of the sequence for testing can be short reads from deep sequencing, or short segments taken from chromosomes or est sliding windows. along with mirna prediction and prediction confidence level, the putative precursor is also produced ready for folding and visualization. if used to scan a chromosome region, this predictor will reveal areas of high or low mirna density. if applied to deep sequencing data the predictor reveals how often in a genome the short read exists, how many are predicted mirnas, and how many unique precursors contain that short - read mirna. | micrornas (mirnas) are nonprotein coding rnas between 20 and 22 nucleotides long that attenuate protein production. different types of sequence data are being investigated for novel mirnas, including genomic and transcriptomic sequences. a variety of machine learning methods have successfully predicted mirna precursors, mature mirnas, and other nonprotein coding sequences. mirtools, mirdeep2, and miranalyzer require read count to be included with the input sequences, which restricts their use to deep - sequencing data. our aim was to train a predictor using a cross - section of different species to accurately predict mirnas outside the training set. we wanted a system that did not require read - count for prediction and could therefore be applied to short sequences extracted from genomic, est, or rna - seq sources. a mirna - predictive decision - tree model has been developed by supervised machine learning. it only requires that the corresponding genome or transcriptome is available within a sequence window that includes the precursor candidate so that the required sequence features can be collected. some of the most critical features for training the predictor are the mirna : mirna duplex energy and the number of mismatches in the duplex. we present a cross - species plant mirna predictor with 84.08% sensitivity and 98.53% specificity based on rigorous testing by leave - one - out validation. |
muscle cramps are painful, local, tangible, and involuntary skeletal muscular contractions that usually affect leg muscles. muscle cramps are one of the common symptoms of pregnancy, especially during the third trimester that are often unidirectional. leg cramps mostly happen twice a week or less frequently, usually at night, last a few seconds to a few minutes, and mostly disappear by themselves. the mechanism for cramping is not known yet and is likely to be idiopathic, but it could be due to physiological changes in neuromuscular performance, weight gain, joint laxity in the final stages of pregnancy, impaired blood supply to lower body organs, and increased pressure on leg muscles during pregnancy. pressure on blood vessels and nerves resulting from the enlarged uterus, imbalances between the intake and output of electrolytes and vitamins, and insufficient minerals intake might be other reasons for cramping. hence, could increase glomerular filtration and greater needs of the fetus for receiving minerals from the mother (which reduces her serum calcium and magnesium levels). cramps during pregnancy are not related to pregnancy complications and are not accompanied by undesirable consequences for the fetus, but the american sleep association considers leg cramps as one of the reasons for sleep disorders during pregnancy. sleep disorder resulting from cramps influences performance of daily activities and may lengthen the duration of pregnancy and the type of childbirth. moreover, the findings of studies have suggested that women with snoring in pregnancy have a higher risk for growth retardation of the fetus ; and women with sleep deprivation have a higher risk for preterm births. various treatments have been suggested for preventing and curing leg cramps during pregnancy including taking vitamins b1, b6, e, and c and magnesium. the effects of the calcium supplement on improving leg cramps during pregnancy were investigated in many studies. some of these studies reported relative or complete improvements of symptoms when calcium supplements were taken while others stated these supplements were ineffective. vitamin d is a fat - soluble vitamin found in very small quantities in natural food materials. taking calcium supplement in food materials is necessary during pregnancy to prevent depletion of calcium in the mother 's body. leg cramps are very prevalent in pregnant women and have negative influences on pregnant women 's sleep performance and quality. moreover, the mechanism of the effects of vitamin d on calcium is not known and no study has been conducted on the effects of vitamin d on leg cramps. furthermore, vitamin d is present in very quantities in food materials, vitamins are cheap, and pregnant women are very willing to take vitamins. that is why we decided to conduct a study on the therapeutic effects of vitamin d and calcium plus vitamin d supplement on leg cramps during pregnancy. this study was conducted as a double - blind randomized controlled clinical trial on pregnant women who were referred to health - care centers in tabriz - iran, 2013. a 1835-year - old, being in 2530 week of pregnancy and having minimum of two cramps during the week. exclusion criteria included history of chronic diseases, kidney problems, osteomalacia, active thyroid or parathyroid diseases, endocrine disorders, hypertension, use of diuretics, calcium - blockers intake, chronic hypertension, and history of allergy to the study supplements. since = 0.05, = 0.02, m1 = 49.17 (the overall mean score of cramp intensity before prevention), m2 = 35 (the overall mean score of cramp intensity after the intervention), sd1 = 20.6, and sd2 = 23, the calculated number of participants for each group was 38. however, 42 participants were placed in each group to allow for dropouts. the tools used for collecting data included sociodemographic and midwifery questionnaires and checklists to record weekly information on leg cramps in the 3 and 6 week of the intervention. the participants marked information on the frequency, intensity (in the visual analog scale [vas ], graded from 0 to 10 with zero for lack of pain and ten for very severe pain, for each cramping event), length (in minutes), and the time leg cramps happened in the checklists during the 3 and 6 week of the intervention. at the end of the 3 and 6 week of the intervention, the participants marked the checklist table related to side effects of taking the vitamin d and vitamin d plus calcium supplements. furthermore, to control for the potentially confounding factor of diet, a dietary record questionnaire including foods containing calcium and vitamin d was also completed by the participants during 1 week before intervention. sociodemographic and midwifery questionnaire as well as checklists for recording number and length of leg cramps were designed by research team and the content and face validity of them were confirmed by ten faculty members of tabriz university of medical sciences. sampling started after obtaining ethical code from the ethics committee of research and technology deputy of tabriz university of medical sciences under the number 91230 and registering the trial in the irct website under the code irct2013040810324n12. thirty - three health centers from various areas in tabriz, with different sociocultural situations that were visited by the largest numbers of women were first selected from the total of eighty health centers in the city. health centers in the country are public, nonreferral, and governmental that all primary care services, including prenatal care, are provided free of charge. a list of all pregnant women in their 2530 week of pregnancy who visited the selected centers was prepared. they were then contacted by telephone and invited to take part in the study. during their first visit to the center, the goals and method of the study were explained and the checklists for recording characteristics of leg cramps including the frequency, length, and intensity of leg cramps were given to them to record these during the week before the intervention started. at the end of this week, those who had recorded two and more cramps during the week and had other eligibility criteria entered the study and completed their informed written consent to participate in the research. the participants were allocated to three groups using a randomized block design with block sizes of three and six with the allocation ratio 1:1:1. to hide the allocation, each participant received two small envelopes, each with enough medicine for 3 weeks, inside a large matte - colored envelope of the same shape that were serially numbered. pills containing vitamin d (1000 units) or vitamin d plus calcium complements (300 mg of calcium carbonate and 1000 units of vitamin d) were of the same shape, size, and weight as the placebo pills. the pills were prepared by the faculty of pharmacy in tabriz university of medical sciences, iran. at the start of the intervention, each participant received a small envelope containing calcium plus vitamin d or vitamin d or the placebo, a checklist for recording leg cramp characteristics, and a checklist for recording daily multivitamins and pills taken during 3 weeks. they were asked to mark the checklists every time they took the pills. at the end of the 2 week of the study, the participants were called and asked to record the characteristics of leg cramps during the third week in the related checklists. they were reminded to bring the medicine envelope and the checklist for leg cramps during the 3 week and the checklist of medicines taken with them at the next follow - up that was at the end of the 3 week. during this visit, each of the participants received a second envelope with the same content as the first, together with the checklists for the second 3-week period. at the end of the 5 week, the participants were called and reminded to record the characteristics of the leg cramps that happened during the 6 week in the related checklists. they were asked to bring these checklists and the checklists related to the medicines taken, together with the second envelopes, with them to the second follow - up visit at the end of the 6 week. chicago). the k - s test was used to investigate the normality of the quantitative data. the variables of the length of cramps before the intervention, during the 3 week of the intervention, and during the 6 week of the intervention were not normal. the chi - square, chi - square for trend, fisher exact test, and one - way anova were used to examine the consistency of study groups. to compare the average pain severity of leg cramps between three groups, the baseline (preintervention) value of pain severity as possible confounder factor was entered to repeated measures anova model as covariate. kruskal wallis and friedman tests were used, respectively, for inter- and intra - groups comparisons of the number and the length of leg cramps before, during the 3, and 6 after the start of the intervention. this study was conducted as a double - blind randomized controlled clinical trial on pregnant women who were referred to health - care centers in tabriz - iran, 2013. a 1835-year - old, being in 2530 week of pregnancy and having minimum of two cramps during the week. exclusion criteria included history of chronic diseases, kidney problems, osteomalacia, active thyroid or parathyroid diseases, endocrine disorders, hypertension, use of diuretics, calcium - blockers intake, chronic hypertension, and history of allergy to the study supplements. since = 0.05, = 0.02, m1 = 49.17 (the overall mean score of cramp intensity before prevention), m2 = 35 (the overall mean score of cramp intensity after the intervention), sd1 = 20.6, and sd2 = 23, the calculated number of participants for each group was 38. however, 42 participants were placed in each group to allow for dropouts. a 1835-year - old, being in 2530 week of pregnancy and having minimum of two cramps during the week. exclusion criteria included history of chronic diseases, kidney problems, osteomalacia, active thyroid or parathyroid diseases, endocrine disorders, hypertension, use of diuretics, calcium - blockers intake, chronic hypertension, and history of allergy to the study supplements. considering research carried out by yaghmaei. and since = 0.05, = 0.02, m1 = 49.17 (the overall mean score of cramp intensity before prevention), m2 = 35 (the overall mean score of cramp intensity after the intervention), sd1 = 20.6, and sd2 = 23, the calculated number of participants for each group was 38. the tools used for collecting data included sociodemographic and midwifery questionnaires and checklists to record weekly information on leg cramps in the 3 and 6 week of the intervention. the participants marked information on the frequency, intensity (in the visual analog scale [vas ], graded from 0 to 10 with zero for lack of pain and ten for very severe pain, for each cramping event), length (in minutes), and the time leg cramps happened in the checklists during the 3 and 6 week of the intervention. at the end of the 3 and 6 week of the intervention, the participants marked the checklist table related to side effects of taking the vitamin d and vitamin d plus calcium supplements. furthermore, to control for the potentially confounding factor of diet, a dietary record questionnaire including foods containing calcium and vitamin d was also completed by the participants during 1 week before intervention. sociodemographic and midwifery questionnaire as well as checklists for recording number and length of leg cramps were designed by research team and the content and face validity of them were confirmed by ten faculty members of tabriz university of medical sciences. sampling started after obtaining ethical code from the ethics committee of research and technology deputy of tabriz university of medical sciences under the number 91230 and registering the trial in the irct website under the code irct2013040810324n12. thirty - three health centers from various areas in tabriz, with different sociocultural situations that were visited by the largest numbers of women were first selected from the total of eighty health centers in the city. health centers in the country are public, nonreferral, and governmental that all primary care services, including prenatal care, are provided free of charge. a list of all pregnant women in their 2530 week of pregnancy who visited the selected centers was prepared. they were then contacted by telephone and invited to take part in the study. during their first visit to the center, the goals and method of the study were explained and the checklists for recording characteristics of leg cramps including the frequency, length, and intensity of leg cramps were given to them to record these during the week before the intervention started. at the end of this week, those who had recorded two and more cramps during the week and had other eligibility criteria entered the study and completed their informed written consent to participate in the research. the participants were allocated to three groups using a randomized block design with block sizes of three and six with the allocation ratio 1:1:1. to hide the allocation, each participant received two small envelopes, each with enough medicine for 3 weeks, inside a large matte - colored envelope of the same shape that were serially numbered. pills containing vitamin d (1000 units) or vitamin d plus calcium complements (300 mg of calcium carbonate and 1000 units of vitamin d) were of the same shape, size, and weight as the placebo pills. the pills were prepared by the faculty of pharmacy in tabriz university of medical sciences, iran. at the start of the intervention, each participant received a small envelope containing calcium plus vitamin d or vitamin d or the placebo, a checklist for recording leg cramp characteristics, and a checklist for recording daily multivitamins and pills taken during 3 weeks. they were asked to mark the checklists every time they took the pills. at the end of the 2 week of the study, the participants were called and asked to record the characteristics of leg cramps during the third week in the related checklists. they were reminded to bring the medicine envelope and the checklist for leg cramps during the 3 week and the checklist of medicines taken with them at the next follow - up that was at the end of the 3 week. during this visit, each of the participants received a second envelope with the same content as the first, together with the checklists for the second 3-week period. at the end of the 5 week, the participants were called and reminded to record the characteristics of the leg cramps that happened during the 6 week in the related checklists. they were asked to bring these checklists and the checklists related to the medicines taken, together with the second envelopes, with them to the second follow - up visit at the end of the 6 week. chicago). the k - s test was used to investigate the normality of the quantitative data. the variables of the length of cramps before the intervention, during the 3 week of the intervention, and during the 6 week of the intervention were not normal. the chi - square, chi - square for trend, fisher exact test, and one - way anova were used to examine the consistency of study groups. to compare the average pain severity of leg cramps between three groups, the baseline (preintervention) value of pain severity as possible confounder factor was entered to repeated measures anova model as covariate. kruskal wallis and friedman tests were used, respectively, for inter- and intra - groups comparisons of the number and the length of leg cramps before, during the 3, and 6 after the start of the intervention. in this study, 126 pregnant women with leg cramps from july 2013 to january 2014 entered the study and their conditions were followed up to april 2014. flowchart of the study there were no statistical differences between groups regarding consuming of foods containing calcium and vitamin d during 1 week before intervention (p = 0.204) and sociodemographic characteristics (p > 0.05) except of prepregnancy body mass index (bmi) (p = 0.024) and bmi during pregnancy (p = 0.008) [table 1 ]. demographic and obstetrical characteristics of the participants by study groups before the intervention, there was no statistically significant difference between the groups in terms of length (p = 0.160) and pain severity of leg cramps (p = 0.990), but there was statistically significant difference between the groups in terms of weekly number of leg cramps (p = 0.012) [tables 24 ]. comparison of the leg cramps number in pregnancy in three groups of study comparison of the leg cramps length (min) in pregnancy in three groups of study comparison of the severity of leg cramps pain (visual analog scale) in pregnancy in three groups of study according to kruskal wallis test, there was significant difference between the groups in terms of the weekly number of leg cramp during the 3 week after the start of the intervention (p = 0.033) and the number of leg cramp was less in the control group. however, there was no significant difference between the groups during 6 week after the start of the intervention (p = 0.098) [table 2 ]. furthermore, there was no significant difference between the groups in terms of the length of leg cramps during the 3 week (p = 0.308) and 6 week after the start of the intervention (p = 0.079) [table 3 ]. in addition, the weekly number of leg cramp and the length of leg cramps were significantly decreased in each three groups according based on intra - groups comparison by friedman test (p < 0.05) [tables 2 and 3 ]. based on the repeated measure anova test with adjusting for baseline value, there was no statistically significant difference between the vitamin d and control groups (p = 0.814), calcium - vitamin d and control groups (p = 0.931), and also between two treatment groups (p = 0.993) [table 4 ]. at the end of the 6 weeks, two participants in the group receiving calcium - vitamin d, 13 in the group receiving vitamin d, and six in the group receiving placebo reported itching. furthermore, six participants in the group receiving calcium - vitamin d, 14 in the group receiving vitamin d, and six in the group receiving placebo reported they experienced dry mouth. results of this research showed that the calcium - vitamin d and vitamin d supplements had no effect on the number, length, and pain severity in leg cramps during the 6 weeks of the study. danesh shahrakiconducted a study on three groups of pregnant women with leg cramps (who received calcium, vitamin e, or magnesium milk) with no control group. they found that prescribing calcium carbonate at 500 mg / day for 45 days reduced the number, length, and pain intensity on the 45 day, but that on the 90 day after the first visit, these figures returned to their previous values. observed that prescription of calcium at 500 mg / day for 40 days reduced the number of leg cramps but had no effect on pain intensity. (1987), prescription of one gram of calcium for 2 weeks clinically improved leg cramps. results of these two studies do not conform to those found in the present study, probably due to the difference in the doses of the prescribed medicines, and because calcium was used together with vitamin d in our research. we suggest that future studies be carried out using higher doses to determine the effective one. according to the study by zhou., there was no difference in the frequency of leg cramps after treatment with calcium versus vitamin c. since the control group received ascorbic acid in their study, and cramps improved relatively or completely in most participants. the results of systematic review by young. showed that calcium can decrease pain in a leg cramps and it has positive effect on treatment. furthermore, the results of cochrane systematic review showed that a greater proportion of women receiving calcium supplements experienced no leg cramps after treatment than those receiving no treatment. this review suggested that large well - conducted randomized controlled trials are needed in this regard due to poor study design and small sample size of trials. the above - mentioned studies have assessed the effects of calcium on leg cramps but no studies have been conducted so far on the effect of vitamin d supplement on cramps. however, calcium and bone metabolisms greatly depend on vitamin d concentration and on its metabolically active form, 1,25(oh)2d, and the human body can not absorb sufficient ca and p without the help of 1,25(oh)2d. moreover, the 1,25(oh)2d produced in the kidneys stimulate the calcium reserve in bone tissues and in the intestines and helps ca and p absorption ; and finally, the combination of these activities increases serum ca and p. increased levels of serum ca and p cause improvements in metabolic performances, bone health, and neuromuscular functions. as vitamin d and ca had no effect on leg cramps in our study, and because the main reason for, and the mechanism of leg cramps in pregnancy are not clear yet, leg cramps during pregnancy may not be due to calcium deficiency and could have other causes. lack of visual observation to investigate pain intensity in leg cramps was one of the limitations of this study. furthermore, the impossibility of measuring blood calcium and vitamin d levels was another limitation. it is recommended that future clinical trials should investigate the effects of vitamin d and calcium - vitamin d supplements with different doses of these materials, intervention should take place using a larger sample, and serum calcium and vitamin d levels ought to be assessed to obtain results that are more accurate. results of this study indicated that taking 1000 iu vitamin d and 300 mg calcium plus 1000 iu vitamin d every day for 6 weeks has no effect on leg cramps. one of the limitations in this study was the short follow - up period, so that a follow - up period longer than 6 weeks in the future researches was suggested. furthermore, the serum levels of calcium and vitamin d did not determined in this study due to cost restrictions. therefore, these supplements should be administrated by assessing their serum levels before and after the intervention. am contributed in the conception of the work, conducting the study, drafting, and agreed for all aspects of the work. mm contributed in the conception of the work, conducting the study, analyzing the data, revising the draft, and agreed for all aspects of the work. sm contributed in the conception of the work, conducting the study, revising the draft, and agreed for all aspects of the work. mn contributed in the conception of the work, synthesis of drugs, revising the draft, and agreed for all aspects of the work. | background : this study intended to determine the effects of vitamin d and calcium - vitamin d in treating leg cramps in pregnant women.materials and methods : this study was conducted as a double - blind randomized controlled clinical trial on 126 participants, 1835-year - old pregnant women with a minimum of two leg cramps per week who were referred to health - care centers in tabriz - iran in 2013. the participants were allocated to three 42 member groups using a randomized block design. for 42 days, the intervention groups took a 1000 unit vitamin d pill or 300 mg calcium carbonate plus a 1000 unit vitamin d pill, and the control group received a placebo pill every day. the participants were evaluated with regard to the frequency, length, and pain intensity of leg cramps during the week before and during the 3rd and 6th week of the intervention. the ancova and repeated measurement test were used to analyze the data.results:results showed that controlling for the effects before the intervention, calcium - vitamin d, and vitamin d supplements had no effect on the frequency, length, and pain intensity of leg cramps.conclusion:the results of this study showed that the calcium - vitamin d and the vitamin d supplements have no effect on the frequency, length, and pain intensity of leg cramps during the 6 weeks of the study. |
protein phosphorylation can modulate protein activity, turnover, subcellular localization, complex formation, folding and degradation. dynamic phosphorylation plays a pivotal role in almost all biological processes including cell division, differentiation, polarization and apoptosis. the importance of this post - translational modification (ptm) for cell biology has driven the development of novel mass spectrometric tools for sensitive and global detection of phosphorylation. however, the analysis of phosphorylated peptides by mass spectrometry is still not as straightforward as for regular, unmodified peptides. one of the major challenges in phosphoproteomics is to improve ms level representation since phosphopeptides are usually present at substoichiometric levels. hence, an enrichment step is necessary to enable deeper penetration of the phosphoproteome. enrichment is typically performed by chromatography, antibodies or metal - ion / metal oxide affinity - based techniques. two other main challenges are the identification of phosphopeptides and confident localization of the corresponding phosphosite. the challenge is caused by the higher lability of the phosphate group when compared to the amide bond. a number of strategies have been proposed to circumvent poor fragmentation and improve sequence and site diagnostic fragmentation, including the use of neutral loss - triggered ms / ms / ms and multistage activation (msa) in ion traps, the use of beam type cid fragmentation, and electron capture / transfer dissociation or a combination of some of these approaches. once phosphopeptide identification is feasible through sufficient peptide backbone fragments, it can still be challenging to pinpoint the true phosphosite. this becomes more difficult as the number of potential phosphorylation sites within the peptide sequence increases. in principle direct validation is feasible through detection of a fragment ion that carries the phosphate group. neutral loss fragment ions can be used as well ; however, since they exhibit the same mass as a water loss from an unmodified residue they do not directly confirm the correct site. diagnostic phosphosite - specific fragments facilitate pinpointing the correct phosphosite. several algorithms and programs these software tools are based on distinct but similar approaches and they all aim to provide a metric that allows for assessment of the confidence in phosphosite localization. recently, taus. have reported on a new algorithm, coined phosphors, which presently is uniquely compatible with cid, hcd and etd fragmentation and was optimized for both low- and high - resolution ms / ms spectra. generally, all scoring tools depend on the quality of the ms / ms spectra. the more site - determining ions are detected, the higher the confidence in phosphosite localization. we have recently introduced a novel fragmentation scheme combining electron - transfer and higher - energy collision dissociation, termed ethcd. this method employs dual fragmentation to generate both b / y and c / z ions which leads to very fragment ion- and thus data - rich ms / ms spectra. compared to hcd and etd, we found a substantial increase in peptide backbone fragmentation, which translated into a remarkable average peptide sequence coverage of 94% for tryptic peptides. we reasoned that localization of post - translational modifications could also highly benefit from ethcd spectra. here, we systematically assessed the impact on phosphosite localization using ethcd. in this work we evaluate the performance of ethcd in comparison to etd and hcd using a defined synthetic phosphopeptide mixture and also on a larger data set of ti - imac enriched phosphopeptides, all in combination with a modified version of phosphors. all chemicals were purchased from sigma - aldrich (steinheim, germany) unless otherwise stated. protein from hela cells was harvested and digested with trypsin, as previously described. briefly, gel - loader tips that were plugged with c8 material (3 m, zoeterwoude, the netherlands) were filled up to 1 cm with ti - imac beads. peptides were reconstituted in loading buffer, loaded onto the columns and washed with washing buffer 1 (50% acn, 0.5% tfa, 200 mm nacl) and subsequently washing buffer 2 (50% acn, 0.1% tfa). phosphopeptides were eluted with elution buffer 1 (10% nh3 in h20) followed by elution buffer 2 (80% acn, 2% fa). eluate was acidified and diluted with formic acid to a final acetonitrile concentration of 99%, which corresponds to a very high confidence in site localization (table 1). together, these findings suggest that ethcd generates ms / ms spectra that contain sufficient fragment ions for the unambiguous and sensitive phosphorylation site localization. next, we assessed the performance of ethcd for phosphosite localization on a larger data set. we used ti - imac material for the enrichment of phosphopeptides from a tryptic digest of hela cells and analyzed equal amounts (corresponding to enriched phosphopeptides from 100 g of protein) by lc ms / ms with etd, hcd and ethcd, respectively (supplementary figure 1a, supporting information). the etd data was also searched with mascot because we found sequest to perform poorly for doubly charged phosphopeptides. note that other search engines such as omssa or spectrummill might provide larger number of identifications for etd data. however, these algorithms are currently not compatible with ethcd data and phosphors analysis within the proteome discoverer software environment. all identified psms were then filtered for 99%. in the hcd data set we found that 89% of all phosphosites were assigned with a confident site localization probability > 99%, while this was only 81% for etd data set. we recalculated these number for all peptides that contain > 2 residues that can be phosphorylated because singly phosphorylated peptides with only one potential phosphorylation site could bias the results toward hcd. of all phosphosites from this subset of peptides 97% (etcad), 93% (ethcd) and 87% (hcd), respectively, were assigned a localization probability > 99%. figure 1 shows an ms / ms spectrum of a doubly phosphorylated peptide upon ethcd fragmentation. the overall sequence coverage is 89% taking b / y- and c / z - ions into account. six out of 18 amino acid bond cleavages are represented by c- and b - ions (referred to as golden pairs). additionally, we observed 11 z / y - ion pairs, which strengthens the argument that ethcd provides extensive sequence information that facilitates pinpointing the correct phosphorylation site. more than 95% of the phosphosites from all doubly phosphorylated peptides were assigned with a site localization probability > 99%, highlighting that ethcd performs equally well with singly and doubly phosphorylated peptides. a known limitation of etd is its inability to cleave the n c bond n - terminal to proline. figure 2 shows the ethcd spectrum of a singly phosphorylated peptide that contains four serine residues. the c- and z - ions derived from the etd step cover only the n - terminal part of the peptide and the site probability is only 50%. the additional y - ions derived from the subsequent hcd activation provide supporting sequence information and cover also the two serine residues next to the prolines which enables unambiguous phosphosite localization. ethcd generates dual ion series that enable phosphorylation site localization with very high confidence (phosphors site probabilities : t(3), 0.0% ; s(6), 100.0% ; s(9), 100.0% ; t(15), 0.0%). this ethcd spectrum of a doubly charged peptide that contains four serine residues, one of which is phosphorylated. etd does not cleave the n c bond n - terminal to proline and the phosphorylation site probability is only 50% based on c- and z - ions alone. dual fragmentation by ethcd generates complementary sequence information from c / z- and b / y - ions (sequest xcorr 4.10). here, the exact phosphosite is revealed by y - ions that cover the corresponding phosphosite (phosphors site probabilitis : s(1) : 0.0 ; s(3) : 0.0 ; s(8) : 99.5 ; s(10) : 0.5). here we have evaluated the potential of ethcd in improving the analysis of phosphopeptides. our data highlights the benefit of dual ion series as generated by ethcd fragmentation. we observed for a defined phosphopeptide mixture average higher sequest xcorr values, higher peptide sequence coverage and more confident phosphosite localization in ethcd compared to etd and hcd. this finding was confirmed when we analyzed a complex phosphopeptide sample resulting from a ti - imac enrichment of peptides from a cellular lysate. this is in line with recent reports that showed that confidence in phosphorylation site localization increases when multiple separately acquired ms / ms spectra (e.g., etd / cid or msa / etd) are combined for scoring. for this larger data set, we observed that the identification success rate was slightly lower for ethcd compared to hcd. this can be attributed to the use of conventional database search engines that are not optimized for spectra that contain dual ion series. however, the fact that both peptide sequence coverage and the percentage of localized phosphosites are higher for ethcd than for hcd suggests that once a peptide was identified, further analyses such as site localization benefit from the more data - rich ethcd spectra. in ethcd often c / b- and z / y - ion pairs are observed that increase the confidence in a particular peptide backbone cleavage. we speculate that the identification success rate of ethcd for phosphopeptides can be improved by novel or optimized data analysis tools. finally, we reason that ethcd can also be beneficial and used to improve the localization of other post - translational modifications such as ubiquitination, glycosylation or acetylation. | we recently introduced a novel scheme combining electron - transfer and higher - energy collision dissociation (termed ethcd), for improved peptide ion fragmentation and identification. we reasoned that phosphosite localization, one of the major hurdles in high - throughput phosphoproteomics, could also highly benefit from the generation of such ethcd spectra. here, we systematically assessed the impact on phosphosite localization utilizing ethcd in comparison to methods employing either etd or hcd, respectively, using a defined synthetic phosphopeptide mixture and also using a larger data set of ti4 + -imac enriched phosphopeptides from a tryptic human cell line digest. in combination with a modified version of phosphors, we observed that in the majority of cases ethcd generated richer and more confidently identified spectra, resulting in superior phosphosite localization scores. our data demonstrates the distinctive potential of ethcd for ptm localization, also beyond protein phosphorylation. |
severe acute respiratory syndrome (sars) is a new emerging infectious disease, which was first reported in china in 2002 and was rapidly spreading all over the world in 2003 [1, 2 ]. patients develop persistent fever, dry cough, progressive radiographic changes of chest, and lymphopenia once infected. despite treatment, about 1015% of the patients would die due to the acute respiratory distress [36 ]. a novel coronavirus (sars - cov) was isolated from sars patients [79 ]. sars - cov is a positive - stranded rna virus with an envelop. comparative genomic studies using the in silico annotated proteins have suggested that sars virus belongs to a new group of coronavirus. according to the genomic sequence of sars - cov, it is predicted that there are several structural proteins can be produced by sars - cov including spike (s), envelop (e), membrane (m), and nucleocapsid. spike protein is very important in the binding and fusion of coronavirus to the host cells [11, 12 ]. the s protein of sars - cov has 1255 amino acids in length and 23 potential n - linked glycosylation sites. the amino terminus of the sars - cov s protein contains a short type 1 signal sequence composed of hydrophobic amino acids that are presumably removed during cotranslational transport through the endoplasmic reticulum. the carboxyl terminus consists of a transmembrane domain and a cytoplasmic tail rich in cysteine residues. the majority of protein (residues 121195) is outside the virus particle, which can be divided into amino - terminal s1 and carboxyl - terminal s2 domain. the s1 domain (residues 12672) binds to the host cell receptor, angiotensin - converting enzyme 2 (ace2), while the s2 domain is responsible for membrane fusion [1315 ]. monoclonal antibodies against s1 domain can block the receptor binding and contain potent neutralization activity against sars - cov. however, peptides derived from s2 domain can also inhibit sars - cov infection. molecular mimicry, which is defined as similar structures shared by molecules from dissimilar genes or by their protein products, is a general strategy for pathogens to infect host cells and has been proposed as a pathogenic mechanism for autoimmune disease. therefore, identification of the molecular mimic regions of pathogen may be helpful to understand the disease induced by that pathogen. at present, it is unclear whether molecular mimicry occurs between sars - cov s proteins and human peptides. we have approached this question using computer to analyze the sequence of spike protein of sars - cov and select regions that share the sequence homology with human proteins. the criteria for the selection of potential regions include antigenic analysis and surface accessibility. in this study, we find that several regions of the s protein share sequence homology with human proteins. synthetic peptides, which represent some of these regions, were synthesized to understand their roles in sars - cov infection. publically available human and coronavirus genome sequences at the national center for biotechnology information (md, usa) were used for in silicoprediction. algorithms predicting immunogenicity, second structure prediction, protein topology analysis, and hydrophobicity were conducted to design the tested peptides. immunogenic viral peptides were calculated based on the algorithm developed by kolaskar and tongaonkar. the algorithm is based on a table constructed from the occurrence of amino acid residues in experimentally known antigenic epitopes. secondary structural analyses of spike protein were performed based on phd and predator algorithms. similarity searches between s protein and human genome database were performed by using blastp with blosum 62. extra amino acid residues were added at either n- or c - terminus to keep the hydrophobic amino acid content below 50%. peptides with high hydrophobicity are difficult to be tested in biochemical experiments since most of in vitro assays are conducted in aqueous buffers. also, on average, one charged residue is added for every five amino acids. multiple antigen peptides were synthesized by cytomol corp (mountain view, calif, usa). in addition, bradykinin and angiotensin i (ang i) were purchased from sigma (st. louis, mo, usa). sars patient sera were collected by the center for disease control, department of health (taipie, taiwan) from march to june, 2003. diagnosis of sars was based on the clinicalcriteria established by the world health organization (who). patients with sars - cov were confirmed by laboratory methods, including viral antigen detection, rt - pcr, and serologic methods. ten sars patient sera collected at the convalescent stage (20 days after disease onset) were included in this study. antibodies against peptides in human sera were detected by solid - phase capture technique using individual peptide - coated plates. elisa plate was coated with or without 50 l peptides (100 g / ml) per well and blocked by 1% bovine serum albumin (bsa) in 0.05% tween-20 in phosphate - buffered saline (pbs) for 1 hour at room temperature. test serum samples were 1:100 diluted and added to the plate for 2 hours at room temperature. after incubation, the elisa plate was washed with 0.05% tween-20 in pbs for three times. the bound antibodies were detected by horseradish peroxidase- (hrp-) conjugated antihuman immunoglobulin antibodies (sigma aldrich, st. louis, mo, usa) and peroxidase substrate, tmb (promega, madison, wiss, usa). the absorbance was measured using the vmax microplate reader (molecular devices corporation, sunnyvale, calif, usa) at 450 nm. antibodies against peptides in mouse sera were assayed by elisa as in human sera except hrp - conjugated antimouse immunoglobulin antibodies (sigma aldrich) was used to detect bound antibodies. human lung adenocarcinoma cell line a549 and vero cells were grown in dmem supplemented with 10% heat - inactivated fcs, 2 mm l - glutamate, and 50 ng / ml gentamycin. cells were incubated in co2 incubator at 37c with 5% co2 in a humidified atmosphere. for immunofluorescent microscopy observation, six- to eight - week - old female balb / c mice were used in this study. these mice were originally purchased from jackson laboratory (bar harbor, me, usa) and bred in the laboratory animal center, national cheng kung university (tainan, taiwan). synthetic peptides (1 mg / ml) were emulsified with complete freund 's adjuvant and injected intraperitoneally into balb / c mice. mice were boosted with the same peptide in pbs (50 g / mouse) intraperitoneally two weeks after priming. sera were collected from the axially plexus of the mice at different time intervals and tested for the presence of antibody against peptides by elisa as mentioned above. significant increase of antibody titer (greater than 4 folds) against immunized - peptide was found in mouse hyperimmune sera as compared to normal sera after boosting. mouse hyperimmune sera against peptide d08 were incubated with a549 cells at 4c for 1 hour. after washing three times with pbs, cells were incubated with 1 ml of 1 g / ml fitc - conjugated antimouse igg (jackson immunoresearch laboratories inc., west grove, pa, usa) at 4c for 1 hour and washed again with pbs. proteins in the cell lysate of a549 were separated by 12% sds - page and transferred to nitrocellulose sheets as described previously. proteins recognized by normal or peptide d08 hyperimmune mice sera were detected using hrp - conjugated antimouse immunoglobulin antibodies (sigma aldrich) and substrate. vero e6 (4104) and a549 cells (5103) were incubated with different doses of synthetic peptides as indicated for 72 hours. cell proliferation was detected using commercial xtt assay (roche diagnostics, indianapolis, ind, usa). the il-8 production was assessed by commercial elisa kits (r&d systems, minneapolis, minn, usa) according to the manufacturer 's instructions. briefly, a549 cells (1105) were cultured alone or with different doses of peptides for 48 hours. culture supernatants were collected after incubation, added to precoated elisa plates, and incubated for 2 hours at 37c. the developed color was read by the vmax microplate reader (molecular devices, calif, usa). the levels of significance for the differences between groups were analyzed using student 's t - test. a value of p <.05 was considered to be significant. the whole amino acid sequence of spike protein was analyzed to find out the potential immunogenic regions and the regions shared sequence homology with human proteins, which is defined as the pathogenic regions. region 1 (residues 199254), region 2 (residues 658715), region 3 (residues 893951), and region 4 (residues 11271184) have shared sequence homology with hydroxyacid oxidase, human golgi autoantigen, angrgm-52, and pallidin, respectively. among these regions, region 3 with homology to human angrgm-52 has the highest score (with 34% identities and 48% similarity of conservative substitutions). in addition, because des - arg bradykinin and ang i are the substrates for ace2, we also compared the sequence of s protein against bradykinin (rppgfspfr) and ang i (drvyihpfhl) and found that residues 490502 (gyqpyrvvvlsfee) of s protein showed sequence homology with bradykinin as indicated by bold letters here and in figure 2(b). the identity score is 27% ; and the similarity score from conservative substitutions is 36%. eleven peptides (d01d11, see table 1), which represent those pathogenic regions were synthesized as well as bradykinin and ang i were tested to see whether those peptides can be recognized by sars patients ' sera. the peptides were designed based on the algorithms predicting immunogenicity, second structure, protein topology, and hydrophobicity. our goal is to select for peptides with high immunogenicity, with location on the protein surface, and with low hydrophobicity. the designed peptide sequences were synthesized and tested with clinical samples of sars patient sera. a significant increase of sars patients ' sera binding to peptide d01, d07, and d08 was found as compared to the binding of normal sera (see figure 3). to test whether synthetic peptides d01, d07, and d08 can induce antibodies crossreacted with human proteins, we immunized mice with these peptides to generate hyperimmune sera against these peptides. we found hyperimmune sera against d08 can bind to the cytoplasmic region of human a549 cells as demonstrated by immunofluorescent stain (see figure 4). using western blot analysis, hyperimmune sera against d08 could recognize more bands in a549 cell lysate as compared to normal mice sera (see figure 5). in addition, hyperimmune sera against d07, but not d01, showed similar crossreactivity to a549 cells as hyperimmune sera against d08 did (data not shown). to test whether synthetic peptides d10, indeed, can induce antibodies crossreactive with bradykinin and ang i, we immunized mice with d10 peptides to generate hyperimmune sera against this peptide. significant increase of antibodies against d10 was found in d10 hyperimmune sera, which could crossreact with bradykinin, but not with ang i - coated plates (see figure 6). to understand whether d10 has similar biological activity as ang i, we incubated vero cells and lung epithelial a549 cells with d10, ang 1, or control peptide d11. both vero and a549 cells were induced to proliferate in the presence of d10 and ang i but not the control peptide (see figure 7). in addition, d10 and ang i could also induce chemokine il-8 production of a549 cells (see figure 8). in this study, we have identified four pathogenic regions of sars - cov s protein which share sequence homology with different human proteins. among them, pathogenic region 3 (residues 893941), which shares sequence homology with angrgm-52 (genbank accession no. aal62340), a novel gene upregulated in human mesangial cells stimulated by angiotensin ii, may deserve further investigation. peptides d07 and d08 of this region were recognized by the sera of sars patient indicating that this region is immunogenic and can be recognized by the immune system during sars - cov infection. murine hyperimmune sera against peptides d07 or d08 were able to bind to recombinant s2 but not s1 domain of s protein (data not shown). in addition, hyperimmune sera against d07 or d08 also bounded to the cytoplasmic region of a549 cells and recognized several proteins in the a549 cell lysate. these results indicate that regions represented by d07 and d08 are immunogenic and may induce autoantibodies. however, further study is required to understand the biological function of these regions and the role of their antibodies in the pathogenesis of sars - cov infection. in addition to d07 and d08 peptides, we also noticed that d10 peptide which represents residues 490502 of s1 domain contained some interesting activities. the d10 peptide, which shared sequence homology with bradykinin, was able to generate antibodies crossreactive with bradykinin. in addition, d10 peptide could stimulate a549 to produce il-8 and proliferation as ang i did. these results suggest that the region of d10 in s protein may bind to ang i receptor, ace2, and may be involved in the binding of sars - cov to ace2. this is consistent with the previous report, which indicates that residues 318510 of s1 domain can bind to ace2 and is similar to the receptor binding domain of the hcov-229e, which is within a fragment containing residues 407 to 547. therefore, region 490502 of s1 domain may be involved in the receptor binding domain of sars - cov. in summary, our results suggest that molecular mimicry occurs between sars - cov and host proteins. motifs shared sequence homology with host proteins of sars - cov may be involved in the binding and fusion of sars - cov to host cells. antibody against these motifs may contain neutralization activity against sars - cov infection or participate in the immunopathogenesis induced by sars - cov. as reported previously, sars - cov, like influenza, can inhibit the host 's corticosteroid stress response via a molecular mimicry strategy. our studies on the mimicry motifs of s protein, which is involved in the virus, entry may provide alternative approaches to disrupt the infection of sars - cov, similar to the previous studies on the virus entry [28, 29 ]. | molecular mimicry, defined as similar structures shared by molecules from dissimilar genes or proteins, is a general strategy used by pathogens to infect host cells. severe acute respiratory syndrome (sars) is a new human respiratory infectious disease caused by sars coronavirus (sars - cov). the spike (s) protein of sars - cov plays an important role in the virus entry into a cell. in this study, eleven synthetic peptides from the s protein were selected based on its sequence homology with human proteins. two of the peptides d07 (residues 927937) and d08 (residues 942951) were recognized by the sera of sars patients. murine hyperimmune sera against these peptides bound to proteins of human lung epithelial cells a549. another peptide d10 (residues 490502) stimulated a549 to proliferate and secrete il-8. the present results suggest that the selected s protein regions, which share sequence homology with human proteins, may play important roles in sars - cov infection. |
simultaneous cardiac and pericardial rupture secondary to blunt chest trauma is a highly lethal combination with a rarely reported survival. we present a young patient with a right atrioventricular groove injury, pericardial rupture and a unique and previously undescribed coronary sinus avulsion. a 17-year - old male presented to our hospital after a high - speed motor vehicle crash that required prolonged extrication followed by rapid - sequence intubation. he required one unit of packed red blood cells for hypotension. on arrival to the trauma bay, he was normotensive with a heart rate of 70 beats / min. a focused abdominal sonogram for trauma (fast) was negative for fluid in the abdominal and cardiac windows. full - body computed tomography (ct) scan revealed a retained left hemothorax and subtle irregularity of the proximal descending aorta concerning for aortic disruption [figure 1 ]. following imaging, cardiothoracic surgery was called to the operating room due to concern for aortic disruption. upon exploring the left chest through a thoracotomy the pericardium was traumatically ruptured, but the avulsed and skeletonized left phrenic nerve was intact [figure 2 ]. chest computed tomography scan image showing aortic irregularity (arrow) and retained left hemothorax traumatic rupture of the pericardium with exposed left phrenic nerve (arrow) control of bleeding was attempted by placing a large sheet of surgifoam along the atrioventricular groove at the base of the heart. shortly thereafter, the heart distended and the patient arrested requiring emergent cardiopulmonary bypass achieved by cannulating the left apex and right atrium. further, inspection revealed avulsion tears of the coronary sinus extending to the inferior vena cava. there was a separate avulsion of the right atrial appendage near the right coronary artery. post - operatively the patient recovered without further cardiac events and has been discharged from rehabilitation. autopsy studies show that cardiac injury in up to 32% of blunt trauma deaths with 64% of those injuries being cardiac rupture. reported only twenty - two pericardial ruptures out of over 20,000 blunt trauma admissions with a mortality rate of 63.6%. in the setting of cardiac rupture, cardiac rupture carries a high mortality rate of 50 - 86% and when combined with pericardial rupture, the mortality is nearly 100%. the clinical signs of these combined injuries are often subtle, but rapid diagnosis is paramount as exsanguinating hemorrhage can quickly ensue. isolated cardiac rupture often presents with cardiac tamponade, but with simultaneous pericardial rupture, the pericardium is decompressed preventing these classic signs. ultrasonography and ct scan can be misleading since pericardial fluid may be absent. in our case, fast revealed no pericardial fluid and chest ct led to a misguided concern for aortic injury. may. proposed an algorithm to aid in an early diagnosis of combined pericardial and cardiac rupture. ct scan and/or pericardial window with lavage are indicated for patients with no pericardial fluid on fast with otherwise unexplained hypotension or enlarging mediastinal hematoma. if no blood is initially identified in the pericardial window, pericardial lavage is performed by instilling 100 - 200 ml of saline into the pericardial sac., median sternotomy is preferred allowing for excellent mediastinal exposure and potential placement on cardiopulmonary bypass or extension to laparotomy. we selected a posterolateral thoracotomy due to concern for an aortic injury since the remainder of the ct was unremarkable. historically, a cardiopulmonary bypass is rarely required and is reported in only 10% of cardiac repairs for blunt trauma ; however, in our case the patient arrested in the operating room requiring emergent cardiopulmonary bypass. for cardiac repair, we report a rare survival of combined pericardial and cardiac rupture and a unique coronary sinus avulsion following blunt chest trauma. in addition, we propose an algorithm for its diagnosis and management [figure 3 ]. traditional diagnostics such as fast and ct are often unhelpful due to the unique combination of injuries repair of this spectrum of injuries requires early involvement of cardiac surgery expertise and possibly placement of patient on cardiopulmonary bypass. | simultaneous cardiac and pericardial rupture from blunt chest trauma is a highly lethal combination with rarely reported survival. we report of a case of young patient with a right atrioventricular groove injury, pericardial rupture and a unique description of a coronary sinus avulsion following blunt chest trauma. rapid recognition of this injury is crucial to patient survival, but traditional diagnostic adjuncts such as ultrasound, echocardiography and computed tomography are often unhelpful. successful repair of these injuries requires high suspicion of injury, early cardiac surgery involvement of and possible even placement of the patient on cardiopulmonary bypass. |
unlike the structure of somatic cells, which are relatively free of chromatin (dna and histones), sperm chromatin is compact and stable in the nucleus. the nuclear condensation in sperm cells is due to the replacement of about 85% of lysine - rich histones associated with dna, with transition proteins that are rich in arginine and protamine. contrary to histones, which form a ring with dna (nucleosomes), protamines are linked to the grooves of the dna helix, wrapping tightly around the strands of dna (approximately 50 kb of dna for protamine), to form tight and highly organized loops. moreover, inter- and intramolecular disulfide bonds between the cysteine - rich protamines are also responsible for the compaction and stabilization of the sperm nucleus. the result of these specific associations is an extreme nuclear condensation with the reduction of approximately 10% of the nucleus size. the key protein that mediates the chromatin compaction is the brdt (bromo domain testisspecific) protein which is able to promote nuclear remodeling, thus ensuring the transition between the histone chromatin organization, or somatic, and the protaminic nucleus, typical of mature sperm. in fact, physiological and environmental stress, as well as genetic mutations and chromosomal abnormalities may interfere with the mechanisms of spermatogenesis. these changes may lead to abnormal chromatin structure that is incompatible with fertility [figure 1 ]. the defects of genomic material found in mature sperm may impair packaging (defective histone and protamine replacement) and maturation of the nucleus, leading to dna fragmentation (i.e. single - or double - strand breaks) and defects of dna integrity or chromosomal sperm aneuploidy. causes of sperm oxidative stress in the case of atypical and immature sperm, the dna can fragment and lose its functional integrity, and thus lead to functional defects of the sperm. the p53 molecule, so called because of its molecular mass is a sequence - specific transcription factor that responds to a wide range of stress signals and cellular functions, acting as a coordinator of cell destiny. it is closely involved in the cellular response to dna damage, by controlling cell cycle arrest, apoptosis and the transcription of genes induced by dna damage. under normal conditions, the level of p53 protein is low in most cells of the body. however, when normal cells are deprived of oxygen or exposed to dna damaging treatments, such as uv light or gamma rays, there is an increase in the concentration of p53 via a reduction of the rapid degradation process of the molecule. it is not surprising that p53 the guardian of the genome seems to play a role in spermatogenesis. it has been suggested, in fact, that the role of the p53 ancestral gene was to ensure the integrity of the genomic germline and the fidelity of the development processes. it is possible that p53 plays different roles in dna repair, depending on the type of damage, on the phase in which the cell has been damaged and on the possible recovery ways available. in brief the aim of this study was to analyze and compare the p53 values (dna damage marker) measured by elisa assay and the sdfi (sperm dna fragmentation index) values obtained by the acridine orange (ao) test, in human sperm samples of seminal fluid from men with normal and pathological parameters. the ultimate goal of this work was to verify the usefulness of the p53 measurement as an objective and repetitive method in the evaluation of sperm dna compared to the current laboratory techniques. the elisa method could, within the medically assisted procreation, identify subjects with low fertility and be predictive on the outcome of in vitro fertilization. a total of 103 samples of human semen were examined in the period from january 2011 to june 2012. subjects were aged between 27 - 35 years, and the ejaculate volume varied from 0.7 to 5.4 ml. the samples were divided according to their diagnostic evaluations in : normospermia, 44/103 (42.7%) ; mild oligospermia, 10/103 (9.7%) ; medium oligospermia, 23/103 (22.3%) ; severe oligospermia, 26/103 (25.2%). the subjects examined did not have chronic diseases, they had not used medicines or drugs in the last 3 months prior to the collection of semen, their work did not involve exposure to environmental stresses and the preliminary doppler results did not indicate any symptoms caused by pathological varicocele. the subjects signed the informed consent form for the processing of personal and sensitive data, as well as of genetic and biological samples collection in compliance with the applicable laws. the procedures followed were in accordance with the ethical standards of experimentation (institutional or regional) and with the helsinki declaration of 1975, as revised in 2000. a makler chamber (makler counting chamber, sefi - medical instruments ltd, haifa, israel) was used for the evaluation of sperm concentration, the total number of spermatozoa and for the study of the cellular component of non - sperm cells (leukocytes, erythrocytes, and germ line cells). the samples were then aliquoted into two parts one of which was immediately analyzed and the other frozen at 20c for future investigations. the acridine orange test allows the calculation of the percentage of mature spermatozoa containing the normal and double - stranded native dna, using the acridine orange dye and by observation with fluorescence microscopy. it is based on the principle that dna possesses a different susceptibility to partial denaturation induced by heat shock or by contact with an acidic solution. the test utilizes the metachromatic properties of acridine orange, a specific fluorochrome for nucleic acids that, when binding to the dna double helix (the native form) emits green fluorescence, whilst when binding to single - stranded dna (denatured form) emits red fluorescence. the staining protocol was applied to each sample after 30 min from the liquefaction of semen at 37c. the coloration of acridine orange was performed according to the tejada and coll. method. the nuclei of at least 300 spermatozoa from each individual were examined and classified as fluorescent green, red, orange, or yellow. spermatozoa with green / yellow fluorescence were considered as having intact dna, while those showing red / orange fluorescence were considered as having damaged dna, like those showing green and red simultaneously [figure 2 ]. the threshold for green fluorescence was set at 75%, and < 25% values were considered positive in this test. fluorescence was reported as sdfi, which expresses the percentage of sperm with fragmented dna (single - strand red fluorescence) over the total sperm counted (with single and double strand dna, red fluorescence and green) to perform the isolation of sperm from semen, the differex system for use with the differex magnet, and the dna iq system - small sample casework protocol kits were used (promega corporation, madison wi, usa). in 1985 gill and coll. developed a method to separate spermatozoa from epithelial cells in a sample of human semen. it requires about 150 min to obtain the complete separation and purification of the sperm dna. the number of sample and reagents quoted has been calculated for a single sample and a single experiment in the following protocol. one hundred microliters of sample were placed in a 1.5 ml tube with 400 l of digestion solution containing 6 microliters of diluted proteinase k and 364 microliters of digestion buffer. the tube was vortexed for 30 s at 14,000 rpm, incubated for 90 min at 56c and then centrifuged for 10 min at 14,000 rpm in a microcentrifuge at room temperature, not before having marked the position where the pellet would form. then 3.5 l of dna iq resin were added in the opposite position of the pellet and the tube was placed on differex magnet so that the resin, attracted by the magnet, would coat the pellets. the yellow liquid layer, containing epithelial cells, was then removed. the sample was then washed three times with 500 l of nuclease free water and the last washing volume was not removed. the tube was centrifuged again at 14,000 rpm for 10 min and 3.5 l of dna iq resin were added in a position opposite the pellet and positioned in differex magnet so that the resin would coat the pellets. after three washes, a further 500 l of nuclease free water together with 100l of separation solution were added, so that the resin would coat the pellets. the washing and separation solutions were then removed and the pellet was resuspended by adding 400 microliters of 0.9% sodium chloride solution. three hundred microliters of the solution thus obtained were aliquoted (no lysis sample). to extract the sperm dna, 250 microliters of lysis solution (containing 2.5 microliters of dtt and 252.5 l of lysis buffer were added to the tube, which was then vortexed for 3 sec at high speed and incubated for 5 min at room temperature. after having vortexed again for 3 s, the tube was positioned in the differex magnet so that the separation would occurs instantaneously and the supernatant was then removed and stored in another tube (lysed sample). then 100 l of lysis solution were added and the tube was removed from the differex magnet. after vortexing for 2 s, the tube was put back in differex magnet and the entire lysis solution eliminated. to perform the washing 100 l of wash buffer were added after removing the tube from the differex magnet and the sample was vortexed for 2 s at high speed. once the tube was repositioned in differex magnet, the entire wash solution (containing 500l of wash buffer, 250 l of isopropyl alcohol and 250 l of ethanol) was eliminated. the resin was allowed to air dry for 5 min, leaving the tube with the cap open. subsequently, 100 l of elution buffer were added (10 mm tris ph 8.0 and 0.1 mm edta), and the tube was vortexed for 2 s. incubated for 5 min at 65c vortexed again and placed immediately on differex magnet. the solution containing the dna was carefully transferred in a new tube (lysed sample and refined dna). a direct and quantitative elisa assay was used to measure p53 (duoset ic, human total p53 r and d systems inc. briefly, 100 l of the capture antibody, appropriately diluted, were pipetted into each well of a 96 well microplate, which was then sealed and incubated overnight at room temperature. the next day the plate was washed three times with 400 l wash buffer (0.05% tween 20 in pbs, ph 7.2 - 7.4, filtered at 0.2 l). each well was blocked with the addition of 300 l of stop solution (sample diluent concentrate : 5x pbs, 5 mm edta, and 2.5% triton x-100). the standards were prepared by diluting in ic diluent # 4 (1 mm edta, 0.5% triton x-100 in pbs, ph 7.2 - 7.4.) and using ic diluent # 4 like standard zero. then 100 microliters of sample or standard were added (no lysis, lysed, and lysed and purified dna cell preparations), the plate was sealed and incubated for 2 h at room temperature. after the incubation, extraction and washing were repeated and 100 l of detection antibody (total p53 detection antibody), appropriately diluted, were pipetted in each well. the plate was sealed and incubated for 2 h at room temperature. after the incubation, extraction and washing were repeated. one hundred microliters of streptavidin - hrp were then added and the plate was incubated for 20 min at room temperature. after the incubation, extraction and washing steps were repeated and 100 l of substrate solution (1:1 mixture of reagent a and reagent b) were added to each well and the plate was incubated for 20 min at room temperature. the optical density was analysed using a microplate reader set at 450 nm with a software that automatically calculated the concentrations expressed in pg/100 microliters. (a dimensionless index ranging between 1,0 and + 1,0, which reflects the extent of a linear relationship between two data sets) was used for the statistical analysis of the groups. x was used to perform a hypothesis test on the correlation coefficient. finally, for the development of probability and data significance, the student 's t test was used. these statistical calculations were performed using microsoft excel 2008 (microsoft corporation, redmond wa, usa). a total of 103 samples of human semen were examined in the period from january 2011 to june 2012. subjects were aged between 27 - 35 years, and the ejaculate volume varied from 0.7 to 5.4 ml. the samples were divided according to their diagnostic evaluations in : normospermia, 44/103 (42.7%) ; mild oligospermia, 10/103 (9.7%) ; medium oligospermia, 23/103 (22.3%) ; severe oligospermia, 26/103 (25.2%). the subjects examined did not have chronic diseases, they had not used medicines or drugs in the last 3 months prior to the collection of semen, their work did not involve exposure to environmental stresses and the preliminary doppler results did not indicate any symptoms caused by pathological varicocele. the subjects signed the informed consent form for the processing of personal and sensitive data, as well as of genetic and biological samples collection in compliance with the applicable laws. the procedures followed were in accordance with the ethical standards of experimentation (institutional or regional) and with the helsinki declaration of 1975, as revised in 2000. a makler chamber (makler counting chamber, sefi - medical instruments ltd, haifa, israel) was used for the evaluation of sperm concentration, the total number of spermatozoa and for the study of the cellular component of non - sperm cells (leukocytes, erythrocytes, and germ line cells). the samples were then aliquoted into two parts one of which was immediately analyzed and the other frozen at 20c for future investigations. the acridine orange test allows the calculation of the percentage of mature spermatozoa containing the normal and double - stranded native dna, using the acridine orange dye and by observation with fluorescence microscopy. it is based on the principle that dna possesses a different susceptibility to partial denaturation induced by heat shock or by contact with an acidic solution. the test utilizes the metachromatic properties of acridine orange, a specific fluorochrome for nucleic acids that, when binding to the dna double helix (the native form) emits green fluorescence, whilst when binding to single - stranded dna (denatured form) emits red fluorescence. the staining protocol was applied to each sample after 30 min from the liquefaction of semen at 37c. the coloration of acridine orange was performed according to the tejada and coll. method. the nuclei of at least 300 spermatozoa from each individual were examined and classified as fluorescent green, red, orange, or yellow. spermatozoa with green / yellow fluorescence were considered as having intact dna, while those showing red / orange fluorescence were considered as having damaged dna, like those showing green and red simultaneously [figure 2 ]. the threshold for green fluorescence was set at 75%, and < 25% values were considered positive in this test. fluorescence was reported as sdfi, which expresses the percentage of sperm with fragmented dna (single - strand red fluorescence) over the total sperm counted (with single and double strand dna, red fluorescence and green) to perform the isolation of sperm from semen, the differex system for use with the differex magnet, and the dna iq system - small sample casework protocol kits were used (promega corporation, madison wi, usa). in 1985 gill and coll. developed a method to separate spermatozoa from epithelial cells in a sample of human semen. it requires about 150 min to obtain the complete separation and purification of the sperm dna. the number of sample and reagents quoted has been calculated for a single sample and a single experiment in the following protocol. one hundred microliters of sample were placed in a 1.5 ml tube with 400 l of digestion solution containing 6 microliters of diluted proteinase k and 364 microliters of digestion buffer. the tube was vortexed for 30 s at 14,000 rpm, incubated for 90 min at 56c and then centrifuged for 10 min at 14,000 rpm in a microcentrifuge at room temperature, not before having marked the position where the pellet would form. then 3.5 l of dna iq resin were added in the opposite position of the pellet and the tube was placed on differex magnet so that the resin, attracted by the magnet, would coat the pellets. the yellow liquid layer, containing epithelial cells, the sample was then washed three times with 500 l of nuclease free water and the last washing volume was not removed. the tube was centrifuged again at 14,000 rpm for 10 min and 3.5 l of dna iq resin were added in a position opposite the pellet and positioned in differex magnet so that the resin would coat the pellets. after three washes, a further 500 l of nuclease free water together with 100l of separation solution were added, so that the resin would coat the pellets. the washing and separation solutions were then removed and the pellet was resuspended by adding 400 microliters of 0.9% sodium chloride solution. three hundred microliters of the solution thus obtained were aliquoted (no lysis sample). to extract the sperm dna, 250 microliters of lysis solution (containing 2.5 microliters of dtt and 252.5 l of lysis buffer were added to the tube, which was then vortexed for 3 sec at high speed and incubated for 5 min at room temperature. after having vortexed again for 3 s, the tube was positioned in the differex magnet so that the separation would occurs instantaneously and the supernatant was then removed and stored in another tube (lysed sample). then 100 l of lysis solution were added and the tube was removed from the differex magnet. after vortexing for 2 s, the tube was put back in differex magnet and the entire lysis solution eliminated. to perform the washing 100 l of wash buffer were added after removing the tube from the differex magnet and the sample was vortexed for 2 s at high speed. once the tube was repositioned in differex magnet, the entire wash solution (containing 500l of wash buffer, 250 l of isopropyl alcohol and 250 l of ethanol) was eliminated. the resin was allowed to air dry for 5 min, leaving the tube with the cap open. subsequently, 100 l of elution buffer were added (10 mm tris ph 8.0 and 0.1 mm edta), and the tube was vortexed for 2 s. incubated for 5 min at 65c vortexed again and placed immediately on differex magnet. the solution containing the dna was carefully transferred in a new tube (lysed sample and refined dna). a direct and quantitative elisa assay was used to measure p53 (duoset ic, human total p53 r and d systems inc. briefly, 100 l of the capture antibody, appropriately diluted, were pipetted into each well of a 96 well microplate, which was then sealed and incubated overnight at room temperature. the next day the plate was washed three times with 400 l wash buffer (0.05% tween 20 in pbs, ph 7.2 - 7.4, filtered at 0.2 l). each well was blocked with the addition of 300 l of stop solution (sample diluent concentrate : 5x pbs, 5 mm edta, and 2.5% triton x-100). the standards were prepared by diluting in ic diluent # 4 (1 mm edta, 0.5% triton x-100 in pbs, ph 7.2 - 7.4.) and using ic diluent # 4 like standard zero. then 100 microliters of sample or standard were added (no lysis, lysed, and lysed and purified dna cell preparations), the plate was sealed and incubated for 2 h at room temperature. after the incubation, extraction and washing were repeated and 100 l of detection antibody (total p53 detection antibody), appropriately diluted, were pipetted in each well. the plate was sealed and incubated for 2 h at room temperature. after the incubation, extraction and washing were repeated. one hundred microliters of streptavidin - hrp were then added and the plate was incubated for 20 min at room temperature. after the incubation, extraction and washing steps were repeated and 100 l of substrate solution (1:1 mixture of reagent a and reagent b) were added to each well and the plate was incubated for 20 min at room temperature. the optical density was analysed using a microplate reader set at 450 nm with a software that automatically calculated the concentrations expressed in pg/100 microliters. pearson correlation coefficient r (a dimensionless index ranging between 1,0 and + 1,0, which reflects the extent of a linear relationship between two data sets) was used for the statistical analysis of the groups. x was used to perform a hypothesis test on the correlation coefficient. finally, for the development of probability and data significance, the student 's t test was used. these statistical calculations were performed using microsoft excel 2008 (microsoft corporation, redmond wa, usa). the sdfi values obtained in the ao tests were reinterpreted, as the sdfi is a parameter which alone has a limited prognostic value for in vivo procreation, except when correlated to sperm counts. in fact, a high sdfi value (pathological) and a high sperm count do not indicate that the subject is potentially infertile. likewise, a low sdfi (normal) and a low sperm count do not indicate potentially fertile subjects. this is why it was considered appropriate to evaluate the sdfi / tsc value for each sample instead of the simple sdfi, were tsc (total sperm count) is the result of the ejaculate volume multiplied by the sperm count / ml. the p53 values were also corrected (corrected p53), because the dosage of p53 was performed on 100 microliters of sample. the p53 values were therefore related to 1/10 of the value of sperm counts and the corrected p53 is a reliable estimate calculated for 1 ml of semen. the value of p53 was measured in three different cell preparations (no lysis, lysed, and lysed and purified dna) for each sample. given the physiological localization of p53 on the dna, there is a considerable difference of concentration in the 3 cell preparations. to make data interpretation more visible, the sdfi / tsc were plotted in ascending order, while the p53 values were correlated to the sample location associated with the sdfi / tsc value. for all semen samples, we correlated the corrected p53 values and sdfi / tsc in the three different cell preparations (no lysis, lysed and lysed and purified dna). this showed that the cell preparations lysed and purified dna has the best correlation among the parameters (r = 0.964, p < 0.025) [figure 3 ]. in the case of atypical sperm, whether immature or subject to external stress, the dna can fragment and lose its functional integrity. in addition, several studies have shown that the p53 molecule is involved in maintaining the integrity of the dna sperm. today, the ao test is one of the most popular methods used to verify the integrity of sperm dna. however, it is not an objective methodology as it is linked to the individual operator 's experience. in order to achieve greater objectivity, it is advisable to use an alternative method. in this study we analyzed 103 samples of seminal fluid with both the ao test and the elisa methodology for the quantitative p53 assay. the evaluation of the data shows a correlation between sdfi / tsc and corrected p53. we therefore suggest that the p53 elisa method for assessing sperm dna damage may be a viable alternative to the ao test and all interpretation tests of dna sperm integrity currently used by laboratories. in fact, this method looks promising for routine use in the laboratory, as it enables to analyse multiple samples simultaneously, ensuring greater precision, repeatability and accuracy [figure 4 ]. although our findings need to be confirmed by further studies, the corrected p53 value could be really useful in the assessment of dna sperm damage. the test could represent a powerful tool in order to prove the efficacy of treatment protocols, in view of a medically assisted procreation and as a predictive test for procreation in vivo. | background : sperm dna integrity is considered an important parameter to assess seminal fluid quality and can be used as a predictive test of potential fertility. amongst the various tests to determine sperm dna integrity, one is the acridine orange test. recent studies have demonstrated the importance of p53 in maintaining sperm dna integrity. the aim of this study was to assess if a p53 elisa assay could be a new indicator of dna damage in human spermatozoa.materials and methods:103 human semen samples were evaluated using both acridine orange test and p53 elisa and results were compared.results:a clear correlation between the values measured by two methods was obtained.conclusions:if this hypothesis will be confirmed by further studies, the p53 elisa assay could become a new and more precise indicator of dna damage in human spermatozoa. |
the bureau of alcohol, tobacco, firearms and explosives (atf), a law enforcement agency within the united states department of justice, has been in the business of investigating the cause and origin of fires and explosions since the 1970s. atf typically collaborates with other federal, state, and local authorities to provide this service through several venues. at the local level, criminal investigators with atf, especially ones holding special designations as certified fire investigators (cfis), work side - by - side with their state and local counterparts to assist with investigation of post - fire and post - blast scenes. these individuals may also serve on city - based federal arson task forces, in partnership with other federal, state, and local investigators, to address acute arson problems. at the national level, atf maintains a well - trained national response team (nrt) which provides comprehensive response to assist these other jurisdictions with onsite investigation of major arson and bombing incidents. the nrt is a multispecialty team comprised of criminal investigators with post - fire and post - blast investigation expertise, explosives enforcement specialists, forensic chemists, fire protection engineers, and other technical experts. atf established the nrt program in 1978 and subsequently instituted a program for certification of criminal investigators as fire investigators (cfis) in 1986. in 1995, atf commenced a voluntary medical surveillance program for members of the nrt to monitor the health of participants working in the potentially hazardous environment of the post - fire / post - blast scene. the agency extended the voluntary program to all cfis in 1997. by 2000, three white male participants of the program had reported diagnoses of bladder cancer between the years 1994 and 1999. this information raised concern that a bladder cancer cluster was occurring among scene investigators. in response to this concern, in 2002 atf mandated participation in the medical surveillance program for all nrt members, all explosives enforcement specialists, and all criminal investigators, including those not involved in post - fire / post - blast investigations. to facilitate future epidemiologic evaluation of the significance of the apparent cancer cluster, atf also committed medical surveillance data entry into an electronic database. by 2006, four additional white male program participants had reported diagnoses of bladder cancer between 2001 and 2005. combined, the seven reported cases ranged in age from 32 to 53 in the year of diagnosis. from a job title perspective, all cases were among criminal investigators, who comprised 96% of the employees participating in the surveillance program. in addition, six of the seven cases reported working post - fire and post - blast scenes while employed with atf. none of the seven cases reported work histories which involved investigation of such scenes prior to their employment with atf. first, was this group of employees, predominantly criminal investigators, experiencing a greater than expected incidence of bladder cancer from a demographic perspective ? second, was post - fire / post - blast scene investigation associated with increased risk for bladder cancer ? part 1 of this study addresses the question on cancer incidence and part 2 addresses the question on cancer risk associated with investigation of these scenes. analyses for both parts use data collected through the medical surveillance program. in the united states, bladder cancer is expected to be the sixth most commonly occurring cancer, excluding basal and squamous cell skin cancers, in 2012. as reported by the american cancer society, the estimated number of new bladder cancer cases in 2012 is 73,510, which equates to approximately 4.5% of all new cases of cancer. the demographic characteristics associated with greatest risk for bladder cancer include male gender, white race, and increasing age. according to the surveillance epidemiology and end results (seer) age - adjusted incidence data for the period 20052009, bladder cancer occurs four times more often in men than in women. in this data set, it ranks as the fourth most common cancer among men, but only the twelfth most common cancer among women. the incidence among whites is 1.78 times greater than the incidence among blacks, while the incidence among hispanics is 0.90 times the incidence among blacks [2, 3 ]. bladder cancer incidence is the lowest among asian / pacific islanders and american indian / alaska natives. the risk of onset increases with age over the age of 40 and is greatest during the ninth decade of life. about 90% of cases occur in individuals over the age of 55, with the average age being 73 at the time of diagnosis. in the atf cancer cluster, all the cases of bladder cancer represented both the gender and the race with the greatest incidence of bladder cancer, but the ages of the cases at the time of diagnosis were relatively young for bladder cancer as all cases occurred in individuals less than 55 years of age. recognized nondemographic risk factors include cigarette smoking, bladder birth defects, chronic bladder inflammation, genetic predisposition, use of herbal remedies containing aristolochic acid, drinking water containing arsenic and chlorination by - products, prior history of bladder cancer, chemotherapy and radiation therapy, and specific industries and occupations with exposures to known or suspect bladder carcinogens [2, 413 ]. many studies have explored the association of fluid intake and type of fluid intake with risk for bladder cancer, but findings for both total fluids and specific fluids have been inconsistent, likely due to influences of confounding variables such as the presence of bladder carcinogens in tap water [1424 ]. two studies have demonstrated that increased frequency of urination is associated with reduced risk for bladder cancer [18, 21 ]. smoking, the number one risk factor for bladder cancer, is estimated to cause approximately 50% of bladder cancer cases in men and 30% of cases in women. second to smoking, occupational exposures to carcinogens may account for as few as 10% of cases in men and five percent of cases in women to as many as 2025% of all bladder cancers [6, 11 ]. established at risk industries include the manufacturing of products such as synthetic dyes and paints, cables, textiles, leather works, and aluminum and the petrochemical, coal tar, and rubber industries [57, 12, 2530 ]. a number of specific occupations have also been identified to be associated with increased risk of bladder cancer. these include, but are not limited to, cooks and kitchen workers, electricians, hairdressers, leather workers, machinists, petroleum workers, rubber workers, coal miners, truckers, and vehicle mechanics, as summarized by schulte. in 1987, as well as coke oven workers, roofers, dry cleaners, chimney sweeps, and painters, as addressed by others in more recent literature [57, 25, 3134 ]. exposure to both cigarette smoke and occupationally related bladder carcinogens may work synergistically to increase further the risk for bladder cancer [2, 7 ]. as an occupation, the broad job category of law enforcement is generally not recognized for being at increased risk for bladder cancer. in 1987, schulte. did include protection guards on their summary list of occupations associated with risk for bladder cancer, but acknowledged that the potential etiologic agent was unknown. as the rate of occurrence of bladder cancer is almost five times greater than the associated death rate in the us, with 80% of cases surviving for five or more years after diagnosis, cancer incidence is a more sensitive measure of occupational risk for bladder cancer than related mortality and it is important to differentiate between epidemiologic studies looking at cancer incidence and those looking at cancer mortality. many epidemiologic studies on the association of bladder cancer incidence and occupations have not found police officers, guards, and related categories, such as protective services and government worker inspection / investigation occupations, to be associated with increased risk for bladder cancer [11, 3546 ]. one exception was a study by howe. which showed guards and watchmen to have a statistically significant age - adjusted relative risk for bladder cancer of 4.0. in reulen. 's 2009 meta - analysis on the association between bladder cancer incidence and occupation, summary relative risks for bladder cancer were obtained for protective service occupations from the findings of 23 studies and for police officers and guards from the findings of 14 studies. these summary relative risks were only marginally elevated and not statistically significant, at 1.07 (95% confidence interval (ci) 0.961.19) for protective services and 1.10 (95% ci 0.951.29) for police officers and guards. three mortality studies on police officer cohorts also did not find statistically significant increases in mortality from bladder cancer overall [4850 ]. one of these studies did demonstrate that policemen who were professional drivers had significantly increased bladder cancer - related mortality and another showed a higher than expected mortality rate for police officers with 1019 years of service. as some, but not all, epidemiology studies have demonstrated an increased risk for bladder cancer in jobs with exposure to diesel or traffic fumes [6, 40, 43, 51, 52 ] and one meta - analysis has shown increased cancer risk for several types of vehicle drivers, the use of broad law enforcement job categories in studies on cancer risk and occupation assumes a degree of uniformity in exposures to potential bladder carcinogens and risk for bladder cancer, which may not always be the case. part 1 of this study investigates the statistical significance of the incidence of bladder cancer in the atf employee population comprised of criminal investigators and members of the nrt. the time interval of this study is 1993, the year preceding diagnosis of the first bladder cancer case, through 2007. for this period, a full roster cohort was constructed from the annual staffing rosters provided by atf for each calendar year from 1993 through 2007. the annual staffing rosters included all criminal investigators, explosives enforcement specialists, forensic chemists, fire protection engineers, and a small number of other specialists typically affiliated with nrt work, regardless of individual membership on the nrt or eligibility for participation in the medical surveillance program, who were currently working for atf in each respective calendar year. atf provided the demographic study parameters of gender, race, and age, and the job series and titles for all members of the full roster cohort. members of the full roster cohort are dispersed throughout the united states and its territories puerto rico and guam and may move around within this geographic area during their employment with atf. with the advent of the medical surveillance program in 1995, a subset of the full roster cohort, comprised of employees participating in the program, was created. as explained in the introduction, the program was initially voluntary and offered to members of the nrt, but became mandatory in 2002 for all criminal investigators, explosives enforcement specialists, and members of the nrt. setup in partnership with federal occupational health (foh), united states department of health and human services, the program consisted of an annual evaluation which included a medical history and tobacco use questionnaire, physical examination, and laboratory and ancillary tests. collection of detailed work history information, including job series and titles, began in 2003 with the institution of a work history questionnaire. the use of foh 's electronic database, the occupational health information management system (ohims), to facilitate future epidemiologic evaluation of the bladder cancer cluster began in 2002. data for key study variables from pre-2002 exams were retrospectively retrieved and also entered into ohims. for the cohort of employees participating in the medical surveillance program from 1995 to 2007, pertinent data collected with each annual evaluation included the demographic variables, gender, race, and age, as well as cancer history and tobacco use history. data on job series and titles were also provided by members of the cohort participating in the program between 2003 and 2007. the demographic data and the job series and titles data provided by atf for the full roster cohort were subsequently cross - referenced with the same data provided by employees participating in the medical surveillance program. any data inconsistencies between the two sources were resolved by medical record review or phone contact with employees. as stated in the introduction, all bladder cancer cases were initially identified by employees self - reporting the diagnosis and year of diagnosis at the time of the annual medical surveillance evaluation. the self - reported cases were subsequently contacted by the occupational medicine physician overseeing the medical surveillance program, who informed them of the bladder cancer cluster and of the plans to evaluate the significance of the cluster through epidemiologic analysis and requested their assistance with voluntary provision of a pathology report for verification of case diagnosis. requests for pathology reports were accompanied by provision of medical release forms for completion by cases. this is a cohort study in which standardized incidence ratios (sirs) were calculated for four defined populations of atf employees to compare the observed bladder cancer case numbers in each atf population with the expected cancer case numbers based on incidence rate in the us general population, appropriately adjusted for age and stratified by sex and race, as relevant to each of the four atf populations. the us general population was chosen as the best reference population for analysis due to the nation - wide dispersion of atf employees under study. as all the bladder cancer cases occurred among white male criminal investigators, the populations chosen for analysis included : (1) the full roster cohort comprising all males and females, (2) all white males in the full roster cohort, (3) all white males in the full roster cohort with examinations (cancer and tobacco use histories), and (4) all white males in the full roster cohort with both examinations and work histories who were also criminal investigators. determination of the expected cancer incidence rate was based on data from the surveillance epidemiology and end results (seer) program for the us population for the period 19932007. the sir estimates the relative risk of bladder cancer incidence in the atf population compared to the us population adjusted for age and stratified by race and gender. computations for each population included determination of the person - year distribution during the study period, which served as the denominator for the respective sir analysis. one person - year was counted for each year an individual was a member of the cohort. the person - years were arranged by five - year age increments and three five - year time intervals, 19931997, 19982002, and 20032007. table 1 shows the distribution of individuals by gender and race for the full roster cohort, for the subset of employees with surveillance examinations, for the subset of employees with surveillance examinations and work histories, and for the subset of criminal investigators with surveillance exams and work histories. the percent distribution of individuals by gender and race for each of four of these populations is comparable. criminal investigators, with job series 1811, accounted for 96% of all employees in the full roster cohort and 96% of all white males in the full roster cohort. within the full roster cohort, only 18.2% of members were non - white and only 12.6% of members were female. of the full roster cohort, 2,712 (72%) members participated in the medical surveillance program between 1995 and 2007 and had data for one or more examinations in the program 's electronic database (table 1). the percentage of full roster cohort members with examinations was not greater than 72% due in part to the medical surveillance program being voluntary and only open to members of the nrt and to cfis prior to 2002. since 2002, when the program became mandatory for all criminal investigators, explosives enforcement specialists, and members of the nrt, the percentage of currently employed cohort members who obtained annual examinations ranged from a low of 50% in 2003 to a high of 67% in 2006. despite this variance in annual rate of participation in the mandatory program, 2697 of 3136 (86%) full roster cohort members who were employed for one or more years between 2002 and 2007 obtained at least one medical exam. job history data, collected from employees between 2003 and 2007 at the time of the annual exam, were available for 2549 (68%) members of the full roster cohort. of these individuals, 2478 (97%) were criminal investigators (table 1). during the study period, seven individuals reported bladder cancer diagnoses. at the time of the analysis, five of the seven cases had provided medical documentation (pathology reports) verifying the diagnosis of bladder cancer and diagnosis year. another case provided verifying documentation following the completion of the analysis. as affirmed from review of pathology reports of the urinary bladder biopsies of the five cases verified at the time of the analysis, four of the cases were low grade papillary transitional cell carcinomas and one was transitional cell carcinoma in situ. the subsequent sixth case verified after analysis was also low grade papillary transitional cell carcinoma. the first case was diagnosed in 1994 and the most recent case was diagnosed in 2005. as already known from the medical surveillance program, table 1 includes the distribution of reported bladder cancer cases by gender and race for each defined employee population. white males comprised 72% (2723/3768) of the full roster cohort and 69 - 70% of each of the three subset populations, including criminal investigators with medical surveillance examinations and work histories (69% (1715/2478)). the cases ranged from 32 to 53 years of age the year of diagnosis, with three individuals being in their 30s, three being in their 40s, and one being in his early 50s. table 2 shows the distribution of the self - reported bladder cancer cases at the time of diagnosis in the same five - year age increments and three five - year time intervals as used to establish the person - year distributions for each sir analysis. two cases were diagnosed in 19931997, four cases were diagnosed in 19982002, and one case was diagnosed in 20032007. table 3 summarizes the total person - years calculated for each of the four study populations undergoing incidence analysis. the number of total person - years ranged from a high of 34,818 for the full roster cohort to a low of 17,976 for the population of white male criminal investigators with exams and work histories. the pattern of distribution of person - years was similar for all four study populations and is illustrated in table 2 for the population of white males in the full roster cohort. the vast majority of employees for each study population fell within 25 and 54 years of age for each of the five - year time intervals. this distribution pattern reflects atf 's practice of hiring criminal investigators with prior work experience and the mandated retirement age of 57 years for federal criminal investigators. table 3 presents the standardized incidence ratios (sirs) calculated for each of the four defined study populations : the full roster cohort, all white males in the full roster cohort, all white males with examinations (cancer and tobacco use histories), and all white males with examinations and work histories who were also criminal investigators. to access the effect of the two unverified cancer cases on sir outcomes, sirs were performed for the scenario with seven reported cancer cases and for the scenario with five verified cases for each of the four study populations. when computed with all seven cases, the sir is 2.41 (95% ci 1.174.96) for the entire roster cohort, 2.93 (95% ci 1.426.07) for the white male cohort, 6.08 (95% ci 2.9412.54) for white males with exams, and 7.63 (95% ci 3.7015.75) for white male criminal investigators with exams and work histories (table 3). when recalculated with only the five verified cases, the sir is 1.72 (95% ci 0.734.02) for the entire roster cohort, 2.09 (95% ci 0.901.91) for white males, 4.34 (95% ci 1.8510.16) for white males with exams, and 5.45 (95% ci 2.3312.76) for white male criminal investigators with exams and work histories (table 3). the elevated sirs are statistically significant for all four of these populations when all seven cases are included in the analysis and remain statistically significant for white males with exams and white male criminal investigators with exams and work histories when only the five verified cases are used. age - specific cancer incidence rates in the white male atf population were greater than the rates for the adjusted u.s. reference seer population for each age group in which cases occurred (table 4). this finding is expected since 90% of bladder cancers occur in individuals over the age of 55 and all seven cases in the atf population were younger than 55 years at the time of diagnosis. the highest age - specific relative risk for bladder cancer in the atf population compared to the reference population, and the only one of statistical significance, was seen in the 3034 age group, the youngest age group experiencing bladder cancer within the atf population. recognition of a bladder cancer cluster among white male criminal investigators participating in an atf medical surveillance program raised concern that the employee population was experiencing a greater - than - expected incidence of bladder cancer and that post - fire / post - blast scene investigation might be associated with increased risk of bladder cancer. part 1 of this epidemiologic study determined that bladder cancer incidence in the white male study population was significantly elevated statistically for the period 19932007. this study illustrates the twofold utility of using a medical surveillance program to monitor the health of an employee population potentially exposed to hazardous agents and to perform epidemiologic analysis of the significance of cancer occurring within the population. although the atf medical surveillance program evolved over time in several ways, which included changing from voluntary to mandatory participation, and despite an annual participation rate of 5067% after the program became mandatory, the requisite data were sufficient to perform sirs for the atf study population which showed white males to be at increased risk for bladder cancer when compared to white males in the us reference population. all bladder cancer cases in the atf cohort were reported by white males, who constituted about 72% of the full roster cohort. in the incidence analyses of the two larger atf populations, the full roster cohort and white males in the full roster cohort, the cancer risk was elevated for analyses performed with both seven reported and five verified cases, but was only statistically significant for the analyses with the seven reported cases. since these two atf populations included individuals who had not participated in the medical surveillance program and whose cancer history was unknown, which amounted to 28% of the full roster cohort and 31% of white males in the full roster cohort, the actual number of cases of bladder cancer within these two populations could be greater than the observed seven reported and five verified cases, which would lead to even higher sirs than the ones calculated with the seven and five cases. with the two smaller atf populations, white males with exams and white male criminal investigators with exams, the computed sirs were elevated and statistically significant with both seven reported and five verified cancer cases. the sirs of the two smaller populations, understandably higher than those of the two larger populations, might best depict the true cancer risk for the atf cohort as the reported cancer history is known for all individuals in these two smaller populations. the finding that white male criminal investigators in the atf population are at increased risk for bladder cancer is in contrast with the findings of prior epidemiologic studies of bladder cancer incidence in law enforcement occupations, in which law enforcement and related job categories were generally found not to be at increased risk for bladder cancer [11, 32, 3539, 41, 42, 4446 ]. in reulen 's meta - analysis, the summary relative risk was 1.10 (95% ci 0.951.29) for police officers and 1.07 (95% ci 0.961.19) for protective service occupations. what may make this population of atf criminal investigators unique from other populations of law enforcement specialists is the presence of a sizable subset of atf criminal investigators who specialize in investigation of post - fire and post - blast scenes. what we know from the medical surveillance program is that six of the seven bladder cancer cases in the current study had occupational histories of working these scenes while employed with atf. thus, the increased incidence of bladder cancer identified among white male criminal investigators in the current study appears to be associated with the performance of post - fire / post - blast investigations. if this is the case, the increased risk for bladder cancer in this subset of specialized criminal investigators is sufficiently strong to influence the bladder cancer risk within the entire atf study population. the magnitude of the sirs computed in this study deserves some discussion. for the population of white male criminal investigators with exams, the sirs were 7.63 (95% ci 3.7015.75) for seven reported cases and 5.45 (95% ci 2.3312.76) for five verified cases, and for the slightly larger population of all white males with exams, the sirs were 6.08 (95% ci 2.9412.54) for seven reported cases and 4.34 (95% ci 1.8510.16) for five verified cases. in individual epidemiologic studies of bladder cancer incidence in other occupations and industries, statistically significant elevations in relative risk have been found with a 1.1-fold to fivefold increase [11, 32, 33, 39, 4144, 46, 5254 ] and even with a sixfold to tenfold increase for some occupations such as chemical workers [41, 47 ], dye manufacturing [55, 56 ], railroad workers, and physicians. in one epidemiologic study on firefighters, thus, some epidemiologic studies on other occupations have obtained elevated relative risk for bladder cancer of the same order of magnitude as that found in the present study on criminal investigators. in reulen 's meta - analysis on the association between bladder cancer and occupations, however, statistically significant summary relative risks which were found for several occupations only fell in the 1.11.3 range. these occupations included miners, rubber workers, leather workers, four types of professional drivers, and mechanics, but not chemical workers, firefighters, police officers, protective service occupations, or health care professionals. the elevated risk for bladder cancer in the current study cohort also approximates the increased risk for bladder cancer seen in smokers compared to nonsmokers, as demonstrated in various epidemiologic studies which show smokers to have a twofold to sixfold increased risk for bladder cancer [1, 6, 5759 ]. further corroborating study is advised to verify the magnitude of the increased risk found among criminal investigators in the current study. although the elevated sirs for the two populations with exams remained statistically significant when analyzed with only the five verified cases, having two of the seven reported cases (28%) unverified at the time of statistical analysis constitutes a weakness of the study and illustrates a limitation of using data from a medical surveillance program to conduct a cancer incidence analysis. with so few total cases, the difference in number between reported and verified cases can impact the significance of study outcomes, as demonstrated by comparing the sirs computed with both the seven reported and the five verified cases for the full roster cohort and for white males in the full roster cohort. since the study analysis was completed, ongoing effort to verify the unverified cases was successful in verifying one of the two cases. another point to make is that the sirs on the populations with exams could in actuality be artificially high, as only 72% of individuals in the full roster cohort underwent physical examination and individuals without medical issues may have selectively avoided coming in for exams. since the institution of the mandatory program in 2002, however, 86% of individuals employed by atf between 2002 and 2007 obtained at least one exam during this time frame and these 2697 employees accounted for 99% of the 2712 individuals in the full roster cohort with exams. with this level of participation in the mandatory program since 2002, potential for bias due to exam avoidance is likely very limited. in conclusion, white male members of the atf cohort experienced statistically significant increased risk for bladder cancer when compared to white males in the us population for the study period 19932007. among white males with exams and white male criminal investigators with exams, the elevated risk was demonstrated for computations with both seven reported and five verified cancer cases. there was no observed bladder cancer risk for nonwhite males and all females in the cohort study. with six of the seven cases in the bladder cancer cluster, having known histories of investigating post - fire and post - blast scenes while employed with atf, scene investigation work appeared to be linked with the observed increase in bladder cancer incidence. part 2 of the study will evaluate the association of post - fire / post - blast scene work history and risk for bladder cancer, while controlling for tobacco use history. | this study investigated a bladder cancer cluster in a cohort of employees, predominately criminal investigators, participating in a medical surveillance program with the united states bureau of alcohol, tobacco, firearms and explosives (atf) between 1995 and 2007. standardized incidence ratios (sirs) were used to compare cancer incidences in the atf population and the us reference population. seven cases of bladder cancer (five cases verified by pathology report at time of analysis) were identified among a total employee population of 3,768 individuals. all cases were white males and criminal investigators. six of seven cases were in the 30 to 49 age range at the time of diagnosis. the sirs for white male criminal investigators undergoing examinations were 7.63 (95% confidence interval = 3.7015.75) for reported cases and 5.45 (2.3312.76) for verified cases. white male criminal investigators in the atf population are at statistically significant increased risk for bladder cancer. |
leiomyomatosis peritonealis disseminata is an exceedingly rare benign disorder characterized by multiple vascular leiomyomas growing along the submesothelial tissues of the abdominopelvic peritoneum. it is commonly described in women of reproductive age and is rarely seen in men and postmenopausal women. a 65-year - old female patient with a history of abdominal surgery for gastrointestinal stromal tumor presented with abdominal pain, weakness, weight loss, and vomiting. an examination revealed a chronically sick looking, emaciated patient with a long midline abdominal scar, and tenderness on deep palpation all over the abdomen., innumerable masses were found to arise from the outer walls of whole small intestine and mesentery, and there was a soft, 810 cm size liver mass. histology showed highly cellular interlacing bundles of proliferating smooth muscle cells not associated with nuclear atypia or mitotic figures, and there was no necrosis seen, suggesting cellular leiomyoma. leiomyomatosis peritonealis disseminata is a very rare condition, especially in men and postmenopausal women. it should be considered as a differential in patients with disseminated intra - abdominal masses arising in mesentery, peritoneum, and on walls of the intestine. leiomyomas are histologically benign tumors of the uterus that very rarely occur in extrauterine sites.1 extrauterine leiomyomas are a diagnostic challenge and are often confused for malignancy during imaging. their clinical presentation depends on their site of origin, number and size, and rapidity of growth. their signal intensity is similar to that of smooth muscles in t1- and t2-weighted mr imaging and they have a whorled appearance on ultrasound.2 these extrauterine or atypical leiomyomas include leiomyomatosis peritonealis disseminata (lpd), benign metastasizing leiomyoma, retroperitoneal leiomyoma, etc.3 factors usually associated with lpd are pregnancy, long - term contraceptive use, and previous surgery for uterine leiomyoma, especially laparoscopic, and it is generally seen in women of reproductive age.4 knowledge of the presence of such pathologies, their different clinical spectrum, and unusual presentation is helpful for proper management.5 multiple leiomyomas arising submesothelially in the pelvic and abdominal peritoneum characterize lpd.5 lpd is an exceedingly rare benign disorder characterized by multiple vascular leiomyomas growing along the submesothelial tissues of the abdominopelvic peritoneum. a 65-year - old female patient with previous abdominal surgery in a regional hospital in bahir dar 6 months prior to this presentation presented with diffuse abdominal pain, weakness, and occasional vomiting of ingested material of 6 months duration. there was no history of abnormal uterine bleeding, oral contraceptive use, or gynecologic operation. the biopsy result of her surgery in bahir dar was suggestive of gastrointestinal stromal tumor. on examination, she looked chronically sick, was emaciated and slightly pale and had a long midline abdominal scar ; there was also mild tenderness all over the abdomen on deep palpation. investigation revealed a hemoglobin value of 9.3 g / dl and a wbc count of 6,900/l. she had o + ve blood group and had normal liver and renal function as well as a normal chest x - ray. ultrasound showed enumerable hypoechoic intra- abdominal masses of different sizes (figure 1) and a large hypoechoic liver mass measuring 7.54.7 cm in the right lobe (figure 2). laparotomy later revealed innumerable globular masses arising from the mesentery and walls of whole small bowel, some pedunculated but most sessile (figures 3 and 4). in addition, there was a large liver mass of approximately 810 cm in the right lobe with normal pelvis. multiple biopsies were taken from the mesenteric and superficial small intestinal masses but not from the liver mass for fear of bleeding and abdomen closure. biopsy specimens were sent to two centers, and both reported a similar finding of highly cellular interlacing bundles of proliferating smooth muscle cells not associated with nuclear atypia or mitotic figures, and there was no necrosis seen. the patient was discharged with no major complication, and a diagnosis of lpd with possible liver leiomyoma was made. the patient was followed up for 3 months, and she still has some abdominal pain but was doing fine in general. lpd is a rare benign proliferative process that develops from the mesothelial or submesothelial layers of the peritoneum. a differential diagnosis of intra - abdominal carcinomatosis, lymphoma, soft - tissue sarcoma, etc was considered in our patient, but the absence of mitosis, atypia, and necrosis ruled out malignancy. the differential diagnosis should also include those conditions often confused with this such as benign metastasizing leiomyomas, intravenous leiomyomatosis of the uterus, parasitic myoma, or diffuse leiomyomatosis of the uterus. a finding of highly cellular interlacing bundles of smooth muscles cell proliferation not associated with nuclear atypia or mitotic figure and absence of necrosis as reported by the two centers and the laparotomy finding of subperitoneal nodules varying in size from a few millimeters to several centimeters on the intestine and mesentery confirms the diagnosis of lpd.6 in 1952, willson and peale7 reported the first case of lpd. lpd is a specific type leiomyomatosis that is rarely identified by clinical evaluation. to date, only a few hundred cases have been reported in the literature. it is very difficult to reach a diagnosis prior to surgery as diagnosis relies on medical history, observations during surgery, and pathological results.8 although it commonly occurs in women of reproductive age, few cases have also been reported in postmenopausal women and in men. lpd is benign ; however, it exhibits recurrent and malignant tendencies.2,4 lpd generally presents with no symptoms or nonspecific symptoms such as abdominal pain or is suspected at ultrasound or ct scan and after laparotomy for an unrelated problem. this patient also presented with nonspecific symptoms such as abdominal pain, discomfort, nausea, and occasional vomiting of ingested material. the similar ultrasound findings of the intra - abdominal masses and masses in the liver suggest similar pathology. there is a similar report from india, by tun,9 regarding liver leiomyoma in a patient with lpd. some of the masses not included in the biopsy could have degenerated into leiomyosarcoma that could also be the other explanation of the liver mass found in our patient.10 her presentation was nonspecific, and the diagnosis was based on operative findings and pathologic report (as is the case with most patients with lpd). thus, lpd should be considered as differential diagnosis in patients with disseminated intra - abdominal conditions such as carcinomatosis, lymphoma, tuberculosis, etc. | backgroundleiomyomatosis peritonealis disseminata is an exceedingly rare benign disorder characterized by multiple vascular leiomyomas growing along the submesothelial tissues of the abdominopelvic peritoneum. it is commonly described in women of reproductive age and is rarely seen in men and postmenopausal women.case detailsa 65-year - old female patient with a history of abdominal surgery for gastrointestinal stromal tumor presented with abdominal pain, weakness, weight loss, and vomiting. an examination revealed a chronically sick looking, emaciated patient with a long midline abdominal scar, and tenderness on deep palpation all over the abdomen. ultrasound revealed diffuse intra - abdominal masses and a big liver mass. on laparotomy, innumerable masses were found to arise from the outer walls of whole small intestine and mesentery, and there was a soft, 810 cm size liver mass. histology showed highly cellular interlacing bundles of proliferating smooth muscle cells not associated with nuclear atypia or mitotic figures, and there was no necrosis seen, suggesting cellular leiomyoma.conclusionleiomyomatosis peritonealis disseminata is a very rare condition, especially in men and postmenopausal women. it should be considered as a differential in patients with disseminated intra - abdominal masses arising in mesentery, peritoneum, and on walls of the intestine. |
a 46-year - old female visited chonbuk national university dental hospital with the complaint of swelling in the left upper lip area. a clinical examination found a bony and hard gingival swelling that extended from the left maxillary incisors to the premolar regions, as well as displacement of the left maxillary lateral incisor and canine. neither tenderness nor pus discharge was observed upon palpation. a panoramic radiograph (fig. 1) and a periapical radiograph (fig. 2) showed an ill - defined multilocular radiolucency with a large cystic lesion extending from the left upper central incisor to the premolar area. thick sclerotic trabeculae were observed in the lesion, as well as displacement of the left upper lateral incisor and canine. 3) revealed an ill - defined multilocular lesion with a cystic lesion and thick trabeculae on the left anterior maxilla. thinning, expansion, and perforation of the buccal and palatal cortical plates were noted. the lesion expanded into the left maxillary sinus with destruction of the anteromedial wall and mucosal thickening. with the tentative diagnosis of an odontogenic myxoma or ameloblastoma, the patient underwent partial osteotomy. after an operation, a biopsy was performed, and the histologic analysis (fig. 4) revealed a non - encapsulated mass with small, scattered tumor nests of epithelium in the fibrous stroma. the patient has been disease - free for three years since the operation and is under routine follow - up care. the present case differed from the lesions described in previous case reports in that it presented with a multilocular radiolucency that had thick sclerotic trabeculae, and showed no root resorption of the involved teeth. ameloblastoma is a benign but locally aggressive polymorphic neoplasm that consists of proliferating odontogenic epithelium.7 three types of intraosseous ameloblastomas exist : the conventional or solid / multicystic variant, the unicystic variant, and the desmoplastic variant.10 in 1984, eversole.11 first described the desmoplastic variant of ameloblastoma, which is distinguished from conventional ameloblastoma based on its tendency to involve the anterior maxilla5 and its unique appearance.2 desmoplastic ameloblastoma also differs from other types of ameloblastoma in that it is located in the anterior or premolar regions of the maxilla or mandible, and its radiographic appearance is often more typical of a mixed lesion. some authors have suggested that desmoplastic ameloblastoma tends to exhibit rapid growth345 and progressive behavior.8 desmoplastic ameloblastoma accounts for 4%-5% of all ameloblastomas.68 it occurs in patients in the third through seventh decades of life.678 a clinicopathological analysis of the 68 cases of desmoplastic ameloblastoma that have previously been reported found that more than 70% of the cases occurred in the anterior and premolar regions of the jaw.7 contrary to reports arguing that desmoplastic ameloblastoma has an anterior maxillary predilection, effiom and odukoya12 reported that 81% of cases showed a mandibular predilection, with 82% of cases showing radiolucency. conventional ameloblastoma predominantly occurs in patients in the third through fifth decades of life, comprising 92% of cases in this age group.13 unilocular forms have been found to be significantly more common in younger patients.14 it has been well established that the majority of multilocular ameloblastomas arise in the mandible, with most in the molar and ramus regions. the present case showed multilocular radiolucency with a large cystic lesion of the anterior maxilla. desmoplastic ameloblastoma appears as a diffuse, mixed radiolucent and radiopaque lesion with a honeycomb or soap - bubble pattern.2 kawai.2 reported that 25% of cases showed a mixed radiolucent and radiopaque pattern with new bone formation. macdonald - jankowski.14 found a significantly higher proportion of unilocular lesions with well - defined margins in conventional ameloblastomas, but poon.15 reported that only 16% of cases showed unilocular lesions. however, kaffe.7 reported that desmoplastic ameloblastoma appeared as a multilocular lesion in 20% of cases and as a unilocular lesion in 33% of cases, while the remaining 47% of lesions were not loculated. unlike other ameloblastomas, desmoplastic ameloblastoma has poorly defined borders,716 and it may be mistaken for a benign fibro - osseous lesion.67 a ct scan is used to obtain a three - dimensional image of the lesion and to better examine the soft tissues.10 although ameloblastoma is more likely than odontogenic myxoma to appear as a unilocular lesion,14 these two tumors have no significant differences regarding locularity. lo muzio.17 reported that six of 10 odontogenic myxomas were multilocular and four were unilocular. macdonald - jankowski.14 reported that ameloblastoma was significantly better defined than odontogenic myxoma. the unique radiographic features of the desmoplastic variant of ameloblastoma, which often give the impression of a fibro - osseous lesion, seem to reflect the characteristic aggressive behavior of the tumor in many cases. this may also explain the ill - defined border of desmoplastic ameloblastoma.718 kaffe.7 and li.16 reported tooth displacement in 48%-92% of desmoplastic ameloblastomas, whereas root resorption was found in 8.7%-33% of desmoplastic ameloblastomas. however, lo muzio.17 reported root resorption in 20% of odontogenic myxomas. one advantage of ct over magnetic resonance imaging is the ability to distinguish desmoplastic ameloblastoma from other fibro - osseous lesions through the detection of thick bony trabeculae.19 in several reports, tumors were incorrectly diagnosed as fibro - osseous lesions based on the history, clinical findings, and radiographic appearance.7 the present case showed an ill - defined expansile multilocular radiolucency with perforation of the cortical plate, unlike the encapsulated mixed radiolucent and radiopaque pattern previously described for these lesions. desmoplastic ameloblastoma appears as compressed islands and thin cords of ameloblastic epithelial cells embedded in a dense collagenized stroma.7 areas of stellate reticulum are rare to absent.10 the present case involved a non - encapsulated mass with small, scattered tumor nests of epithelium in the fibrous stroma. an invasive growth pattern of this type into marrow spaces and the lack of demarcation with fibrous connective tissues are correlated with the indistinct border observed in desmoplastic ameloblastoma.221 the non - encapsulated nature of the mass seems to be related to the ill - defined border, and is an important difference from well - defined fibro - osseous lesions. histologically, cystic changes in the tumor mass of desmoplastic ameloblastoma have been reported.61920 however, no cystic changes were found in the large cystic lesion of the present case. a desmoplastic ameloblastoma with features of other histologic types is considered to be a hybrid lesion.5 a hybrid ameloblastoma is composed of desmoplastic ameloblastoma and conventional follicular or plexiform ameloblastoma.2122 the present case showed no histological findings characteristic of conventional follicular or plexiform ameloblastoma. desmoplastic ameloblastoma often shows prominent bone formation.619 thompson.19 suggested that the production of new bone results from an attempt to repair the damaged laminated bone trabeculae resorbed by the tumor expansion. kawai.2 proposed that the adjacent bone trabeculae tend to persist because osteoblastic activity is more vigorous than osteoclastic activity and may be induced by newly formed bony trabeculae. the present case showed prominent new bone formation with osteoblastic rimming in the solid mass. the new bone formation may have been reflected by the thick sclerotic trabeculae seen on the radiographs. most cases of desmoplastic ameloblastoma have been described as having an ill - defined border and a propensity to recur at least as often as conventional ameloblastoma.21 following surgical treatment, the patient has remained disease - free for three years and is currently under routine follow - up care. the careful analysis of clinicopathological and radiological findings is recommended to ensure an accurate diagnosis of desmoplastic ameloblastoma. | the desmoplastic variant of ameloblastoma is a rare form of ameloblastoma characterized by unique radiographic and histologic features. a 46-year - old female was referred to our hospital, complaining of swelling in the left upper lip area. radiographic findings revealed an ill - defined multilocular lesion with a large cystic lesion and thick sclerotic trabeculae on the left anterior maxilla. after the patient underwent partial osteotomy, histologic analysis revealed a desmoplastic ameloblastoma with no evidence of a hybrid lesion or cyst formation. the radiographic findings in the present case were different from those described in previous case reports. these findings are of special importance due to the unfamiliar radiographic and histologic features of this lesion. |
lupus nephritis (ln) is a major clinical manifestation of systemic lupus erythematosus that occurs in 15% of patients at diagnosis and in approximately 40% during the course of the disease. renal biopsy is the gold standard for the diagnosis and follow - up, whereas the measurement of proteinuria identifies patients with overt renal failure, but fails to detect early silent disease. thus, a better definition of the pathogenetic mechanisms leading to ln is required to identify effective markers of renal inflammation. ln is generally attributed to an intriguing interplay between renal parenchymal cells and inflammatory cells recruited in consequence of the deposition and/or in situ production of immune complexes (ics). ics increase the production of cytokines, chemokines, and adhesion molecules which allow the progressive infiltration of macrophages, dendritic cells (dcs) and t - cells resulting in chronic renal failure. moreover, cytokines and chemokines secreted by cells infiltrating glomeruli further promote the migration of other inflammatory cells that are attracted toward the inflammatory sites in response to a concentration gradient [3, 4 ]. notwithstanding sle is considered a t helper- (th-) 2 driven disease [57 ], experimental models of ln proved the primary role of th1 cytokines for its development and severity, since large amounts of both interleukin- (il-) 12 and il-18 have been found within glomeruli of humans as well as in murine models of glomerulonephritis [811 ]. in parallel, high amounts of th2 cytokines as il-6 and il-10 were found in sera of sle patients with active disease, although they were not clearly associated with renal damage. macrophages and dcs are major producers of cytokines within glomeruli and their interaction with resident t - cells amplifies the renal inflammation. in this context, the impaired t - cell activation as altered function of dcs has been demonstrated in sle, whereas dcs activate nave t - cells and regulate the cytokine production, and the t - cell polarization. it has been recently described as a defect of circulating dcs in parallel with their increased migration toward the kidney due to attractive stimuli promoted by glomerular il-18, il-1, and chemerin. thus, while glomerular il-18 is nephritogenic since it recruits il-18r dcs, these cells locally produce il-12, interferon- (ifn-) and cxcr4 thus amplifying the immune - mediated glomerular damage. in addition, expansion of th-17-producing cells and defective number and function of t - regulatory (treg) cells have been demonstrated in ln. here, we review recent data on the key role of both th1 and th2 cytokines in ln and focus the defect of th17 and tregs in the modulation of inflammatory signals leading to the worsening of sle renal function. derangement of t - cell function has been demonstrated in sle in parallel to abnormal cytokine production associated to loss of immune tolerance, increased antigenic load, and defective b - cell suppression. a large number of studies suggested that sle is a th2-driven disease [57 ]. however, elevation of both th1 and th2 cytokines occurs in both humans and mice suggesting that sle is a complex disease driven by different lymphocyte subsets with high heterogeneity of clinical manifestations and organ involvement (figure 1). t - cells play a crucial role in the pathogenesis of experimental and human ln, since they activate b - cell functions including the production of nephritogenic antibodies and the modulation of t helper immune response. moreover, t - cells infiltrate the glomeruli and promote either direct cytotoxicity or recruit other inflammatory cells as macrophages and dcs. many studies suggested an imbalance in t - cell subsets, and reported an increase of cd4 cells with respect to cd8, altered th1/th2 cytokine production, and raise of the circulating cd4/cd8 subset. in this context, murine / and / cd4/cd8 cells induce b - cells to produce both anti - dsdna and antichromatin antibodies and thus have been considered the major inducer of renal failure in experimental sle [1719 ]. moreover, disruption of t - cell signals in experimental ln by depletion of / t - cells or by blocking the cd40/cd40 ligand cascade, delays the inflammatory state of ln and improves the proteinuria with reduced glomerular accumulation of both inflammatory cells and nephritogenic antibody. in addition, / t - cells and cd16/cd56 natural killer cells cooperate in lupus, since mlr / lpr mice lacking these populations develop severe nephritis associated with polyclonal cd4 t - cell expansion. in fact, peripheral cd4/cd25 tregs are reduced in patients with active ln, and expansion of this population through tolerogenic peptides strongly delays nephritis as well as the production of auto - antibodies by b - cells. since t - cell subsets produce different cytokines in response to inflammatory stimuli, their measurement is considered an effective tool for explaining the pathogenetic mechanisms of ln. most of information concerning the pathogenetic role of cytokines in ln derives from murine models relevant for investigating the disease mechanisms under defined experimental conditions. in particular, knock - out mice for either cytokines and/or cytokine - receptors emphasized the role of ifn- and il-12, since treatment with relative neutralizing antibodies prevents renal disease in nzb / w mice, while ifn- receptor mrl / lpr are protected from glomerulonephritis and show defective anti - dsdna production. moreover, these mice strains show high il-12 production, whereas those defective for the cytokine are protected from ln through inhibition of both ifn- and il-18 production. other studies defined the il-18 functions in mrl / lpr mice and demonstrated that its glomerular accumulation occurs in patients with proliferative glomerulonephritis and correlates with the infiltration of il-18r dcs [8, 10, 25 ]. however, the majority of therapeutic approaches in humans with biologic or genetic modulation of cytokine production provided inconsistent results. this was at least in part attributed to the identification that il-23 and il-27, additional members of the il-6/il-12 family may amplify the th1 immune response through expansion of the cd4 th17 cells. th17 cells develop under the influence of il-6, tgf-, and il-23, and produce il-17. recently, the urinary expression of th17 related genes has been demonstrated in patients with ln and thus further studies might be relevant for explaining its role to human ln. we have recently provided evidence that production of th1 over th2 cytokines occurs in active ln, since a defect of both il-6 and il-10 counterbalances elevations of ifn-, il-12, and il-18 in both sera and urine in the majority of patients. il-6 is a pleyotropic cytokine produced by a large variety of cells that primes the differentiation of b - cells into antibody - producing cells as well as the differentiation of t - cells to effector cells and the activation of macrophages. il-6 promotes the proliferation of renal mesangial cells in mice and several studies report il-6 as a critical mediator of tissue damage for its ability to stimulate the production of nephritogenic antibodies in humans. moreover, it has been reported that urinary excretion of il-6 is elevated in patients with active ln, whereas it declines during the treatment. these studies, however, have not demonstrated a definite relationship between serum and/or urinary il-6 overproduction in renal disease. as shown in figure 2(a), we demonstrated in patients with sle a remarkable increment of serum il-6 with respect to normal subjects (p <.05). however, both serum and urinary il-6 levels derived from ln patients were not associated with the presence of renal injury, although they were increased in patients with high levels of anti - dsdna and antinuclear antibodies (figure 2(b)). this suggests the potential role of il-6 for the stimulation of auto - antibody production by b - cells, rather than acting as direct mediator of glomerular inflammation. it is a key factor in regulating auto - antibody - secreting b - cell activity and promotes b - cell differentiation. elevated levels of serum il-10 occur in sle, although the treatment with anti - il-10 monoclonal antibody failed to restore the disease activity in the majority of patients. moreover, il-10 elevation occurs in different clinical conditions associated to sle and a specific association with organ damage has not been definitively elucidated. our data are in agreement with these results for the high levels of serum il-10 (p <.05) in both patients with and without ln, whereas elevations of urinary il-10 were revealed in those with minimal glomerular lesions. thus, we concluded that overexpression of il-10 in sle may reflect an intrinsic defect of the cytokine homeostasis. however, it remains unclear whether elevations of il-10 are a cause or, rather, an effect of this dysregulation. measurement of th1 cytokines (figure 2(a)) provided different results, since both il-12 and il-18 were strongly overexpressed in ln (410 90 and 618 105 pg / ml), correlated with ifn- overproduction (p <.05), and increased in their concentrations within the kidney of patients with proliferative glomerulonephritis. parallel analyses revealed a large number of il-18r dcs in glomeruli infiltrated by il-18 cells, thus suggesting that the cytokine also exerts a potent chemoattraction of inflammatory cells expressing the functional receptor. additional studies also demonstrated a peculiar association between both serum and urinary il-12 with serum ifn- (p < context, presence of urinary neopterin as a bioactive marker of serum ifn- production further supported the th1 predominance in kidney inflammation. il-17 is a 17-kda transmembrane protein that includes six members and five receptors mostly produced by activated t - cells. although the downstream signals activated by il17r remains to be elucidated, it has been recently shown that its stimulation activates both the nuclear factor kb and map kinase cascades. il-17 exerts a potent proinflammatory activity in parallel with functional recruitment of macrophages and neutrophils within inflamed tissues through the overproduction of il-8 and monocyte chemoattractant protein-1. this cytokine also allows the expression of adhesion molecules by t - cells as well as the production of il-6 and gm - csf. lastly, il-17 synergizes with il-1, tumor necrosis factor- (tnf-) and ifn-, and amplifies the th1 immune response in autoimmune disorders. il-17 is mostly produced by cd4, cd8, cd4/cd8, and / t - cells, and the cd4 t - cell effector subset, namely the th17 cells, has been distinguished from other t helper populations for the specific origin, differentiation, and cytokine production. recent studies demonstrated the involvement of th17 cell in the pathogenesis of sle, since nzb / nzwf1 mice produce relevant amounts of the cytokine while il-17 producing t - cells largely infiltrate the nephritic kidneys of these mice. by contrast, treatment with anti - cd3 monoclonal antibodies delayed the renal damage with concurrent defect of il-17 production in both serum and glomeruli. parallel studies in lupus - prone mice described that presence of il-23 as necessary for the development of a pathogenic th17 functional immune response. in fact, il-23 increases the il-17 production by memory t - cells, thus suggesting a novel t helper functional axis formed by il23/il-17 interaction, almost similar to the il-12/ifn- system. moreover, it has been demonstrated that the majority of il-17 producing t - cells arises from the cd3/cd4/cd8 subset, that is the typical population revealed in nephritic glomeruli from mrl / lpr mice. therefore, this cell population is considered an active mediator of renal cytotoxicity [37, 38 ]. it is conceivable that il-17 t - cells amplify the inflammatory signals activated by other th1 cytokines. in this context, its inhibition by targeted therapies should represent a novel option for the treatment of patients with overt renal disease. regulatory t - cells (tregs) are a heterogeneous population of cd4/cd8/cd25 cells differentiated in two subsets in relation to the expression of foxp3 transcription factor. the foxp3 population includes both il-10-producing tregs and th3 cells that express the transforming growth factor- (tgf-). by contrast, foxp3 tregs play a protective role in autoimmunity since they regulate the efficiency of the immune response. notwithstanding foxp3 is not necessary for the differentiation of tregs, its expression prevents t - cells to differentiate into th17 proinflammatory effector t - cells. this is consistent with the accelerated autoimmune manifestations observed in mice depleted of foxp3 tregs or in humans characterized by a genetic mutation of the foxp3 gene. similarly, natural tregs exert a protective effect in lupus - prone mice, since depletion of cd4/cd25 cells in nzb / nzwf1 accelerates the onset of ln, whereas their transfer into cd4/cd25 knock - out mice delays the glomerulonephritis development. moreover, high il-6 production may interfere with treg function and promote their switch toward th17-producing cells. in addition, stimulation and activation of dcs in nephritic glomeruli are promoted by interferons while limiting both expansion and protective functions of tregs in sle. thus, although tregs play an apparent primary role for the homeostasis of the immune system in sle, the mechanisms leading to their depletion are unclear. it has been suggested that defective number of peripheral tregs in lupus promotes t- and b - cell hyperactivity while recent studies have attributed this defect to dysfunctional dc activation. for this reason, the restoring of the balance between immunogenic and tolerogenic dcs may represent a mechanism for the inhibition of il-17 cytotoxicity and the correction of treg defect during the active phases of ln. defective dc function and glomerular accumulation of these cells in ln are regarded as a consequence of their increased recruitment from peripheral blood in response to chemotactic stimuli [10, 43 ]. dcs interact with renal parenchymal cells through the activation of toll - like - receptors, fc - receptors, c - type lectins, and scavenger receptor, that, once stimulated by either cell - to - cell contact or paracrine signals, accelerate the enrollment of nk, t - cells, and / lymphocytes in inflammatory sites. furthermore, dcs exhibit the hallmark of well - equipped antigen - presenting cells in presence of peculiar costimulatory molecules and chemokine profile. in this scenario, presence of dcs within nephritic glomeruli supports their pathogenic effect. this aspect may thus explain why recent therapeutic approaches aimed to their functional disabling, ameliorate nephritis in rodents. recent data from experimental and human ln provided new insights to the role of cytokine interplay in the pathogenesis of renal damage. in this context, a cutting - edge on the th1 deregulation has been demonstrated and both il-12 and il-18 exert a major nephritogenic role. however, further studies are needed to clarify the interplay of other mechanisms including both th17 expansion and defective treg function within inflamed glomeruli. therefore, development and progression of renal failure in sle is an event that involves multiple players whose regulation by novel - therapeutic strategies might prevent the progressive worsening of ln. | lupus nephritis (ln) occurs in more than one - third of patients with systemic lupus erythematosus. its pathogenesis is mostly attributable to the glomerular deposition of immune complexes and overproduction of t helper- (th-) 1 cytokines. in this context, the high glomerular expression of il-12 and il-18 exerts a major pathogenetic role. these cytokines are locally produced by both macrophages and dendritic cells (dcs) which attract other inflammatory cells leading to maintenance of the kidney inflammation. however, other populations including t - cells and b - cells are integral for the development and worsening of renal damage. t - cells include many pathogenetic subsets, and the activation of th-17 in keeping with defective t - regulatory (treg) cell function regards as further event contributing to the glomerular damage. these populations also activate b - cells to produce nephritogenic auto - antibodies. thus, ln includes a complex pathogenetic mechanism that involves different players and the evaluation of their activity may provide an effective tool for monitoring the onset of the disease. |
as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors | by replacing the central thiophene unit of an anionic pentameric oligothiophene with other heterocyclic moities, a palette of pentameric thiophene - based ligands with distinct fluorescent properties were synthesized. all ligands displayed superior selectivity towards recombinant amyloid fibrils as well as disease - associated protein aggregates in tissue sections. |
in the last decade, the large availability of the internet and the embracing of new digital technologies like smartphones are changing people 's way of life and introducing new social dynamics [1 - 7 ]. social networks allow immediate communication with just one click, by searching, reaching and sending any kind of verbal messages, videos or images. thus, the use of social networks facilitates virtual contacts and meetings with other people, replacing many personal relationships and commitments [1 - 5 ]. with its 1.4 billion active users, facebook is now considered the most popular social network worldwide [8, 9 ] and, as a consequence, researchers have started to study some features of its use [10, 11 ], as well as its excessive use [12, 13 ]. caplan developed an overall theory about the misuse of the internet, according to which an online communication type allows to avoid negative feelings, such as loneliness and anxiety [14, 15 ]. griffiths stated that an addictive behavior is characterized by the six core components of addiction : salience, mood modification, tolerance, withdrawal symptoms, conflict, and relapse [16, 17 ]. he argued that any behavior that fulfills these six criteria can be considered as an addiction, including social networking. furthermore, the addiction on social networks, as facebook, has also been considered only when the excessive use damages personal, family and/or professional life [18, 19 ]. some reports indicate that the excessive use of social networks increases isolation in real life, bringing harms to relationships. it is worthily highlighted that a growing complaint in mental health services from patients or even parents worried about their children s is increased social isolation, levels of anxiety and worsening in school performance due to excessive social networks use. griffiths described the social network sites (snss) addiction by 6 pillars : usage patterns, motivations, dependency and typical profiles, negative consequences, evidence of dependence and comorbidity [17, 22 ]. furthermore, there is also a review evaluating snss addiction in 17 studies which shows a growing interest on this issue, although findings are limited because of the methodology used. as a result, internet addiction and social networks addiction are not well - defined constructs yet, mainly because there is no gold standard measures of these conditions, nor is there any widely accepted theory [4, 12 ]. on the other hand, some authors [5, 24, 25 ] introduced specifically the facebook addiction disorder, or more generally snss addiction disorder, on the basis of these six addiction criteria : (1) neglect of personal life ; (2) mental preoccupation ; (3) escapism (an entertainment that allows people to forget about the real problems of life) ; (4) mood modifying experiences ; (5) tolerance and (6) concealing the addictive behavior. despite many researchers defending the hypothesis that the excessive use of the internet and social networks as facebook can cause addiction, the concept is still controversial [5, 12, 17, 23, 26, 27 ] and the dsm-5 did not include them as addiction disorders. within this context, the aim of the present paper is to summarize the state of the art about the use and excessive use of facebook and to explore how facebook usage could become addictive. statistics provided by facebook in 2015 reveal that, worldwide, there are over 1.44 billion monthly active facebook users and at least 936 million people log in every day. among those daily users, 745 million check the site by their mobile devices. according to a recent review, there are cultural and socio - demographic differences in facebook use : females and ethnic minorities seem to use facebook more than males and caucasians. furthermore, a cross - culture study examined differences in facebook use among people from usa, uk, italy, greece and france, founding that, compared to usa users, the uk users classified groups as being more relevant, italian users rated both groups and games / applications as most relevant, whereas greek users considered status updates being less relevant. a recent study on 100 swedish students about the use of facebook shows that 85% of them log in facebook at least one time every day and 70% admitted that they logged in whenever they started their computer. furthermore, the participants spent an average of 75 minutes a day on facebook, with men spending 64 minutes and women 81 minutes every day. in this study, the average user logs in on facebook 6.1 times / day and almost half the participants mentioned that they feel it is hard to keep up socially without facebook. another survey conducted on a sample of 1605 us adults aged between 18 and 54 years old, shows that 34% of girls aged between 18 - 34 log in to facebook before they go to the toilet every time they wake up in the morning, 21% wake up in the middle of the night to read their texts and 39% identify themselves as facebook addicts. furthermore, hofmann and colleagues, in a survey on 205 german facebook and twitter users aged between 18 and 85, showed that the desire for being daily on social networks reported by participants is superior to the desires for sleep and rest. they concluded that social networks addiction is more harmful than smoking and drug - addiction because social media are widely available and cheaper. even with an increasing amount of evidence focusing on social network addiction and a few studies indicating that the prevalence of facebook addiction ranges from 8.6% to 41.9% [12, 32 - 34 ], limited research has examined how facebook use could become addictive [17, 23 ]. the biopsychosocial framework for the etiology of addictions and the syndrome model of addiction state that people addicted to social networks have similar symptoms to those reported by people who suffer from substances addiction. xu and tan proposed that the shift from normal to social networking misuse arises when social networking is considered as an important (or even exclusive) instrument to cope with stress, loneliness and depression. regarding internet addiction, griffiths argued that it is not well established if people become addicted to the platform or to the contents of the internet. users addicted to the internet can not give up several aspects of online use. thereby, the author postulates three subtypes of internet addicts, on the basis of the object of the addiction : on - line games, sex, and e - mail or text messages. social networks are an online activity in which texting or e - mailing are predominant, in spite of being used for game playing and even sex - related purposes. according to the model proposed by nadkami and hofman, facebook use is mainly driven by two basic social needs : (1) sense of belonging to, and (2) self - presentation. the need to belong arises from the basic drive to affiliate with others and obtain social acceptance, whereas the need for self - presentation is steadily required for the process of impression management. these motivational drives often co - exist and could explain the facebook use. several demographic and cultural factors are associated with the need to belong, whereas personality traits such as neuroticism, narcissism, shyness, self - esteem and self - worth are associated with the need for self - presentation. tamir and michell described an increased neural activation underling the cognitive mechanisms associated with gratification upon talking about oneself. using fmri, the authors explored how brain activity while people talked about themselves was related to a pleasant experience, in comparison to other natural rewards such as sex or food. authors argued that babies aged 9 months try to catch other 's attention to the parts of the environment that they view as the most important, which could be considered the first form of self - exposure ; adults, on the other hand, want to give forward information to others about themselves. thus, human beings have an intrinsic drive for self - exposure, and this behavioral pattern is forced in social networks because of the brain 's reward system : " people dedicate close to 40% of their time talking about themselves. this number reaches 80% in social networks with the possibility of feedback and immediate rewards ". similarly to many addictions, the activation of the reward system through self - exposure [39, 40 ] can increase the level of dopamine in the reward system, generating a dependence framework for social networks excessive use. finally, some studies [17, 23 ] evaluated the tendency to develop a social network addiction based on personality traits such as : extroversion, socialization, awareness, neuroticism and openness to experimentation. furthermore, the potential excessive use of social networks seems to be related to high narcissism, high neuroticism and low awareness [5, 42 ]. several authors attempted to define internet addiction as a syndrome with a set of symptoms that includes : a) preoccupation with the internet activities ; b) increasing tolerance ; c) development of psychological dependency and withdrawal symptoms ; d) inability to reduce internet use ; e) internet use to cope with negative mood and reduce stress ; f) replacing other activities and relationships with recurrent internet use despite awareness of the bad consequences [43 - 45 ]. the internet addiction scale, a 20 item self - report questionnaire, is a revised version of the earlier 8 item scale young s internet addiction diagnostic questionnaire and it was developed by adapting dsm - iv criteria for pathological gambling, a diagnosis classified as an impulse - control disorder. actually, this instrument is the most widely used alternative in the field of internet addiction and it measures the degree to which all types of online activities disturbs aspects of one s daily life : daily routine, sleep pattern, productivity, social life, feelings. with regards to facebook addiction, the systematic review of ryan and colleagues analyzed eight self - report questionnaires used to its evaluation in several studies [8, 48 - 53 ]. authors concluded that research addressing this salient area is still in its infancy and highlighted the overall lack of construct validity surrounding these instruments, developed from existing measures of internet addiction without in - depth exploratory research with facebook addicted individuals. internet addiction and excessive social networks use are already important issues for treatment and research. even if facebook is the number one tool to promote entertainment, maintaining relationships and occupying time, some people could develop addictive behavior based on the sensation to feel better or more self - assured (increased level of excitement or escape) upon navigating social networks. facebook addiction could be related to brain reward and gratification mechanisms and it seems more prevalent in persons with some personality traits and mood states, such as anxiety, depression, narcissism, low self - esteem, seeking for an increased mood elevation. despite being a current topic,. however, specialized clinics and programs already target these kinds of addictions although there is still a need for further research to determine if facebook excessive use can be considered as a specific online addiction disorder or an internet addiction subtype. | facebook is notably the most widely known and used social network worldwide. it has been described as a valuable tool for leisure and communication between people all over the world. however, healthy and conscience facebook use is contrasted by excessive use and lack of control, creating an addiction with severely impacts the everyday life of many users, mainly youths. if facebook use seems to be related to the need to belong, affiliate with others and for self - presentation, the beginning of excessive facebook use and addiction could be associated to reward and gratification mechanisms as well as some personality traits. studies from several countries indicate different facebook addiction prevalence rates, mainly due to the use of a wide - range of evaluation instruments and to the lack of a clear and valid definition of this construct. further investigations are needed to establish if excessive facebook use can be considered as a specific online addiction disorder or an internet addiction subtype. |
, a wasting of bilateral leg muscles was evident, and she wore a foot brace. the motor weakness and wasting were more severe in the lower distal extremities than in the upper extremities. muscular stretch reflexes were absent, and vibratory sense and pain appreciation were impaired distally in both upper and lower limbs. patient 2 (proband 's mother, ii-4) was a 45-year - old woman born after a normal delivery. all early developmental milestones were achieved. at the age of 27 years, she experienced weakness of both upper extremities that lasted for 3 months after giving birth. she experienced sensory impairment of the right upper extremity after working hard. also, her mother (i-2) and two sisters (ii-1 and -2) experienced similar recurrent episodes of weakness and sensory impairments after the application of minor pressure. the proband showed markedly reduced motor nerve conduction velocities (ncvs) with prolonged distal motor latencies. the median motor ncvs of the proband and her mother were 15.4 and 52.3 m / s, respectively (normal value in our laboratory 50.5 m / s). stimulation at the wrist, elbow, and above the elbow evoked motor compound muscle action potentials with normal amplitudes, and no conduction block was observed in either patient (fig. six microsatellite markers within 17p11.2-p12 (d17s921, d1s9b, d17s9a, d17s918, d17s4a, and d17s2230) were genotyped to determine the presence of duplication or deletion. we identified a 17p11.2-p12 deletion in patient 2 (ii-4), and diagnosed her as hnpp. however, the proband (iii-1) was determined as having a de novo duplication of paternal origin. the duplication contained both paternal haplotypes, indicating that the de novo mutation was caused by an unequal nonsister chromatid crossover at paternal meiosis. no other causative mutation in pmp22, mpz, gjb1, egr2, nefl, prx, or mfn2 was identified in this family. cmt disease and hnpp are genetically and phenotypically heterogeneous peripheral neuropathies.1 cmt1a duplication and hnpp deletion are due to a reciprocal unequal crossover event between flanking cmt1a - rep repeats and the pmp22 gene locates within the 17p11.2-p12 region.2 there are several lines of evidence that alterations in the gene dosage of pmp22 are the main cause of the cmt1a phenotype.3 namely, patients carrying one extra copy of pmp22 develop cmt1a, while patients with hnpp deletion have only one copy of pmp22.3 this is reflected in reduced mrna and protein levels in sural nerve biopsy samples from hnpp patients.6 myelin plays an important role in the saltatory impulse transmission along neuronal extensions and communication between neurons, and the level of pmp22 expression must be maintained within the critical range for proper peripheral nerve function.7 it is believed that cmt1a and hnpp are both demyelinating neuropathies, but their clinical, electrophysiological, and histopathological features are quite distinct.6 cmt1a patients with duplication develop slowly progressive sensorimotor length - dependent neuropathies associated with uniformly slow ncvs, and hnpp is characterized by recurrent episodes of focal entrapment neuropathies occurring as a result of trivial trauma or pressure.8 however, most hnpp patients exhibit conduction slowing only at sites of mechanical compression, but mild overlap of clinical features with cmt may lead patients with hnpp to be misdiagnosed as having cmt1a.6 moreover, all heterozygous patients with a pmp22 point mutation (t118 m) had clinical and electrophysiological features of a neuropathy similar to hnpp by a partial loss of pmp22 function.7 cmt is frequently classified into type 1 (the demyelinating form ; cmt1) and type 2 (the axonal form ; cmt2).1 the primary defect in cmt2 patients is neuronal, with them exhibiting slightly reduced (> 38 or even normal ncvs.9 recent studies have defined intermediate - type cmt, which has electrophysiological and pathological features of both cmt1 and cmt2.10 before performing the mutation analysis, we considered this family to be of intermediate - type cmt, because the median ncv of the proband (iii-1 ; fig. 2-a) was markedly reduced, whereas that of her mother (ii-4 ; fig. however, it was subsequently revealed that the heterogeneous phenotypes were due to different genetic defects : a duplication of 17p11.2-p12 was detected in the proband (iii-1), whereas a deletion of the same region was found in her mother (ii-4). although the occurrence of cmt1a duplication and hnpp deletion in this family might be purely coincidental, the presence of both of them in the same inherited neuropathy family might have resulted as misdiagnosis of intermediate - type cmt. in addition, patient 2 (ii-1) had a de novo cmt1a duplication, and this was attributed to unequal nonsister chromatid exchange during spermatogenesis.4 the prevalence of de novo duplications in cmt1a is much higher for those of paternal origin than for those of maternal origin, which may be due to the recombination fractions for the region being larger in females.5 | charcot - marie - tooth disease type 1a (cmt1a) is associated with duplication of chromosome 17p11.2-p12, whereas hereditary neuropathy with liability to pressure palsies (hnpp), which is an autosomal dominant neuropathy showing characteristics of recurrent pressure palsies, is associated with 17p11.2-p12 deletion. an altered gene dosage of pmp22 is believed to the main cause underlying the cmt1a and hnpp phenotypes. although cmt1a and hnpp are associated with the same locus, there has been no report of these two mutations within a single family. we report a rare family harboring cmt1a duplication and hnpp deletion. |
mushrooms have been considered as ingredient of gourmet cuisine across the globe ; especially for their unique flavor and have been valued by humankind as a culinary wonder. more than 2,000 species of mushrooms exist in nature, but around 25 are widely accepted as food and few are commercially cultivated. mushrooms are considered as a delicacy with high nutritional and functional value, and they are also accepted as nutraceutical foods ; they are of considerable interest because of their organoleptic merit, medicinal properties, and economic significance [1, 2 ]. however, there is not an easy distinction between edible and medical mushrooms because many of the common edible species have therapeutic properties and several used for medical purposes are also edible. the most cultivated mushroom worldwide is agaricus bisporus, followed by lentinus edodes, pleurotus spp., and flammulina velutipes. mushrooms production continuously increases, china being the biggest producer around the world [1, 4, 5 ]. however, wild mushrooms are becoming more important for their nutritional, sensory, and especially pharmacological characteristics. mushrooms could be an alternative source of new antimicrobial compounds, mainly secondary metabolites, such as terpenes, steroids, anthraquinones, benzoic acid derivatives, and quinolones, but also of some primary metabolites like oxalic acid, peptides, and proteins. lentinus edodes is the most studied species and seems to have an antimicrobial action against both gram - positive and gram - negative bacteria. they have a great nutritional value since they are quite rich in protein, with an important content of essential amino acids and fiber, poor fat but with excellent important fatty acids content (table 1). moreover, edible mushrooms provide a nutritionally significant content of vitamins (b1, b2, b12, c, d, and e) [7, 8 ]. thus, they could be an excellent source of many different nutraceuticals and might be used directly in human diet and to promote health for the synergistic effects of all the bioactive compounds present [913 ]. a large variety of mushrooms have been utilized traditionally in many different cultures for the maintenance of health, as well as in the prevention and treatment of diseases through their immunomodulatory and antineoplastic properties. in the last decade, the interest for pharmaceutical potential of mushrooms has been increased rapidly, and it has been suggested that many mushrooms are like mini - pharmaceutical factories producing compounds with miraculous biological properties [5, 14 ]. in addition, the expanded knowledge of the molecular basis of tumorigenesis and metastasis has given the opportunity for discovering new drugs against abnormal molecular and biochemical signals leading to cancer. more than 100 medicinal functions are produced by mushrooms and fungi and the key medicinal uses are antioxidant, anticancer, antidiabetic, antiallergic, immunomodulating, cardiovascular protector, anticholesterolemic, antiviral, antibacterial, antiparasitic, antifungal, detoxification, and hepatoprotective effects ; they also protect against tumor development and inflammatory processes [1619 ]. numerous molecules synthesized by macrofungi are known to be bioactive, and these bioactive compounds found in fruit bodies, cultured mycelium, and cultured broth are polysaccharides, proteins, fats, minerals, glycosides, alkaloids, volatile oils, terpenoids, tocopherols, phenolics, flavonoids, carotenoids, folates, lectins, enzymes, ascorbic, and organic acids, in general. polysaccharides are the most important for modern medicine and -glucan is the best known and the most versatile metabolite with a wide spectrum of biological activity [5, 16, 17, 20 ]. a balanced diet is the supporting treatment for the prevention of illness and especially against oxidative stress. in this context, mushrooms have a long history of use in the oriental medicine to prevent and fight numerous diseases. nowadays, mushroom extracts are commercialized as dietary supplements for their properties, mainly for the enhancement of immune function and antitumor activity [3, 9, 11, 17, 2126 ]. in this work, we aimed to review the nutritional value as well as the chemical and nutraceutical composition, and commercial potentialities of the most cultivated edible mushrooms worldwide. the nutritional value of edible mushrooms is due to their high protein, fiber, vitamin and mineral contents, and low - fat levels [8, 10 ]. they are very useful for vegetarian diets because they provide all the essential amino acids for adult requirements ; also, mushrooms have higher protein content than most vegetables. besides, edible mushrooms contain many different bioactive compounds with various human health benefits [27, 28 ]. it is important to remark that the growth characteristics, stage and postharvest condition may influence the chemical composition and the nutritional value of edible mushrooms. also, great variations occur both among and within species [29, 30 ]. mushrooms contain a high moisture percentage that ranges between 80 and 95 g/100 g, approximately. as above mentioned, edible mushrooms are a good source of protein, 200250 g / kg of dry matter ; leucine, valine, glutamine, glutamic and aspartic acids are the most abundant. mushrooms are low - calorie foods since they provide low amounts of fat, 2030 g / kg of dry matter, being linoleic (c18:2), oleic (c18:1) and palmitic (c16:0) the main fatty acids. edible mushrooms contain high amounts of ash, 80120 g / kg of dry matter (mainly potassium, phosphorus, magnesium, calcium, copper, iron, and zinc). carbohydrates are found in high proportions in edible mushrooms, including chitin, glycogen, trehalose, and mannitol ; besides, they contain fiber, -glucans, hemicelluloses, and pectic substances. additionally, glucose, mannitol, and trehalose are abundant sugars in cultivated edible mushrooms, but fructose and sucrose are found in low amounts. mushrooms are also a good source of vitamins with high levels of riboflavin (vitamin b2), niacin, folates, and traces of vitamin c, b1, b12, d and e. mushrooms are the only nonanimal food source that contains vitamin d and hence they are the only natural vitamin d ingredients for vegetarians. wild mushrooms are generally excellent sources of vitamin d2 unlike cultivated ones ; usually cultivated mushrooms are grown in darkness and uv - b light is needed to produce vitamin d2 [3, 8, 2934 ]. in addition to the nutritional components found in edible mushrooms, some have been found to comprise important amounts of bioactive compounds. the content and type of biologically active substances may vary considerably in edible mushrooms ; their concentrations of these substances are affected by differences in strain, substrate, cultivation, developmental stage, age, storage conditions, processing, and cooking practices [810 ]. the bioactive substances found in mushrooms can be divided into secondary metabolites (acids, terpenoids, polyphenols, sesquiterpenes, alkaloids, lactones, sterols, metal chelating agents, nucleotide analogs, and vitamins), glycoproteins and polysaccharides, mainly -glucans. new proteins with biological activities have also been found, which can be used in biotechnological processes and for the development of new drugs, including lignocellulose - degrading enzymes, lectins, proteases and protease inhibitors, ribosome - inactivating proteins, and hydrophobins. in china, many species of edible wild - grown mushrooms, that is tricholoma matsutake, lactarius hatsudake, boletus aereus, are appreciated as food and also in traditional chinese medicine. the rich amount of proteins, carbohydrates, essential minerals, and low energy levels contributes to considering many wild - grown mushrooms as good food for the consumer, which can virtually be compared with meat, eggs, and milk. numerous bioactive polysaccharides or polysaccharide - protein complexes from medicinal mushrooms appear to enhance innate and cell - mediated immune responses and exhibit antitumor activities in animals and humans. a wide range of these mushroom polymers have been reported previously to have immunotherapeutic properties by facilitating growth inhibition and destruction of tumor cells. several of the mushroom polysaccharide compounds have proceeded through clinical trials and are used extensively and successfully in asia to treat various cancers and other diseases. polysaccharides are the best known and most potent mushroom derived substances with antitumor and immunomodulating properties. data on mushroom polysaccharides have been collected from hundreds of different species of higher basidiomycetes ; some specific carbohydrates with these properties have been quantified in different mushrooms : rhamnose, xylose, fucose, arabinose, fructose, glucose, mannose, mannitol, sucrose, maltose, and trehalose (table 2) [11, 15, 38, 39 ]. the antitumor polysaccharides isolated from mushrooms are acidic or neutral, with strong antitumor action and differ significantly in their chemical structures. a wide range of glycans extending from homopolymers to highly complex heteropolymers exhibits antitumoral activity. mushroom polysaccharides have antitumor action by activation of the immune response of the host organism, in other words, mushroom polysaccharides do not directly kill tumor cells. these compounds prevent stress on the body and they may produce around 50% reduction in tumor size and prolong the survival time of tumor bearing mice [39, 40 ]. -glucans are the main polysaccharides found in mushrooms and around half of the fungal cell wall mass is constituted by -glucans. this is important for the industry because many of them are excreted into the cell growth medium, making their recovery, purification and chemical characterization very simple [4143 ]. -glucans are responsible for anticancer, immunomodulating, anticholesterolemic, antioxidant, and neuroprotective activities of many edible mushrooms. also, they are recognized as potent immunological stimulators in humans, and it has been demonstrated their capacity for treating several diseases. natural products with fungal -glucans have been consumed for thousands of years and they have long been considered to improve general health. -glucans are not synthesized by humans and they are not recognized by human immune systems as self - molecules ; as a result they induce both innate and adaptive immune responses. fungal -glucans are notably beneficial to humans ; they markedly stimulate the human immune system and protect from pathogenic microbes and from harmful effects of environmental toxins and carcinogens that impaired immune systems. they also protect from infectious diseases and cancer and aid patients recovery from chemotherapy and radiotherapy. besides, these compounds are also beneficial to middle - age people, people with active and stressful lifestyles, and athletes. a large variability can be observed in mushroom species and their concentration ranges from 0.21 to 0.53 g/100 g dry basis [20, 50 ]. -glucans are well known for their biological activity, specifically related to the immune system. hence, activating and reinforcing the host immune system seem to be the best strategy for inhibiting the growth of cancer cells [17, 51 ]. bioactive proteins are an important part of functional components in mushrooms and also have great value for their pharmaceutical potential. mushrooms produce a large number of proteins and peptides with interesting biological activities such as lectins, fungal immunomodulatory proteins, ribosome inactivating proteins, antimicrobial proteins, ribonucleases, and laccases. lectins are nonimmune proteins or glycoproteins binding specifically to cell surface carbohydrates and in the past few years many mushroom lectins have been discovered. they have many pharmaceutical activities and possess immunomodulatory properties, antitumoral, antiviral, antibacterial, and antifungal activity. some of them exhibit highly potent antiproliferative activity toward some tumor cell lines (human leukemic t cells, hepatoma hep g2 cells, and breast cancer mcf7 cells) [52, 54 ]. fungal immunomodulatory proteins are a new family of bioactive proteins isolated from mushrooms, which have shown a potential application as adjuvants for tumor immunotherapy mainly due to their activity in suppressing tumor invasion and metastasis. xu. published an extensive and comprehensive review about bioactive proteins in mushrooms. polyunsaturated fatty acids are mostly contained in edible mushrooms ; thus, they may contribute to the reduction of serum cholesterol. it is noteworthy that transisomers of unsaturated fatty acids have not been detected in mushrooms (table 3) [3, 9 ]. it has been observed that a diet rich in sterols is important in the prevention of cardiovascular diseases. tocopherols, found in the lipidic fraction, are natural antioxidants because they act as free radical scavenging peroxyl components produced from different reactions. these antioxidants have high biological activity for protection against degenerative malfunctions, cancer, and cardiovascular diseases. linoleic acid, an essential fatty acid to humans, takes part in a wide range of physiological functions ; it reduces cardiovascular diseases, triglyceride levels, blood pressure, and arthritis [11, 30, 38, 56 ]. phenolic compounds are secondary metabolites possessing an aromatic ring with one or more hydroxyl groups, and their structures can be a simple phenolic molecule or a complex polymer. they exhibit a wide range of physiological properties, such as antiallergenic, antiatherogenic, anti - inflammatory, antimicrobial, antithrombotic, cardioprotective, and vasodilator effects. the main characteristic of this group of compounds has been related to its antioxidant activity because they act as reducing agents, free radical scavengers, singlet oxygen quenchers, or metal ion chelators [11, 38, 57 ]. phenolic compounds provide protection against several degenerative disorders, including brain dysfunction, cancer, and cardiovascular diseases. this property is related to their capacity to act as antioxidants ; they can scavenge free radicals and reactive oxygen species. the process of oxidation is essential for living organisms ; it is necessary for the production of energy. evaluated total phenolic and flavonoid contents in eight types of edible mushrooms (agaricus bisporus, boletus edulis, calocybe gambosa, cantharellus cibarius, craterellus cornucopioides, hygrophorus marzuolus, lactarius deliciosus, and pleurotus ostreatus). these authors concluded that mushrooms contain 16 mg of phenolics / g of dried mushroom and the flavonoid concentrations ranged between 0.9 and 3.0 mg / g of dried matter ; the main flavonoids found were myricetin and catechin. b. edulis and a. bisporus presented the highest content of phenolic compounds, while l. deliciosus showed a high amount of flavonoids and a. bisporus, p. ostreatus, and c. gambosa presented low levels. reported protocatechuic, p - hydroxybenzoic, p - coumaric and cinnamic acids in the phenolic fraction in five wild mushrooms from northeastern portugal. a. bisporus, from the agaricus genera, is the most cultivated mushroom worldwide (figure 1). this group of edible mushrooms is nowadays widely used and studied for its medicinal and therapeutic properties [40, 63, 64 ]. a lectin from a. bisporus and a protein from a. polytricha have been found to be potent immune stimulants ; thus, these macromolecules may be considered for pharmaceutical utilization and these fungi may be classified as healthy food. a. bisporus extract has been shown to prevent cell proliferation in breast cancer [5, 65, 66 ]. a. blazei is an edible mushroom native to brazil and it has been cultivated especially in japan. it is a very popular basidiomycete known as sun mushroom, and at these days it is consumed globally as food or in tea due to its medicinal properties. its fruit bodies exhibit antimutagenic, anticarcinogenic, and immunostimulative activities [67, 68 ] ; its extracts have also shown immunomodulatory, anticarcinogenic, and antimutagenic properties. additionally, it has been reported that this mushroom blocks the liver lipid peroxidation. al - dbass. concluded that a. blazei is a natural source of antioxidant compounds and has hepatoprotective activities against liver damage. on the other hand, hakime - silva. reported that the aqueous extract of this fungus is a possible source of free radical scavengers and stated that this fungus can be used as a pharmacological agent against oxidative stress and as a nutritional source. also, it is known that this fungus is rich in -glucans, steroids, tocopherols, and phenolic compounds [30, 63, 71 ]. moreover, liquid extracts of this fungus inhibit cell proliferation in prostate cancer cells and oral supplementation suppressing significantly tumor growth without inducing adverse effects. a. blazei has been used as an adjuvant in cancer chemotherapy and various types of antileukemic bioactive components have been extracted from it [5, 67 ]. in 2013, carneiro. reported powder formulations from a. blazei and l. edodes with proteins, carbohydrates, and unsaturated fatty acids. these formulations may be used in low - calorie diets and have shown high antioxidant activity with high content of tocopherols and phenolic compounds. in view of the previous studies, this fungus has been used as a healthy food for the prevention of a range of illnesses including cancer, diabetes, arteriosclerosis, and chronic hepatitis [70, 72 ]. a. subrufescens is called the almond mushroom for its almond taste, and it is cultivated in the us and has been incorrectly referred as a. blazei. it produces various bioactive compounds that have potential to treat many diseases and has been used as a medicinal food for the prevention of cancer, diabetes, hyperlipidemia, arteriosclerosis, and chronic hepatitis. some of its beneficial properties are the reduction of tumor growth, antimicrobial and antiviral activities, immunostimulatory and antiallergy effects. the bioactive compounds isolated from this mushroom are mainly based on polysaccharides such as riboglucans, -glucans, and glucomannans. the antitumor activity has been found in lipid fractions, that is, ergosterol [63, 72, 73 ]. shiitake mushroom has been used for many years to investigate functional properties and to isolate compounds for pharmaceutical use ; this is because of its positive effects on human health (figure 2). it has been utilized to alleviate the common cold for hundreds of years and some scientific evidence has supported this belief. have provided experimental information about the aqueous extracts of l. edodes as potential sources of antioxidant and anticancer compounds. pizzoferrato reported that l. edodes contains high levels of -glucans in the soluble fraction of dietary fiber. shiitake produces lentinan and -glucan that suppress leukemia cell proliferation and have antitumor and hypocholesterolemic activity [5, 7478 ]. lentinan is used in clinic assays as adjuvant in tumor therapy and specifically in radiotherapy and chemotherapy. on the other hand, it has been reported that lentinan enhances host resistance against infections by bacteria, fungi, parasites, and virus ; it also promotes nonspecific inflammatory responses, vascular dilation, hemorrhage - inducing factors activation, and generation of helper and cytotoxic t cells [17, 74, 79, 80 ]. in other studies, l. edodes exhibited capacity to inhibit the growth of mouse sarcoma, probably due to the presence of an unspecified water - soluble polysaccharide. another edible mushroom is l. polychrous, found in northern and north - eastern thailand, which is used as medicine in diseases like dyspepsia or envenomation caused by snake or scorpion. the methanolic extract and crude polysaccharides have antioxidative activity and inhibitory effect on cell proliferations of breast cancer [8183 ]. additionally, mycelial extracts from this mushroom have antiestrogenic activity, resulting from a new polyhydroxyoctane and several ergostanoids. this genus, also known as oyster mushrooms, has approximately 40 species (all are commonly edible and available) (figure 3). in addition to their nutritional value, they possess medicinal properties and other beneficial effects and health - promoting effects. pleurotus species have been used by human cultures all over the world for many years [17, 8589 ]. these species have been used as medicinal mushrooms for long time since they contain several compounds with important pharmacological / nutraceutical properties. some of these substances are lectins with immunomodulatory, antiproliferative, and antitumor activities ; phenolic compounds with antioxidant activities ; and polysaccharides (polysaccharopeptides and polysaccharide proteins) with immunoenhancing and anticancer activities. -glucans isolated from pleurotus pulmonarius demonstrated an anti - inflammatory response in rats with colitis, and p. ostreatus inhibited leukocyte migration to acetic acid - injured tissues. pleurotus has also been reported with hematological, antiviral, antitumor, antibacterial, hypocholesterolic and immunomodulatory activities, and antioxidant properties [17, 86, 9094 ]. reported the stimulation of macrophages with different concentrations of the heteroglycan isolated from p. ostreatus, and lavi. and tong. reported antiproliferative and proapoptotic effects on colon cancer cells from an aqueous polysaccharide extract. in addition, jedinak. concluded that the edible oyster mushroom may be considered a functional food due to its anti - inflammatory activity and potential to control inflammation. moreover, p. ostreatus exhibits hypocholesterolemic effect on rats with normal cholesterolemia or hypercholesterolemia and hereditary cholesterol disorders. other authors reported some species of pleurotus with this hypocholesterolemic effect as well. according to manzi and pizzoferrato they also concluded that -glucans in mushrooms are distributed in the soluble and insoluble dietary fraction. p. citrinopileatus, p. djamor, p. eryngii, p. flabellatus, p. florida, p. ostreatus, and p. sajor - caju were evaluated by mishra.. the authors concluded that p. eryngii had the highest contents of phenolics, followed by p. djamor. besides, p. eryngii had a better antioxidant activity and p. citrinopileatus had more ascorbic acid and chelating activity. kanagasabapathy. reported antitumor effects and antioxidant properties by p. sajor - caju. the aqueous and butanol extracts exhibited the highest antioxidant activity and corresponded to the total phenolic content. also, a ribonuclease from p. sajor - caju presented antimicrobial, antimutagenic, and antiproliferative activities. however, the antiproliferative activity of this fungus may result from its specific proteins, terpenoids, steroids, fatty acids, and phenolic compounds. on the other hand, finimundy. reported evidence that p. sajor - caju is a potential source of antioxidant and anticancer compounds. water - soluble polysaccharides extracted from p. tuber - regium, a novel edible mushroom, showed effective antiproliferative activity against human leukemia cells and induced apoptosis in hl-60 cells [5, 99 ]. besides, li. it contains 15.4 g of protein and 33.3 g of dietary fiber/100 g of mushroom (dry weigh basis) and it also has important amounts of carbohydrates. it is rich in minerals like magnesium (67.64 mg/100 g dry weight) and potassium (1,345.7 mg/100 g dry weight). the aqueous and ethanolic extracts from p. giganteus have shown antioxidant, genotoxic, and liver protective properties and have a high effect on neuronal differentiation and neurite outgrowth. the high potassium level in the fruiting bodies and the presence of bioactive compounds, mainly triterpenoids, could be responsible for the neuroactivity [101, 102 ]. the mushroom of immortality, commonly known as lingzhi or reishi, has been used in traditional chinese medicine to improve health and longevity for thousands of years, as well as in the treatment of neurasthenia, hypertension, hepatopathy, and carcinoma (figure 4). it is one of the most popular medicinal mushrooms in china, japan, and korea. it has been under modern biochemical and pharmacological research during the last decades [103, 104 ]. modern pharmacological tests have also demonstrated some important characteristics of this fungus, such as immunomodulating, antiallergic, antiradiation, antitumor, anti - inflammatory, antiparasitic, and antioxidant properties. some benefits for the cardiovascular, respiratory, endocrine, and metabolic systems have also been described [40, 105, 106 ]. in asia, ganoderma has been administered for centuries as treatment for cancer ; it exhibits anticancer effect alone or in combination with chemotherapy and radiotherapy. ganoderma decreases viability of human cancer cells, induces cell apoptosis, inhibits cell proliferation, suppresses the motility of invasive breast and prostate cancer cells, and prevents the onset of various types of cancer [107111 ]. also, chen and zhong reported the inhibition of tumor invasion, metastasis and cell adhesion, promotion of cell aggregation, and suppression of cell migration in human colon tumor cell lines. additionally, ye. reported antitumor action in vitro against mouse lymphocytic leukemia, and lai. water - soluble polysaccharides from ganoderma act over more than 20 types of cancer and strongly inhibit tumor growth. the major biologically active polysaccharides from ganoderma are -glucans, and the anticancer and antimetastatic activities are due to its polysaccharides and triterpenoid components. it also contains a large number of proteins and peptides with biological activities, such as lectins, ribosome inactivating proteins, antimicrobial proteins, ribonucleases, and laccases, which are important for life activity and show immunomodulatory and antitumor effects as well [39, 40, 52, 104, 106, 115 ]. first, it does not produce any toxicity or side effects ; second, it does not act on a specific organ ; and third, it promotes the improvement of normalization of the organ function. modern pharmacological and clinical trials have demonstrated that this fungus shows a significant effect on the prevention and treatment of various diseases, especially cancer, including immunomodulation, induction of cytokine production, antiallergic, antiradiation, antitumor, anti - inflammatory, antiparasitic, and antioxidant effects, as well as benefits for the cardiovascular, respiratory, endocrine, and metabolic systems [40, 104106 ]. a large collection of scientific information on bioactive components and pharmacological properties, mainly on the anticancer potential of ganoderma, is available ; it is focused on the anticancer effect, regulation of cell cycle, and cell signaling [52, 103, 106, 116120 ]. moreover, weng and yen studied the inhibitory activity against invasive and metastatic behaviors (i.e., adhesion, migration, and angiogenesis) in various cancer cells in vitro or implanted in mice. nowadays, ganoderma is recognized as an alternative adjuvant in the treatment of leukemia, carcinoma, hepatitis, and diabetes, as well as an immune system enhancer with health benefits. in general, it is safe to be used for a long period of time. the dried powder and aqueous / ethanol extracts of g. lucidum are used worldwide as dietary supplement. boh studied around 270 patents for fruit bodies and mycelia cultivation methods of ganoderma lucidum, basidiomycete mushroom with strong anticancer effects. boh concluded that the anticancer activity of this fungus may be attributed to at least five groups of mechanisms : (1) activation / modulation of the immune response of the host, (2) direct cytotoxicity to cancer cells, (3) inhibition of tumor - induced angiogenesis, (4) inhibition of cancer cells proliferation and invasive metastasis behaviour, and (5) carcinogens deactivation with protection of cells. u. maydis belongs to the ustilaginales order that includes semiobligate biotrophic plant pathogenic fungi that infects only maize and its progenitor plant teosinte (zea mays). it is a heterothallic fungus with a dimorphic life cycle, saprophytic and a parasitic phase ; in nature, the pathogenic and sexual development are inseparable. also, u. maydis has been established as a robust pathogenic model for studying fungi and fungi - plant relationships, especially because the morphological transitions throughout its life cycle, easy culture, genetic manipulation in the laboratory, mating type, biotrophic host interaction, genetic properties to elucidate the molecular mechanisms of the interaction between plant and pathogen, and the severe disease symptoms that it induces in infected maize. on the other hand, u. maydis is responsible for the corn smut, characterized by the formation of galls or tumors, mainly in ears. these ear galls have been used as food in mexico since pre - columbian times. cuitlacoche or huitlacoche is the aztec name given to these young, fleshy, and edible galls (figure 5). in mexico, it has been traditionally prized and many hundreds of tons of fresh, prepared, or processed huitlacoche are sold annually. nowadays, it is a culinary delight for international chefs and has been accepted as a food delicacy in several countries and introduced into countless worldwide markets in countries like japan, china, and some of the european community, as france, spain, and germany. also, in the united states there has been a great interest to produce huitlacoche due to an emerging acceptance by the north american public, who noticed it as a gourmet food and now can be purchased on the internet at high prices. in addition to its unique flavor, huitlacoche has been identified as a high - quality functional food and could be included into the daily diet for its attractive characteristics, selected nutrients, valuable compounds, and nutraceutical potential. the protein content of huitlacoche varies from 9.7 to 16.4% (wet basis) and it is similar or sometimes superior to other edible mushrooms and definitely superior to the maize protein content (10%). therefore, huitlacoche may be proposed as an alternative protein source for vegetarian diets in the same way as other edible mushrooms have been suggested. huitlacoche contains almost all essential amino acids, lysine (6.37.3 g/100 g protein) being one of the most abundant. other abundant amino acids include serine, glycine, aspartic and glutamic acid, which collectively account for 44.3 to 48.9% of total amino acids. the high content of essential fatty acids also suggests an interesting nutritional value for huitlacoche ; some important fatty acids are oleic and linoleic acids (54.5 to 77.5%) [124, 125 ]. huitlacoche produced under different conditions had high concentrations of selected nutrients and compounds with nutraceutical potential, which showed variations due to maize genotype, stage of development, and cooking process. valdez - morales. identified eight monosaccharides and eight alditols in huitlacoche ; glucose and fructose were the most abundant, constituting approximately 81% of the total carbohydrates. also, huitlacoche contains, within its dietary fiber, homoglycans and heteroglycans, similar to those found in other edible mushrooms (table 4). the content of -glucans in huitlacoche is higher (20120 mg / g of huitlacoche, in dry weight) than that reported for corn (0.53.8 mg / g) and similar to other edible mushrooms. they can also be antioncogenic due to their protector effect against genotoxic compounds and because of their antiangiogenic effect. these authors also analyzed different maize genotypes to produce huitlacoche and found differences in -glucans concentrations and concluded that creole corn showed the highest amounts ; this maize was proposed for huitlacoche production in mexico. besides, they concluded that the amount of -glucans in huitlacoche is higher than that reported in corn and it is similar to other edible mushrooms. it has been confirmed that higher basidiomycetes contain bioactive substances that possess hyperlipidemic, antitumoral, immunomodulating, anti - inflammatory, antimutagenic, antiatherogenic, hypoglycemic, and other health - promoting properties. valdez - morales. also reported antimutagenic capacity (41.0 to 76.0%) in huitlacoche, but without assessing the compounds that confer this activity. they also revealed that the total phenol concentration in huitlacoche is elevated and within that reported for other edible fungi (table 5). huitlacoche has been characterized as a high - quality nutraceutical food as well as an attractive ingredient to enrich other dishes, mainly for its extraordinary flavor and exceptional quality. the introduction of this food into the international market requires the development of techniques for massive production during the whole year, particularly because this parasitic fungus only grows in maize ears. an efficient method to inoculate maize plants with u. maydis began in the 18th century when it was unsuccessfully attempted to demonstrate the causal relationship between common smut and maize. many studies have been focused on ear infection, and the most important finding was observed in the silk - channel inoculation procedure, resulting with a much higher incidence of ear galls than natural infection. however, many factors are involved in this process and the efficient production of huitlacoche by inoculating silks with u. maydis may require accurate timing of inoculation and control of pollination to maximize the number of kernels infected and the huitlacoche yield. trametes versicolor has been shown to promote chemopreventive potential ; it inhibits growth of several human cancer cell lines, acts as adjuvant in breast cancer prevention and has a significant ic50 value [127, 128 ]. grifola frondosa is promoted as anticancer agent, particularly on human gastric carcinoma, such effect results from the induction of cell apoptosis and could significantly accelerate the anticancer activity [129, 130 ]. in this context, it could be mentioned that cordyceps militaris has several beneficial effects and it is used for multiple medicinal purposes. it acts as an antitumor, antiproliferative, antimetastatic, insecticidal, and antibacterial compound. more than 21 clinically approved beneficial effects for human health have been found in this mushroom [131, 132 ]. extracts of c. militaris have been used for its immnunomodulatory and anti - inflammatory effects. besides, it is also a cancer preventive material and is effective against chronic bronchitis, influenza a, and viral infections. cordyceps sinensis contains substances called cordycepin, cordycepic acid, with therapeutic applications like the effects of increased oxygen utilization, atp production, and stabilization of blood sugar metabolism. besides, it has antibacterial function, reduces asthma, and lowers blood pressure. on the other hand, it has been reported as organ protector, as well as with a protective effect for heart, liver, and kidney diseases. also, c. sinensis has sedative effect on the central nervous system. antrodia cinnanomea is a medicinal mushroom native to taiwan with various functional compounds and a total of 105 taiwan patent applications. different commercial products are made with this mushroom and it has been used to treat food and drug intoxication, diarrhea, abdominal pain, hypertension, skin itching, and cancer. panellus serotinus (mukitake) is extremely appreciable in japan as one of the most delicious edible mushrooms. the use of this fungus helps to prevent the development of nonalcoholic fatty liver disease. a. polytricha has potential medicinal properties and is considered effective to reduce ldl cholesterol and aortic atherosclerotic plaque ; it also has antitumor and anticoagulant activities. besides, a. auricula - judae is a popular ingredient in many chinese dishes ; it has been used as a blood tonic and has shown antitumor, hypoglycemic, anticoagulant, and cholesterol - lowering properties [137, 138 ]. flammulina velutipes is available as fresh or canned product and it is traditionally used for soups in china. it contains biologically active components such as dietary fiber, polysaccharides, and antioxidants, which reduce blood sugar, blood pressure, and cholesterol. several mushroom species have been pointed out as sources of bioactive compounds, in addition to their important nutritional value. the inclusion of whole mushrooms into the diet may have efficacy as potential dietary supplements. the production of mushrooms and the extraction of bioactive metabolites is a key feature for the development of efficient biotechnological methods to obtain these metabolites. it has been shown by a wide range of studies that mushrooms contain components with outstanding properties to prevent or treat different type of diseases. they present a low fat content and can be used in low - calorie diets, just like the mushrooms fruiting bodies. future studies into the mechanisms of action of mushroom extracts will help us to further delineate the interesting roles and properties of various mushroom phytochemicals in the prevention and treatment of some degenerative diseases. in view of the current situation, there are numerous potential characteristics and old and novel properties, provided by mushrooms with nutraceutical and health benefits, which deserve further investigations. | mushrooms have been consumed since earliest history ; ancient greeks believed that mushrooms provided strength for warriors in battle, and the romans perceived them as the food of the gods. for centuries, the chinese culture has treasured mushrooms as a health food, an elixir of life. they have been part of the human culture for thousands of years and have considerable interest in the most important civilizations in history because of their sensory characteristics ; they have been recognized for their attractive culinary attributes. nowadays, mushrooms are popular valuable foods because they are low in calories, carbohydrates, fat, and sodium : also, they are cholesterol - free. besides, mushrooms provide important nutrients, including selenium, potassium, riboflavin, niacin, vitamin d, proteins, and fiber. all together with a long history as food source, mushrooms are important for their healing capacities and properties in traditional medicine. it has reported beneficial effects for health and treatment of some diseases. many nutraceutical properties are described in mushrooms, such as prevention or treatment of parkinson, alzheimer, hypertension, and high risk of stroke. they are also utilized to reduce the likelihood of cancer invasion and metastasis due to antitumoral attributes. mushrooms act as antibacterial, immune system enhancer and cholesterol lowering agents ; additionally, they are important sources of bioactive compounds. as a result of these properties, some mushroom extracts are used to promote human health and are found as dietary supplements. |
although various training modalities can be applied in patients with chronic obstructive pulmonary disease (copd) (eg, resistance, in - water, and tai chi), aerobic stimulus has been investigated in several studies, predominantly due to the ease of application, prescription and, consequently, monitoring of this training modality.1 aerobic training provides clear improvement in the quality of life and increases in aerobic parameters and functional capacity, thereby improving factors such as dyspnea, exercise intolerance, and reduced quality of life, which are common symptoms in patients with copd.13 mucociliary clearance is deficient in patients with copd4,5 mainly due to mucus hypersecretion, which is related to a significant decrease in forced expiratory volume in the first second and severe coughing. this situation increases the risk of hospitalization.6 improvements in mucociliary clearance have been observed after acute aerobic stimulus, but the chronic effects are still controversial in healthy individuals.7,8 although knowledge about the response of mucociliary clearance to aerobic stimulus appears to be extremely important, no studies have investigated this parameter longitudinally in patients with copd. aerobic training has been related to significant improvements in autonomic modulation, suggesting better adaptation and efficiency of the cardiovascular system.9,10 in addition, relations between autonomic control and aerobic variables were also observed acutely in copd patients,11 highlighting that good aerobic conditioning is related to an increase in the vagal activity of heart rate control. however, the possible effects of different aerobic models on autonomic control remain unclear. in this context, aerobic training in patients with copd can be performed with continuous or interval efforts.1,12 continuous efforts are frequently applied 34 times per week, with different intensities and durations, whereas interval training sessions are applied 23 times per week, using effort periods lasting 30180 seconds, separated by passive or active recovery. when these aerobic training modalities (ie, continuous and interval) are applied separately, the training adaptations appear to be similar in patients with copd.12 moreover, to our knowledge, no studies have investigated the use of continuous and interval sessions in the same periodization in patients with copd. this approach may be more effective, principally by enabling the adaptations related to both models of aerobic training stimulus (ie, continuous and interval). in summary, aerobic exercise can be considered a good intervention in pulmonary rehabilitation, but more studies on mucociliary clearance responses and autonomic control adaptations from aerobic training are necessary, specifically when training is applied with continuous and interval stimulus in the same periodization. thus, the aim of this study was to investigate the effects of a 12-week aerobic training protocol with continuous and interval sessions on autonomic modulation, mucociliary clearance, and aerobic parameters in patients with copd. this study was characterized as a nonrandomized clinical trial which considered the effects of 12 weeks of aerobic training on autonomic modulation (evaluated through heart rate variability [hrv ]), mucociliary clearance, and aerobic function in patients with copd, according to the criteria established by the global initiative for obstructive lung disease (gold).13 the study was conducted in a public rehabilitation center, and patients presenting any of the following conditions were excluded : 1) not having been in smoking cessation for at least 1 year ; 2) the presence of severe pathological conditions and/or unstable heart diseases that could influence the physical activity ; 3) presenting diseases that could interfere in the systemic inflammatory process ; 4) unstable copd (ie, exacerbations and medication changes in the previous 30 days) ; 5) the use of home oxygen therapy ; and 6) individuals who had performed any kind of physical training program before participating in this study. the initial evaluation was performed in 31 patients, after which they were divided into two separate groups : an aerobic training group (at) and a control group (cg). the cg consisted of ten patients who chose not to participate in the training protocol due to transportation difficulties in arriving at the rehabilitation center, living in faraway places, or not being able to attend the 3-day weekly training. among them, four patients did not attend the final evaluations, one due to exacerbation and three patients for personal reasons. the aerobic training protocol started with 21 patients, eleven of whom did not complete the 12-week protocol as five patients exacerbated and six gave up for personal reasons (figure 1). medications used by the patients during the study were : 2 agonists (n=12), anticholinergics (n=6), diuretics (n=4), antagonists of angiotensin receptors (n=5), -blockers (n=1), benzodiazepines (n=1). all participants were previously informed about the procedures and aims of this study, and signed a consent form. all procedures utilized in this study were approved by the institutional ethics research committee (caae : 01114912.0.0000.5402). the experimental protocol consisted of patient identification, anthropometric evaluation, pulmonary function through spirometry, a cardiopulmonary test, evaluation of autonomic function by hrv, a mucociliary clearance test using the saccharin transit time (stt) test, and realization of 12 weeks of aerobic training. the participants were conventionally divided into at and no - training cg. evaluation periods were performed at baseline (m0) and after 12 weeks (m1) for both groups. to minimize interference with circadian rhythm and external factors, evaluations were individually performed in a room with temperature ranging from 21c to 23c9 and relative humidity between 50% and 60%, always in the morning period between 8 am and 12 pm. for all sessions, patients were instructed to : 1) avoid consuming caffeine for 24 hours before procedures ; 2) eat a light meal 2 hours before the tests ; 3) avoid drinking alcoholic beverages for at least 4 hours ; 4) avoid strenuous physical exercises the day prior to the session ; and 5) wear suitable and comfortable clothes for physical exercises. body weight and height were measured to obtain body mass index through the following formula : body weight (in kilograms) divided by height (meters). r / i 200, welmy, sao paulo, brazil) according to the recommendations described by lohman.14 pulmonary function assessment was carried out by a spirometry test (mir - spirobank 3.6 version spirometer ; mir, rome, italy). the results were interpreted according to the american thoracic society and european respiratory society rules.15 normality values were related to the brazilian population.16 for the prescription of aerobic exercise and evaluation of aerobic function, patients performed a maximal cardiopulmonary exercise test (inbrasport atl 2000 ; inbrasport, rio grande do sul, brazil) with an initial speed of 2.0 kmh, constant slope of 3%, and increments of 0.5 kmh every 2 minutes. the test was performed until voluntary exhaustion was reached.17 none of the patients presented clinical or electrocardiographic changes that prevented them from finishing the test. the following variables were monitored continuously : 1) heart rate (polar s810i, polar electro, kempele, finland) ; 2) arterial oxygen saturation (spo2%) (mindray pm 50 oximeter, mindray, sao paulo, brazil) ; and 3) subjective perception of effort.18 furthermore, ventilatory variables were obtained through the gas analyzer (vo2000, medical graphics, st. paul, mn, usa), which was calibrated before every test according to the supplier s specifications. the flow of average air was utilized in all tests, and gas samples were obtained every 10 seconds (aerograph, mi, usa). the peak oxygen uptake (vo2peak) was considered the highest oxygen consumption average of the final 30 seconds of the exercise (vo2). the speed related to vo2peak (vvo2peak) was considered to be the highest intensity reached during the test. in cases where the patient demonstrated exhaustion before the end of the stage, the vvo2peak was adjusted by the equation proposed by kuipers.19 moreover, the gas exchange threshold (get) was determined using the v - slope method, described by sue for copd patients. in the present study, the breakpoint of the vco2vo2 relationship was assumed as the get, observed during the incremental test. for the hrv analysis, heart rate was captured beat by beat through a heart rate monitor, polar s810i (polar electro), which had been properly validated.21 after explaining the necessary procedures for data collection, an elastic strap was positioned on the chest of each patient at the xiphoid process, and a heart rate receiver was placed on the wrist (polar electro). heart rate at rest was recorded for 20 minutes with the patient in a seated position. for the analysis of hrv indexes, 256 consecutive rr intervals were used. they were selected from the most stable portion and, in order to eliminate premature ectopic beats and artifacts, were submitted to digital filtering through polar precision performance sw software (version 4.01.029) supplemented by manual procedures. only series with more than 95% sinus beats were included in this study.22,23 indexes in the time and frequency domains were calculated. in the time domain, the following indexes were used : standard deviation of normal rr intervals (sdnn), expressed in milliseconds, and root mean square of the difference between the adjacent normal rr intervals in a time interval (rmssd), expressed in milliseconds.22,24 for hrv analysis in the frequency domain, low frequency (lf : 0.040.15 hz) and high frequency (hf : 0.150.4 hz) spectral components were used, in ms and normalized units (nu), which represent the relative value of each spectral component in relation to total power, less the very low frequency component and the ratio between these components (lf / hf ratio).22,24 in order to calculate these indexes, kubios version 2.0 software25 was used. for mucociliary clearance evaluation, the patients were seated with their heads in a straight position at 10. the stt started with the introduction of ~2.5 mg of granulated saccharin sodium through a plastic straw, under visual control, ~2 cm into the right nostril. at this moment, the timer was started, and patients were instructed not to walk, talk, cough, sneeze, scratch, or blow their nose. they were also instructed to swallow as little as possible until they could feel the flavor in their mouth. at this point, the patient advised the examiner, who then registered the time.2628 for the stt at baseline condition, the patients remained at rest for 20 minutes before starting the test in order to minimize the effects of the external environment on the nasal ciliary beat. all sessions were performed on a treadmill (inbrasport atl 2000, porto alegre, brazil). the training sessions were divided into three intensity zones : z1 : 50-minute sessions at an intensity corresponding to 60% of vvo2peak (continuous effort) ; z2 : 30-minute sessions at an intensity corresponding to 75% of vvo2peak (continuous effort) ; and z3 : formed by five efforts of 3 minutes performed at 100% of vvo2peak, separated by 1 minute of passive recovery (interval effort). the intensity of each mesocycle was adjusted according to the incremental test performed every 4 weeks. the first training mesocycle predominated in the sessions z1 (85.9% of total sessions ; 344 minutes), with a shorter time in z2 (14.1% of total sessions ; 57 minutes). in the second mesocycle, sessions were applied in z1 (34.5% of total sessions ; 138 minutes), but the predominance was in z2 (65.5% of total volume ; 262 minutes). the third mesocycle had sessions in z2 (32.5% of total volume ; 130 minutes) with predominance in z3 (67.5% of total volume ; 270 minutes). the intensity and different session durations between mesocycles were chosen on the basis of previously published studies.2931 figure 2 demonstrates the time spent in each intensity zone during the 12-week aerobic training protocol. initially, the analysis of data normality was performed and certified by the shapiro wilk test. asymmetric distribution was observed for vo2peak, normalized by weight (m0 in both at and cg ; p0.37). all aerobic variables increased significantly after 12 weeks for the at group (p0.21). at m1, the at group presented higher values compared to the cg for vo2peak (absolute and relative to weight values ; p=0.02 and 0.01, respectively), vvo2peak (p=0.04), and vo2 observed at get (p=0.01). table 3 shows the results of autonomic modulation evaluation in the time and frequency domains. an increase in the hf (ms) index after 12 weeks can be observed in the at (p=0.042). however, although a tendency was observed for rmssd (p=0.08), there was no difference for the other hrv parameters (p>0.13) in the at group. in addition, no changes were observed during 12 weeks either in the at (p=0.94) or in the cg (p=0.69) group. the main findings of this study demonstrate that 12 weeks of aerobic training applied with continuous and interval sessions induced a significant increase in aerobic parameters obtained through an incremental test. moreover, a positive influence on autonomic modulation was observed, evidenced by a significant increase in parasympathetic modulation. vo2peak is considered an index of maximum aerobic power, while vvo2peak can be seen as the index that better represents the association between aerobic power and movement economy.32 get is an important submaximal aerobic variable, mainly because it represents the highest intensity where lactate presents equilibrium in the blood.33 in addition, this index is frequently used in training prescription, monitoring, and performance prediction.34,35 several studies have demonstrated improvement in these parameters after execution of aerobic training,9,3638 which is essential in all copd stages due to its capacity to reduce dyspnea, improve functional capacity, and offer better quality of life for these patients.2,3 in this study, an important improvement in all aerobic variables was observed after the aerobic training. although on a smaller scale, a similar response was found in studies that applied traditional training methods.9,3638 it was also observed that after 12 weeks of aerobic training, there was a significant increase in hf (ms), which represents the parasympathetic component of autonomic modulation.39 in relation to other evaluated indices, no significant difference was observed before or after training. however, it was observed that hf (nu) increased after 12 weeks in the at and the lf (nu) index was reduced in relation to the lf / hf ratio, representing an increase in parasympathetic activity, a reduction in sympathetic activity, and an improvement in the sympathetic vagal balance. these data indicate that there was a positive impact on autonomic modulation in patients submitted to periodized aerobic training, suggesting better adaptation and efficiency of the cardiovascular system in these patients, considering the environmental and physiological stimulus required at every moment of daily life activities. this includes better respiratory capacity and performance responses during physical activity, which is essential to the rehabilitation process in the copd population.39 previous studies have also shown significant improvements in autonomic modulation after completion of physical training in patients with copd. camillo applied a protocol of aerobic resistance training combined with high intensity, after which a significant increase in the rmssd index was observed, with no changes in the spectral indices. borghi - silva9 after a 6-week aerobic training protocol, obtained significant increases in the rmssd index, a decrease in lf, and an increase in hf, analyzed in normalized units. a high level of aerobic fitness has been associated with hrv, probably due to increased vagal activity in controlling the heart rate.9,11 in relation to mucociliary clearance, there was no significant difference between periods. this finding corroborates the results found by salzano who concluded that aerobic training did not significantly affect mucociliary clearance. the study only demonstrated an acute effect, ie, an increase in mucociliary activity 15 minutes after the aerobic exercise, which can be correlated with increased ventilation and autonomic nervous system activity during the execution of exercise. nevertheless, this increase was not sustained when analyzed 75 minutes after the training, when it was observed that stt had returned to values close to the initial values. the chronic effects of aerobic training on mucociliary clearance in patients with copd are still not well established in the literature. salh conducted a study in lung disease patients with cystic fibrosis submitted to aerobic training on an ergometer cycle, and analyzed the quantity of mucus expectoration, which is directly related to mucociliary activity and clearance, after 2 months of home physical training. the results demonstrated weight increases in the collected mucus, although not a significant amount. the results shown by salh were similar to those in the present study as it was not possible to observe any influence of aerobic training on mucociliary clearance in patients with copd. previous studies have demonstrated that patients with copd presented loss and significant alterations in mucociliary clearance.4,5 afonso found that the mucus relative velocity of ciliary transport on frog palate was greater in the healthy individuals than in the copd and bronchiectasis groups : healthy group = 1.00.19 seconds, copd group = 0.910.17 seconds, and bronchiectasis group = 0.760.23 seconds.4 in contrast, such alterations were not detected in the present study, where patients presented preserved mucociliary function with normal baseline values.41 it is suggested that these levels are associated with the smoking cessation period of these patients, who were included in this study only after 1 year of smoking abstinence ; the patients presented a mean time of 9.5 years without smoking. it is known that there is repair and remodeling of the tissue of the respiratory tract in patients with copd, although mechanisms for such restructuring are still not clear.42 ramos found that there is reversibility of mucociliary function in smokers 15 days after smoking abstinence. therefore, smoking cessation is of the utmost importance as a fundamental component of pulmonary rehabilitation programs. the present proposal of linear periodized aerobic training resulted in significant aerobic improvements, in addition to positively influencing autonomic modulation in patients with copd. this enriches and provides additional support to discussions on the theme of training modalities for these individuals. furthermore, the present training proposal consisted of an additional option when compared to other copd training methods, as it allowed better adaptation of the neuromuscular system to support the burdens imposed by the variations in volume and training intensities. in this way, it differentiates itself from pulmonary rehabilitation programs widely recommended as part of copd treatment. this study concluded that 12-week periodized aerobic training positively influenced autonomic modulation and aerobic power in patients with copd ; however, no effect was observed on mucociliary clearance in these patients. in contrast to previous studies, the aerobic training applied was composed of both continuous and interval sessions. this approach was predominantly chosen because we expected lower levels of monotony due to the decrease in time expended in each zone of training (ie, z1 : easy continuous ; z2 : hard continuous ; z3 : heavy interval). thus, we believe that this study contributes to the training prescription applied to copd patients. however, the main limitation was the high loss of participants in both groups during the experiment, which highlights the need for more studies using this aerobic training prescription. | introductionpatients with chronic obstructive pulmonary disease (copd) exhibit aerobic function, autonomic nervous system, and mucociliary clearance alterations. these parameters can be attenuated by aerobic training, which can be applied with continuous or interval efforts. however, the possible effects of aerobic training, using progressively both continuous and interval sessions (ie, linear periodization), require further investigation.aimto analyze the effects of 12-week aerobic training using continuous and interval sessions on autonomic modulation, mucociliary clearance, and aerobic function in patients with copd.methodssixteen patients with copd were divided into an aerobic (continuous and interval) training group (at) (n=10) and a control group (cg) (n=6). an incremental test (initial speed of 2.0 kmh1, constant slope of 3%, and increments of 0.5 kmh1 every 2 minutes) was performed. the training group underwent training for 4 weeks at 60% of the peak velocity reached in the incremental test (vvo2peak) (50 minutes of continuous effort), followed by 4 weeks of sessions at 75% of vvo2peak (30 minutes of continuous effort), and 4 weeks of interval training (53-minute effort at vvo2peak, separated by 1 minute of passive recovery). intensities were adjusted through an incremental test performed at the end of each period.resultsthe at presented an increase in the high frequency index (ms2) (p=0.04), peak oxygen uptake (vo2peak) (p=0.01), vvo2peak (p=0.04), and anaerobic threshold (p=0.02). no significant changes were observed in the cg (p>0.21) group. neither of the groups presented changes in mucociliary clearance after 12 weeks (at : p=0.94 and cg : p=0.69).conclusiontwelve weeks of aerobic training (continuous and interval sessions) positively influenced the autonomic modulation and aerobic parameters in patients with copd. however, mucociliary clearance was not affected by aerobic training. |
developmental dysplasia of the hip comprises of disorders of hip development that present in different forms at different ages. the common etiology is excessive laxity of the hip capsule, which fails to maintain the femoral head within the acetabulum. in newborns, the syndrome consists of instability of the hip such that the femoral head can be displaced from the acetabulum. the hip may also rest in a dislocated position and be reducible. over time, the femoral head becomes fully dislocated and can not be reduced by changing the position of the hip. the syndrome may manifest later in childhood as a developmental dislocation of the hip (ddh) or in adolescence as a hip with poorly developed acetabular coverage ; the latter is termed acetabular dysplasia (ad). in general, infants are screened for ddh in the first 4 months of life by clinical examination. if the infant demonstrates limited abduction of the affected hip, further physical examination, plain radiography, and an ultrasound are performed. infants diagnosed with ddh are usually treated with a pavlik harness at the time of diagnosis. in addition, traction, closed reduction, and open reduction procedures are also selected as treatment options depending on the patient 's age. from 8% to 60% of patients with a history of treatment for ddh osteoarthritis (oa) is an age - related degenerative disease that is common in both middle - aged and older women. primary oa of the hip is an extremely rare condition in japan ; most patients have secondary oa due to ad and ddh. japanese patients with oa of the hip have been reported to have higher rates of dysplasia than american patients (46% vs 4.5%), and significantly worse dysplasia compared with that in british populations (mean center - edge angle of 37 vs 31). patients with ad have been reported to exhibit an abnormal morphology of the pelvis and have a high rate of comorbid spinal congenital anomalies, such as spina bifida occulta. in a recent study, bone mineral density was reported to be higher in patients with ad than that in normal controls. these reports suggest there is a genetic link to the development of ad. in this study, to investigate the relationship between ad and ddh, we examined the percentage of ad patients with hip pain at skeletal maturity also having a history of ddh in infancy and the correlation between the severity of ad at skeletal maturity and a history of treatment for ddh. this retrospective diagnostic study received permission for publication from the institutional review board of our institute. for this study, we selected patients diagnosed with ad of the hip as well as pre - arthritis or early - stage osteoarthritis. patients with advanced or end - stage osteoarthritis were excluded from the study so that we could more accurately evaluate ad by excluding osteophyte formation. patients less than 16 years of age were not included to exclude premature hip joints and those older than 60 years were excluded because of the difficulty of confirming a history of treatment for ddh. in addition, patients with a history of hip osteotomy, hip dislocation into the gluteal muscles, secondary post - traumatic osteoarthritis, inflammatory rheumatic disease, osteonecrosis, or infectious diseases were excluded from the study. all patients in the study visited our hospital for consultation regarding hip joint pain between 2009 and 2012. a total of 245 patients were radiographically examined and questioned for any history of ddh treatment in infancy. the study population comprised 226 women and 19 men with a mean age at examination of 40.7 years (range 1759 years). study parameters evaluated were center - edge angle (ce angle), acetabular head index (ahi), acetabular angle, and acetabular roof angle (figure 1). ad was defined as a ce angle less than 20, ahi less than 75%, acetabular angle more than 45, or acetabular roof angle more than 15 on anteroposterior radiographs. when bilateral ad was observed in a patient, the joint with a higher degree of ad diagram showing the radiological parameters (a, center - edge angle ; b, acetabular angle ; c, acetabular roof angle ; acetabular head index (ahi) = a / b 100). osteoarthritis of the hip was classified into the following four stages : pre - arthritis stage with no osteoarthritic change ; early stage with narrowing of the joint space associated with sclerosis of the subchondral bone ; advanced stage with partial disappearance of the joint space and evidence of cystic radiolucencies and osteophytes, and end stage with almost a total disappearance of the joint space and marked osteophyte formation. anteroposterior radiographs were taken using a source - to - film distance of 110 cm. the patient 's feet were internally rotated with the toes at 15 5 to ensure that the x - ray beam was centered on the superior aspect of the pubic symphysis. to analyze the relationship between the severity of ad at skeletal maturity and history of treatment for ddh, patients were divided into three groups (mild, moderate, and severe ad), comprising equal number of patients (table 1). variables in mild, moderate, and severe acetabular dysplasia groups to test the reproducibility of the radiographic measurements, three authors (ko, ky, and yn) measured the ce angle, ahi, acetabular angle, and acetabular roof angle in five randomly selected hips. each hip was measured three times with an interval of 1 week between measurements, and the values were subsequently averaged. the data were analyzed for intra- and inter - observer variances, and the coefficient of variation was calculated to be less than 5%. differences between values in ddh and non - ddh groups were tested using the mann differences in the percentage of ddh patients between the mild and severe dysplasia groups were tested using fisher exact test. the cochran armitage test was used to test for trends in the percentage of ddh patients according to the three groups. logistic regression analysis was used to evaluate the simultaneous effect of various factors on ddh. the significance level of the hypothesis test was chosen as p < 0.05. all statistical analyses were performed with ezr (saitama medical center, jichi medical university). a total of 88 patients (36%) had a past history of treatment for ddh (ddh group) and the remaining 157 patients (64%) had no history of ddh (non - ddh group). although the average age was lower and the acetabular angle was larger in ddh group, no significant differences of height, weight, ce angle, ahi, and acetabular roof angle were observed between the ddh and non - ddh groups (table 2). younger age was significantly associated with ddh after adjustment for ce angle, ahi, acetabular angle, and acetabular roof angle [odds ratio (or) = 1.09, 95% confidence interval (ci) = 1.061.13, p < 0.0001 ]. variables in total, ddh, and non - ddh group there were significant differences in percentages of ddh and non - ddh patients between the mild and severe dysplasia groups classified by acetabular angle (p = 0.048, figure 2c) and acetabular roof angle (p = 0.013, figure 2d) ; however, no differences were observed on the basis of ce (p = 0.12) (figure 2a) and ahi (p = 0.28) (figure 2b). there were significantly increasing trends in the percentage of ddh patients according to severity of ad classified on the basis of ce (p = 0.039) (figure 2a), acetabular angle (p = 0.017) (figure 2c), and acetabular roof angle (p = 0.0066) (figure 2d). however, no trend was observed among the three groups classified on the basis of ahi (p = 0.12) (figure 2b). graph showing the number of ddh (black square) and non - ddh (white square) patients in three groups (mild, moderate, and severe dysplasia) classified by ce angle (figure 2a), ahi (figure 2b), acetabular angle (figure 2c), and acetabular roof angle (figure 2d). previous reports have described that 8 to 60% patients treated for ddh in infancy have residual ad at skeletal maturity. in this study, we investigated the relationship between ddh in infancy and the later development of ad. we observed that 64% patients with ad at skeletal maturity have no history of treatment for ddh. although there was an increasing trend in the percentage of ddh patients associated with severity of ad at skeletal maturity, more than half of the patients with severe ad at skeletal maturity had no history of treatment for ddh. our study had several limitations with regard to obtaining the treatment history of ddh patients. first, with regard to questioning patients and their parents on the type of treatment received for ddh during infancy, although we were able to obtain treatment history from all patients in the ddh group, we were not able to confirm certain details, such as the side of the dislocated hip or the exact treatment method employed. these patients could describe the treatment history, but were unable to provide detailed aspects of the treatment methods used. second, there is a possibility that the non - ddh group in the present study may have included patients with untreated ddh that spontaneously improved. in this study, we observed several ad patients at skeletal maturity with no history of treatment for ddh. patients with ad have been reported to exhibit an abnormal morphology of the pelvis, a high rate of coexisting spinal congenital anomalies, such as spina bifida occulta, and high bone mineral densities of the lumbar spine, ultradistal radius, and calcaneus. as previously mentioned, primary oa of the hip is an extremely rare condition in japan. japanese patients with oa have been reported to have higher rates of dysplasia than american patients (46% vs 4.5%) and significantly worse dysplasia than british populations (mean ce angle 37 vs 31). these reports and our data suggest that one - third of patients with ad in japan have residual ad affected by treatment of ddh, and the remaining two - third patients came from a genetic background characteristic of japanese individuals from that of caucasians. some previous studies described a natural course of osteoarthritis of the hip in patients with ad. hasegawa evaluated 86 hips in 59 patients with pre- or early stage osteoarthritis (average age 29.9 years) and 31 hips (66%) with more progressive disease (average age 7.8 years). small values for ce angle and ahi and large values for acetabular roof angle were observed in the hips of patients with pre - osteoarthritis, which progressed to an advanced stage. however, no differences in these radiographic parameters were observed in the hips of patients in early stages of osteoarthritis. hisatome reported that 7 of 61 (11%) hips (average age 38.2 years) with pre- or early stage osteoarthritis progressed to advanced stages within an average of 10.1 years. no differences in radiographic parameters were observed between the hips with maintained and progressive disease. neither of these previous studies analyzed existing treatment for ddh in infancy. in this study, the average age of patients initially examined for hip pain was 40.7 years, and lower ages were observed in the ddh group (36.2 years) compared with the non - ddh group (43.3 years). in addition, a significant difference was still observed after adjustment for radiographic parameters of ad. complications of dislocated hip joints in infancy (elongated ligamentum teres, everted labrum, and a stretched hip capsule) may accelerate development of pain in patients with ddh at skeletal maturity, prompting them to seek care for the pain earlier. there were significant differences between the mild and severe dysplasia groups, and an increasing trend in the percentage of patients with ddh associated with severity of ad classified using acetabular angle and acetabular roof angle ; however, no differences were observed in cases of severity of ad classified on the basis of ce angle and ahi. ce angle and ahi evaluations represent the position of the femoral head to the acetabulum. in contrast, acetabular angle and acetabular roof angle evaluation represents the whole structure and slope of the weight - bearing area of the acetabulum (figure 1). the labrum of a non - dislocated hip is a thin fibrocartilaginous rim around the periphery of the acetabular cartilage. this vital cartilaginous acetabular analogue is essential for normal growth and development of the acetabulum. at the fibrocartilage hyaline junction of the labrum and acetabulum, there may be eversional or inversional hypertrophic changes (neolimbus) in the dislocated hip. neolimbus, which was formed during dislocation of the hip, may affect acetabulum growth even after reduction. in conclusion, we observed that 64% patients with ad at skeletal maturity have no history of treatment for ddh. ad patients with a history of treatment for ddh have hip joint pain at a younger age and severe dysplasia, particularly when evaluated by acetabular angle and acetabular roof angle. our data suggests that ad in patients without a history of ddh has different characteristics from ad in patients with a history of ddh. | abstractprevious reports demonstrated 860% patients treated for developmental dislocation of hip (ddh) in infancy have residual acetabular dysplasia (ad) at skeletal maturity. ad patients reportedly exhibit abnormal morphology of the pelvis, high rates of comorbid spinal congenital anomalies and high bone mineral density. these physical findings suggest that ad patients have genetic background. we examined the percentage of ad patients with hip pain at skeletal maturity having a history of ddh in infancy and the correlation between the severity of ad at skeletal maturity and history of ddh treatment to investigate the relationship between ad and ddh.a total of 245 patients were radiographically examined for any history of ddh treatment in infancy. the study included 226 women and 19 men with a mean age at examination of 40.7 years (range 1759 years).eighty - eight patients (36%) had a history of ddh treatment (ddh group) and the remaining 157 patients (64%) had no history of ddh treatment (non - ddh group). the average age was lower and acetabular angle was larger in the ddh group. there was a significant increasing trend of the percentage of ddh patients associated with the severity of ad classified with ce, acetabular angle, and acetabular roof angle.our data suggest that there are several ad patients without a history of ddh in japan, and ad in patients without a history of ddh has different characteristics from ad in patients with a history of ddh. |
originally recognized as a cause for oral candidiasis among hiv - infected patients reports of severe fungal infections caused by candida dubliniensis are emerging in recent years. c. dubliniensis shares many phenotypic characteristics with the predominant fungal pathogen candida albicans like the ability of germ tube- and chlamydospore - production. both species are able to switch between yeast- and filamentous form and are able to form biofilms on biotic and abiotic surfaces. severe systemic infections by c. dubliniensis, however, have only been seen in immunocompromised patients, which indicates lower pathogenic potential of this candida species. candida spondylodiscitis, often associated with significant morbidity, is rarely reported and predominantly caused by c. albicans. so far only one case of spondylodiscitis caused by c. dubliniensis in an intravenous drug addict with chronic hepatitis c virus (hcv) infection has been recently reported in the medical literature. a 47-year old male was admitted with exacerbation of low - back pain radiating to the groin and the right leg to the bernhard - nocht - clinic of the university medical center hamburg - eppendorf, germany (day 0). low - back pain and radicular symptoms started approximately one month prior to presentation with exacerbation by movement. physical examination showed paraesthesias and a weakness of the right lower leg with a decreased patellar- and achilles tendon reflex. he was diagnosed as hiv-1-positive 19 years ago and is currently on antiretroviral therapy with lamivudine 300 mg once daily, atazanvir 400 mg once daily and raltegravir 400 mg twice daily. at the time of presentation his cd4 t - cell count was 237/l (normal range : 5001350/l) and viral load was undetectable (hiv-1 taq - pcr : < 20 copies / ml). furthermore he was diagnosed as hcv - positive genotype 1a 13 years ago. at time of admission he presented with a hcv - related liver cirrhosis (child pugh a) without any treatment up to now and a viral load of 800 000 iu / ml. the patient had a history of intravenous drug abuse (ivda) with cocain and benzodiazepines and is now following methadone substitution programme (methadone 120 mg once daily), any illicit drug abuse is excluded. results of laboratory testing showed a decreased thrombocyte count with 8910 cells/l (15040010 cells/l) and a c - reactive protein level of 118 mg / l (reference value<5 mg / l). initial magnetic resonance imaging (mri) of the lumbar spine showed spondylodiscitis of vertebral bodies and intervertebral discs from l4 to s1 with complete destruction of l5 and contiguous epidural abscess markedly narrowing the spinal canal (fig. 1). due to the obstructive nature with progressive neurological impairment the abscess had to be drained twice in the first two weeks of hospitalization (day 3 and day 10). culture of the abscess fluid on sabouraud dextrose agar showed white, cream - coloured colonies corresponding to candida spp. after 24 h of incubation at 37 c on the two occasions. the isolate was identified as c. dubliniensis by maldi - tof mass spectrometry (day 5). identification was confirmed by sequencing of the its2 region of the ribosomal dna with primers its3 and its4 and sequence comparison to the yeast reference database at the centraalbureau voor schimmelcultures (cbs) fungal biodiversity centre (utrecht, netherlands) according to clinical laboratory standards institute guideline mm18-a, (http://www.cbs.knaw.nl). the its2 region showed 100% sequence identity to c. dubliniensis type strain cbs7987 (genbank accession number ab049123.1). furthermore three pairs of blood cultures drawn at the beginning of hospitalization (day 0) were incubated at 37 c (bactec 9240, becton dickinson, heidelberg, germany) and showed microbial growth after 26 h of incubation. blood from positive blood culture bottles was gram - stained and subcultured on specific agars. further identification by maldi - tof mass spectrometry revealed also c. dubliniensis (day 4). c. dubliniensis strains were tested against amphotericin b, fluconazole, voriconazole and caspofungin using e - test strips according to the manufacturer 's instructions (ab biotest, solna, sweden) and eucast guidelines. the results of the susceptibility tests were : mics of 0.064 g / ml to amphotericin b, 0.008 g / ml to voriconazole, 0.25 g / ml to fluconazole and 0.125 g / ml to caspofungin. candida spp. was not detectable by urine culture and fundoscopy of the eyes showed no pathological abnormalities. antifungal treatment was initiated with fluconazole 400 mg once daily (day 4). sultamicillin 3 g three times daily was added to cover potential co - pathogens even though bacterial pathogens were never detected in abscess or blood cultures. antifungal treatment was continued. as an adjunct treatment the patient had to observe strict bed rest for one month. mobilization was achieved with a surgical corset of the affected spinal region. under this regimen repeat mri after one month of treatment showed marked reduction of the abscess without any spinal stenosis and marked reduction of the inflammation in the spinal bodies (day 34). antifungal treatment was continued for one further month with oral fluconazole 200 mg once daily. herein, we report a severe case of spondylodiscitis and spinal abscess in a hiv-1 and hcv - coinfected patient following hematogenous dissemination of the emerging fungus c. dubliniensis. fungal infections caused by this dimorph fungus have been reported with increasing frequency in the medical literature. reports of other clinical manifestations such as spondylodiscitis and osteomyelitis, however, are extremely rare. recently oksi and colleagues described the first case of spondylodiscitis caused by c. dubliniensis in an immunocompetent intravenous drug addict (ivda) with chronic hcv infection. it remains unclear, however, if the patient had a hcv - related liver cirrhosis. this would contribute to an increased susceptibility to fungal infections as cirrhosis is considered an immunocompromised state. we suggest that liver cirrhosis played a pivotal role in our patient to develop spondylodiscitis and spinal abscess following hematogenous dissemination. furthermore, it remains unclear, if the patient reported by oksi and colleagues, with a history of ivda, was tested on hiv since an unrecognized and therefore, untreated hiv infection may compromise the immune status of the patient additionally. in our patient cd4 t - cell count was 237/l (normal range : 5001350/l) at time of presentation with an undetectable viral load indicating sustaining adherence to antiretroviral therapy. oksi and colleagues successfully treated their patient with liposomal amphotericin b for four weeks, followed by 32 weeks of fluconazole. optimal antifungal treatment for c. dublinienis spondylodiscitis and spinal abscess remains unclear, however, suggested antifungal drugs for c. dubliniensis are comparable to c. albicans and are susceptible to antifungal drugs commonly used. in our patient antifungal susceptibility testing for amphotericin b, voriconazole, fluconazole and high - level resistance to azoles, however, are reported ranging from 2.5% to 41%. fungal spondylodiscitis predominantly caused by c. albicans typically involves the intervertebral disc space with narrowing of the disc cartilage, followed by destruction and lysis of the vertebral endplates and underlying vertebral bones. so far, wellinghausen and colleagues reported the only case of multifocal osteomyelitis following disseminated c. dubliniensis infection in a patient after haematopoietic stem cell transplantation (pbsct). in line with our case, this patient reported was severely immunocompromised and showed a delayed t - cell reconstitution mediated by a lack of sufficient numbers of cd4 t - cells (< 400/l) until 6 month after the pbsct. this indicates that c. dubliniensis is less virulent compared to its closest known relative c. albicans, although it expresses hyphal - specific virulence factors like aspartyl proteinases sap4 and sap5 and the invasin als3, but it seems that c. dubliniensis is more susceptible to environmental stressors and hyphal formation is less efficiently compared to c. albicans. furthermore, c. dubliniensis triggers stronger early neutrophil responses such as neutrophil migration, phagocytosis, and the release of antimicrobial cytokines. neutrophils internalize c. dubliniensis cells more efficiently than cells of c. albicans. the innate immunity represented by neutrophils and macrophages is believed to serve as the first line of defense by phagocytosis and direct killing of candida. furthermore, the antifungal immunity - related il-17a, which is considered an important component in host defense against candida infections, produced by peripheral blood mononuclear cells is significantly lower when challenged with c. dubliniensis in vitro. recently, krause and colleagues even showed that candidemic patients had significantly higher il-17a levels compared to non - candidemic patients. to conclude we report the second case of spondylodiscitis and spinal abscess following hematogenous dissemination of c. dubliniensis in an immunocompromised patient. therefore this case indicates that the pathogenicity of c. dubliniensis may be higher than previously believed. differentiation by standard laboratory methods may be difficult and may often leads to misidentification. considering that it is likely that c. dubliniensis infections are misdiagnosed or not recognized, the burden of infection caused by c. dubliniensis may still be underestimated. | we report a case of spondylodiscitis and spinal abscess following haematogenous dissemination of the emerging yeast candida dubliniensis in a human immunodeficiency virus-1 (hiv-1) and hepatitis c virus (hcv)-coinfected patient. although c. dubliniensis is considered less virulent compared to its closest known relative candida albicans, reports of severe fungal infections are increasing. this case indicates that the pathogenicity of c. dubliniensis may be higher than previously believed. therefore fungal infections caused by this dimorph fungus should be kept in mind in immunocompromised patients with spondylodiscitis and spinal abscess. |
herbal medicines are valuable and available resources of primary health care for thousands year in the traditional medicine including the thai folk medicine. generally, the bioactive compounds in herbal medicines are secondary metabolites which act as various pharmacological properties.1, 2, 3 they are used as the substances of modern drugs e.g. morphine from papaver somniferum for pain treatment,4, 5 colchicine from colchicum autumnale for treatment in pericardial disease. some studies have reported antibacterial activity from various herbs7, 8, 9 ; however, sufficient evidence to support their active compounds and their effectiveness for antibacteria and antibiofilm, particularly on oral pathogens is limited. an imbalance of bacteria in the dental biofilm can cause dental caries and periodontitis which are major oral infections. the high doses of antimicrobial agents are need for removing the biofilm in clinical approach.11, 12 moreover, in long term use of chlorhexine (chx) mouth - rinse can cause side effects ; disturbance in taste sensation, brown discoloration at dorsum of tongue and desquamative lesions of oral mucosa.13, 14 our previous study has presented anticandidal and antibiofilm of some thai herbs in thailand. herbal medicines may be an alternative way for being antibacterial agents to prevent oral infectious diseases. this extended study was to evaluate the antibacterial and antibiofilm activities of 12 ethanol extracts of thai traditional herb included alpinia galanga, curcuma longa, curcuma zedoaria, piper betle, piper chaba, piper nigrum, piper sarmentosum, mentha cordifolia, ocimum africanum, ocimum basilicum, ocimum sanctum and zingiber officinale, and to investigate the active compounds of the most effective extract. twelve herbs were purchased from the local market, and the details were shown in table 1. the herbs were identified by an expert in the department of pharmacognosy and pharmaceutical botany, faculty of pharmaceutical sciences, prince of songkla university, songkhla, thailand, where voucher specimens (table 1) were deposited in the herbarium. dried plants were macerated with ethanol for 3 days, and then filtrated through whatman no. 4 filter paper. each filtrate was dried using a rotary evaporator at 40 c and kept at 20 c. for testing, 0.1 mg of each dried extract was initially dissolved with 100 l of dimethyl sulfoxide (dmso) and then adjusted to 1 ml by adding sterile distilled water, giving a final concentration of 10% (w / v) extract in 10% (v / v) dimethyl sulfoxide (dmso). a total of 7 oral microorganisms were employed in the study including 5 gram positive cariogenic bacteria, enterobacter faecalis atcc 19433 (ef), lactobacillus fermentum atcc 14931 (lf), lactobacillus salivarius atcc 11741 (ls), streptococcus sobrinus atcc 33478 (ss) and streptococcus mutans atcc 25175 (sm), and 2 gram negative periodontopathogenic bacteria, aggregatibacter actinomycetemcomitans atcc 33384 (aa) and fusobacterium nucleatum atcc 25586 (fn). microorganisms were maintained on either brain heart infusion agar (bha) with 5% (v / v) blood for facultative bacteria, and supplemented with 0.5% (w / v) yeast extract, hemin and vitamin k for anaerobic bacteria. the strains were grown under aerobic or anaerobic (10% h2, 10% co2 and 80% n2) conditions as appropriate. one hundred microliters of inoculum, equivalent to 10 cfu / ml, was mixed with 20 ml of warm melted bha. the mixture was then poured into the plate with a 6 mm diameter metal cup. after solidifying of bha, the metal cups were removed and the well was added with 100 l of each plant extract. the plate was incubated at 37 c for 24 h. a 10% dmso were taken as a negative control. the antimicrobial activity of each plant extract was determined by measuring the diameter of the zone of the inhibition in millimeters. minimum inhibitory concentration (mic) and minimum bactericidal concentration (mbc) were carried out as recommended instruction of the clinical and laboratory standards institute (clsi). briefly, 10% stock solution of each extract was diluted in the brain heart infusion broth (bhi) in two - fold serial dilutions to obtain concentrations from 0.02 to 25 mg / ml at a total volume of 100 l per well in 96-well microtiter plates. each tested strain (100 l) at a final concentration of 1 10 cfu / ml was added to each well and incubated at 37 c in appropriate conditions. the medium, 0.1% (w / v) chx and 10% dmso were used as the non - treated, positive and negative controls, respectively. mic was defined as the lowest concentration of the extract that completely inhibited growth in comparison with the non - treated control. mbc was defined as the lowest concentration of wells that did not allow visible growth when 10 l of the well contents was plated on agar and grown at 37 c in appropriate conditions. as the results of the screening of herb extracts revealed that p. betle leaves extract gave the strongest antibacterial activity. the growing cultures (10 cfu / ml) of representative strains, s. mutans atcc 25175 and a. actinomycetemcomitans atcc 33384, were incubated in bhi broth supplemented with the extract at concentrations equivalent to 1, 2, and 4 mic at 37 c. surviving bacteria were observed at 0, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h by culturing on agar plates. the procedure was repeated in triplicate, and the log10 cfu / ml was plotted against time. a 0.1% chx and extract free medium were used as positive and non - treated controls, respectively. betle extract on biofilm formation of each representative strain, s. mutans atcc 25175 and a. actinomycetemcomitans atcc 33384, was examined by using the modified microdilution method. betle extract were prepared, with final concentration ranged from 0.02 to 25 mg / ml. a cell suspension of the tested strains was prepared as described in the mic assay, and 100 l (1 10 cfu / ml) were inoculated in each of a 96-well plate. a 0.1% chx, phosphate buffered saline (pbs) and extract free medium were used as the positive controls, non - treated and blank controls, respectively. after incubation at 37 c for 24 h, supernatants were discarded and washed 3 times with pbs. biofilm formation was quantified by using a 3-[4,5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2h - tetrazolium - bromide (mtt) assay. the numbers of surviving bacteria were determined by measuring their ability to reduce the yellow tetrazolium salt to a purple formazan product at 570 nm. percentage inhibition was calculated by using the equation [1 (a570 of the test / a570 of non - treated control) ] 100. the minimum biofilm inhibition concentration (mbic) was defined as the lowest concentration that showed 50% and 90% inhibition of biofilm formation (mbic50 and mbic90). betle extract was also examined using the minimum biofilm eradication concentration (mbec) assay. briefly, a 200 l in early log phase (10 cfu / ml) of each representative strain, s. mutans atcc 25175 and a. actinomycetemcomitans atcc 33384, was inoculated into each well of a 96-well plate and incubated for 24 h at 37 c for the development of a multilayer biofilm. the culture was then blotted out, and the well carefully washed 3 times with sterile pbs in order to remove non - adherent cells. these biofilms were then exposed to a 200 l of various concentrations of p. betle extract ranged from 0.02 to 25 mg / ml, incubated for 24 h at 37 c in appropriate conditions. at the end - point of the treatment of the biofilms with p. percentage eradication was calculated by using the equation [1 (a570 of the test / a570 of non - treated control) ] 100. the mbec value was defined as the concentrations that showed 50% and 90% eradication of biofilm formation. the 0.1% chx, pbs and the extract free medium were used as the positive, non - treated and blank controls, respectively. the active antibacterial compounds of p. betle extract were analyzed using the high performance liquid chromatography (hplc) (agilent 1100 series, palo alto, ca). the reverse column was apollo c18, 250 mm 4.6 mm, 5 m particle size (alltech associates inc., the conditions were set as following ; mobile phases (methanol : water 70:30 v / v), flow rate at 0.7 ml / min and uv detector at 280 nm. a injection volume (20 l) of each sample, 1 mg of p. betle extract, 5 l of pure eugenol (the agent controller) and 0.5 mg of 4-chromanol (the agent controller), were prepared in 1 ml of 95% (v / v) ethanol. each prepared sample was taken to analyze by hplc, and curves were prepared using the microsoft excel software package (excel 2007 ; microsoft corporation, redmond, wa, usa). for tlc - fingerprinting study, 100 mg of p. betle extract, 5 l of pure eugenol (the agent controller) and 1 mg of 4-chromanol (the agent controller) were prepared in 1 ml of 95% ethanol. a 10 l of each sample was applied onto the alumina silica gel gf254 tlc - plate, and was run through a series of solvent systems using a mixture of toluene and ethyl acetate (90:10 v / v) as the mobile phase. the plate was air dried, and was stained with a 20% (w / v) of phosphomolybdic acid in ethanol. tlc - plates were placed on plate contained 6 ml of bha, and 10 ml of top agar with 100 l of either s. mutans atcc 25175 or a. actinomycetemcomitans atcc 33384 inoculum (10 cfu / ml) was covered on tlc plate. after overnight incubation, the inhibited zone was observed as clear zone. the relative front values (rf) was calculated as : rf = distance traveled by solute / distance traveled by solvent. data were expressed as mean and standard deviation (sd) by computational analysis from the three experiments with duplicate or triplicate independent experiments. antibacterial activity of 12 thai traditional herb extracts against various oral bacteria was revealed in table 1. betle leaves showed a good result of antibacterial activity against all tested microorganisms using agar well diffusion assay (table 1). the mic of p. betle extract ranged from 1.04 to 5.21 mg / ml and mbc ranged from 2.08 to 8.33 mg / ml, respectively (table 2). time kill assay of p. betle extract against representative strains, s. mutans atcc 25175 and a. actinomycetemcomitans atcc 33384, is demonstrated in fig. the completed killing of s. mutans atcc 25175 treated with 1, 2, and 4 mic of the extract occurred within 12, 8, and 6 h, respectively. at 1, 2, and 4 mic, a. actinomycetemcomitans atcc 33384 was killed after 6, 2, and 1 h, respectively. the killing of positive control (0.1% chx) was observed within 30 min. the present study demonstrated that commonly used thai herbs could exhibit antimicrobial activity against various oral bacteria. betle extract revealed the best antibacterial activity against both gram positive bacteria and gram negative bacteria. the inhibition and eradication of the biofilm formation of p. betle extract against s. mutans atcc 25175 and a. actinomycetemcomitans atcc 33384 is demonstrated in fig. the concentrations of p. betle extract required to inhibit for 50% biofilm formation (mbic50) of s. mutans atcc 25175 and a. actinomycetemcomitans atcc 33384 were 0.39 0.20 and 0.10 0.03 mg / ml, respectively. and for 90% biofilm inhibitions (mbic90) were 1.56 0.11 and 0.39 0.65 mg / ml, respectively. the concentrations of p. betle extract to eradicate for 50% biofilm formation (mbec50) of s. mutans atcc 25175 and a. actinomycetemcomitans atcc 33384 were 0.78 0.74 and 0.78 0.11 mg / ml, respectively. and for 90% biofilm eradication (mbec90) were 6.25 0.58 and 3.13 0.28 mg / ml, respectively. chx could completely (100%) inhibit or remove biofilm formation of both tested strains. due to its interesting results, the further study focused on the active compounds of p. betle leaves extract. 3b) and the mixture of p. betle extract and the standards (fig. 3a c was identified as 4-chromanol with retention times of 5.486 0.00 min, 5.654 0.04 min and 5.610 0.00 min, respectively. 3a c was eugenol with retention times of 7.853 0.01 min, 7.966 0.09 min and 7.757 0.01 min, respectively. it indicated that at least 4-chromanol and eugenol were the components of p. betle leaves extract. there were several bands of p. betle extract components appeared on the tlc - plate (fig. 4a). one major band with rf at 0.38 matched to the standard 4-chromanol gave the large inhibited clear zone against s. 4b), and this band also gave inhibited clear zone against a. actinomycetemcomitans atcc 33384 (data not shown). another minor band with rf at 0.74 matched to the standard eugenol did not show any inhibited zone. p. betle (family piperaceae) is known as phlu, which has been extensively used in traditional herbal remedies in thailand and many other countries in tropical asia. it is reported having various pharmacological activities such as antimicrobial, immunomodulatory, and anti - inflammatory. a significant antimicrobial activity against a wide range of medical bacteria such as streptococcus pyogenes, staphylococcus aureus, proteus vulgaris, escherichia coli, pseudomonas aeruginosa etc. was found in the leaves. in this study, it has shown that p. betle extract also exhibited antimicrobial activity against various oral microorganisms including cariogenic and periodontogenic pathogens e.g. s. mutans and a. actinomycetemcomitans. our results agree with the study of deshpande and kadam who demonstrated the finding of antibacterial activity of ethanol and aqueous crude extract of p. betle leaves against s. mutans. in that report, zone of inhibition of ethanol extract (20.6 1.12 mm) was found to be larger than aqueous extract (18.3 0.53 mm). the mic value for the aqueous and ethanol extract was 10 mg / ml and 5 mg / ml, respectively. the p. betle leaves ethanol extract in this study was found to be more potent with mic value of 1.56 mg / ml. this may be due to ecological and geographical conditions, age of herb and time of harvesting. however, there was no information of antibiofilm was mentioned in that study. the antibiofilm is a desired property that expected from herbs extracts. in this study, betle leaves extract has been revealed having dual actions in preventing and eradicating biofilm formation. it may be worth for considering the p. betle leaves extracts to replace chx mouth - rinse. chx is a chemical - based antimicrobial agent which is used extensively in the mouth - rinse to maintain dental biofilm at a level compatible with oral health. however, it was reported on some undesired local side effects for long term use.13, 14 results of both planktonic and biofilm form indicated that p. the similar results were also reported in citrus bergamia, woodfordia fruticosa, and artocarpus lakoocha showing that gram negative bacteria were more susceptible to some plant extracts than gram positive. this contrasted to previous studies showed that plant extracts were more active against gram positive bacteria than gram negative bacteria.24, 25 the gram negative bacteria are considered to be more resistant due to their outer membrane acting as a barrier to many environmental substances including antimicrobial substances. moreover, they could stimulate the membrane - associated mechanisms of resistance via the expression of efflux pumps to control the antibacterial agents.26, 27 deshpande and kadam reported that 4-chromanol was a predominant compound in p. betle leaves aqueous extract, which responded for antibacterial activity. our previously study demonstrated that the major constituents of p. betle leaves extract were 4-chromanol (62.33%) and eugenol (17.10%) in gc - ms analysis, and the 4-chromanol showed good anticandidal activities. in this study, the 4-chromanol also exhibited the strong antibacterial activity by observing a clear inhibition zone against oral pathogens in the tlc - bioautography assay. thus, it indicated that the 4-chromanol could overcome the barrier and membrane - associated resistance of gram negative bacterial membrane. this study suggested that the ethanol extract of p. betle leaves is a potential source of natural antibacterial and antibiofilm agents. the major constituents of p. betle leaves extract was 4-chromanol exhibiting a good antibacterial and antibiofilm properties that may be used for oral infectious diseases. for further study, the mechanisms of antibacterial activity of 4-chromanol should be investigated in more detail as well as its activity in vivo. rawee teanpaisan : project initiation and design, data analysis, writing and preparing the manuscript, editing the manuscript. pajaree kawsud : project initiation and design, laboratory performance, data analysis, writing and preparing the manuscript. | to evaluate the antibacterial activity of 12 ethanol extracts of thai traditional herb against oral pathogens. the antibacterial activities were assessed by agar well diffusion, broth microdilution, and time - kill methods. antibiofilm activity was investigated using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2h - tetrazolium - bromide (mtt) assay. high performance liquid chromatography (hplc), thin layer chromatography (tlc) fingerprinting, and tlc - bioautography were used to determine the active antibacterial compounds. piper betle showed the best antibacterial activities against all tested strains in the minimal inhibitory concentration and minimal bactericidal concentration, ranged from 1.045.21 mg / ml and 2.088.33 mg / ml, respectively. killing ability depended on time and concentrations of the extract. p. betle extract acts as a potent antibiofilm agent with dual actions, preventing and eradicating the biofilm. the major constituent of p. betle extract was 4-chromanol, which responded for antibacteria and antibiofilm against oral pathogens. it suggests that the ethanol p. betle leaves extract may be used for preventing oral diseases. |
the world health organization projected that 300 million people will suffer from diabetes and 1.5 billion from hypertension by 2025. according to the diabetes atlas 2006 published by the international diabetes federation, the number of people with diabetes in india currently around 40.9 million, is expected to rise to 69.9 million by 2025 unless urgent preventive steps are taken. the incidence of hypertension in patients with t2 dm is approximately two - fold higher than in age - matched subjects without the disease. hypertension has been identified as a major risk factor not only for the development of diabetes but also for the development of micro and macro vascular complications, that is, neuropathy, nephropathy, retinopathy, coronary artery disease (cad), stroke, peripheral vascular disease (pvd) in diabetic patients. it has been evident from framingham heart study, ukpds (united kingdom prospective study-39), hypertension optimal treatment (hot), systolic hypertension in the elderly programme (shep), systolic hypertension in europe (syst - eur), hypertension in the very elderly trial (hyvet - pilot) that reduction in either isolated systolic or systolic - diastolic hypertension significantly reduces the risk of micro and macro vascular complications and cardiovascular (cv) death or diabetes - related death [511 ]. lowering blood pressure (bp) in patients with diabetes mellitus is more cost effective than tight blood glucose control, and beneficial results are apparent earlier. therefore, all of the hypertension management guidelines, that is, seventh report of joint national committee on prevention, detection, evaluation, and treatment of high blood pressure-2003 (jnc-7), american diabetes association (ada) 2010, european society of hypertension (esh), who guideline [8, 10, 12 ] focused aggressively on blood pressure (bp) control in diabetic patient to below 130135/8085 mmhg [8, 9, 12 ]. given the importance of the blood pressure control in diabetic patients, there has been much needed data on achievement of target control in real world diabetic hypertensive patients ; however, the data on prescribing pattern as well as blood pressure control is scare especially in asian - indian subcontinent. thus, this study was conducted to assess blood pressure control and the prescribing patterns of antihypertensive in hypertensive diabetic patients in real world clinical settings. this cross - sectional study was carried out from june 2008 to may 2009 at the nehru hospital, postgraduate institute of medical education and research, chandigarh. patients were confirmed by physician diagnosed t2 dm patients, were further examined consecutively for social, demographical, and clinical variables. informed and written consent was obtained from all the participants after full explanation of the procedure. standing body height (to the nearest 0.5 cm) was measured with a commercial stadiometer. a digital scale, with an accuracy of 100 g, the waist circumference (wc) was measured in a horizontal plane, midway between the inferior margin of the ribs and the superior border of the iliac crest. hip circumference (hc) was measured at the fullest point around the buttocks with a metallic tape. wc (cm) was divided by hc (cm) to calculate waist to hip ratio (whr). body mass index (bmi) (kg / m) was calculated by dividing weight (in kilograms) by the square of height (in meters), as a measure of total adiposity. percent body fat (% bf) was evaluated by impedance plethysmography (bioelectrical impedance meter (omron bf 302, tokyo)). systolic and diastolic blood pressure (sbp & dbp) were measured twice at an interval of 3 min in the sitting position after a 15 min rest, and the mean was taken. further, hypertension conformation was based on any of following criteria : at least two outpatient visit diagnosis of hypertension ; at least 1 prescription of antihypertensive drug plus at least 1 outpatient diagnosis of hypertension ; at least 2 elevated bp measurements plus one outpatients diagnosis of hypertension ; at least two elevated blood pressure measurement. according to jnc-7 report (joint national committee on prevention, detection, evaluation and treatment of high blood pressure), elevated blood pressure was defined as higher than or equal to 130/80 mmhg. blood samples (3 ml) were drawn after 812 h overnight fasting for the measurement of lipid profile (total cholesterol, high density lipoprotein (hdl) cholesterol, and triglycerides) and fasting plasma glucose levels. plasma glucose was measured using the glucose oxidaseperoxidase method, serum total cholesterol, and triglycerides by standard enzymatic procedures and hdl cholesterol by direct assay method. uncontrolled hyperglycemia was defined as hba1c (glycosylated heamoglobin a1c) > 7% or fpg (fasting plasma glucose) > 110 mg / dl or ppg (postprandial glucose) > 140 mg / dl. neuropathy was evaluated by history of numbness, paraesthesias, tingling sensation, burning sensation, and confirmed by touch sensation using 10 gm monofilament, vibration sense by biothesiometer (vpt at great toe > 25 mv were considered significant) and ankle reflex. incipient nephropathy was diagnosed by micral test and it was presumed to be present if any two readings out of three of urinary albumin and creatinine ratio were ranging from 30 to 300 g / mg. clinical nephropathy was evaluated by the estimation of 24 hr urine protein, and it was present if urine proteins were more than 500 mg / total volume of urine. ophthalmologist diagnosed retinopathy by detailed fundus examination and was classified according to diabetic retinopathy study (drs) and early treatment diabetic retinopathy study (etdrs). coronary artery disease was diagnosed by history of angina or myocardial infarction or documented by previous treatment records. pathological q wave (major q wave abnormalities) in an ecg recording (minnesota codes 1.1.11.2.7), st segment depression (codes 4.1 - 4.2), t wave abnormalities (codes 5.15.4), and chest x - ray was done to assess cardiac size. peripheral vascular disease (pvd) was diagnosed by definitive history of intermittent claudication or if one or more peripheral pulses were absent in both feet. the grading was done according to ankle brachial pressure index (abpi) by doppler study [multi duplex (r)-ii (huntleigh diagnostics - uk) ]. all type 1 diabetic patient with hypertension, pregnant or breast feeding women, were excluded. proportion of patients using 5 different classes of antihypertensive was calculated. for prescription pattern analysis, immunotherapy was considered as a single drug at any frequency of any antihypertensive class, while polytherapy was considered as a combination of two antihypertensive drugs from two different classes at any dose and frequency. prescribing pattern with five different classes of antihypertensive were analyzed in blood pressure control and diabetic complications. data were validated after double entry and then analyzed using the statistical package for social science (spss) (version 16 usa). binary logistic regression model was used to examine association between predictor variables (as captured by data capturing form) and prescription of a particular class of drug. the main model consists of the following predictor variables : demographic variables, age, gender, obesity, clinical variables like diabetic complications, blood pressure and duration of disease, and so forth. out of 1358 diabetic patients, 1186 (87%) patients were found to have hypertension. the average age (sd) of type diabetic hypertensive patients was 55.6 (10.1) years comprising 620 (52%) male and 566 (48%) female patients. the average duration of diabetes (dod) (sd) was 9.6 (7.5) years and median duration of hypertension was 4 (110) years. a total of 224 (19%) was newly diagnosed to be hypertensive with average age 53 (11) and dod of 7.4 (7). male had higher dod and dhtn to their counterpart female (p 55 years were more likely to receive diuretics compared to patients with 55 years. prescription of any hypertensive drugs were not associated with gender or duration of diabetes, however polytherapy was more predominant in elderly (p 1.2 mg / dl) ace were less likely to be prescribed (or : 0.84, 95% ci 0.710.99). patients with cad were more likely to be prescribed polytherapy (p 65 years of age with isolated systolic hypertension (i.e., > 140 mmhg systolic and < 80 mmhg diastolic blood pressure), pharmacological treatment should be initiated. earlier recommendations to treat a systolic blood pressure < 160 mmhg have been reduced based on the increased cardiovascular risk of these patients and the results of the shep study, in which a systolic blood pressure of 144 mmhg was achieved. when drug therapy is intensified, patients should be monitored carefully for adverse effects, such as orthostatic hypotension. diuretics were more utilized in combinations with other antihypertensive drugs in elderly compare to nonelderly patients a meta analysis has suggested that in elderly, diuretics have more pronounced preventing effects on cardiovascular mortality. in trials of isolated systolic hypertension first line comprised diuretics or calcium channel blocker [8, 38, 39 ]. subgroup analysis of trials on isolated hypertension shown efficacy of arbs [40, 41 ]. it has been shown that aceis as well as conventional antihypertensive delays progression of nephropathy. available evidence show that the presence of microalbunuria is not only early marker of renal diasease but also indiactor of increased cardiovascular risk. hence, all recent guidelines [11, 2931, 43 ] accentuate on use of acei as a first choice for diabetic hypertension. in present study, among 219 overt nephropathy patients 120 were on arbs (or : 1.693, 95% ci : 1.1682.463) and 116 on aceis (or : 1.094, 95% ci : 0.7561.582). a meta analysis has shown aceis to be effective for the primary prevention of kidney disease in diabetes and with aceis, a rr reduction of 42% has been demonstrated (95% ci 16% to 60%). aceis alone or with low dose of diuretics delay end stage renal disease or prevent microalbunuria or proteinuria [11, 41 ]. ada guidelines recommends that proteinuric patients, especially those with diabetes mellitus, need aggressive bp control and use of aceis and/or arbs. in addition, in those with type 2 diabetes, hypertension, macroalbuminuria (300 mg / day), and renal insufficiency an arb should be strongly considered. ace and arb were found to be major category for two - drug combination use. recent studies found that combination of ace and arb compared with ace inhibitors alone, was associated with significant increases in renal dysfunction and hyperkalemia, poorly tolerated, patients less adhere to combination therapy due to adverse effect. the current diabetes guidelines did not clearly avoid mentioning use of this combination, instead suggested use of arb for diabetic nephropathy in addition to ace. our studies results showed that negative outcomes of ace plus arb use in those studies did not influence the prescribing patterns. in patients with chf, arbs were superior to calcium channel blocker for reducing heart failure [43, 45 ], hence ada recommends it as first line drug. treatment with beta blocker has a protective effect on cardiovascular mortality after myocardial infraction, as this is a major cause of disease. the rate of successful blood pressure control was 26% compared higher hypertensive patients receiving treatment, and despite the inadequacy of monotherapy to control blood pressure, many of the patients received this treatment regimen. obviously, bp control is multifactorial, with factors such as age, comorbidity, and patient adherence to medication regimens affecting this outcome, and our study does not attempt to examine all other parameters like adherence to the method. we found that however prescribing patterns was consistent with the evidence - based guideline, only one fourth of diabetic patient had blood pressure within the target. however, the result is encouraging as it is better compared to previous report from the same hospital which suggested only 11% t2 dm patient have hypertension under control. | background. hypertension management is of a paramount importance in diabetic patients for cardiovascular risk reduction. aim. to evaluate prescribing pattern of antihypertensive in t2 dm (type 2 diabetes) patients and compare with existing recent guidelines. methods. a cross - sectional study involving evaluation of all t2 dm patients referred to endocrinology unit at tertiary care centre for hypertension, comorbid complications, and recording prescription. utilization of 5 different antihypertensive drug classes was compared for all patients receiving 1, 2, 3, 4, or more drugs. logistical regression was used to assess likelihood of prescription of drugs and/or therapy for specific conditions mentioned in the guidelines. results. out of 1358, t2 dm enrolled patients 1186 (87%) had hypertension (males 52%, females 48%). the median duration (iq) of hypertension diabetics was 4 (110) years. a total of 25% patients had controlled bp and 75% with uncontrolled blood pressure (13% isolated systolic hypertension, 6% isolated diastolic hypertension, and 55% both elevated). overall, ace inhibitors (aceis) were prescribed the highest (59%) followed by angiotensin receptor blockers (arbs) (52%), calcium channel blockers (ccbs) (29%), diuretics (27%), and beta - blockers (14%). overall, 55% of t2 dm patients were on polytherapy, 41% on monotherapy, and 4% had no antihypertensive treatment. polytherapy was more predominant with age, duration of diabetes, duration of hypertension, and comorbid complications. conclusion. although prescribing pattern of antihypertensive showed adherence to existing evidence - based guidelines, higher proportion of uncontrolled hypertensive patients was found. |
eczema is an inflammatory skin reaction characterized histologically by spongiosis with varying degrees of acanthosis. however, eczema is divided into two groups ; exogenous eczemas, e.g. contact dermatitis, are related clearly to defined external triggering factors, although inherited tendencies can also play a part, whereas endogenous eczema, e.g. atopic dermatitis, seborrhoeic dermatitis, is not a result of exogenous or external environmental factors, but is mediated by processes originating within the body. zliten is located in the northwest of libya, and its population is about 170,000. there is only one general district hospital (with a dermatology clinic) and two private dermatological practices in the area. therefore, the aim of this study was to provide preliminary data about the burden of eczema among libyan dermatologic patients by assessing the following : the proportion of various types of eczema, the distribution of eczema according to gender and age, and its incidence according to the month or season. this retrospective case study describes the clinical patterns of endogenous and exogenous eczema in patients seen in three dermatology clinics in the zliten area (zliten central hospital dermatology clinic and two private practices) from 1st january 2006 to 31st december 2006. the study also describes the clinical pattern of eczema, its relation to age and gender of patients, and its seasonal variation. diagnosis of eczema was based on internationally accepted criteria for the diagnosis of atopic dermatitis based on clinical features of the disease. the uk original criteria is that to make the diagnosis of atopic dermatitis in presence of itch for the preceding 12 months is required plus two or more of flexural dermatitis, onset before the age of two years, a personal history of asthma or hay fever, and dry skin. the diagnosis of cases was mainly clinical but skin scrapings and biopsies were taken and examined in doubtful cases. all data was coded, stored and analyzed by spss (statistical package for the social sciences). we screened 8,228 dermatologic patient records, out of which 1055 (12.8%) were found to be affected by at least one type of eczema. eczema affected all age groups, including infancy, but its contribution declined after the fifth decade of life (fig. female male to ratio was 1.02:1 age distribution of patients the number of eczema cases was higher from march to august, after which it declined to reach the lowest point in january (fig. unexpectedly, the number of cases in july was lower than in the adjacent months. the number of cases in spring and summer months combined (from march to august) significantly higher than those in winter and autumn months combined (from september to february) (p<0.01). monthly variation of the contribution of eczema to dermatologic conditions endogenous eczema was far more common than exogenous eczema (72.6% versus 24.9%, respectively, p<0.001) ; 2.5% of cases were unclassified. the most common type of eczema was contact dermatitis (22.7%) while asteatotic eczema was the least frequent type (0.7%). other types included pityriasis alba (10.5%), and hand eczema (9.1%) (table 1). the number of eczema cases was higher from march to august, after which it declined to reach the lowest point in january (fig. the number of cases in spring and summer months combined (from march to august) significantly higher than those in winter and autumn months combined (from september to february) (p<0.01). endogenous eczema was far more common than exogenous eczema (72.6% versus 24.9%, respectively, p<0.001) ; 2.5% of cases were unclassified. the most common type of eczema was contact dermatitis (22.7%) while asteatotic eczema was the least frequent type (0.7%). other types included pityriasis alba (10.5%), and hand eczema (9.1%) (table 1). carried put on a representative sample of over 20,000 people in the us, the prevalence of all forms of eczema was found to be 1.8%. a study in the uk examining the details of 6819 dermatological consultations of 3500 in a general practice from 1958 to 1985 showed that eczema patients comprised 19% of the consultations. most cases of eczema that are seen in infants and young children are atopic in type. discoid eczema occurs particularly in elderly males in winter, and asteatotic eczema of the legs is common among both elderly males and females. in an investigation of the pattern of endogenous eczema in the northern frontier, kingdom of saudi arabia, 1224 patients were studied over a three - year period from january 1991 to december 1993. atopic eczema was the most common type of endogenous eczema, with male to female ratio was about 1.1:1, which is similar to our findings. our study is in agreement with both of these findings. in the saudi study, seborrheic eczema occurred in 18% of patients, lichen simplex in 3.4%, pityriasis alba in 2.2%, and pompholyx in 2%, while in our study in zliten, libya, the respective frequencies were 17.2%, 5.9%, 10.5% and 0.9%. whereas in our study eczema accounted for 12.8% of dermatologic conditions, in egypt it accounted for 19.8%, and pityriasis alba was the most common eczema (13.5%), as compared to 10.5% in our study. hand dermatitis in the uk accounted for 15% of dermatologic conditions, check carefully while in zliten it accounted for 9%. however, in zliten contact dermatitis is much more common (22.7%) than in ipswich, uk (12%), and the higher frequency in zliten could be attributed to the rapid change in life style among libyans, and specially those living outside the major cities. use of medically unapproved body cleansers, e.g. hair and face lotions or cosmetics of unknown brands imported from china or arab countries is common. there is also a cement factory in the zliten area, which likely contributes to the increasing rate of this type of dermatitis. this hospital - based study provides some evidence that eczema is becoming a common public health problem requiring a proper strategy for care and prevention. epidemiological studies at the community level are needed to determine the incidence and prevalence of this important skin problem in zliten and in other libyan cities. | the life style and demographic structure of libyan society is changing, and this could affect the epidemiology of certain diseases, including eczema. the aim of this study was to assess the burden of eczema among a selected patient population in the zliten area in the northwest of libya. we conducted a retrospective study by reviewing case notes and records in public and private dermatology practices in the zliten area. the frequency of eczema among patients attending dermatology clinics in the zliten area was 12.8%, and the male to female ratio was almost 1:1. the most affected age group among patients was 20 - 29 years. eczema represented a larger proportion of dermatologic conditions during spring and summer. of all cases of eczema, 72.6% were endogenous and 24.9% were exogenous (p<0.001). the most common type of eczema was contact dermatitis (22.7% of all cases), followed by atopic dermatitis (19.7%) and pityriasis alba (10.5%). in conclusion, eczema is a public health problem in zliten - libya, and this necessitates prospective studies to determine its incidence and prevalence. |
crystals of angiotensinogen and the complex of human angiotensinogen with de - glycosylated renin - asp292ala were grown at room temperature under conditions listed in supplementary table 1. datasets were collected at 100k at the daresbury and diamond synchrotron light sources and processed with the ccp4 program suite. the structures of human, rat and mouse angiotensinogen were solved by molecular replacement in phaser27, using an ensemble of distantly related serpins as the search model with additional phase information obtained from a gdcl3 derivative of human angitensinogen. the structure of the complex with renin was solved by molecular replacement using renin from pdb entry 2bks28 and its peptide complex from pdb entry 1smr7. further details related to crystal structures and refinement, recombinant expression, thiol titration and nitrosylation and the assessment of sulphydryl blockage and oxidation, the determination of redox potentials, renin kinetics and the demonstration of reduced and oxidised forms of angiotensinogen, are given in methods. | blood pressure is critically controlled by angiotensins1, vasopressor peptides specifically released by the enzyme renin from the tail of angiotensinogen, a non - inhibitory member of the serpin family of protease inhibitors2,3. although angiotensinogen has long been regarded as a passive substrate, the crystal structures solved here to 2.1 resolution show that the angiotensin cleavage - site is inaccessibly buried in its amino - terminal tail. the conformational rearrangement that makes this site accessible for proteolysis is revealed in a 4.4 structure of the complex of human angiotensinogen with renin. the co - ordinated changes involved are seen to be critically linked by a conserved but labile disulphide bridge. we show that the reduced unbridged form of angiotensinogen is present in the circulation in a near 40:60 ratio with the oxidised sulphydryl - bridged form, which preferentially interacts with receptor - bound renin. we propose that this redox - responsive transition of angiotensinogen to a form that will more effectively release angiotensin at a cellular level contributes to the modulation of blood pressure. specifically, we demonstrate the oxidative switch of angiotensinogen to its more active sulphydryl - bridged form in the maternal circulation in pre - eclampsia - the hypertensive crisis of pregnancy that threatens the health and survival of both mother and child. |
. a myoepithelioma classically presents as an asymptomatic mass that slowly enlarges over a long period of time. however, recurrent and malignant ones have also been described. they are more likely to occur in the parotid gland, but rare cases arising from minor salivary glands are also on record.[35 ] the authors present an exceptionally rare case of sinonasal myoepithelioma with recurrent and resistant to treat clinical course. to our knowledge and regarding the medline database, the patient was a 57-year - old iranian male living in dargaz, a small district in north east of iran. he was admitted to the e.n.t ward, ghaem hospital, mashhad university of medical sciences in june 2010. first, at 1993, he accidentally discovered a small painless mass of the palate, which was excised in an outpatient care facility. thereafter, the patient had five more debulking surgeries. at the time of admission, the patient was complaining of nasal fullness, hyponasal speech, snoring, oral breathing, and loss of hearing on left side. on oral examination, the hard palate in the right side had been removed and a nasal mass was obvious through it. both computed tomography scan and magnetic resonance imaging demonstrated a huge bilateral lobulated sinonasal mass with extension to cribriform plate and near the dura, medial walls of both orbits, and upper gingival area [figure 1 ]. t1-weighted transverse (left) and sagittal (right) planes show a huge bilateral sinonasal mass the patient underwent a functional endoscopic sinus surgery to debulk the tumor. the removed specimen was stored in 10% buffer formalin and sent to the pathology department. the specimen was made by multifragmented irregular - shaped brownish soft tissue with the dimensions of 3.5 3 2 cm. the whole tissue was processed, cut, and stained with h and e. microscopically, the specimen showed a hypercellular tumoral tissue composed of plasmacytoid cells with eccentrally located round - ovoid nuclei and acidophilic cytoplasms. the neoplastic cells were arranged in nests, cords, and sheaths intermixed with partially mucoid and hyalinized collagenous stroma. scattered gland - like structures bordered by neoplastic cells were seen constituting less than 5% of the tissue. dispersed foci of spindle cells were also present surrounded by plasmacytoid cells [figure 2 ]. (b) plasmacytoid cells in the background of mucoid material on immunohistochemistry staining, the specimen showed strong and diffuse reactivity for vimentin, cytokeratin, smooth muscle actin and s-100 confirming the myoepithelial nature of the tumoral cells [figure 3 ]. (a) the specimen strongly expresses vimentin, (b) cytokeratin, (c) smooth muscle actin, and s-100 (d) confirming its myoepithelial nature the patient refused to undertake any more treatments. after 15 months of follow - up myoepithelioma is a generally benign neoplasm that constitutes approximately 11.5% of all salivary gland neoplasms. they are more likely to occur in the parotid gland, but rare cases arising from minor salivary glands such as in palate, gingiva, lips, nasopharyngeal space, orbit, and middle ear are also on record.[35 ] exceptionally, they can occur in the sinonasal cavity, with only seven reports, including our case, present in the literature.[611 ] histologically, myoepitheliomas are classified as spindle, plasmacytoid, reticular, epitheliod, and clear cell types. the most common is the spindle type, whereas the others are very rare. all the previously reported sinonasal myoepitheliomas were composed mostly of spindle cells and to a lesser extent of plasmacytoid/ epithelioid cells.[611 ] however, the present case was almost entirely composed of plasmacytoid cells, with dispersed foci of spindle cells. the most important differential diagnosis of myoepitheilomas includes pleomorphic adenoma. there is a general tendency to put myoepitheliomas at one end of the histologic spectrum that includes pleomorhic adenomas at the other end. this claim is supported by the fact that these two entities are very similar in biologic course, distribution, morphology, and even immunohistochemical aspects. world health organization proposes that if the neoplasm contains less than 5% of ductal components they must be named myoepithelioma. furthermore, several mesenchymal neoplasms such as fibrosarcoma, leiomyoma, schwannoma, and myxoma are in the differential diagnoses of the spindle cell myoepitheliomas, but they all lack the immunoreactivity for cytokeratin. however, in the presented case, the non- spindle (plasmacytoid) appearance of the neoplastic cells can easily rule out these entities. myoepitheliomas are characteristically immunoreactive for cytokeratins, s-100, calponin, smooth muscle actin, myosin, vimentin, glial fibrillary acidic protein, and carcinoembryonic antigen. in the presented case, conventional myoepitheliomas are not more aggressive than pleomorphic adenomas, but invasive myoepitheliomas that infiltrate the adjacent tissues have been described. the malignancy is assisted by the infiltrative margins, necrosis, marked pleomorphism, and prominent mitotic activity, which were all absent in the presented case. moreover, recurrence is associated with involved surgical margins at the first excision. in the present case, the tumor had a benign morphology with a progressive, invasive, and recurrent clinical course, as the tumor has been recurred six times within 17 years. therefore, the surgical excision must be done with wide healthy margins and an appropriate follow - up is also recommended. myoepitheliomas are generally well - circumscribed solid masses that measure less than 3 cm in diameter. however, in our case, the tumor was a huge bilateral sinonasal mass, which showed prominent extension to cribriform plate, dura, both orbits, and upper gingiva, so that complete surgical removal was practically impossible. to our knowledge, this is the most extensive myoepithelioma of its kind. some of the malignant myoepitheliomas have been arisen from pleomorphic adenomas. regarding our case, the primary lesion had been diagnosed as a myoepithelioma ; thus, the further transform of a pleomorphic adenoma seems improbable. we reported the seventh case of myoepitheliom of the sinonasal cavity ever diagnosed regarding the medline database until 2012. immunohistochemical investigation may be a useful tool in order to reach to the appropriate diagnosis while a myoepithelioma is in the differential diagnosis of a tumor of the oral / sinonasal cavity. | myoepithelioma is an uncommon benign neoplasm that most likely occurs in the parotid gland. extra - salivary myoepitheliomas are rare, with less than 10 cases reported in the sinonasal cavity. we present a rare case of benign myoepithelioma with recurrent behavior, abundant extensions to adjacent structures, and resistant to treat clinical course, which influenced the patient s quality of life for more than 18 years. histologic, immunohistochemical, and the potential differential diagnoses are discussed. the patient refused to undertake any more treatments, but after 15 months of follow - up, the lesion did not progress further. |
nickel - titanium alloys were developed in the early 1960 's by w. f. buehler, a metallurgist at the naval ordnance laboratory in silver springs, maryland, usa. considering its property of superior elasticity and resistance to torsional fracture, walia. introduced nickel - titanium endodontic rotary files to the field of endodontics. these properties help ni - ti instruments to negotiate and prepare curved root canals without early permanent deformation unlike stainless steel files. the ni - ti alloys used for manufacturing the endodontic files contain approximately 56% (wt) nickel and 44% (wt) titanium. though niti instruments have many advantages, incidence of instrument separations are not uncommon in clinical use. niti rotary instruments fracture in two different ways : fracture because of torsion and fracture because of flexural fatigue. the endodontic files are in constant contact with the irrigants during the cleaning and shaping procedures. even short - term contact with these irrigants can cause alterations in the surface of niti instruments. sodium hypochlorite is the most commonly used endodontic irrigant whose antimicrobial effectiveness is based on its high ph (ph > 11). there is a contact between rotary niti instruments and the naocl solution during clinical use and cleaning and sterilization procedures. nygaard ostby introduced ethylene diamine tetraacetic acid (edta) to the field of endodontics, in 1957, and recommended the use of 15% edta solution with a ph of 7.3. since then, edta has been used as the most common chelating solution, which reacts with calcium ions in dentin and form soluble chelates. the constant increase in antibiotic resistant microbial strains and the side effects caused by the use of synthetic drugs and also the hazards associated with irrigants such as naocl has prompted researchers to look for safer, more biocompatible and patient friendly herbal alternatives. among the herbal alternatives triphala has been proven to be safe and containing active constituents that have beneficial physiologic effect and also having curative properties such as being antioxidant, anti - inflammatory and radical scavenging. triphala is an indian ayurvedic herbal formulation consisting of dried and powdered fruits of three medicinal plants terminalia bellerica, terminalia chebula, and emblica officinalis. scanning electron microscopy has been widely used for surface analysis of niti rotary endodontic instruments. recent studies have been done to evaluate the surface topography of these instruments with the help of atomic force microscope (afm). atomic force microscope facilitates the imaging and analysis of surface topography with little or no sample preparation. energy - dispersive x - ray spectroscopy (eds or edx) is an analytical technique used for the elemental analysis or chemical characterization of a sample. it relies on the investigation of an interaction of x - ray excitation and a sample. it identifies and characterizes semi - quantitatively, the chemical elements present in deposits found on instrument surfaces. many studies have been conducted to analyze the surface of rotary niti endodontic files using eds. the aim of the present study is to evaluate the effects of naocl, edta and triphala on the nano - structure surface of protaper and irace rotary niti endodontic files using afm and eds. a total of eight nickel - titanium rotary endodontic files, four files each of protaper s2 (dentsply, switzerland) and irace r3 (fkg dentaire, switzerland) were used for this study. ltd, india), 17% edta (prime dental products pvt. ltd, india) and triphala (impcops, chennai, india). afm (5500-agilent technologies, usa) belongs to the scanning probe microscopy family and can reconstruct three - dimensional surface topography images in real time. the cantilever deflects in the z - direction as a result of the surface topography during tip scanning over the sample surface. this deflection in the cantilever is detected by a photodiode through a laser beam focused on and reflected from the rear of the cantilever. afm records data of the samples in digital form as sets of x, y and z values. in eds (bruker, usa), a high - energy beam of charged particles such as electrons, or a beam of x - rays, the incident beam may excite an electron in an inner shell, ejecting it from the shell while creating an electron hole where the electron was. an electron from an outer, higher - energy shell then fills the hole, and the difference in energy between the higher energy shell and the lower energy shell may be released in the form of an x - ray. the number and energy of the x - rays emitted from a specimen as the energy of the x - rays is characteristic of the difference in energy between the two shells, and of the atomic structure of the element from which they were emitted, this allows the elemental composition of the specimen to be measured. the cutting flutes of the three files each from protaper and irace were immersed in the three different irrigating solutions separately for 5 minutes. one file each from protaper group and irace group were evaluated without immersion in the irrigants and were kept as the control. nine areas of the surface were analyzed on a 3-mm section taken at the middle portion of the files. the afm images of the tested samples were then recorded using the contact mode of the afm under ambient conditions. a total of nine perfect squares of 2 2, 5 5 and 10 10 micrometers were examined on every sample, which was then unitized to 1 1 micrometer for statistical analysis. the roughness average (ra), root mean square (rms) and mean height of roughness profile elements (rc) of the scanned profiles were then recorded. these parameters depict the vertical topography of the instrument surface and an increase in these value means that there are alterations caused by the irrigants. the weight percentage of the elements present on the surface of the samples was evaluated using the energy - dispersive x - ray diffraction spectroscopy. data was analyzed using the computer software, statistical package for social sciences (spss) version 10. student 's t test was used to compare the mean values between protaper and irace groups. one - way analysis of variance (anova) was performed as parametric test to compare different treatments. duncan 's multiple range test was also performed along with anova as post - hoc test to elucidate multiple comparisons between treatments. for all statistical evaluations, the three - dimensional afm images of the surfaces of all the protaper and irace instruments including new and those immersed in the three different irrigants showed topographic irregularities at the nanometric scale. ra, rms and rc values of new protaper files were significantly lower compared to irace files (p < 0.05). the new and triphala immersed irace files did not differ significantly, but had a very highly significant difference with naocl and edta immersed irace files (p < 0.001) [table 1 ]. though both the protaper and irace show significant surface roughness properties, irace group showed higher values of surface roughness (ra, rms and rc) than the protaper group [tables 1 and 2 ]. among the immersed files, the maximum ra value was observed for naocl immersed irace files and minimum ra value was observed for triphala immersed protaper files. analysis of variance comparing different treatment of irace analysis of variance comparing different treatment of protaper the mechanical instrumentation of root canals alone can not sufficiently disinfect the canal space regardless of whether stainless steel or niti instruments are used. the ideal irrigant or combination of irrigants should destroy the bacteria, dissolve the necrotic tissue, lubricate the canal, remove the smear layer and not irritate the healthy tissues. sodium hypochlorite, as an irrigant encompasses many desirable properties required for an ideal root canal irrigant. edta creates a stable calcium complex with the dentin mud, smear layer or the calcific deposits present along the canal walls. triphala has been proven to be safe, containing active constituents that have beneficial physiologic effect and also having curative properties such as being antioxidant, anti - inflammatory and radical scavenging. the nitinol used in the manufacture of endodontic instruments contain approximately 56% (wt) nickel and 44% (wt) titanium. the manufacturers of nickel - titanium (niti) root canal instruments have come up with different methods, which focus on altering the surface of the alloy as well as altering the alloy microstructure in order to enhance the efficacy and the longevity of the niti instruments. in the present study, middle region of the cutting flutes was selected for evaluation based on the ease of specimen mounting and focusing on the atomic force microscope. since the aim of our study was to evaluate the effect of irrigants on the instrument surface, the region to be evaluated on the cutting flutes was assumed to be less significant. one important surface characteristic, as claimed by the manufacturers of irace, is that of electro - polishing, which is a method commonly employed for corrosion resistance and surface finishing of most metallic medical equipments. the eds analysis of our study specimens revealed the presence of tantalum (ta) on the surface of new irace files, which was not present on the surface of new protaper files. the presence of this noble metal on the surface of irace files may be attributed to the electro - polishing procedure. sodium hypochlorite is known to be corrosive to metals causing, selective removal of nickel from the niti instrument surface leading to micro pitting. it is supposed that these microstructural defects can lead to areas of stress concentration, crack formation and finally leads to the weakening of the instrument surface. the cutting efficiency of stainless steel files is significantly reduced by edta as a result of slight corrosion. in the present study, afm was selected for the present study as it is a sensitive and reliable technique that offers a suitable means for the acquisition of qualitative and quantitative data concerning the surface topography of rotary niti files. afm can reconstruct a three - dimensional image of the surface topography in real time. in the present study, both the untreated protaper and irace files showed surface deterioration at the nanometric scale. it is assumed that the presence of surface irregularities on niti files make them more prone to corrosion and fracture. manufacturing defects present in niti files can also play a role in the process of corrosion. in our study, both irace and protaper files have demonstrated considerable amount of instrument surface deterioration, which can attribute to the milling marks present on the file surface that act as crevices and thereby initiate corrosion. the present study showed higher surface roughness values for new irace files when compared with the new protaper files. the fact that both files were from different manufacturers could be a reason for such minute disparity in the surface roughness values between the files. a significant increase in surface roughness has been shown for naocl and edta immersed irace when compared to the naocl and edta immersed protaper sample files. the initial roughness profile of the study niti files has proportionally changed the intensity due to the immersion in the different irrigants. a previous study done using afm to evaluate edta and sodium hypochlorite immersed protaper files have shown significant surface alterations on the surface of edta immersed files when compared to naocl immersed study files. this result is in accordance with our study signifying high surface deterioration potential of edta when compared to naocl, which may be attributed to the lower ph of edta. as of date, there are no published data regarding the use of afm to evaluate the effect of any herbal irrigant on rotary endodontic files. in our study, sodium hypochlorite and edta immersed irace files have shown a significantly higher surface roughness values when compared to the triphala immersed irace files. the fact that triphala is a less reactive hydrocarbon compound when compared to other study irrigants could be a reason for this difference. in the present study, only the cutting flutes were immersed in the irrigants, so as to avoid any bias that may occur due to the interaction of different metals, which are present in the shank of the instrument. the energy dispersive x - ray microanalysis (eds) has shown that the cutting and non - cutting sections of protaper files was made of niti alloy, while the shank was made of gold - plated brass. the eds analysis showed that the surface of new protaper and irace files had almost the similar amount of nickel, titanium and oxygen components. the sodium and chlorine elements present on the surface of these niti files are due to the immersion in sodium hypochlorite. the eds result also showed a decrease in the weight percentage of nickel and titanium with a corresponding increase in the weight percentage of sodium and chlorine elements. the analysis on the nanoscale supplied evidences of the necessity of manufacturers to adjust processing parameters to minimize and prevent the irregularities on the surface of these instruments. to enhance the performance, durability and safety of root canal instruments, alteration in the inherent metallic properties like heat treatment can also be considered. within the limitations of this study, it can be concluded that : short - term contact with 17% edta and 5% naocl can cause significant surface deterioration on the surface of protaper and irace rotary niti endodontic files.electropolished irace files did not show any resistance to surface deterioration when compared to protaper files on immersion in irrigants.afm can be considered as a suitable method for quantifying and evaluating the surface characteristics of endodontic instruments. short - term contact with 17% edta and 5% naocl can cause significant surface deterioration on the surface of protaper and irace rotary niti endodontic files. electropolished irace files did not show any resistance to surface deterioration when compared to protaper files on immersion in irrigants. afm can be considered as a suitable method for quantifying and evaluating the surface characteristics of endodontic instruments. | aim : to use atomic force microscope and energy dispersive x - ray spectroscopy to evaluate the effect of 5% naocl, 17% edta and triphala on protaper and irace rotary ni - ti instruments.methodology:a total of eight ni - ti rotary files, four files each of protaper - s2 (dentsply) and irace - r3 (fkg dentaire) were used. three out of four files each from protaper and irace were immersed in 5% naocl, 17% edta and triphala separately for five minutes. the roughness average (ra), root mean square (rms) and mean height of roughness profile elements (rc) of the scanned profiles were then recorded using afm and the elemental composition was evaluated with eds. data were analyzed by student 's t test, one way anova and duncan 's multiple range test.results:topographic irregularities at the nanometric scale were observed for all files. files immersed in edta and naocl showed highly significant surface roughness than untreated files.conclusion:short-term contact with 17% edta and 5% naocl can cause significant surface deterioration of protaper and irace rotary niti files. afm proves to be a suitable method for evaluating the instrument surface. |
diabetes mellitus (dm) increases the risk of heart failure development even in the absence of coronary artery disease, hypertension, or other comorbidities1, 2 and could result in a two to fourfold greater mortality in heart failure patients. indeed, dm is capable of impairing the myocardial function : this is initially a clinically silent condition ; nevertheless, if left unrecognized and insufficiently managed, it could beget overt diabetic cardiomyopathy. the right ventricular (rv) function plays a significant role in the overall myocardial contractility.3 nevertheless, most of the previous studies regarding diabetes - induced changes in myocardial dysfunction were dedicated to the left ventricle (lv) at the cost of ignoring the role of the right heart chambers. the existing literature of course does contain a limited number of studies on the rv cardiomyopathies of patients with dm type i ; however, to our knowledge, there is only one recent study probing into dm type ii drawing up on three - dimensional strain and strain rate. the objectives of the present study were to evaluate the rv systolic and diastolic functions using conventional echocardiography, tissue doppler imaging (tdi), and deformation indices (strain and strain rate) in patients with dm type ii and without coronary artery disease or lv dysfunction. the study group was comprised of 22 diabetic patients (9 men and 13 women) aged 55.6 6.0 years. these patients either referred to the dm clinic of rajaei cardiovascular, medical and research center or were amongst the hospital stuff. the control group consisted of 22 healthy individuals (9 men and 13 women). all the diabetic patients enrolled in this study were asymptomatic, without any clinical evidence of either systolic or diastolic heart failure. the control subjects were considered to have a low probability of coronary disease or heart failure based on clinical data. coronary artery disease was excluded if there was a negative dobutamine stress echocardiographic examination or a negative treadmill exercise electrocardiographic test. in a few cases, coronary artery disease was excluded on the evidence of equivocal non - invasive stress tests and normal coronary angiograms. the other exclusion criteria included valvular or congenital heart disease, left ventricular ejection fraction (lvef) less than 50%, rhythms other than normal sinus rhythm, documented pulmonary diseases or pulmonary hypertension, and tricuspid regurgitation more than a mild degree. plasma glucose and hba1c were observed periodically and controlled by the endocrinologist in the aforementioned clinic. none of the patients had such diabetic complications as progressive and complex diabetic retinopathy, neuropathy, and nephropathy. the study participants in the two groups underwent echocardiographic examinations (vivid 7, ge vingmed). the variables measured are presented as follows : the rv dimension and tricuspid annular plane systolic excursion (tapse) were measured from the apical four - chamber view.the rv doppler parameters of the diastolic function (peak early [a ] and peak late diastolic flow velocity [e ], together with their ratio [e / a ] and deceleration time [dt ]) were recorded. the trans - tricuspid flow was measured in the apical four - chamber view using pulsed doppler, with the sample volume positioned at the tips of the tricuspid leaflets. the measurements were averaged from three end - expiratory cycles.tdi was conducted to assess the rv longitudinal myocardial function in the apical four - chamber view. color tdi was used in addition to two - dimensional images, with adjustment of the depth and sector angle to obtain an acceptable frame rate. the tdi variables of peak systolic velocity (sm), peak early diastolic velocity (em), peak late diastolic velocity (am), myocardial performance index (mpi), and acceleration time of the isovolumetric contraction time (ivct) were analyzed online.the deformation indices were measured by adjusting the imaging angle to achieve a parallel alignment of the beam with the myocardial segment of interest. the images were stored on a remote archive of the echocardiography machine so that they could be analyzed offline. the myocardial deformation curves were investigated from the basal segment of the rv free wall, which belongs to the inflow part of the rv, and also from the apical segment, which belongs to the trabecular portion of the rv.the strain rate and stain were measured based on the standard deformation measurement, and the peak of the r on the electrocardiography (ecg) was utilized to define end diastole. end systole was determined using the pulsed doppler velocity profile of the left ventricular outflow tract (lvot) as the aortic valve closure, and the rv end systole was determined using the pulsed wave profile of the right ventricular outflow tract (rvot) at end expiration. the analysis of the myocardial strain and strain rate data included the assessment of the systolic strain (), peak systolic strain rate (srs), and peak early diastolic strain rate (sre). the rv dimension and tricuspid annular plane systolic excursion (tapse) were measured from the apical four - chamber view. the rv doppler parameters of the diastolic function (peak early [a ] and peak late diastolic flow velocity [e ], together with their ratio [e / a ] and deceleration time [dt ]) were recorded. the trans - tricuspid flow was measured in the apical four - chamber view using pulsed doppler, with the sample volume positioned at the tips of the tricuspid leaflets. tdi was conducted to assess the rv longitudinal myocardial function in the apical four - chamber view. color tdi was used in addition to two - dimensional images, with adjustment of the depth and sector angle to obtain an acceptable frame rate. the tdi variables of peak systolic velocity (sm), peak early diastolic velocity (em), peak late diastolic velocity (am), myocardial performance index (mpi), and acceleration time of the isovolumetric contraction time (ivct) were analyzed online. the deformation indices were measured by adjusting the imaging angle to achieve a parallel alignment of the beam with the myocardial segment of interest. the images were stored on a remote archive of the echocardiography machine so that they could be analyzed offline. the myocardial deformation curves were investigated from the basal segment of the rv free wall, which belongs to the inflow part of the rv, and also from the apical segment, which belongs to the trabecular portion of the rv. the strain rate and stain were measured based on the standard deformation measurement, and the peak of the r on the electrocardiography (ecg) was utilized to define end diastole. end systole was determined using the pulsed doppler velocity profile of the left ventricular outflow tract (lvot) as the aortic valve closure, and the rv end systole was determined using the pulsed wave profile of the right ventricular outflow tract (rvot) at end expiration. the analysis of the myocardial strain and strain rate data included the assessment of the systolic strain (), peak systolic strain rate (srs), and peak early diastolic strain rate (sre). the student unpaired t - test was used to evaluate the differences between the groups, and the chi - square test was employed to compare the categorical variables. for the statistical analyses, the statistical software spss version 17.0 for windows (spss inc., there were no significant differences between the two groups in terms of sex, age, body mass index, creatinine, total cholesterol level, low - density lipoprotein cholesterol (ldl), high - density lipoprotein cholesterol (hdl), and being a smoker. the diabetic group, however, showed a higher level of triglyceride (p value = 0.049), but this was statistically, and not clinically, significant. the echocardiographic and doppler data of the studied groups are illustrated in table 2. there were no significant differences between the two groups as regards the right ventricular end diastolic diameter (rvedd) (mm), and tricuspid e velocity, whereas the tricuspid annular plane systolic excursion (tapse), and tricuspid e / a ratio of the diabetic patients (18.9 2.1 vs. 23.2 2.9 and 0.96 0.2 vs. 1.21 0.3, respectively) were significantly lower. the tricuspid a velocity and e wave deceleration time of the diabetic patients (0.5 0.1 m / s vs. 0.4 0.1 m / s and 289.2 67 msec vs. 250.9 57.1 msec) were significantly higher. the tdi data of the study population are shown in table 3, according to which the rv basal segment sm, em, am, and e / em (12 1.9 cm / sec vs. 13.4 2.1 cm / sec, 8.5 2.1 cm / sec vs. 11.6 2.4 cm / sec, 12.6 2.7 cm / sec vs. 14.7 3.3 cm / sec, and 0.05 0.01 vs. 0.04 0.01, respectively) were significantly lower in the case group than in the control group. there were no significant differences between the two groups with respect to the mpi of the rv and the acceleration time of the ivct. the data on the deformation indices of the two study groups are demonstrated in table 4, according to which the rv basal segment systolic strain () and rv apical segment (, srs, and sre) of the case group were significantly lower than those in the control group (p value < 0.01). there were, however, no significant differences in regard to the rv basal segment srs and sre between the two groups. there were no significant correlations between hba1c, duration of diabetes, c - reactive protein (crp), and measures of the rv systolic or diastolic dysfunction (based on conventional doppler, tdi data, or deformation indices). the present study shows that both systolic and diastolic rv functions are impaired in patients with dm type ii and without coronary artery disease or ejection fractions of less than 50%. this finding was demonstrated by significantly lower rv systolic parameters (tapse, rv basal sm, basal and apical rv free wall systolic strains, and apical srs) and lower rv diastolic parameters (e / a ratios, em, am, e / em ratios, and apical early diastolic strain rate [sre ]) in our case group than those in a normal, sex- and age - matched control group. there were no statistically significant differences in the rv basal segment srs and sre between the diabetic and control subjects, which may be due to difficulties in drawing basal rv strain rate curves. our results chime in with those of the kosmala.,4 study, which reported the impairment of both basal and apical segments of the rv free wall performance. in that study, however, the apical segments exhibited more pronounced systolic impairment than did the basal segments and it was concluded that the rv might be divided into the three components of the inflow or sinus, the trabecular, and the outflow portions. geva.,5 stated that the inflow region, compared with the other parts of the rv, had significant predominance in fiber shortening and contribution to the global rv systolic function. as the basal segment of the rv free wall is a part of the inflow component, and the apical segment refers to the trabecular portion of the rv, they suggested that the differences seen in their cohort might be related to the regional inhomogeneity of the rv in patients with dm. the kosmala., study4 reported rv diastolic dysfunction in a non - uniform type i and type ii diabetic cohort using tdi and demonstrated significantly lower values of em and em - to - am ratio in the basal and mid - segments and longer isovolumic relaxation time (irtm) in the mid - segment. nonetheless, that study failed to show any difference in the e / a ratio, em, and systolic parameters (sm and tapse) between the diabetic patients and normal subjects. furthermore, the said study found no significant correlations between the estimated echocardiographic parameters and indices of diabetic control (plasma glucose and hba1c) as well as the duration of diabetes. in another study, kosmala.,4 evaluated non - uniform type i and type ii diabetic groups and showed impairment of the rv systolic function according to deformation studies (rv systolic stain and srs in the basal and apical segments of the rv free wall) and impairment of the rv diastolic function (decreased sre in both rv segments). in a study by karamitsos.,6 type i diabetic patients were found to have an impaired rv diastolic function (trans - tricuspid e velocity, a velocity, e / a ratio, em, and am). consistent with the findings of the present study, gaber.,7 assessed patients with dm type ii and demonstrated impairment of the rv systolic and diastolic functions according to deformation studies via three - dimensional echocardiography (systolic strain, srs, and sre in both rv basal and apical segments). the present study, in agreement with some other similar studies,4, 8, 9 found no importance for the impact of the duration of diabetes on the rv function. whether the rv diastolic abnormalities have prognostic implications in the clinical course of patients with dm type ii remains to be investigated. we believe that serial echocardiography measurements are warranted in this diabetic population if the progression from subclinical rv involvement to symptomatic rv dysfunction is to be followed. in addition, subclinical rv systolic and diastolic abnormalities should be considered when planning pharmacotherapy to prevent the development of symptomatic rv dysfunction. | background : diabetes mellitus is capable of impairing the myocardial function. several studies have documented the influential impact of diabetes mellitus on the left ventricular function. the right ventricular function plays a significant role in the overall myocardial contractility ; hence, this study was undertaken to evaluate the effect of diabetes mellitus type ii on the right ventricular function.methods:twenty-two diabetic patients without any coronary artery disease, hypertension, or left ventricular dysfunction were studied. the right ventricular end diastolic diameter, tricuspid plane systolic excursion, right ventricular inflow doppler parameters, longitudinal myocardial velocities, and deformation indices from the basal and apical segments of the right ventricular free wall of the case group were measured. the control group consisted of 22 healthy individuals.results:the tricuspid annular plane systolic excursion (tapse) and tricuspid peak early to peak late diastolic flow velocities ratio (e / a) in the diabetic patients were significantly lower than those of the control group patients (18.9 vs. 23.2, p value < 0.001 and 0.96 vs. 1.21, p value = 0.012), but there were no significant differences in the right ventricular end diastolic diameter and the right ventricular tei index between the two groups (p value = 0.72). the right ventricular basal peak myocardial systolic velocity (sm) (12 cm / sec vs. 13.4 cm / sec ; p value = 0.03), basal and apical right ventricular free wall systolic strain (13.3% and 18.7% vs. 20.2% and 25.7% ; p value = 0.001), and apical strain rate (1.2 1/s vs. 1.6 1/s ; p value = 0.008) were significantly lower in the study group. there was a weak correlation between the right ventricular function and hba1c as well as the duration of diabetes mellitus and c - reactive protein.conclusion:our results suggest that diabetes mellitus type ii can influence the right ventricular function in the absence of coronary artery disease, diastolic dysfunction, and pulmonary hypertension. |
the development of artificial bioengineered constructs largely depends on adequate vascularization to guarantee sufficient oxygen supply. the arteriovenous (av) loop model is an established method to obtain transplantable bioartificial tissue in vivo. originally based on the findings of erol and spira and subsequently modified by murphy. and tanaka. this model of benign angio - inductive behavior triggers the formation of novel vessel sprouts remarkably without additional extrinsic and angio - inductive factors. in brief, a femoral vein is dissected, harvested, and as an autologous graft, interposed between the contralateral femoral artery and vein. this results in an arteriovenous fistula, which is then embedded in a fibrin - filled teflon chamber. a mature microcirculatory system originates mainly from the venous graft, thereby facilitating cultivation, harvesting, and transfer of rudimentary organ - like structures to a secondary defect site, thus minimizing donor site morbidity [69 ]. as vascularization is crucial to promote clinical implementation of bioengineered constructs, tremendous research efforts have been undertaken to further optimize vessel formation within the av loop. the current understanding of angiogenesis is evolving, as the underlying nature and mediating signaling pathways fundamentally differ in various types of angiogenesis. watson. demonstrated that vascular flow is essential for hypoxia - driven angiogenesis in embryonic development. furthermore, we recently pointed out that distinct av associated changes of the hemodynamic load namely the shear rate are responsible for triggering the angio - inductive behavior of endothelial cells within the venous graft. both of these observations fundamentally rely on the presence of angio - inductive endothelial cells and a viable venous graft. however, polykandriotis. and westerband moreover, zdolsek. demonstrated that utterly decellularized and avital vein allografts are continuously able to maintain angio - inductive properties within the av loop. due to the constant progress in reconstructive surgery and due to improved and standardized microsurgical treatment algorithms, the range of medical indications for reconstructive vascularized tissue transfers has increased dramatically during the last decades. thus, microvascular flaps are exposed to high - dose radiation after defect coverage with increasing frequency. for this reason, the vascular behavior of novel reconstructive approaches, such as intrinsically vascularized av loop - based constructs, allows an excellent evaluation of the accompanying angiogenesis, and is of special interest in evaluating the response to radiation. furthermore, the understanding of the av loop associated angiogenesis is of special translational importance. there is an ongoing discussion as to whether the interposed venous graft is vitally important for the induction of angiogenesis under conditions of high flow. in order to alter the cellular hemostasis specifically within the grafted vein we exposed the graft intraoperatively to 2 gy of ionizing radiation (a level of radiation considered a relevant rat dose - rate). we wanted to demonstrate that the graft possessed vital intrinsic factors that were at least partially responsible for triggering flow - mediated angiogenesis in the av loop model, and we generally wanted to investigate the effect of radiation in the developing av loop microvasculature. beside three - dimensional micro - ct, a previously described observer - independent automatic software algorithm was used to assess vessel number, density, and area by analyzing two - dimensional histological cross sections in order to characterize the differences of angiogenesis in detail. all operations were performed by the same investigator (jmc) using a surgical microscope (magnification 16, opmi ifc, carl zeiss, oberkochen, germany). 2 to 4 month old male lewis rats (n=14) with an average body mass of 340 g were obtained from charles river laboratories (sulzfeld, germany). all experiments were approved by the institutional animal care and use committee of the regierungsprsidium mittelfranken (az 54 - 2532.1 - 34/09) and in accordance with the german animal welfare act. for induction of anesthesia, the rats received 5% isoflurane (baxter, vienna, austria) inhalation. before surgery, they received buprenorphin for analgesia, (0.3 mg / kg body weight, temgesic, essex chemie ag, luzern, switzerland), as well as heparin anticoagulation (80 iu / kg liquemin, ratiopharm ulm, germany). the right femoral vascular bundle was exposed by a mid - ventral incision. the femoral vein was dissected and a 20 mm long venous graft was harvested. the grafts either underwent irradiation or were directly interposed as a loop (figure 1) between the contralateral femoral artery and vein using 11/0 nylon sutures (ethilon, ethicon, norderstedt, germany) after similar incubation in sodium heparin solution (sham treatment, 10,000 ie / l). the av loop was embedded in the isolation chamber, which was filled with 800 l of fibrin sealant (tissucol, baxter) composed of fibrinogen (10 mg / ml), thrombin (2 iu / ml) and aprotinin (1,500 kie / ml) as described previously. the chamber was sutured subcutaneously onto the underlying adductor fascia with 6 - 0 polypropylene (prolene 6/0, ethicon, norderstedt, germany). for wound closure, interrupted vertical mattress sutures with vicryl 4 - 0 (ethicon, norderstedt, germany) were used. all rats received buprenorphin and heparin (80 iu / kg liquemin, ratiopharm ulm, germany) postoperatively. the rats were kept at a 12-hour dark / light cycle in the animal facility of the university of erlangen medical centre with free access to standard chow (sniff) and water. at the end of the experiment, the rats were sacrificed under deep anesthesia (5% isoflurane) by exsanguination, and subsequent reperfusion with india ink or microfil (flowtech, ma, usa). the autologous venous graft was harvested and rinsed in sodium heparin solution (10,000 ie / l). a dose of 2 gy ionizing radiation was applied to the vein graft within 30 seconds using an isovolt titan x - ray machine with a 120 kv power supply (ge sensing & inspection technologies, ahrensburg, germany). after 15 days, an abdominal midline incision was performed and newly formed vessels along the main vessel axis were detected by cannulating the distal descending aorta using a 24-gauge catheter. the descending vascular system was cleansed with 100 ml of prewarmed (37c) isotonic salt solution containing heparin (100 ie / ml) and subsequently perfused with 30 ml of prewarmed (37c) india ink solution [50% v / v india ink (rohrer & klinger) ] in 5% gelatin and 4% mannitol in order to detect additional vessels around the constructed av loop. micro - ct analysis required the reperfusion of the circulatory system with 20 ml of warmed yellow microfil (mv-122) containing 5% of mv curing agent (flowtech inc., carver, usa). incubation at 4c for 24 hours allowed the gelatin and mannitol to solidify before continuing with downstream applications. for histological analysis then 5 m histological sections in standardized planes perpendicular to the longitudinal av loop axis were produced using a microtome (leica microsystems, wetzlar, germany). hematoxylin and eosin (h&e) staining was performed using an autostainer (st5010, leica microsystems) according to standard protocol. isolectin b4 (sigma l 2140, sigma - aldrich, hamburg, germany) staining was used to detect luminal endothelial cells by means of immunohistochemistry as described elsewhere. histological cross sections were visualized using an inversed microscopy (olympus ix81, 10 magnification, olympus corporation, hamburg, germany) and analyzed using an automatic two - dimensional quantification algorithm established previously by our group for superior morphometric quantification. high - resolution micro - ct scans were acquired on a cone - beam micro - ct scanner at the institute of medical physics, university erlangen, germany (forbild scanner). further technical specifications and specialized algorithms for optimization and enhancement of images obtained were described in detail elsewhere. for statistical analysis and figures, originpro 2015 (b9.2.214, originlab corp., for analysis of more than two groups, analysis of variance (one - way anova) was used. all operations were performed by the same investigator (jmc) using a surgical microscope (magnification 16, opmi ifc, carl zeiss, oberkochen, germany). 2 to 4 month old male lewis rats (n=14) with an average body mass of 340 g were obtained from charles river laboratories (sulzfeld, germany). all experiments were approved by the institutional animal care and use committee of the regierungsprsidium mittelfranken (az 54 - 2532.1 - 34/09) and in accordance with the german animal welfare act. for induction of anesthesia, the rats received 5% isoflurane (baxter, vienna, austria) inhalation. before surgery, they received buprenorphin for analgesia, (0.3 mg / kg body weight, temgesic, essex chemie ag, luzern, switzerland), as well as heparin anticoagulation (80 iu / kg liquemin, ratiopharm ulm, germany). the right femoral vascular bundle was exposed by a mid - ventral incision. the femoral vein was dissected and a 20 mm long venous graft was harvested. the grafts either underwent irradiation or were directly interposed as a loop (figure 1) between the contralateral femoral artery and vein using 11/0 nylon sutures (ethilon, ethicon, norderstedt, germany) after similar incubation in sodium heparin solution (sham treatment, 10,000 ie / l). the av loop was embedded in the isolation chamber, which was filled with 800 l of fibrin sealant (tissucol, baxter) composed of fibrinogen (10 mg / ml), thrombin (2 iu / ml) and aprotinin (1,500 kie / ml) as described previously. the chamber was sutured subcutaneously onto the underlying adductor fascia with 6 - 0 polypropylene (prolene 6/0, ethicon, norderstedt, germany). for wound closure, interrupted vertical mattress sutures with vicryl 4 - 0 (ethicon, norderstedt, germany) were used. all rats received buprenorphin and heparin (80 iu / kg liquemin, ratiopharm ulm, germany) postoperatively. the rats were kept at a 12-hour dark / light cycle in the animal facility of the university of erlangen medical centre with free access to standard chow (sniff) and water. at the end of the experiment, the rats were sacrificed under deep anesthesia (5% isoflurane) by exsanguination, and subsequent reperfusion with india ink or microfil (flowtech, ma, usa). the autologous venous graft was harvested and rinsed in sodium heparin solution (10,000 ie / l). a dose of 2 gy ionizing radiation was applied to the vein graft within 30 seconds using an isovolt titan x - ray machine with a 120 kv power supply (ge sensing & inspection technologies, ahrensburg, germany). after 15 days, an abdominal midline incision was performed and newly formed vessels along the main vessel axis were detected by cannulating the distal descending aorta using a 24-gauge catheter. the descending vascular system was cleansed with 100 ml of prewarmed (37c) isotonic salt solution containing heparin (100 ie / ml) and subsequently perfused with 30 ml of prewarmed (37c) india ink solution [50% v / v india ink (rohrer & klinger) ] in 5% gelatin and 4% mannitol in order to detect additional vessels around the constructed av loop. micro - ct analysis required the reperfusion of the circulatory system with 20 ml of warmed yellow microfil (mv-122) containing 5% of mv curing agent (flowtech inc., carver, usa). incubation at 4c for 24 hours allowed the gelatin and mannitol to solidify before continuing with downstream applications. for histological analysis, the chambers with the av loop were explanted. formalin - fixed, paraffin - embedded tissue sections were obtained using standard protocols. then 5 m histological sections in standardized planes perpendicular to the longitudinal av loop axis were produced using a microtome (leica microsystems, wetzlar, germany). hematoxylin and eosin (h&e) staining was performed using an autostainer (st5010, leica microsystems) according to standard protocol. isolectin b4 (sigma l 2140, sigma - aldrich, hamburg, germany) staining was used to detect luminal endothelial cells by means of immunohistochemistry as described elsewhere. histological cross sections were visualized using an inversed microscopy (olympus ix81, 10 magnification, olympus corporation, hamburg, germany) and analyzed using an automatic two - dimensional quantification algorithm established previously by our group for superior morphometric quantification. high - resolution micro - ct scans were acquired on a cone - beam micro - ct scanner at the institute of medical physics, university erlangen, germany (forbild scanner). further technical specifications and specialized algorithms for optimization and enhancement of images obtained were described in detail elsewhere. for statistical analysis and figures, originpro 2015 (b9.2.214, originlab corp., northampton, ma, usa) was used. all data are presented as mean sem and compared using unpaired t - test. for analysis of more than two groups, analysis of variance (one - way anova) all rats survived the experiments and tolerated the anesthesia and surgical procedures well (n=14). one rat in the control group had a significant swelling in the groin in the first week after surgery most likely due to seroma formation. after explantation of the chamber, the peripheral tissue was examined by macroscopic assessment. the matrix embedding the av loop within the chambers had a milky translucent appearance at implantation. stained main vessel axis was visible in all rats indicating a successful reperfusion and patency of the av loop at the time of reperfusion. fifteen days after the loop implantation, angiogenesis was visualized by three - dimensional micro - ct analysis in two rats from each group. in line with previous results, control loops showed homogenous vessel sprouting that originated predominantly from the lumen of the graft (figure 2a). local graft irradiation considerably impaired neovessel formation along the graft (figure 2b). lumina of the microcirculatory system along the graft appeared darkly stained in h&e and lectin stained cross sections of india ink - or microfil - perfused av loop constructs. the microcirculatory system along irradiated grafts was predominantly aligned in close proximity to the main vessel axis. when a non - irradiated control graft was used, however, newly formed vessels extended further peripherally from the main vessel axis (figure 3). the degree of vascular sprouts was assessed by an automatic observer - independent quantification algorithm, as described earlier, and yielded a total amount of 739111 (n=7) vessels per cross section. no significant morphometrical differences were found between the inflow (43183, n=7) and the outflow tract (30843, n=7) of non - irradiated control grafts (figure 4a). the amount of vessels of the graft inflow (17458, n=7) was not statistically different from the graft outflow (13738 n=7). mean vessel area (figure 4b) assessed by cross section analysis within constructs where control grafts were used amounted to 238,92463,126 m and, similar to the vessel number, there were no statistical significant differences among control grafts with an area of 151,73051,975 m (n=7) at inflow segments and 84,33722,964 m (n=7) at outflow segments. irradiated grafts behaved correspondingly with an area of 21,9986,850 m, n=7 (graft inflow) and 21,1405,897 m, n=7 (graft outflow) respectively. although luminal sprouting was strongly impaired, the formation of new vessels from the luminal graft was not thoroughly abolished. single - dose irradiation of 2 gy reduced the mean number of vessel sprouts to 31173 (n=7) and was significantly different from the control (figure 4a). irradiation reduced mean vessel area to 43,13710,224 m (n=7) compared to 238,92463,126 m (n=7) in corresponding control grafts (figure 4b). the mean number of vessels and area of irradiated grafts were significantly lower when compared to non - irradiated control grafts (figure 4a and 4b). while not statistical significant, mean vessel diameter after graft irradiation (141.8 m, n=7) tended to be lower compared to control grafts (192.6 m, n=7, figure 5). densities of neovessels were differently distributed when comparing av loop constructs with control and irradiated grafts. vessel formation aroused primarily within a peripheral radius of 401600 m when normalized to the main vessel axis. in contrast, mean vessel diameter began to differ at radii greater than 400 m, suggesting that radiation may have negative effects on peripheral maturation of newly formed vessels (figure 6b). all rats survived the experiments and tolerated the anesthesia and surgical procedures well (n=14). one rat in the control group had a significant swelling in the groin in the first week after surgery most likely due to seroma formation. after explantation of the chamber, the peripheral tissue was examined by macroscopic assessment. the matrix embedding the av loop within the chambers had a milky translucent appearance at implantation. stained main vessel axis was visible in all rats indicating a successful reperfusion and patency of the av loop at the time of reperfusion. fifteen days after the loop implantation, angiogenesis was visualized by three - dimensional micro - ct analysis in two rats from each group. in line with previous results, control loops showed homogenous vessel sprouting that originated predominantly from the lumen of the graft (figure 2a). local graft irradiation considerably impaired neovessel formation along the graft (figure 2b). lumina of the microcirculatory system along the graft appeared darkly stained in h&e and lectin stained cross sections of india ink - or microfil - perfused av loop constructs. the microcirculatory system along irradiated grafts was predominantly aligned in close proximity to the main vessel axis. when a non - irradiated control graft was used, however, newly formed vessels extended further peripherally from the main vessel axis (figure 3). the degree of vascular sprouts was assessed by an automatic observer - independent quantification algorithm, as described earlier, and yielded a total amount of 739111 (n=7) vessels per cross section. no significant morphometrical differences were found between the inflow (43183, n=7) and the outflow tract (30843, n=7) of non - irradiated control grafts (figure 4a). the amount of vessels of the graft inflow (17458, n=7) was not statistically different from the graft outflow (13738 n=7). mean vessel area (figure 4b) assessed by cross section analysis within constructs where control grafts were used amounted to 238,92463,126 m and, similar to the vessel number, there were no statistical significant differences among control grafts with an area of 151,73051,975 m (n=7) at inflow segments and 84,33722,964 m (n=7) at outflow segments. irradiated grafts behaved correspondingly with an area of 21,9986,850 m, n=7 (graft inflow) and 21,1405,897 m, n=7 (graft outflow) respectively. although luminal sprouting was strongly impaired, the formation of new vessels from the luminal graft was not thoroughly abolished. single - dose irradiation of 2 gy reduced the mean number of vessel sprouts to 31173 (n=7) and was significantly different from the control (figure 4a). irradiation reduced mean vessel area to 43,13710,224 m (n=7) compared to 238,92463,126 m (n=7) in corresponding control grafts (figure 4b). the mean number of vessels and area of irradiated grafts were significantly lower when compared to non - irradiated control grafts (figure 4a and 4b). while not statistical significant, mean vessel diameter after graft irradiation (141.8 m, n=7) tended to be lower compared to control grafts (192.6 m, n=7, figure 5). densities of neovessels were differently distributed when comparing av loop constructs with control and irradiated grafts. vessel formation aroused primarily within a peripheral radius of 401600 m when normalized to the main vessel axis. in contrast, mean vessel diameter began to differ at radii greater than 400 m, suggesting that radiation may have negative effects on peripheral maturation of newly formed vessels (figure 6b). to the best of our knowledge, this is the first study investigating the effects of ionizing radiation within an av loop - based neovascular system. grafts were exposed intraoperatively to a single dose of 2 gy ionizing radiation prior to loop construction in order to elucidate whether they provide angio - inductive features under conditions of high flow that are mandatory for av loop associated angiogenesis. as assessed by three - dimensional micro - ct analysis and two - dimensional quantification of mean vessel number and area, we demonstrated that vessel sprouting was significantly impaired when irradiated vein grafts were used for av loop construction. further dissecting this angio - inductive phenomenon histologically, we provided evidence that the emerging microcirculatory system was homogeneously distributed along the main vessel axis additionally, we illustrated that radiation compromises neovessel stabilization and maturation as mean vessel diameter primarily decreased in more peripheral regions of constructs containing irradiated vein grafts. changes in mechanical load following vein grafting into an arteriovenous shunt flow have been shown to foster angio - inductive properties in the graft endothelium. however, it has also been demonstrated that vascular grafts are exposed to a significant amount of endothelial denudation. notably, even decelluarized and avital vascular allografts seem sufficient to provide angiogenesis in the av loop. thus, it has been proposed that the venous graft merely serves as a connecting prosthesis providing sufficient length to ensure a tension - free av anastomosis. contrary to this believe, we repeatedly observed luminal sprouts emerging from the grafted vein. observed by means of scanning electron microscopy (sem) of corrosion casts, that luminal sprouting was present in the vein and the grafted vein but not in the arterial segment between day 10 and 14 after av loop creation. we previously pointed out that, in contrast to pulsatile flow, increased vascular shear rate induced specific cellular responses linked to angio - inductive behavior. notably, intercellular gap junction protein connexin (cx) 43 is specifically upregulated in vein graft endothelium, and it has been suggested that it is vital for angiogenic signaling and vessel growth. thus, we believe that the interposed graft transduces mechanosensitive stimuli after exposure to high flow. the detrimental effects of ionizing radiation have long been recognized and are widely used in adjuvant and neoadjuvant treatment options worldwide. hydroxyl radicals (oh) alter cellular lipid hemostasis, membrane transport, and constitute significant genotoxic stressors on vascular cells. double - strand dna breaks ultimately activate proto - oncogenes and propel malignant cell transformation. the molecular mechanisms behind radiation - induced genetic alterations are for the time being largely unidentified but may, among others factors, be linked to dysfunctional expression and nuclear translocation of cytoplasmic transcription factors. within blood vessels, endothelial cells are the most vulnerable to irradiation. radiation - induced vascular injury correlates histologically with desquamated endothelial cells, which leads to small vessel obstruction and subsequent tissue ischemia. it has been previously demonstrated that the acute effect of radiation - induced vascular injury depends on vessel diameter. severity of adventitial fibrosis and medial hyalinization are most distinctively recognized in vessels < 500 m. in the presented study, we showed that single - dose graft irradiation with 2 gy impaired the development of a functional microcirculatory system likely due to dysfunctional graft - specific and angio - inductive properties. the clinical management of head and neck tumors and extended extremity sarcomas continue to challenge reconstructive surgeons worldwide ; av loop - based tissue transfer may provide an essential adjunct for vascularized tissue transfer after oncologic resection. as the majority of these patients regularly require adjuvant radiotherapy, the effect of radiation on av loop vascularization is of great clinical importance. although strongly decreased, vessel sprouting was not fully abolished after irradiation in our study. hence, vascular shear rate might constitute a fairly sturdy and vital determinant for loop - associated neoangiogenesis even after isolated and high - dose radiation of the mediating vein. nevertheless, we only examined short - term effects of radiation on av loop associated angiogenesis. as vascular tissue mainly consists of slowly proliferating cells, single - dose local graft irradiation may impede vascular hemostasis of bioengineered constructs primarily in the long term. further analysis of radiation - mediated ramifications of the av loop model may contribute to safe clinical implementation and adequate patient selection. the isolated and homogeneous chamber environment, as well as the established quantification algorithms, render the av loop model eligible for compartment - specific characterization of newly developing vascular networks. as various malignant microenvironments induce different dynamic and angio - inductive behavior, the av loop model may conceivably be a useful tool for further in vivo characterization of anti - angiogenic treatment options. dynamic and molecular interactions of various cancer stem cells with a defined vascular supply, as well as their intrinsic susceptibility to radiotherapy and other novel and locally restricted treatment options, may conveniently and separately be studied within the av loop chamber. in the presented study, we investigated the effects of radiation in a benign angiogenesis rat model. these findings provide a useful tool for further molecular characterization of tumor growth ; especially as various tumor cells differently interact with vascular structures thereby altering radiation sensitivity. taken together, we provided evidence that the venous graft constitutes a vital part of the av loop model and is essential to initiate vascular luminal sprouts. it remains speculative whether reduced graft viability or cellular alterations are responsible, as irradiation induces a plethora of vascular and metabolic changes within the vascular tissue. although not completely impaired, graft - specific irradiation negatively influences av loop associated vascularization. radiation compromises neovessel stabilization and maturation primarily in peripheral regions of av loop - based constructs. these results add to the current understanding of av loop - based neovascularization and suggest clinical implications for patients relying on combined av loop - based tissue transfer and adjuvant radiotherapy. | backgroundthe arteriovenous (av) loop model enables axial vascularization to gain a functional microcirculatory system in tissue engineering constructs in vivo. these constructs might replace surgical flaps for the treatment of complex wounds in the future. today, free flaps are often exposed to high - dose radiation after defect coverage, according to guideline - oriented treatment plans. vascular response of av loop - based constructs has not been evaluated after radiation, although it is of particular importance. it is further unclear whether the interposed venous av loop graft is crucial for the induction of angiogenesis.material/methodswe exposed the grafted vein to a single radiation dose of 2 gy prior to loop construction to alter intrinsic and angio - inductive properties specifically within the graft. vessel loops were embedded in a fibrin - filled chamber for 15 days and radiation - induced effects on flow - mediated vascularization were assessed by micro - ct and two - dimensional histological analysis.resultsvessel amount was significantly impaired when an irradiated vein graft was used for av loop construction. however, vessel growth and differentiation were still present. in contrast to vessel density, which was homogeneously diminished in constructs containing irradiated veins, vessel diameter was primarily decreased in the more peripheral regions.conclusionsvascular luminal sprouts were significantly diminished in irradiated venous grafts, suggesting that the interposing vein constitutes a vital part of the av loop model and is essential to initiate flow - mediate angiogenesis. these results add to the current understanding of av loop - based neovascularization and suggest clinical implications for patients requiring combined av loop - based tissue transfer and adjuvant radiotherapy. |
aging is associated with an increased risk of developing respiratory impairment1. after a stroke, respiratory function lung function tests are not necessarily conducted in rehabilitation, sometimes because there is no examination equipment. (mainly for the pectoralis major muscle) is to improves the ratio of dyspnea in chronic obstructive pulmonary disease with respiratory dysfunction. therefore, it appears thought that the shoulder joint horizontal adductor muscles on the rib cage are involved in respiratory function. shoulder joint horizontal adductor muscle strength measurements could be used as an alternative evaluation of cases for whom lung function tests are difficult. accordingly, this study examined a measurement method of shoulder joint horizontal adductor muscle strength using geriatric and stroke patients. muscle strength measurements using a handheld dynamometer (hhd) consist of two types : a make test and a break test. one limitation of using an hhd is that the make test can not measure the maximum muscle strength of a subject if the examiner can not resist the subject s movement. another limitation of an hhd is that the break test can not measure the maximum muscle strength of a subject if the subject can not maintain the position. previous studies have reported that 30 kg (300 n) is the fixed limit for an hhd in the make test4, 5. katoh. devised a method of using a belt as a countermeasure to the fixed hhd limit when measuring lower limbs with high muscle strength values and reported on the reliability of this method for a variety of subjects6,7,8,9. our research group has also reported on the usefulness of the belt with a fixed hhd limit for shoulder joint horizontal adductor muscle strength measurements of young healthy japanese adults10 and its reliability11. however, the reliability of this method in the measurement of geriatric and non - healthy individuals has not yet been reported. few measurements and high reliability are necessary to reduce the patient burden in the evaluation of geriatric patients and stroke patients presenting with motor paralysis. the purpose of this study was to examine the appropriate number of measurements and the intrarater reliabilities of shoulder joint horizontal adductor muscle strength measurements using an hhd with a belt for geriatric and stroke patients. the subjects were 40 inpatients over the age of 65 years (onset days, 3103). they were divided into two groups : 20 stroke patients in the stroke group (sg), and 20 geriatric patients in the no - stroke group (n - sg). subjects were included in the sg only if the movement of shoulder joint horizontal adduction was possible with a japanese coma scale (jcs) score of 1. subjects were included in n - sg if neurological and orthopedic abnormalities were absent from the upper limbs. table 1table 1.general and medical characteristics of subjects sg (n=20)n - sg (n=20)gender (male, female)(14,6)(11,9)age (years)75.06.6 (6684)80.26.2 (6791)height (cm)159.210.3 (136178)152.28.5 (137170)body weight (kg)58.312.6 (4281)47.79.7 (3164)onset dates (days)13.810.4 (341)24.824.6 (5103)disease statedisease stateintracerebral hemorrhage4pneumonia9cerebral infarction12pelvic fracture1subdural hemorrhage3spinal compression fracture4hemorrhagic infarction 1bruise of head or general body2paralyzed side (right / left)12/8dehydration1sias knee - mouth test score57cancer1412acute renal failure131diabetes mellitus1200meansd (range), sg : stroke group, n - sg : no - stroke group shows the subject characteristics. meansd (range), sg : stroke group, n - sg : no - stroke group all shoulder joint horizontal adductor muscle strength measurements were performed with the subjects lying on an examination table in the supine position. the shoulder joint of each subject was abducted to 90, with 0 internal and external rotation, and the elbow joint was flexed to 90. the subject was positioned such that the shoulder joint was aligned with the bedpost. the elbow on the measurement side was positioned on the edge of the examination table. the equipment used for shoulder joint horizontal adductor muscle strength measurements was a tas f-1 hhd (anima corp., the sensor was placed on a thin rubber pad, which was fixed to the distal part of the upper arm with a surface fastener. the belt was fitted with sensors and inserted between the bedpost and the floor to fix the hhd. the examiner was a 32-year - old male physical therapist, 168 cm in height and 60 kg in weight, with 10-year - experience. the examiner controlled his hand in such a way so as not to move the hhd sensor and placed his other hand on the anterior aspect of the opposite shoulder joint to suppress compensatory movements. subjects were asked to perform isometric contractions in a manner similar to the make test. maximum contractions were attained within 3 s and were maintained for 5 s. the maximum values of shoulder joint horizontal adductor muscle strength were recorded. all measurements were performed after practice and prior orientation. in addition, all measurements were performed on the same day, three consecutive times on each side, at 30-s intervals. measurering method (image) reliability was verified using three measurements, the maximum value obtained during the first and second measurements and the maximum value obtained during the first and second measurements and the third measurement. the difference in mean values was assessed using one - way analysis of variance with the scheffe multiple comparison procedure. reliability was verified using intrarater reliability as assessed by the intraclass correlation coefficient (icc) (1, 1) and bland - altman analysis (baa). baa was performed using the first and second measurements as well as the maximum value obtained during the first and second measurements and the third measurement. a probability (p) value of 0.05no significant difference between the non - paralyzed side vs. paralyzed side, and between the right side vs. left side.. the results of icc (1, 1) were 0.92 (table 3table 3.icc of shoulder joint horizontal adductor muscle strengthgroupmeasurement sideicc (11)95%cisgnon - paralyzed0.937 0.8740.972 paralyzed0.920 0.8430.965n - sgright0.930 0.8610.969 left0.943 0.8860.975sg : stroke group, n - sg : no - stroke group, icc (1,1) : intra - rater correlation coefficients, 95% ci : 95% confidence interval). the results of baa are shown in table 4table 4.bland-altman analysis of shoulder joint horizontal adductor muscle strength measurementsgroupmeasurement sidecomparisonfixed biasproportional biasloamdc95 (kgf / kg)95 cibiasslope of the regression linebiassg (n=20)non - paralyzeda0.01 to 0.03n - ex 0.033 n - ex0.03 to 0.060.08 b0.01 to 0.04ex0.166 ex0.02 to 0.070.08 paralyzeda0.02 to 0.01n - ex0.016n - ex0.04 to 0.040.07 b0.001 to 0.05n - ex0.152 n - ex0.03 to 0.090.10 n - sg (n=20)righta0.03 to 0.001ex0.011 n - ex0.04 to 0.020.05 b0.002 to 0.02n - ex0.142n - ex0.02 to 0.030.05 lefta0.02 to 0.01n - ex0.122n - ex0.04 to 0.030.06 b0.001 to 0.02ex0.071n - ex0.01 to 0.040.04 sg : stroke group, n - sg : non - stroke group, a : comparison is a the 1st and 2nd measurements, b : the maximum obtained during the 1st and 2nd measurements and the 3rd measurrment, loa : limits of agreement, mdc95 : minimal detectable change 95%. : exsist : bias is present, n - ex : not present. in sg, there was no systematic bias on either side between the first and second measurement values. the third measurement value was smaller than the maximum value obtained during the first and second measurements. in n - sg, a systematic bias was observed between the first and second measurement values on the right side. however, no systematic bias was observed between the maximum value obtained during the first and second measurements and the third measurement value, and no systematic bias was observed between the first and second measurement values on the left side. on both sides, the third measurement value was smaller than the maximum value obtained during the first and second measurements. meansd (range), sg : stroke group, n - sg : no - stroke group : vs. the maximum value, p0.05 no significant difference between the non - paralyzed side vs. paralyzed side, and between the right side vs. left side. sg : stroke group, n - sg : no - stroke group, icc (1,1) : intra - rater correlation coefficients, 95% ci : 95% confidence interval sg : stroke group, n - sg : non - stroke group, a : comparison is a the 1st and 2nd measurements, b : the maximum obtained during the 1st and 2nd measurements and the 3rd measurrment, loa : limits of agreement, mdc95 : minimal detectable change 95%. : exsist : bias is present, n - ex : not present the results of this present study indicate that in sg, icc (1, 1) of relative reliability was high at 0.920.94. the results of baa of absolute reliability indicate that there was no systematic bias on either the non - paralyzed side or the paralyzed side between the first and second measurement values. in addition, the maximum value obtained during the first and second measurements was greater than that obtained during the first measurement. therefore, these results suggest that when evaluating stroke patients, the required number of measurements is two for the maximum value of the first and second measurements. in addition, the minimal detectable change in the 95% confidence interval (mdc95) on the non - paralyzed side was 0.08 kgf / kg, and 0.07 kgf / kg on the paralyzed side. in n - sg, icc (1, 1) of relative reliability was high at 0.930.94. the results of baa of absolute reliability indicate there was a systemic bias between the first and second measurement values on the right side. no systematic bias was found between the maximum value obtained during the first and second measurements and the third measurement or between the first and second measurement values on the left side. in n - sg, for both sides, the third measurement value was smaller than the maximum value obtained during the first and second measurements. therefore, these results suggest that when evaluating geriatric patients, the required number of measurements is two for the maximum value of the first and second measurements. in addition, mdc95 on the right side was 0.05 kgf / kg, and 0.06 kgf / kg on the left side. in a study on the reliability of knee extension strength measurements, katoh.8 suggested that the first measurement value or the average value of two measurements could not be used because the icc (1, 1) of measurements using an hhd with a belt was 0.820.92, and sometimes, the second measurement value was greater than the first measurement value. in addition, based on the results of absolute reliability in knee extension strength measurements, they reported that the required number of measurements was three9. in contrast, in a study of the reliability of muscle strength measurements made with an hhd of the upper extremity, schrama.12 reported that the intrarater reliability of an hhd in the assessment of the upper limb muscle strength of healthy subjects was acceptable only for elbow measurements. hirano.11 examined young healthy adults to establish the absolute reliability of shoulder joint horizontal adductor muscle strength measurements and observed a systematic bias between the maximum value obtained during the first and second measurements and the third measurement. the third measurement was smaller than the maximum value obtained during the first and second measurements. as these results showed the limits of agreement (loa), they recommended that the maximum value of two measurements should be used. based on our findings, the relative reliability of shoulder joint horizontal adductor muscle strength measurements of hospitalized geriatric and stroke patients was as high as 0.920.94. similar to previous studies, and considering our results of absolute reliability, it is our recommendation that the adopted values should be the maximum value of two measurements. in addition, to help with the interpretation of the determined effects, mdc95 was evaluated and found to be 0.08 kgf / kg in sg and 0.06 the present study investigated the reliability of shoulder joint horizontal adduction muscle strength measurements of hospitalized patients. the appropriate number of measurements of shoulder joint horizontal adductor muscle strength measurements was two for geriatric and stroke patients. however, the present study had a limitation in that it did not validate the interrater reliability. future studies to investigate the relevance of shoulder joint horizontal adductor muscle strength measurements in relation to activities of daily living and respiratory function of geriatric patients and stroke patients will help to clarify the potential clinical application of the present findings. | [purpose ] this study aimed to verify the appropriate number of measurements and the intrarater reliabilities of shoulder joint horizontal adductor muscle strength measurements using a handheld dynamometer (hhd) for geriatric and stroke patients. [subjects and methods ] the subjects were 40 inpatients, who were divided into two groups : 20 stroke patients in the stroke group (sg), and 20 geriatric patients in the no - stroke group (n - sg). measurements were performed three times using an hhd with a belt. the reliability was verified using bland - altman analysis and the intraclass correlation coefficient (icc). [results ] icc (1, 1) was > 0.9. a systematic bias was not observed between the first and second measurement values except for the right side in n - sg. a systematic bias between the maximum value obtained during the first and second measurements and third measurement value was observed on the left side in n - sg, and on the non - paralyzed side in sg : the third measurement values were small in both cases. [conclusion ] intrarater reliabilities were high for shoulder horizontal adductor strength measurements using an hhd with a belt for geriatric and stroke patients. taking the systematic bias into consideration, these findings suggest that the required number of measurements is two. |
benign schwannoma is a slow growing encapsulated tumor arising from the neuroectodermal sheath of schwann. approximately, 25 - 30% of all reported schwannomas occur in the head and neck and most of these in the eighth nerve. schwannoma of the facial nerve is a rare entity with very few cases reported in the literature. in our case a 7-year - old female was brought to ent out - patient department with right - sided parotid swelling of 1-year duration. initially, the swelling was small in size that gradually and painlessly increased up to its present size [figure 1 ]. the swelling was 10 cm 9 cm 7 cm in size, firm in consistency, had bosselated appearance, was mobile horizontally and vertically and the skin over the swelling was mobile. cytology was advised, and it came out to be a benign parotid tumor. since the mass was large in size and patient was a child, a computed tomography (ct) scan ct scan suggested a well - defined, lobulated, homogenous, mildly enhancing soft tissue lesion with loss of interface with the masseter muscle and multiple small lymph nodes [figure 2 ]. a provisional diagnosis of pleomorphic adenoma was made, and superficial parotidectomy planned [figure 3 ]. peroperatively, the facial nerve trunk was found deeper and inferior due to pushing effect of the tumor. however, on further anterior dissection the tumor was found to originate from the buccal and zygomatic branches of the facial nerve. the patient had postoperative (after 3 months of surgery) facial weakness of grade three (house brackmann classification) [figure 4 ]. female patient with right parotid swelling (preoperative) computed tomography scan showing the parotid mass line of incision of the planned surgery postoperative photograph showing facial weakness the gross appearance of the tumor was like a brain tissue [figure 5 ] and histopathologically, it was confirmed to be an intraparotid shwannoma, showing antoni type a and b cells [figure 6 ]. the lymph node showed reactive changes. resected tumor resembling brain tissue histopathological slide showing salivary and nervous tissue among 802 parotid tumors reported eneroth and hamberger could demonstrate two cases with neurogenic origin and in a review of 700 parotidectomies nussbaum. found only one case of neurilemma of the facial nerve. the intraparotid facial nerve shwanommas are mostly (75%) asymptomatic. the tumor grows eccentrically pushing the nerve fibers away as observed in the present case. the ability of the parotid to accommodate the expanding tumor results in facial nerve palsy in only 20 - 27% of cases. in some cases, radiology may provide preoperative information about facial nerve schwannoma but in many cases it mimics pleomorphic adenoma. the ct scan although not the preferred method of imaging shows a smooth and sharply defined mass in the parotid. fine - needle aspiration cytology is also unreliable for diagnosing these lesions as in most cases results are inconclusive or suggest a pleomorphic adenoma. the diagnosis is, therefore, generally made peroperatively when the surgeon finds difficulty in identifying the facial nerve. in our case, the size of the tumor was quite big and could have complicated into facial nerve palsy very soon. moreover, the patient 's preference to get the tumor operated was the main indication for a surgical intervention. any parotid mass in children should be viewed with suspicion and adequately investigated before any intervention is planned. intraparotid schwannoma should be suspected if the facial nerve can not be found intraoperatively, if the tumor is intimately associated with the facial nerve and if the gross appearance of the tumor is like that of a brain tissue. in cases where schwannoma is suspected, biopsy is recommended while complete resection is postponed to obtain imaging studies to evaluate the extent of disease and to discuss possible outcomes with the patient. | pleomorphic adenomas are the most common tumors which present as parotid masses. shwannoma (peripheral nerve sheath tumor) is a rare entity in this region. very few schwannomas originate from the facial nerve and in the majority of these cases the tumor involves its intratemporal part. the following case is reported because it presented as an asymptomatic parotid swelling with normal seventh nerve function, which masqueraded as pleomorphic adenoma clinically, radiologically and cytologically. however, it turned out to be peripheral nerve sheath tumor on histopathological examination. |
one - dimensional (1d) nanostructures such as wires, rods, belts, and tubes, whose lateral dimensions fall anywhere in the range of 1100 nm, have become the focus of intensive research, owing to their unique applications in mesoscopic physics and fabrication of nanoscale devices [1 - 6 ]. among one - dimensional (1d) nanostructures, nanobelts (or nanoribbons), a relatively new family of 1d nanostructures with a rectangular cross section, have received increasing attention since the discovery of novel oxide semiconductor nanobelts [4 - 8 ]. a variety of functional oxide and sulfide [10 - 17 ] nanobelts have been successfully fabricated by simple thermal evaporation. the methods used in 1d nanostructure synthesis and hydrothermal processes have emerged as powerful tools for the fabrication of anisotropic nanomaterials with some significant advantages, such as controllable particle size and low - temperature, cost - effective, and less - complicated techniques. under hydrothermal conditions, many starting materials can undergo quite unexpected reactions, which are often accompanied by the formation of nanoscopic morphologies that are not accessible by classical routes. in recent years, 1d nanomaterials such as ln(oh)3[19 - 21 ], cdwo4, moo3, and dy(oh)3 cadmium hydroxide, cd(oh)2, is a wide band gap semiconductor with a wide range of possible applications including solar cells, photo transistors and diodes, transparent electrodes, sensors, etc.. cadmium hydroxide is also the precursor to prepare cadmium oxide. as a consequence, numerous techniques have been proposed to synthesize nano - sized cd(oh)2 particles with promising control of properties [25 - 28 ]. however, up to now, to our best knowledge, the synthesis of cd(oh)2 nanobelts by hydrothermal process has not been reported. herein, we report the preparation of cadmium hydroxide nanobelts by the conventional polyol assisted hydrothermal process. in a typical procedure ; cdcl2 2h2o (0.2281 g) was dissolved in 32 ml of distilled water, and then nh3 h2o (25 wt.%, 5 ml) was slowly added into the solution and stirred for about 10 min, and a transparent cd(nh3)4solution was formed. then, the above solution was loaded into a 50-ml teflon - lined autoclave, which was then filled with 8 ml of glycol. the autoclave was sealed, warmed up at a speed of 3 c / min and maintained at 100 c for 6 h, and was then cooled to room temperature on standing. the white precipitate was filtered off, washed with absolute ethanol and distilled water for several times, and then dried in vacuum at 40 c for 4 h. x - ray diffraction (xrd) patterns were carried out on a japan rigaku d / max ra x - ray diffractometer equipped with graphitemonochromatized high - intensity cu ka radiation (= 1.541784). the accelerating voltage was set at 50 kv, with 100 ma flux at a scanning rate of 0.06/s in the 2 range 1080. the x - ray photoelectron spectra (xps) were collected on an escalab mkii x - ray photoelectron spectrometer using nonmonochromatized mg kr x - ray as the excitation source. the field emission scanning electron microscopy (fe - sem) images were taken on a jeol jsm-6700fsem. the transmission electron microscopy (tem) images were characterized by hitachi h-800 transmission electron microscope with a tungsten filament and an accelerating voltage of 200 kv. 1) from the as - synthesized bulk samples reveals the crystal structure and phase purity of the products. all the diffraction peaks can be indexed to the hexagonal cd(oh)2with cell constantsa = 3.4942,c = 4.7102, which are consistent with the values in the literature (jcpds 31 - 0228). the abnormally intensified (100) peak in the xrd pattern also indicates that the belt - like product comprises 1d cd(oh)2crystals preferentially grown along the direction. powder xrd patterns of the products figure 2 shows the xps spectra of the as - obtained cd(oh)2 sample. a survey spectrum shown in fig. 2a, indicates the presence of cd and o as well as c from reference. there are no peaks for other impurities, indicating that the as - obtained product is relatively pure. high - resolution spectra are also taken for the cd 3d region and the o 1s region to determine the valency state and atomic ratio. the binding energies of cd(3d5/2) and o(1s) were found to be 405.30 and 531.25 ev, respectively. all the above observed binding energy values are in good agreement with the reported data. quantification of the xps peaks gives the molar ratio of cd : o as 1:2.02, close to the stoichiometry of cd(oh)2. 3a) shows that the as - synthesized products consist of a large quantity of 1d nanostructures with lengths from several tens to several hundreds of micrometers ; some of them even have lengths of the order of millimeters. a representative high magnification sem image (fig. 3b) of several curved cd(oh)21d nanostructures reveals that their geometrical shape is belt - like, which is distinct from those of previously reported nanowires, and their thickness is about 3050 nm. (b) high - magnification image of curved nanobelts tem and saed studies of the as - synthesized products provide further insight into the belt - like cd(oh)2nanostructures. the nanobelts are uniform in width and thickness, and their typical widths and thickness are in the range of 60250 nm and 1030 nm, respectively. the saed pattern (inset in fig. 4b) taken from the straight section of the curved nanobelt demonstrates that this particular nanobelt is a single crystal. (b) single curved cd(oh)2 nanobelt for the polyol process, glycol was selected as the solvent because of its excellent viscosity, which makes it possible to mix the reagents homogeneously. in the process, glycol can provide reaction conditions adequate to greatly enhance solubility, diffusion, and crystallization, but is still mild to leave molecular building blocks to bring about the formation of the solid - state phase. at reaction temperature, the diffusion of ions in glycol is more rapid than in other polyol, such as glycerine and diethylene glycol ; this leads to acceleration in the solubility of starting materials and in the following crystal growth. the concentrations of glycol of about 2030 vol.%, were found to be favorable for the formation of the cd(oh)2 nanobelts in high yield. such viscosity had a good effect on prohibiting aggregation of cd(oh)2particles and then resulted in a relatively stable suspension. control reactions at a low concentration of glycol (3 ml) would plate out a large amount of the cd(oh)2 nanorods (fig. 5a). at very high concentrations (20 ml glycol), however, only the aggregated particles were observed (fig. when glycerine was used, nanobelts were not obtained due to the high viscosity of solvent., we can clearly see that the viscosity is of importance to the structure of the final product. sem images of cd(oh)2 samples using different concentrations of glycol : (a) 3 ml ; (b) 20 ml the optical absorption spectrum of our sample is shown in fig. compared to other researcher s work, the absorption edge obviously shifts toward shorter wavelength, i.e., blue shift. the absorption band gap eg can be determined by the following equation : (h)= b(h eg), in which h is the photo energy, is the absorption coefficient, b is a constant relative to the material, and n is either 2 for a direct transition or 1/2 for an indirect transition. the (h) ~ h curve for the samples shown in fig. 6 insert reveals that the band gap of the samples is about 4.45 ev, which is larger than the reported value for cd(oh)2 thin film (eg = 2.75 ev), but is less than the reported value for nanostrands, which have a constant width of 1.9 nm (eg = 4.76 ev) due to the quantum size effect. optical absorption spectrum and (h) ~ h curve for the cd(oh)2 nanobelts in summary, cd(oh)2nanobelts with a uniform diameter have been successfully prepared in high yield through a rapid polyol process. it was found that the viscosity of the solvent played an important role in determining the morphology. we believe that it should be possible to synthesize other similar patterns by choosing an appropriate solvent. the optical absorption spectrum indicates that the cd(oh)2nanobelts have a direct band gap of 4.45 ev. this work was supported by a grant - in - aid for scientific research from the japan society for the promotion of science (jsps) and the crest program of the japan science and technology agency (jst). we are grateful to young and middle aged academic leaders of jiangsu province universities blue and green blue project. we are grateful to the electron microscope and x - ray diffraction facilities of university of science & technology of china for assistance in xrd and sem measurement. | cd(oh)2nanobelts have been synthesized in high yield by a convenient polyol method for the first time. xrd, xps, fesem, and tem were used to characterize the product, which revealed that the product consisted of belt - like crystals about 40 nm in thickness and length up to several hundreds of micrometers. studies found that the viscosity of the solvent has important influence on the morphology of the final products. the optical absorption spectrum indicates that the cd(oh)2nanobelts have a direct band gap of 4.45 ev. |
mesoporous sba-15 silica molecular sieves of large pore diameter (up to 30 nm) and area (up to 1000 m g) show excellent homogeneity and stability and can be well controlled for adsorption / desorption processes. mesoporous silica materials, especially mesoporous sba-15 molecular sieves, have been modified with 3-mercaptopropyl, 3-aminopropyl, octyl, or octadecyl groups for the separation and analysis of inorganic ions, organic compounds, and biological molecules [25 ]. magnetic separation is a useful tool because of its fast recovery, high efficiency, and low high cost. inclusion of magnetic components in modified materials allows convenient and economical magnetic separation instead of centrifugation and filtration steps on application of an appropriate magnetic field [79 ]. because of their potential applications in this approach, the preparation of co, co / fe, -fe2o3, -fe2o3, and fe3o4 magnetic sba-15 materials have been reported [1015 ]. we propose a convenient and effective procedure for iron doping of mesoporous sba-15 silica because of its excellent magnetic properties. then fe was transformed to the oxide form by calcination in the air. finally, ferric oxide in sba-15 was reduced with h2 [7, 15, 16 ]. fourier - transform infrared (ft - ir) spectroscopy, powder x - ray diffraction (xrd), mssbauer spectra (ms), surface area analysis, scanning electron microscopy (sem), and transmission electron microscopy (tem) techniques were used to characterize the material. the iron content in fe - sba-15 was determined by atom adsorption spectroscopy (aas). with the development of petroleum refining and chemical engineering, the release of harmful organic chemicals into the environment has attracted global attention because of their toxicity and widespread use, especially aromatic compounds considered as carcinogenic. the aim of the present study was to develop a method for preparing magnetic sba-15 for use as a sorbent to remove benzene and related derivatives from water. the adsorption of benzene, toluene, and ethyl benzene on magnetic sba-15 particles was determined by gas chromatography (gc). benzene, toluene, and ethyl benzene were purchased from sigma (st. louis, mo, usa). deionized water was prepared using a millipore unit (bedford, ma, usa). sba-15 was soaked in 0.1 mol l hcl for 24 h, filtered, washed with deionized water, and dried in an oven at 100c for 8 h. a sample of 2 g of sba-15 was dispersed in 1 mol l fe(no3)3 solution by ultrasonication for 2 h. the material was filtered under low pressure and dried in an oven at 100c. samples were calcined in a muffle furnace at 750c for 6 h to obtain a red powder, which was then reduced under a h2 flow (35 ml min) at 800c for 6 h. the final product was designated fe - sba-15. ft - ir spectra of sba-15 particles were recorded before and after modification on a nicolet (usa) 360 ft - ir instrument. powder xrd patterns were measured on a d / max-2400 (rigaku, japan) instrument using cu k radiation. fe mssbauer spectra were collected using a conventional constant acceleration spectrometer (wissel, german) with a 25 m ci source of co in palladium at room temperature. the percentage of fe, fe(ii), and fe(iii) were calculated and contributor of magnetic behavior was confirmed. nitrogen adsorption - desorption experiments were carried out at 76.53 k on an asap 2010 accelerated surface area and porosimetry system (micromeritics, usa). the brunauer - emmett - teller (bet) surface area (sbet) was calculated from the linearity of the bet equation. the surface area, volume and pore diameter were calculated from pore size distribution curves using the barrett - joyner - halenda (bjh) formula. the surface morphology of powders was observed under a jsm-6380lv scanning electron microscope (jeol, japan). for tem observation, samples were dispersed in ethanol, deposited on a grid of holey copper and transferred to a jem-1230 emission electron microscope (jeol, japan) at an accelerating voltage of 100 kv. a lakeshore (usa) 7304 vibrating sample magnetometer was used to record magnetization curves of the samples. a sample of 10 mg of fe - sba-15 was heated in a mixture of hydrochloric acid and nitric acid to completely dissolve the iron and transferred to a 25-ml volumetric flask and made up to volume with water. the solution was centrifuged at 10000 rpm and the iron concentration was determined on an aa-6800 instrument (shimadzu, japan). standard fe solutions (0, 2.0, 4.0, 8.0, 10.0 g ml) were measured to generate a standard curve. benzene, toluene, and ethyl benzene, were dissolved in deionized water at a concentration of 1.0 ppm each. then 20 mg of fe - sba-15 was added to the aqueous solution and sonicated at room temperature (approx. 20c) for 30 s to form a homogeneous dispersion. after standing for 5 min, fe - sba-15 finally, 1.0 l of the eluate was analyzed on a varian (usa) cp-3380 gas chromatograph and the content of the aromatic compounds was calculated. figure 2 shows ft - ir spectra of fe(no3)3, sba-15, fe(no3)3-sba-15, fe2o3-sba-15, and fe - sba-15. the ir vibration for the no3 ion at 1385 cm is evident in curves a and c, but not in b, indicating that fe(no3)3-sba-15 was prepared successfully. fe2o3-sba-15 was obtained by transformation from the nitrate to the oxide form, as confirmed by the disappearance of the peak at 1385 cm after calcination (curve d). the peak at 1385 cm also disappeared after fe2o3-sba-15 reduction (curve e). the material was red after fe(no3)3-sba-15 calcination at 750c, suggesting that iron oxide is mainly in the form of -fe2o3 (hematite). hematite is extremely stable under ambient conditions and is often the end product of the transformation of other iron oxides. xrd patterns of the materials are shown in figure 3. in the narrow - angle range peaks for sba-15 are evident at 0.8, 1.4 and 1.7. for fe - sba-15, there is one prominent peak at 0.9 and two peaks at 1.5 and 1.8. these results indicate that magnetic fe - sba-15 still has a high degree of hexagonal symmetry [1, 19 ] and retains the structure of sba-15 after calcination at 750c and reduction at 800c, indicating that sba-15 has good hydrothermal stability. peaks for iron are evident at 44.6 and 65 in the wide - angle xrd pattern for fe - sba-15 [20, 21 ]. residual iron oxides may be a result of surface passivation, which can be explained by the following equations for reduction of fe2o3 with hydrogen : (1)3fe2o3+h22fe3o4+h2o, fe3o4 + 4h23fe+4h2o(1x)fe3o4+(14x)h23fe(1x)o+(14x)h2o, fe(1x)o+h2(1x)fe+h2o figure 4 shows mssbauer spectra of fe - sba-15 and table 1 shows mssbauer parameters of fe - sba-15. from the calculation of fe mssbauer spectra, the percentage of fe, fe(ii), and fe(iii) in the prepared materials were 17.0%, 64.5%, and 18.5%, respectively. the above results could also confirm that the reduction process followed the equations and the reduction was not complete under this condition. furthermore, it can be seen that the magnetic behavior of fe - sba-15 was contributed by fe3o4. figure 5 shows nitrogen adsorption - desorption isotherms for the materials, which exhibit a typical type iv isotherm with an h1 hysteresis loop as defined by iupac. however, there was a shift in hysteresis position to lower relative pressure and a decreasing trend. the shift of the sharp inflection from p / p0 0.60 to 0.80 is characteristic of a diameter in the mesopore range [27, 28 ]. the bet surface area changed from 524 to 308 m g and the pore diameter decreased from 72.8 to 72.6 on modification of sba-15 with fe. the decrease in surface area and pore diameter, probably caused by filling of the pores with small iron particles, indicates successful iron doping within the mesoporous channels of sba-15. the pore diameter and d100 data for the two materials indicate that the mesopore uniformity of the parent sba-15 silica was retained in the modified material. large fibrous structures of 3080 m in length and 1020 m in diameter are clearly evident in figure 6. compared to the parent sba-15, the size of the modified material decreased on both a microscopic and macroscopic scale, suggesting that the particle size can change on ultrasonication. the tem images in figure 7 provide direct evidence that the material comprises well - ordered hexagonal arrays of mesopores (1d channel) and a 2d hexagonal structure. the tem image of fe - sba-15 (figure 6(b)) shows that the structure is the same as in the parent sba-15 silica. tem and xrd results also confirmed that modification occurred within pores and that the sba-15 structure did not change. the modified material retained the mesopore uniformity of the original inorganic wall structure of the parent sba-15 silica. figure 8 shows that fe - sba-15 exhibited strong magnetic behavior with a magnetization value of 8.8 emu g, confirming that fe - sba-15 was magnetic with potential as a magnetic adsorbent for removal of aromatic compounds from water. using aas data, the iron content of fe - sba-15 was calculated as a = 0.0794c + 0.0249 (r = 0.9993), where a is the sample absorbance and c is the sample concentration. the iron load was thus determined to be 19.1 wt.% in the modified material, in accordance with its strong magnetic behavior. large iron metallic particles that formed in fe - sba-15 can be observed in figure 6(b). after 5 min, a magnet was fixed to the vessel wall to attract the material. following the absorption process, fe - sba-15 was redispersed in methanol to desorb the aromatic compounds and the efficiency of magnetic separation was assessed by gc. analytical data for benzene, toluene, and ethyl benzene compared to standards revealed removal of 90.6%, 88.7%, and 85.4%, respectively, demonstrating that fe - sba-15 can be used as a sorbent in water. it is worth noting that fe - sba-15 can be reused after washing several times with methanol and vacuum drying. magnetic fe - sba-15 was prepared from mesoporous silica molecular sieves via a simple iron ion doping of sba-15, transformation to fe2o3 and then reduction to magnetic iron particles using h2. results confirm that the modified material retained the well - ordered hexagonal mesoporous structure after calcination at 800c. mesoporous silica materials may have further application potential, such as in sensing materials, solid supports, and nanobioelectronics, especially as magnetic sorbents allow the reuse of adsorbent materials for several cycles. | magnetic fe - sba-15 mesoporous silica molecular sieves were prepared, characterized, and used for magnetic separation. wet impregnation, drying, and calcination steps led to iron inclusion within the mesopores. iron oxide was reduced to the metal form with hydrogen, and the magnetic fe - sba-15 was obtained. fourier - transform infrared spectroscopy confirmed the preparation process from the oxide to metal forms. the structure of magnetic materials was confirmed by mssbauer spectra. powder x - ray diffraction data indicated that the structure of fe - sba-15 retained the host sba-15 structure. brunauer - emmett - teller analysis revealed a decrease in surface area and pore size, indicating fe - sba-15 coating on the inner surfaces. scanning electron micrographs confirmed the decrease in size for modified sba-15 particles. from scanning electron micrographs, it was found that the size of the modified sba-15 particles decreased. transmission electron micrographs also confirmed that modified sba-15 retained the structure of the parent sba-15 silica. fe - sba-15 exhibited strong magnetic properties, with a magnetization value of 8.8 emu g1. the iron content in fe - sba-15 was determined by atom adsorption spectroscopy. fe - sba-15 was successfully used for the magnetic separation of three aromatic compounds in water. our results suggest wide applicability of fe - sba-15 magnetic materials for the rapid and efficient separation of various compounds. |
this special issue of food biophysics journal contains papers originally presented at the 3rd international symposium on delivery in complex food systems, which was held at wageningen university, the netherlands, from october 18 to 21, 2009. as the subtitle already indicates, the purpose of this symposium is to build a better understanding of the physical processes which are involved in creating food products and which impact their consumption, at all relevant length scales. the topic is of interest to scientists from academia, industry and government as it relates to the building of basic understanding, to the production of high - quality foods and to development and maintenance of the regulatory framework to protect consumers, the environment and ascertain a healthy competition in the food industry. this symposium was the third one in what can truly be called a series now, with the first symposium held in lausanne (ch), 20051 and the 2nd one in 2007 in amhurst (ma, usa).2 in this 3rd symposium a number of talks concentrating on, or taking into account the fate, of common and newly designed food structures during their passage through the human gastro - intestinal tract. these steps target the actual delivery of the nutrients to the human body which is critical for its biological functionality. not only are consumers nowadays better informed about the relation between life - style and own health, of which their food consumption pattern is a critical part, there is also a strong development in the regulatory domain to protect the same consumers from misleading claims on food products. foods with attached health claims which go beyond the normal nutritional content and impact, the so - called functional foods, have to existing requirements and consumers expectations with regard to all sensory aspects, convenience and pricing, but also have to actually deliver the functional ingredient which is linked to the claimed health effect. the large number of dossiers submitted to the european authorities to assess the validity of functionality claims put forward by companies signals the great commercial interest in such products. the symposium recognized five different sessions that are to be connected to address the issues above ; in regard to the preparation of foods, one has to meet boundary conditions on health, deliciousness, ingredient availability and cost. in addition, for both foods prepared at home and at industrial scale, flexibility in terms of formulation is additionally of importance. all these boundary conditions apply to one or more steps at the same time during the product life cycle : processing, transport, storage, consumption, and digestion. one is to investigate commonly existing foods on their function in terms of their microstructures and according constituents (molecular sized). the other is to prepare novel (micro-)structures and investigate these regarding their possible functions. the contributions in this session have to be viewed in terms of this latter area. one important class of ingredients is of course proteins, and their assembly is a key area within food development and production. in the session, two excellent contributions were presented in the area of protein / peptide assembly (by professors gazit, hebrew university of jerusalem, israel, and hammarstrom, linkoping university, sweden). in relation to the physics at larger length and time scales, an invited lecture was given by professor cipelletti, universit montpellier ii, on the long - time dynamics of microstructural evolution. the focus in this session was on how techniques and approaches developed in materials science, physics, and biophysics are having an impact in the field of food science and technology. advances in measurement techniques from the single molecule to the mescopic level were highlighted, with emphasizes on the scientific framework in which their results can be used to advance our understanding of complex food systems. understanding processes involved in delivery of the functional ingredient in the human gi tract is relatively new and was the focus of the biophysics of nutrient digestion and absorption session. better understanding of the processes involved methods to model or predict these in an experimental setting and finally meet delivery and regulatory requirements is essential for the functional food area. not only will it help to substantiate the nutritional claims made for the food product, but products which score better on all these aspects are ultimately also the differentiator in the marketplace. there is a whole cascade of events from food assembly, preparation, to digestive breakup starting in the mouth, to the complex stomach and intestinal events, involving biochemical processes to finally present the functional molecules and nutrients to the absorptive sites at the intestinal wall. this can be captured in the term bioaccessability and defines the amount of functional ingredient which is available for absorption. so far, models which mimic the human gastro - intestinal events have been derived from anatomical and enzymatic understanding and less so from, e.g., a fluid dynamics approach. this poses a challenge as for ethical, practical, cost constraint reasons, availability of in vitro models with good in vitro in vivo correlations would be ideal. in order to meet the challenges regarding the understanding of the development, production, consumption and digestion of foods one needs a multiscale approach. this means that one needs to connect many different scientific fields : physics, physiology, and the science of human perception. this session was organized as a first, exploratory attempt to bring together scientific developments in these various fields under the umbrella complexity, which is a well - defined concept in many disciplines such as physics, network theory and sociology. we are convinced that in the food field the concept of complexity will have a major impact in the future, not only because of the complexity of, e.g., the physics or physiology, but in particular as the notion of complexity can help to modulate the way different scientific disciplines may interact and evolve together on a common topic, such as a food, food consumption or food digestion. for the first time in the symposium series, a session emphasizing the relation between food technology and nutrition was organized. in the session, examples were presented of successful products in which physical principles were utilized to create food products and food ingredients with enhanced stability, functionality and release of nutrients. in addition, in the session, conceptual approaches towards the development of new and sustainable food technologies were presented. the conference was attended by about 160 scientists from academia and industry, and the formal and informal discussions were lively and stimulating, integrating old and new approaches. we are very pleased to announce that the next symposium, the 4th edition in this series, will be held in the early autumn of 2011 in guelph, canada. | the wageningen delivery of functionality symposium covered all aspects involved with food structural design to arrive at high - quality foods which meet demanding customer expectations and regulatory requirements. the symposium integrated aspects from the structural organization of foods at molecular and supramolecular scales to dedicated techniques required to describe and visualize such structures, the gastro - intestinal events and how to model these in a laboratory setting, and finally the impact those food structures and ingredients have on the consumer s physiology and on the human perception. as an interdisciplinary platform, bringing together more than 160 researchers from academia and industry, the symposium meanwhile fulfills an important role in the food science community. |
the introduction of imatinib mesylate (glivec/gleevec, novartis, basel, switzerland) has improved outcomes for patients with advanced gastrointestinal stromal tumor (gist), and imatinib is now considered the standard first - line treatment in patients with metastatic or unresectable gist (1, 2). imatinib is generally well tolerated, although some patients have difficulty tolerating the standard dose of 400 mg / day, which may necessitate imatinib dose reductions. however, patients and physicians are cautious and sometimes reluctant to reduce the dose of imatinib because of concerns about disease progression. indeed, results from a pharmacokinetic analysis suggested that adequate drug exposure, as determined by imatinib plasma trough concentration, is correlated with clinical benefit ; a low plasma trough concentration of imatinib (< 1,100 ng / ml) might contribute to reduced efficacy in patients with advanced gist (3). previous pharmacokinetic studies also showed that imatinib blood levels have high interpatient variability (3 - 5) and are affected by several covariates, such as albumin concentration, creatinine clearance, white blood cell count, and major gastrectomy (3, 5). as a result, monitoring imatinib plasma concentration on an individual basis may be important for optimizing clinical outcomes of patients with advanced gist (6, 7). some patients who take the standard dose of imatinib may have abnormally high plasma levels of imatinib due to their individual predisposition to decreased drug metabolism (3 - 5) ; it is known that some imatinib - related toxicities may be related to overexposure to the drug (6). the problem becomes even more complicated when these imatinib - related toxicities confound the evaluation of response to imatinib therapy. for example, when patients do not have tumor size reduction and ascites develops as a toxicity of imatinib, this may be misinterpreted as having disease progression and managed with imatinib dose escalation or switching to second - line sunitinib (8). therefore, adjusting imatinib dosage by plasma level monitoring may help manage patients who experience intolerable toxicities while also achieving sufficient imatinib trough concentrations to maintain efficacy. here we describe two patients with advanced gist whose imatinib - related toxicities were successfully managed with dose modifications guided by imatinib plasma level testing. a 67-yr - old asian man who presented with intermittent melena and significant weight loss was diagnosed with small bowel gist with multiple liver metastases and peritoneal seeding on may 25, 2010. he underwent jejunal resection and anastomosis with palliative intent. the excised jejunal gist measured 7 5 5 cm and had a c - kit exon 9 mutation (1,510 - 1,515 duplication). two months later, the patient developed grade 3 edema, grade 3 ascites, and grade 2 vomiting, with chest radiography revealing layering of a moderate amount of pleural fluid. because the patient had peritoneal seeding, it was hard to determine whether his ascites and pleural effusion were due to imatinib toxicity or the progression of gist. although the follow - up abdomino - pelvic computed tomography (ct) scan showed that the patient 's liver metastases had not changed significantly, disease progression was suspected, which prompted a cessation in imatinib treatment and a commencement of sunitinib treatment. the patient was transferred to our hospital for a second opinion after he had been taking sunitinib for approximately 2 months. a thorough review of his previous serial ct scans showed no definitive evidence of disease progression whilst he was on imatinib treatment. we therefore decided to resume 400 mg / day imatinib and use f - fluorodeoxyglucose (fdg) positron emission tomography (pet)/ct for accurate assessment of response to imatinib treatment. one month after the patient restarted imatinib, a pet / ct scan revealed a significant decrease in maximum standardized uptake value from 4.2 to 2.9 in one of his liver metastases (fig. 1a and b), suggesting the tumor was responding to imatinib. however, the patient required paracentesis once weekly to control his ascites, and he complained of peripheral edema and dyspnea. because imatinib blood level testing revealed that the patient had very high imatinib trough plasma exposure, 4,120 ng / ml and 4,600 ng / ml on two different days (fig. 1c), we decreased his dose to 300 mg / day, which resulted in a steady - state imatinib plasma trough concentration of 3,220 ng / ml (fig. however, the patient still had grade 2 edema, ascites, and dyspnea on exertion due to pleural effusion. as his imatinib trough plasma exposure was sufficiently high to achieve an adequate tumor response (3), we further reduced his dose to 200 mg / day. at this dose, his fluid retention, including ascites, edema, and pleural effusion, was greatly improved, and he had no difficulties in daily life ; in addition, his liver metastases remained stable (fig. five months later, the patient expressed concerns that 200 mg / day of imatinib may be insufficient to control his tumor, as studies have shown that gist patients with the c - kit exon 9 mutation may benefit from higher than normal exposure to imatinib (3, 9, 10). he was able to tolerate ascites and dyspnea with the use of regular diuretics but had some limitation of activities. follow - up ct scans to date showed the patient 's disease remained stable (fig. 1b). a 69-yr - old asian man presenting with melena was diagnosed with duodenal gist and underwent whipple 's operation on may 6, 2004. the excised mass measured 5.5 4.5 2.0 cm and had a low mitotic rate (< 5 mitoses per 50 high - powered fields). mutation analysis showed that the tumor had a kit exon 11 deletion at amino acid 552. twenty - two months after surgery, a liver metastasis was detected on follow - up ct scans. the treatment was effective, with a partial response noted after 3 months (fig. 2a) ; the treatment was also tolerable, with grade 2 edema the only adverse event experienced by the patient. at 26 months following the commencement of imatinib treatment, the patient developed several small, round, mesenteric lymph node enlargements, which were regarded as a sign of disease progression even though the metastatic lesions in his liver remained stable. after 7 months at this dosage, the patient experienced grade 3 dyspnea and grade 3 pericardial effusion. however, an in - depth review of his previous serial ct scans by an experienced gastrointestinal radiologist revealed that the morphology of these enlarged mesenteric lymph nodes suggested reactive changes, instead of lymph node metastases, which are rare in gist (11). ct scans 10 months later showed that the patient 's liver metastasis were stable (fig. although his ascites and pleural effusion gradually improved, the patient complained of dyspnea, and diuretics were required for the control of grade 2 generalized edema. because imatinib plasma monitoring revealed that the patient had high imatinib plasma trough concentrations (3,850 ng / ml and 4,280 ng / ml on two different days ; fig. 2b), we reduced his dose to 300 mg / day, which resulted in imatinib plasma trough concentrations of 2,670 ng / ml and 2,880 ng / ml (fig. however, pericardial effusion was persistently observed, even after his imatinib dose was further decreased to 200 mg / day, which resulted in steady - state imatinib plasma trough concentrations of 3,070 ng / ml and 2,710 ng / ml (fig. 2b). as the patient 's imatinib trough plasma exposure was still sufficiently high (3) and the follow - up ct scan showed that his liver metastasis remained stable (fig. 2a), we further decreased his dose to 100 mg / day, which resulted in imatinib plasma trough concentrations of 1,660 ng / ml and 1,480 ng / ml (fig. to date, the patient has been taking 100 mg / day of imatinib for approximately 1 yr. a 67-yr - old asian man who presented with intermittent melena and significant weight loss was diagnosed with small bowel gist with multiple liver metastases and peritoneal seeding on may 25, 2010. he underwent jejunal resection and anastomosis with palliative intent. the excised jejunal gist measured 7 5 5 cm and had a c - kit exon 9 mutation (1,510 - 1,515 duplication). two months later, the patient developed grade 3 edema, grade 3 ascites, and grade 2 vomiting, with chest radiography revealing layering of a moderate amount of pleural fluid. because the patient had peritoneal seeding, it was hard to determine whether his ascites and pleural effusion were due to imatinib toxicity or the progression of gist. although the follow - up abdomino - pelvic computed tomography (ct) scan showed that the patient 's liver metastases had not changed significantly, disease progression was suspected, which prompted a cessation in imatinib treatment and a commencement of sunitinib treatment. the patient was transferred to our hospital for a second opinion after he had been taking sunitinib for approximately 2 months. a thorough review of his previous serial ct scans showed no definitive evidence of disease progression whilst he was on imatinib treatment. we therefore decided to resume 400 mg / day imatinib and use f - fluorodeoxyglucose (fdg) positron emission tomography (pet)/ct for accurate assessment of response to imatinib treatment. one month after the patient restarted imatinib, a pet / ct scan revealed a significant decrease in maximum standardized uptake value from 4.2 to 2.9 in one of his liver metastases (fig. 1a and b), suggesting the tumor was responding to imatinib. however, the patient required paracentesis once weekly to control his ascites, and he complained of peripheral edema and dyspnea. because imatinib blood level testing revealed that the patient had very high imatinib trough plasma exposure, 4,120 ng / ml and 4,600 ng / ml on two different days (fig. 1c), we decreased his dose to 300 mg / day, which resulted in a steady - state imatinib plasma trough concentration of 3,220 ng / ml (fig. however, the patient still had grade 2 edema, ascites, and dyspnea on exertion due to pleural effusion. as his imatinib trough plasma exposure was sufficiently high to achieve an adequate tumor response (3), we further reduced his dose to 200 mg / day. at this dose, his fluid retention, including ascites, edema, and pleural effusion, was greatly improved, and he had no difficulties in daily life ; in addition, his liver metastases remained stable (fig. five months later, the patient expressed concerns that 200 mg / day of imatinib may be insufficient to control his tumor, as studies have shown that gist patients with the c - kit exon 9 mutation may benefit from higher than normal exposure to imatinib (3, 9, 10). he was able to tolerate ascites and dyspnea with the use of regular diuretics but had some limitation of activities. follow - up ct scans to date showed the patient 's disease remained stable (fig. 1b). a 69-yr - old asian man presenting with melena was diagnosed with duodenal gist and underwent whipple 's operation on may 6, 2004. the excised mass measured 5.5 4.5 2.0 cm and had a low mitotic rate (< 5 mitoses per 50 high - powered fields). mutation analysis showed that the tumor had a kit exon 11 deletion at amino acid 552. twenty - two months after surgery, a liver metastasis was detected on follow - up ct scans. the treatment was effective, with a partial response noted after 3 months (fig. 2a) ; the treatment was also tolerable, with grade 2 edema the only adverse event experienced by the patient. at 26 months following the commencement of imatinib treatment, the patient developed several small, round, mesenteric lymph node enlargements, which were regarded as a sign of disease progression even though the metastatic lesions in his liver remained stable. after 7 months at this dosage, the patient experienced grade 3 dyspnea and grade 3 pericardial effusion. however, an in - depth review of his previous serial ct scans by an experienced gastrointestinal radiologist revealed that the morphology of these enlarged mesenteric lymph nodes suggested reactive changes, instead of lymph node metastases, which are rare in gist (11). ct scans 10 months later showed that the patient 's liver metastasis were stable (fig. although his ascites and pleural effusion gradually improved, the patient complained of dyspnea, and diuretics were required for the control of grade 2 generalized edema. because imatinib plasma monitoring revealed that the patient had high imatinib plasma trough concentrations (3,850 ng / ml and 4,280 ng / ml on two different days ; fig. 2b), we reduced his dose to 300 mg / day, which resulted in imatinib plasma trough concentrations of 2,670 ng / ml and 2,880 ng / ml (fig. however, pericardial effusion was persistently observed, even after his imatinib dose was further decreased to 200 mg / day, which resulted in steady - state imatinib plasma trough concentrations of 3,070 ng / ml and 2,710 ng / ml (fig. 2b). as the patient 's imatinib trough plasma exposure was still sufficiently high (3) and the follow - up ct scan showed that his liver metastasis remained stable (fig. 2a), we further decreased his dose to 100 mg / day, which resulted in imatinib plasma trough concentrations of 1,660 ng / ml and 1,480 ng / ml (fig. to date, the patient has been taking 100 mg / day of imatinib for approximately 1 yr. imatinib is metabolized in the liver, predominantly by cytochrome p450 (cyp) 3a4 (12). as a result, the plasma concentration of imatinib may be affected by intrinsic variability of cyp enzyme activity and other factors, such as albumin concentration (3, 5). although studies have assessed the relationship between imatinib plasma concentration and efficacy (3, 4, 12), less is known about the relationship between imatinib plasma concentration and toxicity (6). one study showed that the occurrence and number of side - effects correlated with imatinib total and free plasma concentration in patients with gist (6). however, the study did not assess imatinib exposure with the grade or type of toxicities, findings that may have been of value in clarifying the dose - dependent toxicities of imatinib. here we described two patients who experienced intolerable toxicities while on standard - dose imatinib treatment. both had high imatinib plasma trough concentrations, which appeared to have contributed to the severe fluid retention (e.g. ascites, edema, and pleural effusion) observed in these patients. through imatinib plasma monitoring, we reduced the dose of imatinib, consequently decreasing these toxicities, while maintaining sufficient imatinib exposure for adequate efficacy of the treatment. another interesting aspect of these two patient cases is that they highlight the difficulties of response evaluation for imatinib therapy in gist patients. toxicity of imatinib or reaction to imatinib treatment may sometimes be mistaken for disease progression. for example, in the first patient who had peritoneal metastases, ascites was regarded as a sign of disease progression ; in the second patient, enlarged reactive mesenteric lymph nodes were mistaken for lymph node metastatses. because both patients had no tumor shrinkage in other nodules, which is quite common in patients with advanced gist on imatinib therapy, it was difficult to determine whether imatinib treatment was effective. fdg - pet / ct scan may be considered for accurate assessment of response to imatinib, especially when rapid readout of activity is necessary. when considering imatinib plasma level monitoring to address intolerable adverse events, it is necessary to also consider covariates that may affect imatinib pharmacokinetics, such as demographics, body weight, and other clinical characteristics. patients of asian descent, for example, might display different imatinib pharmacokinetics compared with those of caucasian descent as a result of different physical characteristics. whilst there has been no clear elucidation of the influence of race on imatinib pharmacokinetics, several reports have suggested a trend towards lower imatinib trough levels in patients with higher body mass index (3, 5, 13). as such it might be assumed that asian patients would have a tendency towards lower imatinib trough levels compared to their caucasian counterparts. however, this assumption should be treated with caution given that the reports were associated with only a weak correlation or tendency towards insignificance. furthermore, it should also be noted that physical attributes alone are unlikely to explain pharmacokinetic differences between racial backgrounds given potential differences in pharmacogenomics and activity of metabolic enzymes such as cyp3a5. therefore, in order to individualize treatment with imatinib in patients with gist, investigation of clinical and demographic covariates correlated with imatinib pharmacokinetics is warranted. the optimal imatinib exposure level in an individual patient maybe defined as the level that is able to sustain maximal tumour response whilst minimizing adverse events. no reliable evidence exists to define a pharmacokinetic threshold for clinical benefit, though an imatinib plasma trough concentration of less than 1,100 ng / ml has been reported to be related to reduced efficacy in patients with advanced gist (3). the results of these case reports demonstrate that imatinib plasma monitoring - guided dose modification can successfully manage imatinib - related toxicities due to overexposure without compromising the efficacy of the treatment. our findings suggest that individual imatinib blood level testing may be a promising approach for fine - tuning imatinib dosage for better tolerability and optimal clinical outcomes in patients with advanced gist. | imatinib, the first - line treatment in patients with advanced gastrointestinal stromal tumors (gist), is generally well tolerated, although some patients have difficulty tolerating the standard dose of 400 mg / day. adjusting imatinib dosage by plasma level monitoring may facilitate management of patients who experience intolerable toxicities due to overexposure to the drug. we present two cases of advanced gist patients in whom we managed imatinib - related toxicities through dose modifications guided by imatinib plasma level monitoring. imatinib blood level testing may be a promising approach for fine - tuning imatinib dosage for better tolerability and optimal clinical outcomes in patients with advanced gist. |
cataract surgery with intraocular lens (iol) implantation is the most common ophthalmic surgical operation. the mean annual incidence of postoperative endophthalmitis (poe) among cataract patients ranges from 0.05 to 0.14% [15 ]. in practice, ophthalmologists apply topical antibiotic drops to prevent this rare but potentially devastating complication. [3, 68 ]. an american society of cataract and refractive surgery (ascrs) survey (n = 1312) showed that fourth - generation fluoroquinolones were preferred by most surgeons (81%) for infection prophylaxis after surgery. the topical fourth - generation fluoroquinolone moxifloxacin has proven advantageous over older fluoroquinolones as well as other topically available antimicrobials. it has a broader spectrum of action and excellent penetration into eye tissues and is able to deliver a concentration thousands of times the minimum inhibitory concentration [1013 ]. topical corticosteroids such as dexamethasone are applied with infection prophylaxis to minimize, if not eliminate, the inflammatory reaction expected after surgery. it has been reported that treatment with combined steroid - antibiotic eye drops was effective in preventing infection and controlling inflammation after phacoemulsification and iol implantation [1416 ]. in our setting, as well as in other developing nations, financial capabilities of patients and expenditure restrictions from health care organizations demand cost effectiveness. a fixed - combination eye preparation not only helps in cutting costs but also improves patient compliance due to convenience in dosing and application. its efficacy and tolerability in ophthalmic surgery has been evaluated [15, 17 ]. however, its clinical use in asian patients with cataract has not been reported. the purpose of the present study was to evaluate the efficacy and tolerability of the combination formulation in the prevention of postoperative inflammation and infection following phacoemulsification in predominantly asian patients. this 15-day, open - label, and single - arm clinical trial was conducted at the american eye center, philippines. the center 's institutional review board approved the study protocol, which followed the principles set forth in the declaration of helsinki. adult patients (18 years) with a diagnosis of cataract underwent clear cornea incision phacoemulsification with iol implantation. patients who presented with the following conditions were excluded : uncontrolled glaucoma, intraocular hypertension, diabetes mellitus, iris atrophy, chronic or recurrent ocular inflammatory disease (i.e., iritis, scleritis, uveitis, iridocyclitis, and rubeosis iridis), intraocular inflammation, or ocular pain in the study eye prior to the surgery. the use of any ocular antimicrobial drug within 30 days prior to enrollment in the study, nonsteroidal anti - inflammatory drugs or systemic or topical steroids within 14 days prior to enrollment, or a topical prostaglandin analogue four days prior to the surgery until the completion of the study also excluded patients from the study. patients were instructed to instill one drop from a labeled bottle of moxifloxacin 0.5%/dexamethasone 0.1% (vigadexa) 4 times a day in the conjunctival sac of the eye to be operated on beginning from day 1 (1 day before the surgery) until day 15 (15 days after the surgery). on day 0 (surgery day), the patient was dosed by the study nurse. the patients underwent phacoemulsification (2.2 mm clear cornea incision) using the infiniti vision system (alcon laboratories, inc., fort worth, tex, usa) followed by implantation of a single - piece aspheric hydrophobic acrylic iol (acrysof iq, alcon laboratories, inc., viscoelastics used were sodium chondroitin sulfate - sodium hyaluronate (viscoat, alcon laboratories) and sodium hyaluronate (provisc, alcon laboratories) and were removed using coaxial irrigation and aspiration with a vacuum level of 600 mm hg. all cases were done using the same surgical technique. preoperative and intraoperative medications included tropicamide / phenylephrine, povidone - iodine local antiseptic and topical proparacaine hcl, and intracameral lidocaine anesthesia. during the screening visit (within 14 days prior to surgery), baseline values of both eyes were recorded for best - corrected visual acuity (bcva) in logmar, and intraocular pressure (iop) was measured by goldman applanation tonometry. patients were examined for the presence of anterior chamber (ac) cells and flare and any pathology of the eyelids, conjunctiva, and cornea through slit - lamp biomicroscopy. a dilated fundus examination was performed to examine the retina, macula, choroid, vitreous, and optic nerve. patients were seen postoperatively on days 1, 3, 8, and 15. at each visit, patients were examined for signs of infection, inflammation, and ocular pain. ac inflammation, a major criterion of effectiveness, was evaluated based on the number of cells per high - power field measured using the narrowest slit beam of the lamp (0.5 at a height of 8 mm) and was recorded on a 04 point scale, where 0 indicates less than 5 cells, 1 = 510 cells (mild), 2 = 1120 cells (moderate), 3 = 2150 cells (marked), and 4 indicates more than 50 cells (hypopyon, severe). ocular pain was scored by patients subjectively (0 = none, 1 = minimum, 2 = mild, 3 = moderate, 4 = moderately severe, and 5 = severe). additionally, structural changes and signs of inflammation in the eyelids / conjunctiva and cornea were evaluated by slit lamp (0 = absence of active inflammation and 1 = presence of active inflammation). those with at least one follow - up visit after the surgery were included in the per - protocol (pp) analysis (n = 60). a fisher 's exact test of independence was employed to evaluate the differences in the percentage of patients with a score of zero for ac cells at each visit before and after treatment. the ocular signs score was analyzed using the cochran - mantel - haenszel (cmh) test with rank score. the replacement of missing values was adopted for the pp population according to the last value option carried forward technique. sixty - four patients (27 male and 37 female) were enrolled in the study. the mean age was 68 years 11.4 years (sd) (range from 34 to 86 years). two were lost to followup, and two were shifted to moxifloxacin (vigamox) and prednisolone acetate (pred forte) on day 1 due to severe ocular inflammation. an increased inflammatory response was observed for the first few days after surgery which gradually declined until day 15 (table 1). on day 1, 85% of patients had ac cells grade 02, while 15% had grade 3. the number of patients with grade 3 ac cells decreased to 1.7% on day 3. on day 15, 91.7% had grade zero ac cells and only 1 patient had moderate inflammation. at the end of the study, 96.9% did not experience eye pain, while 3.1% rated their eye pain as minimum. signs of active inflammation in the eyelid / conjunctiva and cornea significantly decreased from day 1 postoperatively to day 15. at day 1, inflammation was documented in 9.4% (n = 6) of eyes in the eyelid / conjunctiva, while the same was observed in the cornea in 23.4% (n = 15) of eyes (table 2). at the end of the study, signs of inflammation in the eyelid / conjunctiva and in the cornea were seen in only 1 eye and in 2 eyes, respectively. the bcva improved from a mean of 0.5 logmar preoperatively to 0.0 logmar on day 15 (p < the mean iop was recorded at 17 mm hg. the mean iop was maintained postoperatively in the range of 12 - 13 mm hg over the course of the 15-day treatment (figure 2). no abnormality was found in the fundus of the study eyes at exit from the study. our study assessed the efficacy of a fixed - combination moxifloxacin 0.5%/dexamethasone 0.1% formulation (vigadexa) in the prevention of postoperative inflammation and infection following phacoemulsification in mostly asian patients. at the completion of the study, a score of 0 for ac cells less than 5 was found in 91.7% (55/60) of eyes. patients with an ac reaction higher than grade 0 did not complain of any ocular discomfort or blurring of vision. they were, however, given topical fluorometholone four times daily dosing for one week after discontinuing vigadexa with resulting resolution of the inflammation. on the 1st postoperative day, more than half (55%) had moderate to marked ac cell grading, which decreased to 21.7% by day 3 and to 3.3% by day 8 (table 1). this is consistent with the postoperative ac reaction pattern the investigators observed in cases where separate antibiotic and corticosteroid eye drops were given after cataract surgery. the postoperative inflammatory pattern in the eyelid / conjunctiva and cornea was judged by the investigators as consistent with previous observations. all these were reflective of the mild ocular changes expected to occur as a result of surgery. in this small population of 60 eyes, however, the rarity of the event and the size of the study population did not allow us to make statistically significant conclusions about the effectiveness of the medication in preventing poe. prior to our single - arm trial, the efficacy of the fixed combination had already been established in a study by freitas. in a brazilian population. in this randomized, parallel - group trial (n = 139), the combination moxifloxacin 0.5%/dexamethasone 1% was as effective in preventing infection and controlling inflammation postoperatively compared to when its individual components were administered concurrently. the current study also evaluated the safety and patient acceptance of the fixed - combination preparation of moxifloxacin 0.5% and dexamethasone 0.1%. the formulation was well tolerated by patients ; patients did not report any discomfort during or immediately after its application. no corneal or other ocular surface signs attributable to medication toxicity as well as drug - related adverse events were observed during the entire duration of the study. this can be attributed to the tolerability profile of the drug and ease of administration. patients will comply with instilling an eye drop that does not sting, burn, cause redness, or blur vision. furthermore, applying less number of drops makes it easier for patients to remember and adhere to the dosing regimen. with a combination preparation, patients no longer have to wait a minimum of five minutes to instill a drop from a separate medication to prevent a wash - out effect [16, 18, 19 ]. the formulations of netilmicin - dexamethasone, tobramycin - dexamethasone, and neomycin - polymyxin - dexamethasone effectively controlled postoperative inflammation and were well tolerated, as described in comparative trials with caucasian patients [16, 18, 20 ]. the fixed - combination moxifloxacin 0.5%/dexamethasone 0.1% formulation was found to be well tolerated and effective in minimizing inflammation following cataract surgery in asians. because vigadexa is a relatively new medication, further clinical trials with larger number of patients are warranted to further demonstrate and confirm its long - term safety and efficacy profile, particularly in the prevention of endophthalmitis. | background. the use of a fixed - combination antibiotic corticosteroid for infection prophylaxis in asian patients undergoing phacoemulsification has not been reported. methods. a 15-day, open - label, single - arm trial of 64 patients for phacoemulsification with intraocular lens (iol) implantation is described. patients applied moxifloxacin 0.5%/dexamethasone 0.1% (vigadexa) eye drops four times daily before and until 15 days after surgery. anterior chamber (ac) reaction, visual acuity, ocular pain and signs, and intraocular pressure (iop) were assessed at baseline and on postoperative days 1, 3, 8, and 15. results. at day 15, 55 (91.7%) patients scored 0 (0. no drug - related adverse event was reported. conclusion. following phacoemulsification and iol implantation, the topical combination moxifloxacin 0.5%/dexamethasone 0.1% was effective in preventing infection and controlling inflammation and was well tolerated. |
it has been argued that the unaffected hemisphere may contribute to early recovery of motor function after stroke in humans1 and animals.2 a single session of somatosensory stimulation in the form of peripheral nerve stimulation improves motor function in chronic stroke.35 a decrease in short - interval intracortical inhibition (sici) in the affected hemisphere has been reported after somatosensory stimulation and motor training.3 on the other hand, the effects of somatosensory stimulation on cortical excitability in the unaffected hemisphere are largely unknown. different aspects of corticomotor excitability can be evaluated using transcranial magnetic stimulation (tms). resting motor threshold, (i.e., the minimum intensity necessary to elicit an mep in 50% of trials6 in a target muscle using a single tms pulse) sici refers to a decrease in motor evoked potential (mep) amplitude that usually occurs when a conditioning, subthreshold stimulus precedes a suprathreshold stimulus at an interstimulus interval (isi) of 16 ms.7 an increase in mep amplitude (intracortical facilitation) occurs at greater isis (625ms). sici and intracortical facilitation (icf) likely reflect intracortical post - synaptic activity, which depends on the excitability and integrity of separate inhibitory (sici) and excitatory icf neurons with a possible spinal contribution to icf.89 our study measured sici and icf in the unaffected hemisphere of nine hemiparetic, well - recovered, chronic stroke patients to evaluate if somatosensory stimulation of the paretic hand was associated with excitability changes in the ipsilateral unaffected hemisphere. in addition, we evaluated correlations between hand motor function and corticomotor excitability. nine chronic phase patients (> 6 months) with infarcts in the middle cerebral artery territory participated in this study. inclusion criteria were hemiparesis, caused by a single ischemic cortical or subcortical infarction in the middle cerebral artery territory ; age 18 years ; and mild to moderate hand disability, defined in terms of ability to perform all the tasks of the jebsen - taylor test (jtt).10 the jtt scores the time, in seconds, required to perform seven activities that are relevant to common daily activities. the lower the jtt score, the better the functional performance of the upper limb. exclusion criteria were other neurological disorders, use of drugs that influence corticomotor excitability, and contraindications to tms.11 patient characteristics are shown in table 1. the protocol was approved by the local ethics committee and was performed in accordance with the declaration of helsinki. tms was delivered to the unaffected hemisphere at the optimal scalp position12 to elicit motor evoked potentials in the unaffected abductor pollicis brevis (apb) through a figure - of - eight shaped coil (mean diameter, 70 mm) connected to two magnetic stimulators via a bi - stim 2002 module (magstim, uk). electromyography (emg) responses were amplified (1000), filtered (2 hz- 2 khz) and sampled at 5 khz.13 the following tms measurements were performed before and after control or active somatosensory stimulation : - resting motor threshold (rmt), defined as the minimum tms intensity required to elicit at least three out of six meps 50 microv in consecutive trials.6 - sici and icf, measured with paired - pulse tms as previously described.79 the conditioning stimulus (cs) intensity was set to 80% of the apb rmt. the intensity of the test stimulus (ts) was that required to evoke meps of approximately 0.5 to 1 mv (tsmep). the order of presentation of inhibitory (2 ms), excitatory (10 ms) and control trial intervals (test stimulus alone) was randomized. results are expressed as average percentages of mep amplitudes in conditioning trials and in test trials (mepcs+ts / mepts, %). patients were submitted to 2-h somatosensory stimulation, in the form of active median nerve stimulation, and to a control, consisting of subthreshold median nerve stimulation, in separate sessions, as previously described4 in a cross - over design. four subjects were submitted to active stimulation in the first session and five to control stimulation. the interval between active and control sessions was 19.6 3.1 days (mean standard error). surface electrodes were optimally placed to stimulate the median nerve at the wrist in the paretic arm. initially, the minimum intensity of stimulation at which patients reported paresthesias in the median nerve cutaneous territory (sensory threshold) was measured three times. stimulus intensity was increased until the maximum at which patients reported strong paresthesias in the median nerve territory in the absence of pain, while compound muscle action potential amplitudes remained below 100 microv in the apb. in the control session, stimulus intensity was kept below the sensory threshold. the correlation between hand motor function, evaluated by jtt scores, and tms measurements (average of pre - somatosensory stimulation results in control and active somatosensory stimulation sessions) tms results were analyzed with repeated - measures anova with factors time (2 levels : before stimulation and after stimulation) and condition (2 levels : control and active somatosensory stimulation). visual analog scales (vas) to measure attention, fatigue and drowsiness were administered at the beginning of the experiments, and after each set of tms measurements. there was a significant correlation between jtt scores in the affected hand and sici in the unaffected hemisphere (r=0.73 ; p=0.025) (figure 1). no significant correlations were found between jtt scores and rmt or icf (p > 0.05). there were no significant differences in rmt and sici after control or active somatosensory stimulation (table 2). there were no significant effects of time or condition alone, but there was a significant time condition interaction for icf (table 2). there was a significant increase in icf after control somatosensory stimulation (p 0.05). there were no significant differences in rmt and sici after control or active somatosensory stimulation (table 2). there were no significant effects of time or condition alone, but there was a significant time condition interaction for icf (table 2). there was a significant increase in icf after control somatosensory stimulation (p 6 months) after stroke. our results indicate that increased inhibition in the unaffected hemisphere is related to better motor function in these patients. our conclusions are in agreement with the results reported by swayne and colleagues at 3 months after stroke.18 consistent with the concept that increased inhibition of the unaffected hemisphere can be beneficial while decreased inhibition may be maladaptive, liepert and colleagues19 reported disinihibition in the unaffected hemisphere in patients with severe upper limb impairment (strength 01 in the medical research council scale) in the first month after stroke. even though the functional role of the unaffected hemisphere in motor recovery is a matter of controversy, it has been suggested that changes in excitability can occur in the unaffected hemisphere in response to rehabilitative interventions. for instance, icf has been found to increase in the unaffected hemisphere in chronic stroke patients submitted to constraint - induced therapy for ten days.20 kimberley and colleagues1 reported improved jtt performance, and enhanced functional magnetic resonance imaging (fmri) activity in the primary sensory cortex in the unaffected hemisphere of chronic stroke patients submitted to somatosensory stimulation in the form of electrical muscle stimulation of the forearm extensor muscles for three weeks. the increased activation in the primary sensory cortex could be related to increased sensorimotor interactions and enhanced excitability after somatosensory stimulation, but the functional relevance of this finding should be further explored. despite the fact that patients were asked every five minutes about the presence and intensity of paresthesias in the paretic hand in both control and active sessions, vas scores for drowsiness increased after the control somatosensory stimulation. therefore, it is possible that increased icf after the control stimulation, but not after the active stimulation, could reflect differences in non - specific changes in arousal across the two conditions. first, a previous study reported a decrease, instead of an increase, in icf following sleep deprivation (a condition in which drowsiness is usually increased).21 second, the same study reported a decrease in sici after sleep deprivation, but, in our patients, there were no significant changes in sici in either experimental session. however, we can not completely rule out different non - specific effects of active and control interventions in arousal. decreased sici (likely reflecting decreased gabaa activity) and no changes in icf in the affected hemisphere were reported after active somatosensory stimulation in chronic stroke patients3. our results do not support an overt effect of a single session of somatosensory stimulation of the paretic hand on motor cortical excitability of the unaffected hemisphere as measured by rmt, sici or icf. it remains to be determined if other markers of cortical excitability22,23 are modulated by somatosensory stimulation, and if repeated sessions or lesion location may lead to different effects. | introductionsomatosensory stimulation of the paretic upper limb enhances motor performance and excitability in the affected hemisphere, and increases activity in the unaffected hemisphere, in chronic stroke patients. we tested the hypothesis that somatosensory stimulation of the paretic hand would lead to changes in excitability of the unaffected hemisphere in these patients, and we investigated the relation between motor function of the paretic hand and excitability of the unaffected hemisphere.methodstranscranial magnetic stimulation was administered to the unaffected hemisphere of nine chronic stroke patients. patients were submitted to 2-h somatosensory stimulation in the form of median nerve stimulation and control stimulation using a cross - over design. baseline jebsen - taylor test scores were evaluated. resting motor threshold, intracortical facilitation, short - interval intracortical inhibition, and visual analog scores for attention, fatigue and drowsiness were measured across conditions.resultsbetter pre - stimulation baseline motor function was correlated with deeper sici in the unaffected hemisphere. we found no overt changes in any physiological marker after somatosensory stimulation. there was increased drowsiness in the control session, which may have led to changes in intracortical facilitation.conclusionsour results do not support an overt effect of a single session of somatosensory stimulation of the paretic hand on motor cortical excitability of the unaffected hemisphere as measured by motor threshold, short - interval intracortical inhibition or intracortical facilitation. it remains to be determined if other markers of cortical excitability are modulated by somatosensory stimulation, and whether repeated sessions or lesion location may lead to different effects. |
intravenous rtpa remains the only proven treatment for acute ischaemic stroke in the general population. patients with end - stage renal disease (esrd) on dialysis have a 510-fold higher incidence of stroke than the general population with an overall incidence rate of 1333 per 1000 patient - years. this may reflect the bleeding diathesis of uraemia as well as the effects of anticoagulation for vascular access and dialysis circuit patency, the prevalence and degree of hypertension, and the established ethnic variations. patients with esrd have been traditionally excluded from the large prospective randomized controlled trials that form the evidence base for treatment of vascular disease, including stroke, in the general population. renalism is to potentially restrict access to beneficial therapies in a cohort that would have derived the greatest benefit. in addition, extrapolating outcome data derived from studies in the general population to patients on haemodialysis is not always fruitful. in one of the few adequately powered large prospective randomized controlled trials in haemodialysis (hd), atorvastatin did not reduce the incidence of stroke in stark contrast to studies in the general population. at present, the use of thrombolytic therapy for acute ischaemic stroke in hd patients has not been described. he had a prior diagnosis of progressive chronic kidney disease secondary to obstructive uropathy and recurrent urosepsis, and a hydronephrotic left kidney requiring insertion of a j - j ureteric stent in june 2009. in addition, he had type 2 diabetes mellitus, ischaemic heart disease (myocardial infarction march 2009) and a diagnosis of hypertension. he did not have a history of cardiac dysrhythmia, a prothrombotic diathesis or cerebrovascular disease. following emergency admission to the intensive care unit with pulmonary oedema, oliguria and a serum creatinine of 10.2 mg / dl (899 mol / l), he was established on maintenance thrice weekly hd via a right internal jugular tunnelled cuffed central venous catheter (tesiocath, medcomp, harleysville, pa, usa) 2.5-months prior to presentation with an acute stroke. he presented to his local emergency department with acute right hemiparesis and aphasia of a 90-min duration. he had undergone routine hd 24-h with no complications prior to presentation. on arrival, his blood pressure (bp) was 190/87 mmhg, capillary blood glucose was 86.5 mg / dl (4.8 mmol / l) and his glasgow coma score (gcs) was 15/15. an urgent ct scan of his brain revealed an acute ischaemic stroke affecting the superior parasagittal cortex of the left frontal lobe. clinical examination revealed a mild right facial droop, evidence of a right hemiparesis (power 3/5 in the upper limb and power 0/5 in the lower limb) with increased muscle tone in the upper limb, lower limb hyper - reflexia and an upgoing plantar response in the lower limb. laboratory tests at time of presentation there were no absolute contraindications to thrombolysis, and he was eligible for trial enrolment. following informed consent, he underwent randomization and received 54 mg rtpa (0.9 mg / kg weight estimated at 60 kg) as per trial protocol (10% bolus followed by an infusion). rtpa (actilyse, boehringer ingelheim ltd, bracknell, berkshire, uk) was delivered at 4-h after symptom onset. a repeat ct brain scan was performed 24-h after thrombolysis which showed a small area of haemorrhagic transformation within the original infarct and no other interval change (figure 1). he remained clinically stable although required sodium valproate (600 mg b.d.) and clobazam (5 mg o.d.) for intermittent left upper limb myoclonus which responded well to therapy. a 24-h holter during the interdialytic period revealed sinus rhythm with a 1-h paroxysm of asymptomatic atrial fibrillation that terminated spontaneously. four days after admission, he was transferred to our specialist renal stroke rehabilitation unit where he was an inpatient for the following month. during this time, power in his upper limb improved to 4/5 proximally and 3/5 distally, and in his lower limb to 4/5 proximally. he was discharged home 5 weeks after his initial admission and remains stable on maintenance hd. single representative axial section through the brain post - thrombolysis. within the parasagittal cortex of the left frontal lobe in the anterior cerebral artery territory is an ischaemic infarct with a small amount of haemorrhagic transformation (area of increased attenuation marked with the arrow). rtpa (alteplase) is a glycoprotein that becomes activated on binding to fibrin, converting plasminogen to plasmin and leading to fibrinolysis. when administered within 3-h of symptom onset in the seminal placebo - controlled national institute of neurological disorders and stroke (ninds) rtpa study, it resulted in a significantly better neurological outcome at 3 months. analysis of pooled results of six randomized controlled trials of intravenous rtpa showed that the best outcomes occurred in patients treated within 2-h of symptom onset and suggested a benefit extending to 4.5-h. although mortality did not differ between the two groups, 52.4% patients in the rtpa arm recovered with no disability after 90% vs 45.2% in the placebo arm (p = 0.04). there was, however, a higher rate of symptomatic intracerebral haemorrhage in the rtpa arm (2.4% vs 0.2%, p = 0.008) in keeping with prior studies. a cochrane systematic review suggested a benefit associated with rtpa up to 6-h from symptom onset, and this is currently under study. use of rtpa is contraindicated in cases where there is a known haemorrhagic diathesis or severe uncontrolled arterial hypertension (defined as 185/110 mmhg) as well as administration of heparin within the previous 48-h and a thromboplastin time exceeding the upper limit of normal for laboratory. the manufacturer advises caution in all situations where there is a high risk of haemorrhage. the use of warfarin in hd patients with atrial fibrillation or for maintenance of arteriovenous graft patency will preclude some patients from receiving rtpa despite a study demonstrating a higher incidence of stroke in patients on this therapy. high rates of coronary and peripheral arterial disease and its treatment with percutaneous interventions in esrd lead to a significant proportion of patients on single or dual maintenance antiplatelet therapy. clinicians need to be aware of the high rates of occult gastrointestinal bleeding in hd patients as well as considering uraemia as a state of mild haemorrhagic diathesis. although the effect of unfractionated heparin can be monitored using the activated partial thromboplastin time (aptt), this is not the case for low - molecular - weight heparins (e.g. tinzaparin and enoxaparin) that are increasingly used for this indication. to our knowledge, this is the first reported case of intravenous thrombolysis for treatment of acute ischaemic stroke in a haemodialysis patient. the outcome for this patient was favourable ; however, we would advise extreme caution with the use of rtpa (alteplase) and treatment delivered on a case - by - case basis. | stroke is a leading cause of death worldwide and is associated with significant morbidity in survivors. early thrombolytic therapy in acute ischaemic stroke has been shown to dramatically improve patient outcomes. although the age - adjusted incidence of stroke is 510 times greater in haemodialysis patients, the use of thrombolysis for this indication in this group of patients has not been described to date. we present a case where alteplase was used successfully for acute ischaemic stroke in a patient established on maintenance haemodialysis in the setting of an international randomized controlled trial and advocate caution with the use of systemic thrombolytics despite the favourable outcome seen with this case. |
point spread function (psf) is used to define the quality of a retinal image. when an eye is looking at a point source, the luminance distribution on the retina is called the psf. the visual acuity corresponds to the central part of the psf (0~1 min of arc), and the outer part of the psf (> 1) is the straylight. straylight is caused by light that enters the eye and is scattered rather than focused on the retina. straylight can create a veil of light over the retina, which can lead to halo, glare, hazy vision, and night blindness while driving [2, 3 ]. the cornea, iris, sclera, retina, and lens are the five major sources that contribute to intraocular straylight. changes in intraocular straylight after cataract surgery are mainly caused by intraocular lenses (iols). although advances in iol design have improved postoperative visual outcomes, patients often complain of reduced contrast and glare after cataract surgery. a recent study on european drivers showed large variations in straylight values in pseudophakic eyes. the iol material should be considered in studying the influence of intraocular straylight on pseudophakic eyes. this study aims to determine whether there are differences among pmma, hydrophilic acrylic, and hydrophobic acrylic iols in perceived intraocular straylight. patients who underwent cataract extraction with iol implantation may complain about dysphotopsia, entopic phenomena, and photic phenomena. furthermore, we want to assess the differences among the various iol materials when the pupil is dilated with the iol edge exposed. the c - quant straylight meter proposed by van den berg offers a convenient technique that uses the compensation comparison method. moreover, cervio and guber both showed that c - quant possesses excellent reliability and high reproducibility [11, 12 ]. all the procedures followed the tenets of the declaration of helsinki and were approved by the local ethics committee. we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research. seventy - six eyes of sixty - two patients (age 64.86 8.39 years, range 4179 ; 38 women and 24 men) were included in this study. the patients were scheduled for phacoemulsification and iol implantation from may 1, 2012, to december 31, 2012, in tianjin eye hospital. all the surgeries were performed by the same doctor (ya - wen guo). phacoemulsification with a 3.2 mm clear corneal incision in the hydrophobic acrylic and hydrophilic acrylic group and a 5.5 mm sclera tunnel incision in the pmma group were followed by iol implantation in the capsular bag. a hydrophobic acrylic spherical iol (ar40e, amo, usa) was implanted in the hydrophobic acrylic iol group. a hydrophilic acrylic spherical iol (hq201hep, hexavision, france) a pmma spherical iol (pc156c55, henan universe iol r&m co., china) was implanted in the pmma group. the diameters of pupil were measured with wave - front analyzer kr-1w in dark room (topcon, tokyo medical system inc., japan) with natural pupil and dilated pupil. the retinal straylight was measured with c - quant straylight meter (oculus optikgerte, gmbh, wetzlar, germany). it is more suitable for clinical examination than the instrument which works with direct compensation method. the center of the test field is divided in half. when the compensation light is presented to one - half the center is a ring - shaped flickering light source, which serves as the straylight source. when the subject is tested, one - half of the center has counter - phase flickering added, the other has not. the straylight meter will change the luminance of the stimulus and counter - phase modulating light automatically until the two halves are balanced. according to the subjects ' responses all the subjects were examined with natural and dilated pupils one week and one month after surgery. only when the estimated standard deviation (esd) was lower than 0.08 and the quality factor (q) was higher than 1.00, the measurement was accepted. the straylight data were presented as the mean sd. the straylight values of three groups were compared with one - way analysis of variance (anova). the lsd (least significance difference) was used to identify effects among the iol types. in each pseudophakic group a paired t - test was used to compare the straylight values before and after pupil dilation. comparison of the straylight one week and one month after surgery was performed with paired t - test. all pupils involved in this study were round with no iris trauma and showed good responsiveness to light after surgery. the mean ages of the hydrophobic acrylic, hydrophilic acrylic, and pmma iol groups were 67.17 6.82, 62.82 9.50, and 64.92 8.16. there were no significant differences between the three groups (p > 0.05). table 1 shows the characteristics of the rigid pmma, hydrophilic acrylic, and hydrophobic acrylic iols evaluated. table 2 shows the mean diameter of pupil one week after surgery and one month after cataract surgery. there were no statistically significant differences among groups 1 week after surgery and 1 month after surgery (p > 0.05). the hydrophilic acrylic iol showed a significantly lower straylight value than the hydrophobic iol with dilated pupil one week after surgery (p = 0.002). there was not a significant difference between hydrophilic acrylic and pmma iol group (p > 0.05). but the straylight values of hydrophilic acrylic iol were the lowest of the three iol groups. there was no significant difference in retinal straylight between one week and one month after surgery in each iol group with natural pupil (p > 0.05). a similar result was found with dilated pupil (p > 0.05) (figure 1). no significant difference was found in retinal straylight between natural and dilated pupil in each iol group 1 week after surgery (p > 0.05). a similar result was found a month after surgery (p > 0.05) (figure 2). patient complaints regarding dysphotopsia and glare after iol implantations have recently gained a great deal of attention. only the hydrophilic acrylic iols showed a significantly lower straylight value than the hydrophobic acrylic iols in dilated pupils. no significant difference was found in straylight between the other groups. with or without dilated pupil, the straylight value of the hydrophilic acrylic iol was the lowest among the three iol materials. with natural pupil, the square edge of iols can cause clinically significant glare and halo [13, 14 ]. the edge of the iol may have been exposed when the pupil was dilated, thereby increasing light scatter. accordingly, hydrophobic iols induced more scattering. did the hydrophobic acrylic material induce more straylight ? the difference in the optic diameter between the pmma iol and the other two iols should theoretically change the straylight value with dilated pupils. more study is recommended. montenegro. also found that postoperative straylight in the hydrophilic acrylic group was lower than that of the hydrophobic acrylic group. he supposed that the glistenings around the hydrophobic acrylic iol can explain the reason for the higher amount of straylight induced by the hydrophobic acrylic iols. when iols are implanted into the eyes, some fluid - filled microvacuoles form in the hydrophobic iol optics, which are called glistenings. the glistenings were observed from as early as one week after surgery until after several months [16, 17 ]. the high incidence of glistenings was associated with hydrophobic acrylic iols, with an incidence of 55% at one year after the hydrophobic acrylic iol implantation. a japanese study showed that internal light scattering caused by the glistenings within the optic of the acrysof iols increased over time up to three years after surgery. the high incidence of glistenings may be the reason for the higher straylight values in the hydrophobic acrylic iol group. in the present study, we found that the straylight values at one week and one month after hydrophilic acrylic iol surgery were both lower than those of the hydrophobic acrylic iols. in addition, we did not find any apparent glistening in the hydrophobic acrylic iol using a slit lamp. in the present study, we did not find an increase in straylight values with increasing pupil size at one week and one month after surgery. franssen 's study showed that there was no significant difference in straylight between pupils with diameters of 2.0 mm and 7.0 mm of healthy phakic eyes. cervio. also found that straylight values were not associated with the pupil size of the subject. reported that straylight increased with increasing pupil size in pseudophakic eyes at one year after surgery. straylight values were significantly higher in dilated than those in natural pupils, which was contradictory to our study. in van gaalen 's study, the presence of the anterior capsule rim in the pupillary area, not the pupil size, was considered as the main reason for the increase in straylight. van gaalen measured straylight using natural pupils and dilated pupils at one year after surgery. although the eyes that had posterior capsular opacification (pco) within the 3.0 mm diameter central zone of the posterior capsule were excluded, the migration of residual lens epithelial cells may have resulted in the opacification of the peripheral anterior and posterior capsules., we did not find pco in any of the patients during the brief follow - up period. the follow - up period of one month is not enough to elicit changes of light scatter related to anterior capsule opacification. they thought corneal edema could increase straylight values. in the present study, we did not find any marked cornea edema in all the subjects using slit lamp. but corneal thickness was not measured. no significant differences were found in straylight values in natural pupils at one week and one month after surgery ; similar results were obtained in dilated pupils. van der meulen. found no significant differences in straylight values between the patients examined within 30 days after surgery and those examined after 30 days or longer postoperatively. study and van der meulen 's study all subjects were measured 7 and 30 days after surgery. two parameters (esd and q) were used to determine the reliability of the measurement. the measurement results are considered reliable when the esd is lower than 0.08 and the q is higher than 1. moreover, old age did not affect the measurement reliability of c - quant using the compensation comparison method. | purpose. to investigate the intraocular straylight value after cataract surgery. methods. in this study, 76 eyes from 62 patients were subdivided into three groups. a hydrophobic acrylic, a hydrophilic acrylic, and a pmma iol were respectively, implanted in 24 eyes, 28 eyes, and 24 eyes. straylight was measured using c - quant at 1 week and 1 month postoperatively in natural and dilated pupils. results. the hydrophilic acrylic iols showed significantly lower straylight values than those of the hydrophobic acrylic iols in dilated pupils at 1 week and 1 month after surgery (p 0.05). moreover, no significant difference was found in straylight between natural and dilated pupils in each group at 1 week and 1 month postoperatively (p > 0.05). conclusions. although the hydrophobic acrylic iol induced more intraocular straylight, straylight differences among the 3 iols were minimal. pupil size showed no effect on intraocular straylight ; the intraocular straylight was stable 1 week after surgery. |
primary sjogren 's syndrome (pss) is a relatively common autoimmune disease affecting 23% of the adult population. undiagnosed pss presenting with severe hypokalemia and demyelination in the absence of dryness is rare but can occur in 27%. a 36-year - old lady presented with weakness of upper and lower limbs progressing to complete inability to move her limbs over a 12-h period. she did not report diplopia or urinary retention and denied any fever, neck pain, or trauma. she was a homemaker, did not smoke and denied any alcohol or substance abuse. she was diagnosed with postviral encephalomyelitis 3 years ago and treated with steroids with complete resolution of symptoms and signs. she had normal menstrual cycles and had one spontaneous second trimester abortion 8 years ago. she had one living child and had undergone tubal ligation after medical termination of pregnancy 4 months prior to this presentation. on evaluation, she was afebrile, normotensive with a respiratory rate of 25-breaths / min and resting pulse oximetry of 98%. there was flaring of alae nasi during quiet respiration. mini - mental state examination was normal, and there was no evidence of cranial nerve deficits. power was 2/5 on the medical research council scale in the proximal muscles of the upper limbs and 1/5 in the lower limbs throughout. arterial blood gas showed combined respiratory and normal anion gap acidosis (ph 7.23, paco2 46 mmhg, pao2 88 mmhg, hco3 18 meq / l, chloride 105 meq / l, and corrected anion gap 10 meq / l). she was electively intubated and ventilated for impending respiratory failure with deep venous thrombosis prophylaxis, enteral feeding, and evaluation for acute onset quadriparesis. chest radiographs were normal, and electrocardiography showed prolonged pr interval, prominent u waves and t inversions. meq / l) and deranged renal function (serum creatinine 2.1 mg / dl, normal 0.81.2 mg / dl and blood urea 60 mg / dl, normal 2040 mg / dl). serum sodium, calcium, phosphate, magnesium levels, and plasma glucose were normal. liver function tests revealed hypoalbuminemia (2.8 g / dl, normal 46 g / dl) ; enzymes were normal. urinary electrolytes showed potassium level of 45 meq / l and positive urine anion gap. 24-h urinary calcium excretion levels were elevated (550 mg/24-h, normal 20275 mg). the absent tendon reflexes, prior neurological illness, and lack of complete improvement with potassium replacement prompted re - evaluation. cerebrospinal fluid examination was acellular with raised proteins (65 mg / dl, normal 1545 mg / dl) ; oligoclonal bands were negative. computed tomography (ct) head was normal, and nerve conduction tests showed normal sensory and motor conduction. ct -abdomen showed medullary nephrocalcinosis [figure 1, left ] with an incidental suprarenal inferior vena cava thrombus (ivc). magnetic resonance imaging of the head and spine with contrast showed extensive demyelination of the brain and spinal cord [figure 2 ]. anti - aquaporin-4 antibodies were strongly detected by enzyme - linked immunosorbent assay (elisa). pulse methylprednisolone 1 g was administered for 3 days followed by 1 mg / kg / day enteral prednisolone. composite images of the computed tomography of the abdomen (computed tomography, left) at the level of the kidneys showing large chunky calcification due to medullary nephrocalcinosis related to untreated distal renal tubular acidosis and (right, arrow) contrast - enhanced computed tomography - abdomen showing a suprarenal inferior vena cava thrombosis magnetic resonance imaging (left) of the head and spine (right) with contrast revealed t2 hyperintensities in the left middle cerebral peduncle (white arrow), superior medulla and a long segment spinal cord (c7 t3, block arrow) t2 hyperintensity with mild cord swelling suggestive of demyelination she did not report ocular or oral dryness, oral ulcers, malar rash, and photosensitivity or joint pains. in view of distal renal tubular acidosis (rta) with nephrocalcinosis, recurrent central nervous system (cns) demyelination and unprovoked unusual site thrombosis, and antinuclear antibodies (ana) ana was 3 + by immunofluorescence with a speckled pattern ; anti - ss - a (ro) antibodies were positive by elisa (1:1280). schirmer 's i test showed normal tear flow (> 15 mm at 5 min). serum immunoglobulin levels were normal, and the results of hiv and hepatitis c antibodies by elisa were negative. a diagnosis of pss with distal rta and medullary nephrocalcinosis, recurrent cns demyelination with quadriparesis (neuromyelitis optica) and respiratory failure and secondary apla syndrome with ivc thrombosis was made. current classification criteria [table 1 ] are specific but insensitive and may not be fulfilled in up to 1317% of the cohort with pss. salivary scintigraphy showing markedly diminished salivary gland flow, including delayed uptake, reduced concentration, or delayed secretion of the tracer (left) when compared to normal (right) comparison of the revised aecg classification criteria and the acr criteria for sjogren s syndrome dry eyes and dry eyes are the hallmark of this disease and 93% and 98% in large prospective series have evidence of dry eyes and dry mouth, respectively. extraglandular manifestations are seen in a third of patients with ss, including renal involvement in 4%. the reported prevalence of cns manifestations in ss [table 2 ] range from 0% to 60% ; a high index of suspicion is needed. recurrent signs and positive anti - aquaporin-4 antibodies favored ss rather than apla as the cause of cns manifestations. spectrum of central nervous system disease in pss her further course was complicated by pseudomonas - related ventilator - acquired pneumonia and severe weakness with prolonged ventilation. tracheostomy was performed and enoxaparin, prednisolone, piperacillin - tazobactam, pantoprazole, good hydration, enteral shohl 's solution (60 meq / d alkali), and potassium (40 meq / day) were administered. pulse cyclophosphamide 600 mg / m was initiated a week after resolution of pneumonia and continued monthly for 3 months. she was decannulated and discharged with a proximal power of 4/5 and the international normalized ratio of 2.1 by d48 of admission. she is ambulatory with minimal support for activities of daily living, normal renal functions, and potassium at 3 months of discharge. oral azathioprine has been initiated along with warfarin with a plan to continue treatment for 2 years and anticoagulation for life. | we present a middle - aged woman with a prior history of central nervous system (cns) demyelinating disorder who presented with an acute onset quadriparesis and respiratory failure. the evaluation revealed distal renal tubular acidosis with hypokalemia and medullary nephrocalcinosis. weakness persisted despite potassium correction, and ongoing evaluation confirmed recurrent cns and long - segment spinal cord demyelination with anti - aquaporin-4 antibodies. there was no history of dry eyes or dry mouth. anti - sjogren 's syndrome a antigen antibodies were elevated, and there was reduced salivary flow on scintigraphy. coexistent antiphospholipid antibody syndrome with inferior vena cava thrombosis was also found on evaluation. the index patient highlights several rare manifestations of primary sjogren 's syndrome (pss) as the presenting features and highlights the differential diagnosis of the clinical syndromes in which pss should be considered in the intensive care unit. |
reactive oxygen species (ros) are oxygen metabolites that are highly active in terms of oxidative modifications of cellular macromolecules including proteins, lipids, and polynucleotides. superoxide radical (o2) is usually the primal ros species produced and is subsequently converted into hydrogen peroxide (h2o2) through spontaneous or superoxide dismutase (sod)-catalyzed dismutation. and reaction of o2 and nitric oxide (no) generates peroxynitrite (onoo), a ros and reactive nitrogen species (rns) species. of all cellular ros sources, electron leakage from the mitochondrial electron transfer chain (etc) to molecular oxygen generates a steady flux of o2 and thus constitutes the major site of cellular ros production [1, 2 ]. other enzymes, including nadph oxidases, lipoxygenase and cyclooxygenase, cytochrome p450s, and xanthine oxidase, also participate in ros generation. the cellular redox homeostasis is set by a delicate balance between ros production and the antioxidant system. the ros - scavenging enzymes include sods, which convert o2 to h2o2, and catalases, which convert h2o2 to water. collectively, they form an antioxidant pool, while a third category of enzymes such as glutathione peroxidase and thioredoxin reductase catalyze the interconversion and equilibrium among the reduced / oxidized species of different reductants [4, 5 ]. when ros are produced excessively or endogenous antioxidant capacity is diminished, indiscriminate oxidation elicits harmful effects, resulting in oxidative stress. mounting evidence has established strong links between oxidative stress and a wide variety of pathologies including malignant diseases, diabetes mellitus, atherosclerosis, ischemia reperfusion injury, and chronic inflammatory processes as well as many neurodegenerative diseases [3, 614 ]. moreover, the oxidative stress theory of aging states that systematic accumulation of oxidative damage from multiple ros sources constitutes the core process that drives the biological clock of aging [15, 16 ]. nevertheless, homeostatic ros are required to maintain a redox environment optimal to biochemical activities of the cell. it has been shown that acute application of sod mimetics to cardiomyocytes halves the rate of occurrence of spontaneous ca sparks and decreases action potential - elicited ca transients and contraction., a state with too little ros production and/or too strong antioxidant reactivity, can also lead to pathology. for instance, cardiac - specific expression of human alphab - crystallin autosomal - dominant mutant hr120gcryab led to reductive stress causing cardiomyopathy through inducing protein aggregation [19, 20 ]. as an exciting paradigm - shifting development, ros emerge as powerful, ubiquitous, and indispensable cellular messengers, adding to the repertoire of only a handful of second messengers that we know (ca, camp, ip3, and arachidonic acid). the specific ros targets range from ionic channels and transporters, to kinases and phosphatase, and to transcription factors, and the list continues to grow and permeate throughout pivotal pathways in differentiation and organogenesis, cell fate regulation [22, 23 ], stress response, and wound healing [25, 26 ]. as a rule of thumb, the ros effects are multiphasic and bidirectional, depending on the species of oxidants, their concentrations, history of exposure, and cellular context. the proven failure of antioxidant therapies despite decades of industrious efforts [2730 ] serves us a humbling lesson on how much we still do not know about ros signaling. understanding the cell logic and principles of ros signaling and developing efficient and specific antioxidant therapies to constrain ros damages would both hinge on precise and quantitative knowledge of intracellular ros dynamics, concentrations, compartments, and modes of action. first, the trend of ros investigation moving from cell - free preparations to intact cells and even in living animals ; second, the development of a set of novel fluorescent protein - based ros indicators and protein indicator - expressing transgenic animal models, enhanced with cell type and subcellular compartment - targeting ability ; and third, the visualization of exquisite spatiotemporal architecture of intracellular ros dynamics by time - lapse imaging in intact cells or in vivo imaging in transgenic animal models. in this short review, we summarize these recent advances in fluorescent ros imaging in cells and animals with emphasis on novel indicators, genetic animal models, and in vivo imaging technology. depending on ros species and cellular environments, lifetime of a ros molecule in biological systems varies from nanoseconds to seconds. so what is required for a fluorescent ros indicator is that it should compete with the antioxidants for ros and produce fluorescently altered products for visualization and quantification. for an ideal ros indicator, the criteria include selectivity for specific ros species, fast and reversible kinetics, high signal - to - background contrast, and superb signal - to - noise properties as well as ease with intracellular loading and proper subcellular compartmentalization. it is also desirable to be excitable at a visible wavelength, be resistant to photobleaching, and display no toxicity in general and phototoxicity in particular. currently available fluorescent ros indicators fall in two categories, synthetic small - molecule dyes and genetically encoded fluorescent protein - based probes. as will be discussed in the following, major limitations in ros measurements are related to selectivity, kinetics, and ability for quantitative calibration. of the small - molecule fluorescent ros probes, 2-7-dichlorodihydrofluorescein (dcfh), dihydroethidium (dhe), and mitochondrial - targeted dhe (mitosox) are the most popular ones. the diacetate form of dcfh (dcfh - da) is a cell - permeable form that allows ester loading of the dye, resulting in intracellular accumulation of the nonfluorescent dcfh. in the presence of h2o2 and other oxidants, two - electron oxidation of dcfh results in the formation of a fluorescent product, 2-7-dichlorofluorescein (dcf), which can be monitored by fluorescence microscopy and flow cytometry [33, 36 ]. however, severe limitations and potential artifacts are confounding the dcf measurement of ros. apart from its relative nonselectivity to ros species and oxidants, dcfh oxidation can also be catalyzed by cytochrome c and heme peroxidases. worse, the one - electron oxidization product or dcf radical can react with oxygen to produce o2 and subsequently h2o2, thus artificially generating the very ros that it is attempting to quantify. cautions should also be taken to minimize light exposure because dcfh is both susceptible to photo - oxidation (increasing dcf fluorescence) and to photobleaching (loss of dcf fluorescence). kinetically, it is difficult for dcfh to track small and rapid ros transients because dcfh oxidation is irreversible in the intracellular milieu, and the slope of dcf fluorescence rise (df / dt), instead of fluorescence intensity (f) per se, is often used to measure the level of ros. after subtraction of the rising basal fluorescence (fbase), local d(f fbase)/dt has also been used to reflect approximately brief ros transient. this procedure, however, could be complicated because oxidized dcf becomes membrane - permeable. as to its subcellular compartmentalization, dcfh may be enriched in either the cytosol or the organelle mitochondria, depending on loading and experimental conditions. dhe is widely used as a small - molecule fluorescent ros probe specific for o2. the reaction between o2 and dhe generates a highly specific red fluorescent product, 2-hydroxyethidium (2-oh - e(+)), shifting its excitation and emission peaks from 350 and 400 to 518 and 605 nm, respectively [4143 ]. mitosox, a dhe derivative with addition of a positively charged triphenylphosphonium group (tpp+), is highly enriched in the mitochondria [44, 45 ], and the binding of oxidized mitosox to mtdna greatly enhances its fluorescence. the chemical reactivity of mitosox with o2 is similar to the reactivity of dhe with o2, and the particular product 2-oh - e(+) is unique to o2 since several studies have confirmed that 2-oh - e(+) is the only product in the presence of o2 generated by the xanthine xanthine oxidase however, the dhe detection of o2 is still interfered by a prominent reaction : two - electron oxidation of dhe by oxidants other than o2 produces ethidium cation (e+), another red fluorescent product that is bound to nuclear dna and often present at a much higher concentration. it has recently been suggested that selective detection of 2-oh - e+ is possible by excitation at 396 nm because an excitation band between 350 and 400 nm is present for 2-oh - e+ but not e+. however, other studies have reported that e+ can still significantly contribute to the fluorescence intensity even at 396 nm excitation because of high levels of e+ involved. these indicators are also light - sensitive and prone to auto - oxidation [35, 43 ], further constraining and complicating design and data interpretation in time - lapse experiments. additionally, it has been shown that mitosox at high concentration significantly impairs mitochondrial function. particularly, dihydrorhodamine 123 (dhr123) is a nonfluorescent agent that scavenges the oh generated from h2o2 in an iron - dependent fenton reaction and is thereby converted into the fluorescent rhodamine 123. dhr123 reacts also with no2 and hypochlorous acid but is unreactive to o2 or h2o2 in the absence of catalyst [34, 5254 ]. a family of boronate - based indicators (e.g., peroxysensor family) has also been introduced for targeting to the cytosol or the mitochondria [5557 ]. boronate masks the fluorophore ; but, upon exposure to h2o2, it undergoes a nucleophilic attack and its removal unmasks the fluorescence emission. however, the boronate - based indicators are promiscuous as they also react stoichiometrically with onoo, yielding phenols and permitting light emission [58, 59 ]. hkgreen-3, a rhodol - based fluorescent probe, is recently developed by peng. and shows high sensitivity and selectivity for peroxynitrite in both chemical and biological systems. hkocl-1 is a bodipy - based fluorescent probe for detecting hypochlorous acid with high specificity. 4-amino-5-methylamino-2,7-difluorofluorescein (daf - fm) is of popularity for measuring no due to its high sensitivity, ph stability, and relative resistance to photobleaching. since the detection relies on conversion of the parent compound into a fluorescent triazole, the presence of oxidants / antioxidants and reaction with other molecules would affect this fluorescence detection. amplex ultrared is a fluorogenic substrate for horseradish peroxidase that reacts with h2o2 in a 1:1 stoichiometric ratio to produce the fluorescent product resorufin with long - wavelength spectra (excitation / emission maxima, 563/587 nm). in a recent study, amplex ultrared and daf - fm have been successfully used for in vivo measurement of extracellularly released h2o2 and no of superficial lumbar spinal cord of anesthetized mice, respectively. the caveat from above considerations is that ros measurement with small - molecule fluorescent probes is not as straightforward as it seems to be. proper experimental design, careful choice of loading and light illumination parameters, stringent control of experimental conditions, and judicious interpretation of experimental data should all be exercised. evidently, developing small - molecule fluorescent ros probes suitable for faithful measurement of ros dynamics remains a huge challenge to the ros research field. over the last decade, fluorescent protein - based ros indicators have entered the arsenal for ros measurement. while protein chemistry introduces a higher level of complexity as compared to the small - molecule chemistry, it at the same time offers tremendous opportunities for rational design (e.g., redox oxidation - sensitive green fluorescent proteins (rogfps) for redox potential measurement and hyper for h2o2 measurement) [6466 ] as well as serendipitous discoveries (e.g., mt - circularly permutated yellow fluorescent protein (cpyfp) for mitochondrial superoxide flashes). while selectivity is generally improved, reversibility another distinct advantage is that these genetically encoded indicators can be specifically targeted to different type of cells using cell type - specific promoters or to different cellular compartments or microdomains by n- or c - terminal fusion with a specific targeting sequence [68, 69 ]. ros indicator - expressing transgenic animals have been generated [67, 7073 ], allowing for imaging ros ex vivo and in vivo. however, the fluorescent protein indicators have also their own set of limitations. particularly, ph sensitivity is common to most of currently used protein - based ros indicators, due to reversible fluorescence - quenching protonation of the chromophore at pka close to physiological ph. as such, ph changes in the cytosol or other specific compartments, if not judiciously controlled, could lead to erroneous observation and misinterpretation. among all ros sources, the etc consists of intermolecular and intramolecular pathways of increasing redox potential (eh), from 320 mv at the entry point of complex i to + 390 mv at the terminal point of complex iv. however, with its eh = 160 mv, o2 also snaps up 0.152 % of the respiratory chain electrons, one at a time, at places prior to complex iv ; and one - electron reduction of o2 forms o2 [1, 2 ], the primal ros. this constitutive mode of mitochondrial ros production plays an important role in setting the ros and redox homeostasis of the cell. while studying mitochondrial ca signaling with a genetically encoded mitochondrial - targeted ca indicator, pericam, we serendipitously found that the fluorescent moiety of pericam, cpyfp, can reversibly detect superoxide with high selectivity and sensitivity. extensive in vitro characterization indicates that, when excited at 488 nm, cpyfp emission displays a several fold increase in response to superoxide generated by the xanthine xanthine oxidase o2 system, but is insensitive to many other oxidants and metabolites, including h2o2, peroxynitrite, ca, atp, adp, nad(p), and nad(p)h. it is also insensitive to eh varying between 319 and 7.5 mv (controlled by mix of oxidized and reduced dtt in different proportions), while displays a ph sensitivity with a pka 8.5. the reversibility of cpyfp has been evidenced by the fact that sod added after superoxide formation reverses the cpyfp signal in vitro. in addition, mitochondrial - targeted cpyfp (mt - cpyfp) acts as a ratiometric indicator because its signal at 405 nm excitation is essentially ros - independent, allowing for the use of the f488/f405 ratio as the readout. using mt - cpyfp superoxide flashes are sudden, brief, and bursting superoxide - producing events in single mitochondria. in intact cells, spontaneous flashes occur at a low rate in a stochastic manner, but their frequency can be regulated over a broad dynamic range. to date, superoxide flashes are universally found in all cell types examined, including cardiomyocytes, skeletal muscle cells, neurons, glials, fibroblasts, and several types of cancer cells [23, 67, 70, 7582 ]. they are also highly conserved in species ranging from mammals (mouse, rat, and human) and to caenorhabditis elegans (unpublished data). ex vivo and in vivo imaging in transgenic mouse models with cardiac - specific or pan - tissue mt - cpyfp expression have allowed for detection of superoxide flashes in beating hearts under langendorff perfusion and in gastrocnemius muscle and sciatic nerve of living mice under anesthesia (fig. 1) [70, 71 ]. in addition, superoxide flashes of similar characteristics are active in freshly isolated, respiratory mitochondria, indicating that single mitochondria contain the full machinery for the genesis of superoxide flashes. notably, the rate of flash occurrence varies depending on species, tissue and cell types, metabolic states, the presence of stressors, and disease conditions ; the amplitude and duration of the flashes, however, appear to be stereotypical at multiple levels, from isolated mitochondria, to intact or plasma membrane - permeabilized cells, to whole tissues or organs, and to living animals.fig. 1 in vivo detection of mitochondrial superoxide flashes in skeletal muscle. an upright confocal microscope was used to image the hindlimb skeletal muscle in mt - cpyfp transgenic mouse under anesthesia. a superoxide flash in mouse gastrocnemius. the striated pattern reflects that double - row arrays of mitochondria locate at z line regions of sarcomeres. bottom panel enlarged views of this punctiform superoxide flash in the boxed region at 3-s intervals, with dual wavelength excitation at 488 and 405 nm. b time course of the superoxide flash in a. modified from fang. in vivo detection of mitochondrial superoxide flashes in skeletal muscle. an upright confocal microscope was used to image the hindlimb skeletal muscle in mt - cpyfp transgenic mouse under anesthesia. a superoxide flash in mouse gastrocnemius. the striated pattern reflects that double - row arrays of mitochondria locate at z line regions of sarcomeres. bottom panel enlarged views of this punctiform superoxide flash in the boxed region at 3-s intervals, with dual wavelength excitation at 488 and 405 nm. b time course of the superoxide flash in a. modified from fang. sweetlove. reported similar phenomenon in arabidopsis mitochondria, but interpreted its nature as transient alkalization of the mitochondrial matrix [84, 85 ]. recently, we and others have systematically examined the respective contributions of ros and ph to a flash and concluded that ros burst is the dominant signal while ph change, if any, has only a minor contribution. in experiments combining the protein and small - molecule ros indicators, it has been shown that mitosox, which is ph insensitive, reports a concomitant stepwise increase during a mt - cpyfp flash [78, 83, 86 ]. furthermore, owing to its reversibility, mt - cpyfp is able to track the time course of flashes with the briefest duration of only 1 to 2 s. differing from constitutive mitochondrial ros production, the genesis of superoxide flashes involve different but overlapping molecular mechanisms. the flash ignition is tightly coupled to transient opening of mitochondrial permeability transition pore (mptp), evidenced by sudden dissipation of mitochondrial membrane potential and partial and irreversible loss of fluorescent solutes (mw, 752980 da) preloaded to the matrix [67, 75 ]. the involvement of mptp is also supported by the fact that the flash production is partially sensitive to pharmacological inhibition or molecular knockdown of cyclophilin d, consistent with a regulatory, but dispensable, role of this protein in mptp activity [8790 ]. the flash production is abolished by disruption of the etc at any possible site, complex i through v, in a manner distinctly different from etc regulation of constitutive ros production. for instance, antimycin a, an inhibitor - targeting complex iii abolishes the bursting quantal ros production in the form of the flashes while stimulating continuous ros production [1, 91, 92 ]. that inhibition of complex v also suppresses the flash production is consistent with an intimate relationship recently suggested for the atp synthase and mptp [93, 94 ]. by in situ, ex vivo, and in vivo ros imaging, we and others have shown that superoxide flashes represent not only a digital readout of mitochondrial metabolic status but also a novel biomarker of mitochondrial stress. the rate of skeletal muscle superoxide flashes in live anesthetized mice increases after intraperitoneal injection of glucose or insulin, indicating that superoxide flashes are coupled to whole - body dietary glucose metabolism. in isolated skeletal muscle cells with electroporation - mediated transient mt - cpyfp expression, superoxide flashes occur at a markedly elevated frequency and display a similar though less profound response to glucose plus pyruvate stimulation, in the absence of insulin and other whole - body factors. a further elevated superoxide flash activity is observed in skeletal muscle of ryr1 malignant hyperthermia mice which exhibit marked temperature - dependent increases in ros and rns generation. thus, imaging superoxide flashes in vivo exemplifies how both the integrative whole - body metabolic response and the mechanistic single - mitochondrion behavior can be investigated in one single experiment. investigation of superoxide flash production in mice deficient of sod2 has revealed that superoxide flashes negatively regulate neural progenitor proliferation and cerebral cortical development through modulating activation of erk. remarkably, a reversible 20-fold increase of superoxide flashes occurs in response to hyperosmotic stress, due to the synergistic effect mitochondrial ca uniport, and basal ros elevation. the high activity of superoxide flashes, in turn, contributes to activating jnk and p38, essential signals for adaptive cell survival responses. in cultured cardiomyocytes, a flurry of superoxide flash activity occurs in a 510 min window after reoxygenation from hypoxia or anoxia [67, 95 ]. in the pathology of huntington disease, the elevated flash activity induced by elevated mitochondrial ca signaling acts to exacerbate mtdna damage. likewise, superoxide flashes act as early mitochondrial signals mediating the apoptotic response during oxidative stress in hela cells. collectively, these recent advances indicate that superoxide flashes offer a rare window through which we can glimpse into the whole - body metabolic response at the single - mitochondrion level, and gauge a wide variety of stresses converging to the mitochondria. to our knowledge, many types of cpyfp transgenic organisms, from mice, zebrafish, c. elegans, drosophila melanogaster, and yeast, have been generated or are currently being created to address multidisciplinary questions in broad settings. we are eager to see what these new models and approaches can teach us about ros signaling in biology and diseases. hyper is a ratiometric fluorescent indicator of h2o2 in which cpyfp is inserted into the regulatory domain of an escherichia coli peroxide sensor oxyr. naturally used by the bacterium to trigger transcriptional response to oxidative stress, oxyr contains an h2o2-sensitive regulatory domain and a dna - binding domain, and, upon oxidation by h2o2, intramolecular disulfide bond forms between two cysteine residues (cys199 and cys 208) and the resultant conformational change shifts the excitation maximum of the attached cpyfp from 420 to 500 nm (emission maximum at 516 nm). hyper is able to detect nanomolar h2o2 in vitro and, when expressed in cells, responds to micromolar h2o2 added externally or changes of intracellular h2o2 upon growth factor stimulation [66, 96 ]. in an elegant study, niethammer. have exploited hyper expressed in transgenic zebrafish larvae to visualize a regional, graded, and transient h2o2 signal produced by dual oxidase (duox) in response to tail fin injury. functionally, this duox - elicited h2o2 signal is required for rapid recruitment of leukocytes in the process of wound healing. a series of hyper mutants have been developed in order to improve its dynamic range and reaction kinetics for h2o2 detection. in particular, hyper-2, an a406v single - point mutant of hyper, exhibits twice - expanded dynamic range, but the response to h2o2 is much slower, doubling both half - oxidation and half - reduction from 6 and 200 s for hyper to 13 and 400 s for hyper-2, respectively. recently, a h34y mutant of hyper, hyper-3, was developed, which shows expanded dynamic range compared to hyper and faster oxidation - reduction kinetics compared to hyper-2. notably, in hyper-3 transgenic zebrafish, similar h2o2 gradients along the fish tail regions were observed upon wounding ; however, hyper-3 showed a higher fluorescence ratio (f500/f420) than what reported by hyper demonstrating its advantage for h2o2 detection. both hyper and hyper-3 are applicable for fluorescence lifetime imaging microscopy (flim). instead of measuring the fluorescence intensity, the physical parameter measured in flim is the time constant () of the excited fluorophore returning to its basal states and, in this case, the change in is quantified to reflect the redox state of the ros indicator and hence the ros level. because is independent of indicator concentration, flim measurement is essentially insensitive to indicator expression level, non - uniform distribution, and partial photobleaching. it is also advantageous for quantifying signals at different depths in a biological tissue because is less interfered by light scattering and reabsorption (inner filtering). moreover, flim generates absolute quantitative readouts while requiring only a single - wavelength excitation, provided that the indicator is calibrated in situ (e.g., in permeablized cells) or in vitro under conditions closely resembling intracellular environments. as is the case for mt - cpyfp, hyper and its mutants are ph sensitive : a shift of 0.2 ph units is sufficient to change the f500/f420 ratio as much as those corresponding to a full reduction or oxidation. thus, monitoring ph changes should be included as an essential control in hyper measurement. although in vitro data have shown that hyper is insensitive to other oxidants including o2, gssg, nitric oxide, and onoo, events similar to mt - cpyfp flashes were detected with mitochondrial targeted hyper, accompanying a simultaneous stepwise increase of mitosox fluorescence. sypher, a ph indicator and insensitive to h2o2 (by a disruption of the h2o2-sensing cysteine pair (c199s), of hyper), detects similar mitochondrial flash events [66, 86, 98 ]. thus, since hyper and sypher comprise a cpyfp as the fluorophore, it is possible that the cpyfp part in these two indicators reports mitochondrial superoxide flashes, as do mt - cpyfp and pericam [67, 78 ]. as an example of rational design, rogfp1 have been generated by substituting two surface exposed amino acids in gfp with cysteines (s147c and q204c). the introduced cys 147 and 204 are situated next to each other on two adjacent -strands and form disulfide bonds due to significant conformational changes of rogfp1 upon oxidation [64, 65 ]. these cysteines are located near the chromophore of gfp and the formation of the disulfide bond leads to a simultaneous shift of the absorption properties. the rogfps have two fluorescence excitation maxima at about 400 and 490 nm, corresponding to the neutral fluorophore and anionic form of the flurophore, respectively [64, 65 ]. the disulfide formation promotes protonation of the chromophore and increases the excitation peak near 400 nm at the expense of the peak near 490 nm. therefore, they serve as dual - excitation ratiometric indicators for eh measurement in vitro and in vivo [64, 65 ]. the first generation of rogfps with different mutation sites, including rogfp1 - 6, all have midpoint eh of 272 mv or below, which made them most useful in reducing compartments, such as the cytosol and the mitochondrial matrix. in particular, rogfp1and rogfp2 expressed in cytoplasm report a basal eh of 315 to 325 mv and both respond to a variety of oxidant stimuli. interestingly, the mitochondrial matrix of hela cell is highly reducing with a midpoint eh near 360 mv as reported by rogfp1 ; membrane - permeable reductants and oxidants reversibly change the eh in the matrix of mitochondria. recently, transgenic animals of rogfps have been developed and provided very useful tools for investigating the physiology and pathology of ros signaling [73, 99 ]. a combined approach using transgenic mice with mitochondrial - targeted rogfp and two - photon laser scanning microscopic imaging in brain slices have shown that normal autonomous pace - making produced oxidative stress specific to dopaminergic neurons in substantia nigra pars compacta that are usually vulnerable. in mitochondrial - targeted rogfp2 transgenic drosophilae, it has been demonstrated that elevated ros contribute to pathogenesis in a neurodegenerative mutant atpalpha and in a model of mitochondrial encephalomyopathy. it should be cautioned that it takes minutes or longer for current rogfps to equilibrate with the environmental redox potential changes and their reversibility is too slow to detect transient ros events. indeed, rogfp2 expressed in either cytoplasm or the plasma membrane showed no response to stimulation with epidermal growth factor or lysophosphatidic acid, which induces h2o2 production that can be detected by dcfh [65, 100, 101 ]. by measuring the steady - state redox levels in different cellular compartments, future investigations may exploit rogfps to complement the measurements using selective, fast responding, and reversible ros indicators. importantly, to meet the needs of measuring eh in severe oxidative stresses, it would also be desirable to obtain a collection of redox indicators with different midpoint potentials. fluorescence resonance energy transfer (fret)-based ros indicators, which consist of cyan and yellow fluorescent proteins (cfp / yfp) linked by redox sensitive polypeptides, have also been developed. the fret - based ros indicators sense the redox state via their internal disulfide bonds, resulting in a conformation change of the protein leading to a fret response. in this regard, kolossov. developed a series of fret ros indicators with different redox sensitive linkers, which consist of -helical structures in conjunction with redox - sensitive motifs, between cfp and yfp and found that rl5 exhibited a 29 % increase of fret efficiency from its reduced to oxidized states. have developed a fret ros sensor with the 69 amino acid cysteine - containing regulatory domain from redox - regulated heat - shock protein hsp-33 as the linker of cfp and yfp. with this fret indicator, they found that hypoxia - induced ros production requires a functional mitochondrial etc and that is essential for hypoxia - induced hif-1 stabilization. a third type of fret indicators, redoxfluor, comprises a tandem repeat of partial sequence of carboxy - terminal cysteine - rich domain of yap1, a yeast transcriptional factor sensing the intracellular redox state. by expressing redoxfluor to the peroxisome of yeast and chinese hamster ovary cells, yano. have demonstrated that the redox state within the peroxisomes is more reductive than that in the cytosol in wild - type cells, despite the fact that ros are generated within the peroxisomes, and the cytosolic redox state of the of cell mutants for peroxisome assembly is more reductive than that of the wild - type cells. because fret based - ros measurement builds on disulfide bond formation of the redox - sensitive linkers, its specificity and kinetics are subjected to the same constraints for other disulfide bond - based ros indicators discussed above. among all ros sources, mitochondrial etc is the major site of cellular ros production. the etc consists of intermolecular and intramolecular pathways of increasing redox potential (eh), from 320 mv at the entry point of complex i to + 390 mv at the terminal point of complex iv. however, with its eh = 160 mv, o2 also snaps up 0.152 % of the respiratory chain electrons, one at a time, at places prior to complex iv ; and one - electron reduction of o2 forms o2 [1, 2 ], the primal ros. this constitutive mode of mitochondrial ros production plays an important role in setting the ros and redox homeostasis of the cell. while studying mitochondrial ca signaling with a genetically encoded mitochondrial - targeted ca indicator, pericam, we serendipitously found that the fluorescent moiety of pericam, cpyfp, can reversibly detect superoxide with high selectivity and sensitivity. extensive in vitro characterization indicates that, when excited at 488 nm, cpyfp emission displays a several fold increase in response to superoxide generated by the xanthine xanthine oxidase o2 system, but is insensitive to many other oxidants and metabolites, including h2o2, peroxynitrite, ca, atp, adp, nad(p), and nad(p)h. it is also insensitive to eh varying between 319 and 7.5 mv (controlled by mix of oxidized and reduced dtt in different proportions), while displays a ph sensitivity with a pka 8.5. the reversibility of cpyfp has been evidenced by the fact that sod added after superoxide formation reverses the cpyfp signal in vitro. in addition, mitochondrial - targeted cpyfp (mt - cpyfp) acts as a ratiometric indicator because its signal at 405 nm excitation is essentially ros - independent, allowing for the use of the f488/f405 ratio as the readout. using mt - cpyfp superoxide flashes are sudden, brief, and bursting superoxide - producing events in single mitochondria. in intact cells, spontaneous flashes occur at a low rate in a stochastic manner, but their frequency can be regulated over a broad dynamic range. to date, superoxide flashes are universally found in all cell types examined, including cardiomyocytes, skeletal muscle cells, neurons, glials, fibroblasts, and several types of cancer cells [23, 67, 70, 7582 ]. they are also highly conserved in species ranging from mammals (mouse, rat, and human) and to caenorhabditis elegans (unpublished data). ex vivo and in vivo imaging in transgenic mouse models with cardiac - specific or pan - tissue mt - cpyfp expression have allowed for detection of superoxide flashes in beating hearts under langendorff perfusion and in gastrocnemius muscle and sciatic nerve of living mice under anesthesia (fig. 1) [70, 71 ]. in addition, superoxide flashes of similar characteristics are active in freshly isolated, respiratory mitochondria, indicating that single mitochondria contain the full machinery for the genesis of superoxide flashes. notably, the rate of flash occurrence varies depending on species, tissue and cell types, metabolic states, the presence of stressors, and disease conditions ; the amplitude and duration of the flashes, however, appear to be stereotypical at multiple levels, from isolated mitochondria, to intact or plasma membrane - permeabilized cells, to whole tissues or organs, and to living animals.fig. 1 in vivo detection of mitochondrial superoxide flashes in skeletal muscle. an upright confocal microscope was used to image the hindlimb skeletal muscle in mt - cpyfp transgenic mouse under anesthesia. a superoxide flash in mouse gastrocnemius. the striated pattern reflects that double - row arrays of mitochondria locate at z line regions of sarcomeres. bottom panel enlarged views of this punctiform superoxide flash in the boxed region at 3-s intervals, with dual wavelength excitation at 488 and 405 nm. b time course of the superoxide flash in a. modified from fang. in vivo detection of mitochondrial superoxide flashes in skeletal muscle. an upright confocal microscope was used to image the hindlimb skeletal muscle in mt - cpyfp transgenic mouse under anesthesia. a superoxide flash in mouse gastrocnemius. the striated pattern reflects that double - row arrays of mitochondria locate at z line regions of sarcomeres. bottom panel enlarged views of this punctiform superoxide flash in the boxed region at 3-s intervals, with dual wavelength excitation at 488 and 405 nm. b time course of the superoxide flash in a. modified from fang. sweetlove. reported similar phenomenon in arabidopsis mitochondria, but interpreted its nature as transient alkalization of the mitochondrial matrix [84, 85 ]. recently, we and others have systematically examined the respective contributions of ros and ph to a flash and concluded that ros burst is the dominant signal while ph change, if any, has only a minor contribution. in experiments combining the protein and small - molecule ros indicators, it has been shown that mitosox, which is ph insensitive, reports a concomitant stepwise increase during a mt - cpyfp flash [78, 83, 86 ]. furthermore, owing to its reversibility, mt - cpyfp is able to track the time course of flashes with the briefest duration of only 1 to 2 s. differing from constitutive mitochondrial ros production, the genesis of superoxide flashes involve different but overlapping molecular mechanisms. the flash ignition is tightly coupled to transient opening of mitochondrial permeability transition pore (mptp), evidenced by sudden dissipation of mitochondrial membrane potential and partial and irreversible loss of fluorescent solutes (mw, 752980 da) preloaded to the matrix [67, 75 ]. the involvement of mptp is also supported by the fact that the flash production is partially sensitive to pharmacological inhibition or molecular knockdown of cyclophilin d, consistent with a regulatory, but dispensable, role of this protein in mptp activity [8790 ]. the flash production is abolished by disruption of the etc at any possible site, complex i through v, in a manner distinctly different from etc regulation of constitutive ros production. for instance, antimycin a, an inhibitor - targeting complex iii abolishes the bursting quantal ros production in the form of the flashes while stimulating continuous ros production [1, 91, 92 ]. that inhibition of complex v also suppresses the flash production is consistent with an intimate relationship recently suggested for the atp synthase and mptp [93, 94 ]. by in situ, ex vivo, and in vivo ros imaging, we and others have shown that superoxide flashes represent not only a digital readout of mitochondrial metabolic status but also a novel biomarker of mitochondrial stress. the rate of skeletal muscle superoxide flashes in live anesthetized mice increases after intraperitoneal injection of glucose or insulin, indicating that superoxide flashes are coupled to whole - body dietary glucose metabolism. in isolated skeletal muscle cells with electroporation - mediated transient mt - cpyfp expression, superoxide flashes occur at a markedly elevated frequency and display a similar though less profound response to glucose plus pyruvate stimulation, in the absence of insulin and other whole - body factors. a further elevated superoxide flash activity is observed in skeletal muscle of ryr1 malignant hyperthermia mice which exhibit marked temperature - dependent increases in ros and rns generation. thus, imaging superoxide flashes in vivo exemplifies how both the integrative whole - body metabolic response and the mechanistic single - mitochondrion behavior can be investigated in one single experiment. investigation of superoxide flash production in mice deficient of sod2 has revealed that superoxide flashes negatively regulate neural progenitor proliferation and cerebral cortical development through modulating activation of erk. remarkably, a reversible 20-fold increase of superoxide flashes occurs in response to hyperosmotic stress, due to the synergistic effect mitochondrial ca uniport, and basal ros elevation. the high activity of superoxide flashes, in turn, contributes to activating jnk and p38, essential signals for adaptive cell survival responses. in cultured cardiomyocytes, a flurry of superoxide flash activity occurs in a 510 min window after reoxygenation from hypoxia or anoxia [67, 95 ]. in the pathology of huntington disease, the elevated flash activity induced by elevated mitochondrial ca signaling acts to exacerbate mtdna damage. likewise, superoxide flashes act as early mitochondrial signals mediating the apoptotic response during oxidative stress in hela cells. collectively, these recent advances indicate that superoxide flashes offer a rare window through which we can glimpse into the whole - body metabolic response at the single - mitochondrion level, and gauge a wide variety of stresses converging to the mitochondria. to our knowledge, many types of cpyfp transgenic organisms, from mice, zebrafish, c. elegans, drosophila melanogaster, and yeast, have been generated or are currently being created to address multidisciplinary questions in broad settings. we are eager to see what these new models and approaches can teach us about ros signaling in biology and diseases. hyper is a ratiometric fluorescent indicator of h2o2 in which cpyfp is inserted into the regulatory domain of an escherichia coli peroxide sensor oxyr. naturally used by the bacterium to trigger transcriptional response to oxidative stress, oxyr contains an h2o2-sensitive regulatory domain and a dna - binding domain, and, upon oxidation by h2o2, intramolecular disulfide bond forms between two cysteine residues (cys199 and cys 208) and the resultant conformational change shifts the excitation maximum of the attached cpyfp from 420 to 500 nm (emission maximum at 516 nm). hyper is able to detect nanomolar h2o2 in vitro and, when expressed in cells, responds to micromolar h2o2 added externally or changes of intracellular h2o2 upon growth factor stimulation [66, 96 ]. in an elegant study, niethammer. have exploited hyper expressed in transgenic zebrafish larvae to visualize a regional, graded, and transient h2o2 signal produced by dual oxidase (duox) in response to tail fin injury. functionally, this duox - elicited h2o2 signal is required for rapid recruitment of leukocytes in the process of wound healing. a series of hyper mutants have been developed in order to improve its dynamic range and reaction kinetics for h2o2 detection. in particular, hyper-2, an a406v single - point mutant of hyper, exhibits twice - expanded dynamic range, but the response to h2o2 is much slower, doubling both half - oxidation and half - reduction from 6 and 200 s for hyper to 13 and 400 s for hyper-2, respectively. recently, a h34y mutant of hyper, hyper-3, was developed, which shows expanded dynamic range compared to hyper and faster oxidation - reduction kinetics compared to hyper-2. notably, in hyper-3 transgenic zebrafish, similar h2o2 gradients along the fish tail regions were observed upon wounding ; however, hyper-3 showed a higher fluorescence ratio (f500/f420) than what reported by hyper demonstrating its advantage for h2o2 detection. both hyper and hyper-3 are applicable for fluorescence lifetime imaging microscopy (flim). instead of measuring the fluorescence intensity, the physical parameter measured in flim is the time constant () of the excited fluorophore returning to its basal states and, in this case, the change in is quantified to reflect the redox state of the ros indicator and hence the ros level. because is independent of indicator concentration, flim measurement is essentially insensitive to indicator expression level, non - uniform distribution, and partial photobleaching. it is also advantageous for quantifying signals at different depths in a biological tissue because is less interfered by light scattering and reabsorption (inner filtering). moreover, flim generates absolute quantitative readouts while requiring only a single - wavelength excitation, provided that the indicator is calibrated in situ (e.g., in permeablized cells) or in vitro under conditions closely resembling intracellular environments. as is the case for mt - cpyfp, hyper and its mutants are ph sensitive : a shift of 0.2 ph units is sufficient to change the f500/f420 ratio as much as those corresponding to a full reduction or oxidation. thus, monitoring ph changes should be included as an essential control in hyper measurement. although in vitro data have shown that hyper is insensitive to other oxidants including o2, gssg, nitric oxide, and onoo, events similar to mt - cpyfp flashes were detected with mitochondrial targeted hyper, accompanying a simultaneous stepwise increase of mitosox fluorescence. sypher, a ph indicator and insensitive to h2o2 (by a disruption of the h2o2-sensing cysteine pair (c199s), of hyper), detects similar mitochondrial flash events [66, 86, 98 ]. thus, since hyper and sypher comprise a cpyfp as the fluorophore, it is possible that the cpyfp part in these two indicators reports mitochondrial superoxide flashes, as do mt - cpyfp and pericam [67, 78 ]. as an example of rational design, rogfp1 have been generated by substituting two surface exposed amino acids in gfp with cysteines (s147c and q204c). the introduced cys 147 and 204 are situated next to each other on two adjacent -strands and form disulfide bonds due to significant conformational changes of rogfp1 upon oxidation [64, 65 ]. these cysteines are located near the chromophore of gfp and the formation of the disulfide bond leads to a simultaneous shift of the absorption properties. the rogfps have two fluorescence excitation maxima at about 400 and 490 nm, corresponding to the neutral fluorophore and anionic form of the flurophore, respectively [64, 65 ]. the disulfide formation promotes protonation of the chromophore and increases the excitation peak near 400 nm at the expense of the peak near 490 nm. therefore, they serve as dual - excitation ratiometric indicators for eh measurement in vitro and in vivo [64, 65 ]. the first generation of rogfps with different mutation sites, including rogfp1 - 6, all have midpoint eh of 272 mv or below, which made them most useful in reducing compartments, such as the cytosol and the mitochondrial matrix. in particular, rogfp1and rogfp2 expressed in cytoplasm report a basal eh of 315 to 325 mv and both respond to a variety of oxidant stimuli. interestingly, the mitochondrial matrix of hela cell is highly reducing with a midpoint eh near 360 mv as reported by rogfp1 ; membrane - permeable reductants and oxidants reversibly change the eh in the matrix of mitochondria. recently, transgenic animals of rogfps have been developed and provided very useful tools for investigating the physiology and pathology of ros signaling [73, 99 ]. a combined approach using transgenic mice with mitochondrial - targeted rogfp and two - photon laser scanning microscopic imaging in brain slices have shown that normal autonomous pace - making produced oxidative stress specific to dopaminergic neurons in substantia nigra pars compacta that are usually vulnerable. in mitochondrial - targeted rogfp2 transgenic drosophilae, it has been demonstrated that elevated ros contribute to pathogenesis in a neurodegenerative mutant atpalpha and in a model of mitochondrial encephalomyopathy. it should be cautioned that it takes minutes or longer for current rogfps to equilibrate with the environmental redox potential changes and their reversibility is too slow to detect transient ros events. indeed, rogfp2 expressed in either cytoplasm or the plasma membrane showed no response to stimulation with epidermal growth factor or lysophosphatidic acid, which induces h2o2 production that can be detected by dcfh [65, 100, 101 ]. by measuring the steady - state redox levels in different cellular compartments, future investigations may exploit rogfps to complement the measurements using selective, fast responding, and reversible ros indicators. importantly, to meet the needs of measuring eh in severe oxidative stresses, it would also be desirable to obtain a collection of redox indicators with different midpoint potentials. fluorescence resonance energy transfer (fret)-based ros indicators, which consist of cyan and yellow fluorescent proteins (cfp / yfp) linked by redox sensitive polypeptides, have also been developed. the fret - based ros indicators sense the redox state via their internal disulfide bonds, resulting in a conformation change of the protein leading to a fret response. in this regard, kolossov. developed a series of fret ros indicators with different redox sensitive linkers, which consist of -helical structures in conjunction with redox - sensitive motifs, between cfp and yfp and found that rl5 exhibited a 29 % increase of fret efficiency from its reduced to oxidized states. have developed a fret ros sensor with the 69 amino acid cysteine - containing regulatory domain from redox - regulated heat - shock protein hsp-33 as the linker of cfp and yfp. with this fret indicator, they found that hypoxia - induced ros production requires a functional mitochondrial etc and that is essential for hypoxia - induced hif-1 stabilization. a third type of fret indicators, redoxfluor, comprises a tandem repeat of partial sequence of carboxy - terminal cysteine - rich domain of yap1, a yeast transcriptional factor sensing the intracellular redox state. by expressing redoxfluor to the peroxisome of yeast and chinese hamster ovary cells, yano. have demonstrated that the redox state within the peroxisomes is more reductive than that in the cytosol in wild - type cells, despite the fact that ros are generated within the peroxisomes, and the cytosolic redox state of the of cell mutants for peroxisome assembly is more reductive than that of the wild - type cells. because fret based - ros measurement builds on disulfide bond formation of the redox - sensitive linkers, its specificity and kinetics are subjected to the same constraints for other disulfide bond - based ros indicators discussed above. the past decade has witnessed significant progress in understanding physiological and pathological functions of ros, marked by the emergence of a revolutionary concept that ros acts as cellular messengers that permeate pivotal pathways in the network of intracellular signal transduction. this ongoing revolution has been catalyzed, to a significant extent, by the advent of new small - molecule and fluorescent protein - based ros indicators and novel imaging methods, both conferring the ability to visualize and quantify ros in organelle, intact cells, whole tissues and organs, and even live animals (fig. 2). we begin to appreciate that, analogous to ca signaling, spatiotemporal ros dynamics exhibit an exquisite hierarchical architecture in intact cells and organisms, from superoxide flashes as elemental mitochondrial ros signaling events to cell - wide ros oscillations [17, 106, 107 ] supported by the ros - induced ros release mechanism [39, 108 ] and to tissue - level ros gradients for wound healing.fig. 2imaging ros dynamics in vitro and in vivo. by combining transgenic animal models with confocal and multiphoton microscopy, images of high spatiotemporal resolution can now be acquired from isolated mitochondria, cultured cells, intact tissues or organs, and even in living animals imaging ros dynamics in vitro and in vivo. by combining transgenic animal models with confocal and multiphoton microscopy, images of high spatiotemporal resolution can now be acquired from isolated mitochondria, cultured cells, intact tissues or organs, and even in living animals imaging ros in situ and in vivo, with high selectivity, quantitative ability, and spatiotemporal resolution will continue to be our most delicate investigative tool for the analysis of the tremendous complexity and subtlety of ros signaling. to further sharpen the tool, it calls for continued efforts in designing small - molecule fluorescent ros probes with improved selectivity, reversible kinetics and compartment - targeting property. meanwhile, biologically inspired novel ros indicators could be developed as we identify more ros target proteins and understand better the mechanisms hereby they achieve signaling specificity, sensitivity and reversibility at once. combined, the promise is that, by searching the enormous chemical space of small molecules and of proteins, we would greatly extend the current repertoire of ros indicators and ultimately achieve the same level of reliability as we have enjoyed while measuring intracellular ca with small - molecule probes such as fluo-3, fura-2, indo-1, and fluorescent protein probes such as gcamp6 and gecos, the palette with blue, improved green, and or red - shifted indicators. in synergy with the exponentially increasing numbers of indicator - expressing organisms and disease models, the booming technology for super - resolution and single - molecule imaging [113115 ], and the trend for using miniature, plant - in devices to obtain images in conscious, free - moving animals [116120 ], imaging ros in vivo will serve as the most powerful force to transform the landscape and push forward the frontiers in ros signaling. | reactive oxygen species (ros) act as essential cellular messengers, redox regulators, and, when in excess, oxidative stressors that are widely implicated in pathologies of cancer and cardiovascular and neurodegenerative diseases. understanding such complexity of the ros signaling is critically hinged on the ability to visualize and quantify local, compartmental, and global ros dynamics at high selectivity, sensitivity, and spatiotemporal resolution. the past decade has witnessed significant progress in ros imaging at levels of intact cells, whole organs or tissues, and even live organisms. in particular, major advances include the development of novel synthetic or genetically encoded fluorescent protein - based ros indicators, the use of protein indicator - expressing animal models, and the advent of in vivo imaging technology. innovative ros imaging has led to important discoveries in ros signaling for example, mitochondrial superoxide flashes as elemental ros signaling events and hydrogen peroxide transients for wound healing. this review aims at providing an update of the current status in ros imaging, while identifying areas of insufficient knowledge and highlighting emerging research directions. |
in modern society, the incidence of cervical lordosis and thoracic and lumbar kyphosis is increasing, because many people maintain a fixed posture for long periods and number of aged people is increasing. an increase in thoracic kyphosis causes restrictions in chest expansion and respiratory muscle weakness, thereby reducing lung capacity and the thoracic cavity size and deforming vertebral column alignment1,2,3. previous studies have investigated the use of thoracic joint mobilization and thoracic flexibility exercises to improve deformations of the chest and vertebrae and thereby enhance pulmonary function1, 4. these interventions enhanced pulmonary function and thoracic mobility and improved respiratory muscle function, chest expansion, and diaphragm movement by reducing the stiffness of the inter - vertebral discs and surrounding tissues and by improving vertebral extensor muscle stretch and endurance with thoracic flexibility exercises1, 4. as reported in previous studies, chest mobilization exercises and stretching exercises increase thoracic vertebral mobility, chest expansion, and lung capacity. however, these exercises required firsthand therapeutic application by therapists and can not be easily performed by the subjects in a non - clinical environment5. therefore, the present study examined self - mobilization of thoracic vertebrae in healthy adults and investigated the effect of these exercises on pulmonary function and chest expansion. the subjects of this study were 19 healthy adults who understood the purpose of the study and provided written consent to participate. those diagnosed with neurological findings and had undergone operations, or were receiving surgical treatment, or taking medicines on a regular basis to relieve pain, were excluded from the study. the study was approved by the ethics committee of the catholic university of pusan and adhered to the tenets of the declaration of helsinki (1975, revised 1983). the subjects were assigned to one of two groups, the thoracic region self - mobilization group (tsmg ; 2 males and 6 females) or the control group (cg ; 5 males and 6 females). none of the subjects showed restrictive pulmonary disease or obstructive pulmonary disease, as determined by a spirometer pretest. age, height, and weight were 22.50 1.06 years, 164.25 10.60 cm, and 60.12 13.35 kg, respectively in the tsmg, and were 22.36 3.26 years, 165.50 7.37 cm, and 65.09 14.90 kg, respectively in the cg. to measure chest expansion when breathing, the subject s chest wall was measured with a tapeline in an upright sitting position. chest expansion was calculated using the difference in chest wall circumference during the state of maximal expiration and maximal inspiration6, 7. a spirometer (pony fx, cosmed, italy) was used to measure pulmonary function. using a maximal - effort expiratory spirogram, forced vital capacity (fvc), forced expiratory volume in one second (fev1,), fev1/fvc, peak expiratory flow (pef), and predicted pulmonary function (pred fvc, pred fev1, pred fev1/fvc, pred pef) were measured while the subjects were in an upright position1. thoracic region self - mobilization exercise was performed by the intervention group using a method modified and a self - mobilization tool from previous studies for 20 minutes, three times per week for 6 weeks5. if a participant felt pain or inconvenience, the self - mobilization tool was moved toward the transverse process of the spine or the rib, and the exercise was performed again5. before the experiment, the participants were requested to practice 23 times to become familiar with the exercise5. wilcoxon signed - rank test was used to compare differences between the two groups (tsmg and cg). the mann - whitney u test was used to compare the two groups at the significance level of () = 0.05 chest expansion (axillary, sternum, low costal region) measurement results are summarized in table 1table 1.within-group and between - group comparisons for outcome measurestsmg (n=8)difference (post - pre)zcg (n=11)difference (post - pre)zzpre - testpost - testpre - testpost - testax4.312.106.182.011.871.452.31 4.773.243.060.791.703.671.682.99st3.181.095.181.362.001.531.68 3.632.852.901.540.722.481.132.28lc5.062.736.121.951.061.741.553.362.372.071.381.291.492.532.74within - group comparison, between - group comparison, ax : axillary region, st : sternum region, lc : low costal region, unit ; cm,p 0.05). within - group comparison, between - group comparison, ax : axillary region, st : sternum region, lc : low costal region, unit ; cm,p < 0.05, all results presented as mean sd. chest expansion, respiratory function, and thoracic vertebral pain are adversely affected by reduced thoracic vertebral mobility and structural changes in the thoracic and lumbar angles8, 9. although many interventions have been applied in previous studies to address these issues, those interventions have shortcomings because they require firsthand application by therapists1, 4. therefore, the present study aimed to verify whether active thoracic vertebrae self - mobilization methods are sufficient5 and to investigate changes in chest expansion and pulmonary function caused by thoracic region self - mobilization. although engaging in thoracic joint mobilization and self - stretching exercise for 6 weeks improved pulmonary function in a previous study1, another study showed no improvement in pulmonary function after chest region respiratory muscle stretching exercise for 4 weeks7. therefore, the argument that interventions applied to the thoracic region are involved in pulmonary function improvement is controversial. in the present study, no significant change in pulmonary function was observed. this may be attributable to the fact that the intervention was applied to healthy subjects, who were relatively less affected by the intervention. however, the intervention applied in the present study resulted in larger increases in axillary region and sternum region measurements, than in previous studies in which respiratory muscle stretch gymnastics were applied7. interestingly, previous studies have reported that changes in low costal region measurements were relatively larger than changes in axillary region and sternum region measurements when thoracic flexibility exercise was applied. conversely, changes in lower costal region measurements were not significantly larger in the present study4, 7. these results may be attributable to the fact that although thoracic region self - mobilization directly affected ribs 110, which are directly connected to the thoracic vertebrae or to the cartilage, it could not directly affect ribs 11 and 12. the control group showed a statistically significant decrease in low costal region measurements post - test. thus, it could be indirectly concluded that if thoracic vertebrae are not mobilized, chest region muscle stiffness persists and the mobility of the muscles around the chest wall declines owing to over activation of the muscle spindles, potentially leading to respiratory disorders7, 9. therefore, thoracic region self - mobilization is an easy intervention that can be self - applied by patients with low chest mobility in order to improve expansion capability of the entire chest region. | [purpose ] the aim of this study was to determine the effects of thoracic region self - mobilization on chest expansion and pulmonary function in healthy adults. [subjects ] nineteen healthy adults were randomly allocated to either an intervention group (n = 8) or a control group (n = 11). [methods ] subjects in the intervention group performed self - mobilization of the thoracic region 3 times per week for 6 weeks (18 sessions). the outcome measures included chest expansion when breathing, pulmonary function, and predicted pulmonary function. [results ] there was a significant difference in chest expansion between the intervention group and the control group. however, there was no significant difference in pulmonary function between the intervention group and the control group. [conclusion ] thoracic region self - mobilization may be beneficial for increasing chest expansion in healthy adults. |
ethylcellulose, a nonbiodegradable and biocompatible polymer, one of the extensively studied encapsulating materials for the controlled release of pharmaceuticals, was selected as the retardant material for propranolol hydrochloride. ethylcellulose, a polymer to microencapsulate a drug by coacervation phase separation technique, emulsion solvent evaporation technique, and spherical crystallization technique. eudragit rs and eudragit rl are biocompatible copolymers synthesized from acrylic and methacrylic acid ester having similar structure that differs only in the extent of the quaternary ammonium substitution and hence high water permeability and hydrophilicity in eudragit rl as compared to eudragit rs polymer. it is a beta blocker and commonly used for the treatment of hypertension which affects nearly about 1020% of the population [3, 4 ]. the purpose of the present work was to prepare and evaluate oral sustained release microparticulate drug delivery system of propranolol hydrochloride using three different forms of polymers such as ethylcellulose, eudragit rs, and eudragit rl and to evaluate the drug release from microparticles prepared by hydrophobic emulsion solvent evaporation method (o / o) with a high entrapment capacity and extended release of the drug [5, 6 ]. various process and formulation parameters such as a drug polymer ratio and surfactant concentration were optimized to maximize the entrapment efficiency of the drug. these microparticles were evaluated for encapsulation efficiency, drug content, and in vitro drug release. drug - polymer interactions in the solid state were studied by fourier transform infrared spectroscopy (ftir) ; the size and shape were evaluated by motic microscope. the aim of the current work was to encapsulate propranolol hydrochloride with ethylcellulose, eudragit rs 100, and rl 100 microparticles that were prepared by modified hydrophobic solvent evaporation techniques. 1 : 1 combination of acetone and isopropanol was used as an internal phase and paraffin oil as external phase. prepared microparticles were characterized for drug content, particle size, in vitro drug release, and kinetic release studying. the highly water soluble drug, propranolol hcl, was gift samples from micro advanced research center, pvt. the nonbiodegradable polymer, ethylcellulose (300 cps) was purchased from sigma eldritch, usa. the two biodegradable polymers, eudragit rs and eudragit rl, were donated by colorcon asia pvt. ltd.,, mumbai. heavy liquid paraffin, potassium dihydrogen phosphate, and sodium hydroxide were procured from s.d. polymeric microparticles were prepared by dispersing accurately weight quantities of propranolol hydrochloride and polymers individually (ethylcellulose 300 cps, eudragit rs 100, and rl 100) in the primary phase as a solvent (1 : 1 combination of acetone and isopropanol) with continuous stirring at 500 rpm by using magnetic stirrer for 15 min. sustained released microparticles of drug were prepared by modified hydrophobic emulsion solvent evaporation method (o / o). this primary emulsion was slowly added to the external secondary oil phase containing span 80 (0.4% v / v) as an emulsifying agent with constant stirring for 2 hours using a four - blade lab stirrer (remi elektrotechnik ltd., after complete evaporation, stirring was stopped, the n - hexane (20 ml) was added to harden the formed microparticles, and the mixture was vacuum filtered to obtain microparticles. the resulting microparticles were collected and allowed to dry for 24 h at room temperature. all batches of prepared microparticles are in triplicate way and all results are articulated as the mean value of three inspections. the diameter of microparticle of each formulation was measured by spreading a thin layer of microparticles on a glass slide and viewing the microparticles under an optical microscope fitted with an eyepiece having 40x to 100x resolution of motic microscope (b1 series, motic, china). each sample was measured at three times and an average particle size was articulated as mean diameter. the encapsulation efficiency of the prepared microparticles was determined by accurate weighing and added in acetone to dissolve polymer and then add the required volume of distilled water. precipitate solution was filtered and make up the volume up to 100 ml into a volumetric flask. recovered filteration was measured at maximum absorbance at 289 nm by using uv - visible spectrophotometer (uv-1800 shimadzu co. ltd., japan) and encapsulation efficiency was calculated using the following equation : (1)ee (%) = (actual amount of drug)(theoretical amount of drug)100. ir spectra of pure drug and microparticles prepared by using polymers like ethylcellulose, eudragit rs, and eudragit rl - loaded microparticles were obtained with infrared (ir) spectra of the samples that were scanned in the range from 400 to 4000 cm and recorded on a fourier transform infrared spectrometer (shimadzu co. ltd., the microparticles were fixed with carbon - glu and coated uniformly with gold palladium under argon atmosphere. samples were then observed with a hitachi model s4800, japan, scanning electron microscope. in vitro dissolution studies were performed using usp type ii dissolution test apparatus (paddle) at 100 revs./min and temperature at 37c 0.5c. withdrawing 5 ml samples at preselected time intervals up to 12 hours monitored progress of the dissolution. the absorbance of the withdrawn samples was assayed by uv - spectophotometry at the wavelength of maximum absorbance (289 nm). each dissolution study was carried out in triplicate and the mean values were expressed as standard deviation. the in vitro drug release data was analysed according to zero order, first order, higuchi square root, hixon crowel, and korsemeyer model. selecting the most suitable model was preferred on the basis of integrity of fit test [912 ]. in the present paper, ethylcellulose (300 cps) polymer has higher viscosity grade compared with polymethacrylate resins polymers like eudragit rs 100 (rs) and rl 100 (rl). these are two copolymers synthesized from acrylic and methacrylic acid esters, containing a low level of quaternary ammonium groups. rs has a lower content of charged groups (4.56.8%), and it is considered less permeable to water with respect to the more readily permeable rl (8.812% ammonium groups). higher permeability of eudragit rl 100 is due to maximum number of quaternary ammonium substitution present in the structure of eudragit rl 100 compared to rs 100 which affects the release behavior of the drug. in this work, the effect of drug - polymer ratio and surfactant concentrations on particle size, entrapment efficiency, and release pattern of propranolol hydrochloride from ethylcellulose, eudragit rs 100, and rl 100 microparticles prepared by nonaqueous (hydrophobic) solvent evaporation method was examined. due to decrease in solubility of propranolol hydrochloride in external oil phase, we used ethylcellulose polymer having 300 cps viscosity range as drug carrying polymer. due to the high viscosity range it formed a saturated solution with acetone : isopropane organic solvent. ethylcellulose was hydrophobic in nature ; thus, the hydrophobic polymer encapsulates larger amount of the drug. when organic phase was added in external oil phase containing surfactant, propa - ec matrix immediately starts to precipitate because of insoluble in oil and fast diffusion of acetone. one of the objectives of nonaqueous emulsion technique was to entrap maximum amount of propranolol hydrochloride. due to polymer saturated solvent and 1 : 1 combination of acetone and isopropane immiscible with oil, polymeric matrix was immediately precipitated out as solvent starts to evaporate and gives maximum encapsulation efficiency. span 80 is a better surfactant in terms of encapsulation efficiency, drug content, and particle size. so saturated solution of polymer (ec 300 cps) and faster diffusion rate of solvent enhance the encapsulation efficiency. as the ratio of polymer increased particle size, encapsulation efficiency was also increased. this is because of the saturation concentration of organic phase increased with viscosity at maximum ratio which helps to enlarge the size and a maximum encapsulation with a homogeneous matrix. as polymer concentration increased, the binding capacity or matrix forming competency of polymer with drug also increased. due to this the maximum amounts of drug get entrapped in polymeric core and give more encapsulation and percentage yield of recovered microparticles in higher drug polymer ratio than lower ratio. the emulsion droplets containing propranolol hydrochloride get transformed into solid state due to ethylcellulose hydrophobic property and acetone evaporation during stirring. this evaporation rate was maintained by stirring at 1000 rpm and 25c temperature. table 1 shows the influence of drug - polymer ratio, surfactant, and its concentration on particle size, percentage yield, and encapsulation efficiency of different formulation trials. the obtained results concluded that the fact that the particle sizes were directly proportional to polymers concentration may be due to increase in viscosity of the internal phase, and inversely proportional the surfactant concentration may be due to the formation of new surfaces for the small emulsion globules. formulation prepared using ethylcellulose 300 cps showed higher encapsulation efficiency (96.7 0.5) than those prepared using eudragit rs 100 (83.7 0.6) and rl 100 (89.7 0.6). it was also found that release of drug is slower than formulation prepared from eudragit polymers. formulations f4, f6, and f12 which are prepared using higher drug to polymer ratio with 0.4% surfactant concentration showed drug release of 59.08 0.2, 75.10 0.4, and 92.9 0.3 percent, respectively, at 12 h. all recovered microparticles were spherical in shape and slightly porous in nature (figure 1). this porous nature of microparticles is responsible for sustained release of drugs, because the polymer concentration may help to enhance the holding capacity for drug and surfactant decreased the interfacial tension between aqueous drug solution and polymeric organic solution. drug polymer interaction was determined by comparing the ir spectra of propranolol hydrochloride loaded ethylcellulose microparticles with the ir spectrum of pure drug. as shown in figure 2, propranolol hydrochloride gives peaks in the ir spectrum nearby at 2977 cm due to the presence of a secondary amine group, 3372 cm due to the hydroxyl group (secondary), the aryl alkyl ether shows a stretching band at 1354.64 cm and the peak at 782 cm due to a - substituted naphthalene. frequencies of functional groups of pure drug remained intact or overlapped by polymer in a physical mixture containing polymer. hence, there was no major interaction between the drug and polymer used in the study. figure 3 illustrates in vitro release of f4, f6, and f12 formulations, by representing the cumulative percentage of drug released. the formulation f4 was more sustained than formulations f8 and f12, because the maximum number of particles may extend the time to release the drug from f4 formulation. in f4 formulation prepared by using ethylcellulose polymer and 0.4%, v / v span 80 surfactant was used which gives larger size particles than f8 and f12 formulation. at the end of 12 hours f4 formulation released 59.08 0.2, f8 released 75.10 0.4, and f12 released 92.9 0.3 percent propranolol hydrochloride. f8, and f12 are 42.8 0.4, 53.9 0.4, and 63.8 0.4, respectively ; it may be due to the drug present at the surface of particles. the burst release effect may be due to the adsorption of the drug on the surface of the particles or due to concentrating the drug at the surface of the particles because insufficient concentration or ineffectual surfactant was unable to encapsulate drugs at the core of particles and drug moved towards the interface of both phases. the obtained results revealed that the most sustained f8 formulation best fitted in higuchi kinetics. it describes the release of drugs from an insoluble matrix as a square root of time dependent process based on fickian diffusion. in higuchi or square root kinetics, drug diffuses at a comparatively slower rate as the distance for diffusion increases. from all above evaluations, it was concluded that the prepared microparticles were successfully sustained for 12 h. thus from all these results it was discovered that ethylcellulose 300 cps viscosity range polymer can be used to formulate sustained release microparticles at different ratios. from the above results, it was concluded that propranolol hydrochloride was successfully encapsulated into ethylcellulose and eudragit microparticles using o / o hydrophobic emulsion solvent evaporation method. span 80 was more suitable surfactant with a concentration of 0.4%, v / v. the 1 : 7 drug - polymer ratio obtained the highest encapsulation and sustained the propranolol hydrochloride for 12 h. it follows higuchi model release kinetics ; therefore this dosage form maintains the drug level in therapeutic window which may help to minimize the side effects and minimize the frequency of dose which improve patient compliance. | objective. the purpose of the recent study was to prepare and estimate sustained release of ethylcellulose (300 cps) and eudragit (rs 100 and rl 100) microparticles containing propranolol hydrochloride used as a treatment of cardiovascular system, especially hypertension. method. propranolol hydrochloride was microencapsulated with different polymers (ethylcellulose, eudragit rs, and eudragit rl) using modified hydrophobic (o / o) solvent evaporation method using 1 : 1 combination of acetone and isopropanol as the internal phase. obtained microparticles were showing higher batch yield with higher encapsulation efficiency. microparticles were prepared with different ratios of 1 : 1, 1 : 3, 1 : 5, and 1 : 7 (%, wt / wt) using span 80 (%, v / v) as a surfactant. results. the influence of formulation factors like drug : polymer ratio, internal phase, and type of polymers on obtained microparticles was characterized with respect to particle size distribution, encapsulation efficiency, percentage yield, ftir, and fe - sem. higher encapsulation efficiencies were obtained with various polymers like ethylcellulose (96.63 0.5) compared to eudragit rs 100 (83.70 0.6) and rl 100 (89.62 0.6). the in vitro release study was characterized by initial burst. conclusion. the result of study displays that ethylcellulose and eudragit loaded microparticles of propranolol hydrochloride can be effectively prepared using modified hydrophobic emulsification solvent evaporation technique. therefore, the modified hydrophobic emulsion technique can also be applied to the preparation of microparticles for low molecular weight and highly water soluble drugs. |
preparation of solid dispersion of domperidone : domperidone inclusion complexes were prepared with -cd in different ratio (1:1, 1:2 and 1:3) by kneading method. preparation of fast dissolving films : solid dispersion of domperidone and -cyclodextrin (1:3) was selected and dispersed in half quantity of water and to it methanol is added along with tween-80 and heated at 60c. in other half quantity of water, hpmc e15 it is dried in oven at 60c for 5 hrs to obtain the film. morphological properties : properties such as homogeneity, color, transparency and surface of the oral films were evaluated by visual inspection.film mass : the mass of three films were determined by an analytical balance and means.d was calculated.film thickness : film thicknesses were measured at five positions (central and the four corners) using the digital vernier caliper and the mean thickness was calculated.folding endurance study : it was measured manually by repeatedly folding a film at the same place till it broke.in vitro disintegration studies : the film as per the dimensions (3 2 cm) required for dose delivery was placed on a stainless steel wire mesh placed in a petridish containing 10 ml phosphate buffer ph 6.8. time required for the film to break was noted as in vitro disintegration time.dissolution and drug release : dissolution test of films was performed using (900 ml ; phosphate buffer ph 6.8 with usp dissolution apparatus ii at 50 rpm and 370.5c temperature. the drug release was analyzed spectrophotometrically at max 286.5 nm using ultraviolet (uv) spectrophotometer (shimadzu model no : 1800). morphological properties : properties such as homogeneity, color, transparency and surface of the oral films were evaluated by visual inspection. film mass : the mass of three films were determined by an analytical balance and means.d was calculated. film thickness : film thicknesses were measured at five positions (central and the four corners) using the digital vernier caliper and the mean thickness was calculated. folding endurance study : it was measured manually by repeatedly folding a film at the same place till it broke. in vitro disintegration studies : the film as per the dimensions (3 2 cm) required for dose delivery was placed on a stainless steel wire mesh placed in a petridish containing 10 ml phosphate buffer ph 6.8. dissolution and drug release : dissolution test of films was performed using (900 ml ; phosphate buffer ph 6.8 with usp dissolution apparatus ii at 50 rpm and 370.5c temperature. the drug release was analyzed spectrophotometrically at max 286.5 nm using ultraviolet (uv) spectrophotometer (shimadzu model no : 1800). morphological properties : properties such as homogeneity, color, transparency and surface of the oral films were evaluated by visual inspection.film mass : the mass of three films were determined by an analytical balance and means.d was calculated.film thickness : film thicknesses were measured at five positions (central and the four corners) using the digital vernier caliper and the mean thickness was calculated.folding endurance study : it was measured manually by repeatedly folding a film at the same place till it broke.in vitro disintegration studies : the film as per the dimensions (3 2 cm) required for dose delivery was placed on a stainless steel wire mesh placed in a petridish containing 10 ml phosphate buffer ph 6.8. time required for the film to break was noted as in vitro disintegration time.dissolution and drug release : dissolution test of films was performed using (900 ml ; phosphate buffer ph 6.8 with usp dissolution apparatus ii at 50 rpm and 370.5c temperature. the drug release was analyzed spectrophotometrically at max 286.5 nm using ultraviolet (uv) spectrophotometer (shimadzu model no : 1800). morphological properties : properties such as homogeneity, color, transparency and surface of the oral films were evaluated by visual inspection. film mass : the mass of three films were determined by an analytical balance and means.d was calculated. film thickness : film thicknesses were measured at five positions (central and the four corners) using the digital vernier caliper and the mean thickness was calculated. folding endurance study : it was measured manually by repeatedly folding a film at the same place till it broke. in vitro disintegration studies : the film as per the dimensions (3 2 cm) required for dose delivery was placed on a stainless steel wire mesh placed in a petridish containing 10 ml phosphate buffer ph 6.8. dissolution and drug release : dissolution test of films was performed using (900 ml ; phosphate buffer ph 6.8 with usp dissolution apparatus ii at 50 rpm and 370.5c temperature. the drug release was analyzed spectrophotometrically at max 286.5 nm using ultraviolet (uv) spectrophotometer (shimadzu model no : 1800). all the formulation contained varied amount of polymer and hence thickness of each film was varied between the ranges of 0.14 mm-0.36 mm. when the concentration of hpmc e15 is increased from 5%-10%, thickness of the strip increased. folding endurance was measured manually and it was found to decrease with increase in the concentration of film forming polymer hpmc e15. the surface ph of all the formulation was found to be in the range of 6.8 - 7.2 and hence will not cause any irritation to oral mucosa. f1 formulation found to give minimum disintegration time (45 sec) as compared to other formulations. the drug release rate decreases as the concentration of the film forming agent hpmc e15 increases in the formulation. after 15 min interval more than 75% of drug was released from batches as defined in the guidances such as usp30. films were prepared of domperidone--cyclodextrin solid dispersion by the use of hpmc e15 as a film forming agent and peg-400 as a plasticizer. domperidone mouth dissolving films were prepared successfully by the use of solid dispersion of domperidone with -cyclodextrin and it can be used to treat emesis caused by various conditions in geriatric, bedridden and non - cooperative patients due to its ease of production. | the present investigation was undertaken with the objective of formulating mouth dissolving film(s) of the antiemetic drug domperidone to enhance the convenience and compliance by the elderly and pediatric patients. domperidone is a drug of choice in case of nausea and vomiting produced by chemotherapy, migraine headaches, food poisoning and viral infections. it causes dopamine (d2 and d3) receptor blockage both at the chemoreceptor trigger zone and at the gastric level. it shows high first pass metabolism which results in poor bioavailability (10 - 15%). in view of high first pass metabolism and short plasma half - life it is an ideal candidate for rapid release drug delivery system. the solid dispersions of domperidone were prepared with the use -cyclodextrin in various ratios (1:1, 1:2, 1:3) and solubility study was performed to determine the ratio in which solubility of domperidone was highest (1:3). the selected solid dispersions were then utilized for the preparation of film by solvent casting method utilizing hpmc e15 as a film forming agent and peg-400 as plasticizer. five formulae were prepared and were evaluated for their in vitro dissolution characteristics, in vitro disintegration time, and their physico - mechanical properties. the promising film (f1) showed the greatest drug dissolution (more than 75% within 15 min), satisfactory in vitro disintegration time (45 sec) and physico - mechanical properties that are suitable for mouth dissolving films. |
several cognitive domains, including attention, memory, and executive functions are impaired in bipolar disorder (1, 2). meanwhile, in recent decades, there has been an increasing tendency to study neurocognitive deficits like executive functions among researches (3). executive functions are a series of problem - solving tasks to achieve specific goals (4), including abilities like attention, reasoning, planning, working memory, response inhibition, and interfering factors control. disruption in these functions of often ten causes psychiatric or behavioral disorders, suggesting their important role in human complex behavior (5). executive functions are hypothesized in explanation of many disorders such as schizophrenia (6), obsessive - compulsive disorder (7), autistic disorder (8), eating disorders (9), panic disorder (10), and posttraumatic stress disorder (11). in fact, quite enough studies have repeatedly shown that bipolar patients suffer from cognitive impairments both during acute phases (13) and remission / euthymic periods (14, 15). (12) on 26 studies (689 patients and 721 controls), suggested that aspects of executive function had a large effect size ([d 0.8 ] indicating marked impairment), whereas other cognitive domains, including response inhibition and set shifting had medium effect (0.5 d 0.8), and finally verbal fluency, immediate memory, and sustained attention had small effect sizes (0.2 d 0.5). (12), which added studies on first - degree relatives provided the same results on cognitive impairments for euthymic patients and to lesser degrees, but still significantly different from healthy controls, for first - degree relatives (16). because cognitive impairment appears to occur in the early stages of bipolar disorder and increases both during depressive episodes and after periods of mania, some researches consider this defect as a state marker (17, 18). other studies have shown that cognitive deficits exist both after a long symptom - free and controlled subthreshold symptoms (12). a recent research with a considerable follow - up period has also spotted that cognitive deficits are stable features in bipolar patients with normal mood during the next 6 years (19). among cognitive deficits in bipolar disorder, response inhibition impairment accompanies manic symptoms (e.g. impulsivity, psychomotor agitation, over - talkativeness, overspending, and risky sexual behavior). some researchers suggest that impulsivity, which includes some defects in response inhibition, is higher in patients with bipolar disorder even during normal mood periods (20, 21). cognitive impairments have adverse impacts on functions such as daily activities, job, interpersonal relationships, and quality of life (22) ; also they have significant correlation with weak psychosocial functioning in the future (19). on the other hand, due to negative psychosocial consequences of cognitive deficits on both patients ' medication compliance (13) and psychological interventions complementing pharmaceutical treatment (23), identification and rehabilitation of these defects stress their particular importance. in other words, empowerment of bipolar patients, in terms of psychosocial aspects, has been a major concern for clinicians. studies like this can provide mental health practitioners with useful intervention strategies, because investigators soon found that effectiveness of psychosocial interventions in these patients is bound to considering their cognitive limitations (24). as a new approach in last few years, it aims to help these patients by intervening on their basic cognitive components, called neuropsychological remediation or cognitive rehabilitation (25). by manipulation of basic cognitive processes, these interventions are supposed to improve cognitive functions and consequently promote psychosocial functioning of bipolar patients (26). it seems that despite convincing evidence on cognitive impairments during euthymic periods, more researches are still needed with methodologically solid designs to control confounding factors such as medication, and sub - symptoms effects (12) ; and applying measures with high sensitivity and specificity for evaluating cognitive elements of bipolar patients (16) ; as well as using a well - designed battery for assessing cognitive impairments (27). this study aimed to evaluate two executive functions (working memory and response inhibition) in euthymic bipolar patients and comparing them to the control group. our hypothesis is that working memory and response inhibition in euthymic patients are weaker compared to healthy controls. in this case - control study, 30 patients (18 to 45 years) with bipolar diagnosis admitted to roozbeh psychiatric hospital (from may to october 2013) entered the study and were compared to 30 healthy controls. in patients group, 25 were manic in which 17 ones had psychotic features ; the other 5 patients had mixed episodes. the patients were examined within 3 to 4 weeks after symptoms remission (in euthymic period). after psychiatric evaluation and determining their diagnosis, and obtaining their primary consent, the patients were referred to a researcher. then the researcher described the study to them and if they wanted to participate in the study, they would be asked to complete a consent form. after that, they were interviewed using structured clinical interview for dsm - iv for axis (scid - i) disorders. inclusion criteria included age between 18 - 45 years, diagnosis of bipolar disorder based on psychiatrist diagnosis, and scid - i diagnoses. exclusion criteria consisted of any obvious cognitive disorders, major depressive episode, clinical anxiety, substance abuse, and mental retardation. all 30 patients were receiving mood stabilizers ; 22 of them were taking anxiolytics ; and 17 took also anti - psychotic. in addition, the control group was interviewed using scid - i as a diagnostic tool for ruling out psychiatric disorders ; and if they met the criteria for any axis i disorders or a history of these disorders, they were excluded. participants also matched with the patient group based on age, gender, and degree of education. to assess executive functions (working memory and response inhibition), cambridge neuropsychological test automated battery (cantab) was applied, which was originally developed at cambridge university in 1980s (28) and now provided by cambridge cognition (www.cambridgecognition.com). it is a computer - based cognitive assessment system consisting of 22 neuropsychological tests for various cognitive functions. these tests are administered to subjects using a touch screen computer with no specific language or culture bound, it is based on the standard cognitive tests that are routinely used in psychological assessment (29). since the tests have no use in diagnosis or determining disorder and are based on comparison with no need to the mean score like other neuropsychological tests, they can be applied without confirming their reliability and validity to reflect between group differences. these test kits are validated in a sample of 3000 people. in this study, to assess working memory, swm, ssp, were used and response inhibition was obtained through sst test. it assesses the ability to retain spatial information and manipulate remembered items in working memory, while the subject performs the task to achieve goals. the test consists of a series of colored squares (boxes) that are displayed on the screen. according to the guidelines, by touching boxes and using a process of elimination, token in each of boxes and use them to fill up an empty column on the right hand side of the screen. thus it seems that in the next item, searches should be limited to boxes that contain blue signs. this process goes on until all blue signs of the boxes are discovered on the screen. when the trial ends up, a new one begins (with new color and position different from previous trials). this test consists of 4 experimental trials and every trial has 3 boxes for search (the first 4 trials will not be scored), then test continues with 4, 6, and 8 boxes. the variables used in swm test include errors (between - group, within - group, and double errors), strategy, and latency (time from when the task is presented until the participant s first touch for opening the box) (27). in this test, when a subject touches a box that blue signs have already been found in, errors (between - group) are recorded. within - group errors include the number of errors occurring within a search, for example, the frequency of selecting a box that had been found empty in previous searches, (frequency of re - selection the empty box in a trial). double errors are calculated when the participant makes an error (re - selection of an empty box) that can simultaneously fall within group error categories. to start, a predetermined sequence of boxes marked with a blue sign were found to start and when it comes back, the new scouring is to begin. estimated use of this strategy (strategic points) is obtained from the total number of times that participants seek a new one with 6 and 8 boxes. the high score indicates a poor use of these strategies and a low score indicates an efficient use. spatial span assesses working memory capacity, which is a visuospatial analogue of the digit span test. this test measures the ability to remember the location and order of a set of visual stimuli (white squares) displayed on the screen. the number of boxes is increased from 2 at the start of the test to 9 at the end, and sequence and color are changing through the test. the test is stopped when the participant fails in all 3 trials of a stage. time span is defined as the highest level that the subject can remember in a trial. this measurement requires both visual and spatial components representing the ability to store data temporarily or online in order to plan more operations in the future. this test has 5 variables span length, mean time to first response (span length 2), mean time to last response (span length 2), total errors, and total usage errors (30, 31)., the participant should press the arrow that displayed on the screen (according to the arrow s left or right direction, the subject should press the corresponding button). this test consists of two sections : in the first one, the participant is introduced to press the left hand button when he sees a left - pointing arrow, and right hand button when seeing a right - pointing arrow. there is one block of 16 trials for the subject to practice this. in the second section, the participant is told to continue pressing buttons when they see the arrows, as before, but, if they hear an auditory signal (a beep), they should withhold their response and not press the button. this part consists of 4 blocks each containing 16 trials (total trials : 64). the variables of sst include 5 items including direction errors on stop and go trials, proportion of successful stops, median correct reaction time on go trials, stop signal delay, and stop signal rt (31). this scale was developed by young, biggs, ziegler, and meyer (32) for mania evaluation. this measure rates symptoms of bipolar disorder in mania episode and consists of 9 items. the scoring scale is based on severity of symptoms (total score : 60). there was a high correlation between scores of two independent clinicians on both the total score (0.93) and the individual item scores (0.66 to 0.92) (32). reliability and validity of this scale were acceptable in an iranian sample (isfahan province) (33). in this study, concordant validity of ymrs with the world health organization world mental health composite international diagnostic interview (who wmh - cidi) was 0.87. discriminatory analysis results indicated 17.14 and the optimal cut - off point with 98.4% sensitivity and 98.4% specificity. ymrs (young mania rating scale) is a valid instrument with acceptable sensitivity and specificity which can be applied in both clinical and research settings. its test - retest reliability has been reported between 0.48 to 0.86 and a mean of 0.86 (34). internal consistency of bdi - ii was estimated to be 0.73 - 0.93 and the correlation between parallel forms was 0.89. concurrent validity of the questionnaire with the hamilton rating scale for depression (hrsd) and depression subscale of scl-90 has been 0.71 and 0.89, respectively (34). (35) on a persian version of bdi - ii, the test had high internal consistency (cronbach = 0.87) and acceptable test - retest reliability (r = 0.74). this questionnaire consists of 21 multiple choice items (scoring from 0 to 3), which assesses the severity of anxiety. three items relate to anxious mood, 3 ones assess specific phobias and other questions evaluate autonomous hyperactivity symptoms and tension. beck. (36) have reported the internal consistency of this scale as 0.93 and its test - retest reliability as 0.75. this questionnaire was validated in iran by kaviani and mousavi (37). in their study, internal consistency measured by cronbach was 0.92 ; test - retest reliability was 0.83 and within class validity was 0.83. independent t - test was applied to assess differences between means considering p 0.05, df = 58, t = 2.84), and the mean score on ymrs was 7 in patients groups. table 1 presents the results of the sst test. regarding direct errors on stop and go trails, bipolar group scored significantly higher than controls (p 0.05, t = 3.08) and the proportion of successful stops in the bipolar group was significantly lower than the control group (p 0.05, t = -2.02). stop signal reaction time in bipolar group was significantly greater than the control group (p 0.05, t = 4.14). median correct reaction time on go trails was not significant between the two groups (p 0.05, t = 1.16). the delay between the two groups was not significant on stop signal delay (p 0.05, t = -1.13). bipolar group got lower scores than the non - clinical group, and this difference was significant (p 0.05, t = -3.89). the mean latency to first response in the bipolar group was higher than the control group, and the difference was significant (p 0.05, t = 3.40) and the mean response latency in the last item was also significantly greater in bipolar group (p 0.05, t = 3.88). as shown in table 2, the mean of total errors in bipolar group is higher than the control group, but this difference was not significant (p > 0.05, t = -1.14) and the total usage errors was similar between the two groups (p 0.05, t = 0.291).. the total number of between - errors was significantly higher in bipolar group (p 0.05, t = 3.98). between - group errors in 4, 6, and 8 boxes stages were significantly higher in bipolar group compared to the control group (p 0.05, t = 2.25 ; p 0.05, t = 3.30 ; and p 0.05, t = 3.76, respectively). strategies used in clinical and non - clinical groups were significantly different (p 0.05, t = 2.33). the total error was greater in bipolar group compared to the control group (p 0.05, t = 3.76). in 4 boxes step, there was no significant difference in total errors between the two groups (p > 0.05, t = 1.91), but total errors in 6 and 8 boxes levels were higher in bipolar group compared to the control group (p 0.05, t = 3.24 ; p 0.05, t = 3.06, respectively). in double - error and within - group in this study, the performance of working memory and response inhibition of bipolar patients were assessed during remission phase of mania symptoms. sst results showed that the direction errors in start - stop trials were higher in bipolar group ; and in successful stop ratio, the patients were less successful than the controls. in addition, the stop reaction time of bipolar group was greater than the control group, indicating that people with bipolar disorder have more errors in sst even after remission of acute mania symptoms. the results also showed that the time between moving and stopping, considered as response inhibition reaction time (ssrt), was higher in patients than controls. these results are consistent with the findings of a meta - analysis study done by hajek (38). this meta - analysis included 30 studies, consisting of 635 patients and 677 controls. findings of this study showed that the patients performance during periods of normal mood (228 patients and 277 controls) was different from healthy controls, also the accuracy level in bipolar group in doing gng test, ssrt, and stroop attention test was weaker than control group during periods of normal mood. there were similar deficiencies in not paying attention to stroop interferes, and stop signal reaction time (ssrt) was significantly different from matched control group. the patient group had a lower precision and needed more time to inhibit responses. in this study, the involved brain areas were also examined by the functional magnetic resonance imaging (fmri). furthermore, the study of gruber (39) compared 30 patients suffering from major depression, 17 patients with manic bipolar disorder, and 22 patients with depressive bipolar disorder by several neurocognitive tests (memory, attention, executive function, and sst). evaluations were done at admission and 7 weeks after discharge (in recovery period). in the first evaluation, manic patients had lower performance in response inhibition reaction time (sst) compared to depressive bipolar patients (ef = 0.75). although all three groups showed significant improvement after the follow - up period, deficiency in some domains (especially executive function) remained constant. in addition patients with mania had weaker results in inhibition of start responses than patients with major depression (ef = 0.87) and also had poorer performance compared to depressed bipolar patients (ef = 0.58). they also had slower reaction time than patients with bipolar depression at the start of the responses (ef = 0.55). in their study, two groups of bipolar patients (15 ones during manic episode, and 18 patients during normal mood period), were compared to 18 healthy controls using object alternation task. patients during both periods of mania and normal mood showed greater errors in response inhibition compared to the control group. other researches (41, 42) have confirmed deficits in response inhibition during periods of normal mood in patients with bipolar disorder. there are few studies inconsistent with previous ones (43). in this study, 20 patients with bipolar disorder during period of normal mood were compared to 20 controls in response inhibition test (gng) ; they were also examined by fmri during this test. results showed that performance was similar in both groups, while the activity pattern of involved brain areas in response inhibition was different. on the other hand, both the meta - analysis studies by robinson. (12) that examined the patients during periods of normal mood and bora (44) that examined the close relatives of bipolar patients, suggested that response inhibition was the most prominent factor in the phenotype of bipolar disorder. this finding is supported by other researches ; patients who are at risk of bipolar disorder show structural changes in areas associated with inhibition (38). in conclusion, it seems that enough evidence supports the idea that response inhibition deficit is an independent phenomenon existing beyond pathological mood episodes in patients with bipolar disorder. the results of the second hypothesis, in swm test, showed more within - group errors in bipolar patient. in the strategies employed index, there was also significant difference between the two groups, in other words, bipolar patients in normal mood period had more errors on swm test. (45). in their research 63 bipolar patients during normal mood period (27.3 months on average) results showed that patients had more within - group errors compared to healthy group in swm, but in strategies employed by the two groups there was not any significant difference, which contradicts with our finding regarding strategies. in addition, sweeney. (46) using cantab, compared 58 patients with major depression, 21 bipolar patients in depressive phase, 14 ones in mania or mixed episodes, to 51 normal individuals. patients in mania or mixed episodes had lower performance compared to depressed patients and healthy controls. also, in the employed strategies, there were significant differences between groups ; patients in manic or mixed phase had the least stability compared to those in the control group or depressed patients. the third part of our findings regarding ssp test indicated that the span length of clinical group was significantly lower compared to the healthy group. a significant difference existed in the meantime to first response and meantime to last response delays between the two groups, so that patients needed more time to do their first and last touches. (46) that bipolar patients in normal mood period had significantly weaker performance in ssp test compared to the control group. our findings in this section are also compatible with the findings of dittmann. (15), who examined 65 patients with bipolar disorder type i, 38 patients with bipolar disorder type ii (both during the period of normal mood), and 38 healthy controls using a brief neuropsychological battery. results indicated that bipolar patients had poorer performance in some items, such as working memory (measured by the wechsler memory subscale) compared to the control group. finally, it was in agreement with other studies confirmed impaired cognitive functions during periods of normal mood in patients with bipolar disorder (23), such as cavanagh 's study (23), which assessed memory and verbal learning in patients with bipolar disorder in euthmic phase compared to normal group. findings also revealed negative correlation between frequency of mania episodes and memory function (delayed recognition domain). the second limitation is the possible interference of sub - symptoms in cognitive functioning of the patients and the third one is controlling the effect of medications. most of our patients were on medications including mood stabilizers, antipsychotics, and anxiolytics. early evidence relating positive effects of mood stabilizers on neurocognitive functioning of bipolar patients (47) and at the same time some negative effects of antipsychotics on neuropsychological functioning in bipolar patients (48, 49) make it hard to draw conclusion about medication effects. we suggest that further researches with longitudinal designs and using homogenous patients based on their medication regimes can shed light on some of these limitations. we also suggest that future studies should do more with these confounding factors. in conclusion, current research suggests that bipolar patients had some inabilities in working memory ability and need more time to inhibit their responses compared to the control group. these findings have some clinical implications for practitioners such as paying more attention to cognitive symptoms that seems to be impaired even during remission. also they would be better to consider neuropsychological remediation in their bipolar patients treatment protocols. future studies should answer other remaining questions such as what cognitive function in which type of bipolar disorder is stable or transient, and how much these stable cognitive dysfunctions take part in patients specific psychosocial areas of functioning. also they should clearly respond to questions regarding severity of the illness, and specific clinical features like presence of psychosis, substance abuse, and so on. | background : several cognitive domains, including attention, memory, and executive functions are impaired in bipolar disorder.objectives:this study aimed to investigate two executive functions (working memory and response inhibition) in patients with bipolar i disorder during remission of the symptoms.patients and methods : in this case - control design, 30 bipolar i patients (18 to 45 years old) were matched with 30 ones in the control group in terms of age, gender, and education. the patients were selected from roozbeh psychiatric hospital (a hospital affiliated to tehran university of medical sciences) from may to october 2013. they were evaluated and contrasted using working memory (spatial span and spatial working memory (ssp and swm)) and response inhibition (stop signal task (sst)) tests.results:we used independent t - tests for comparing and contrasting 2 groups on total and sub - scales scores of these 3 tests. in terms of swm test there was a significant difference in between - group error between the two groups (p = 0.05) ; there was also a meaningful difference between the strategies used by two groups (p = 0.05). in ssp test, a significant difference appeared between averages of span length of the two groups. in the first and last item delays, there was also a clear difference, but the total error index was not noticeably different. in sst test, the direction error indicator in start - stop trials indicated a major difference, while in successful stops ratio, the case group had a lower ratio. in addition, reaction time to stop signs in bipolar group was meaningfully lower than the control group.conclusion:in conclusion, even during remission phase, executive dysfunction is detectable at least in some areas in patients with bipolar disorder. |
chronic hepatitis c virus (hcv) infection remains a global health threat with 175 million carriers worldwide. approximately 3% of the worldwide population is infected with the hepatitis c virus (hcv). the prevalence of hcv infection varies throughout the world, with the highest prevalence reported in egypt. the natural targets of hcv are hepatocytes and, possibly, b lymphocytes [4, 5 ]. a new form of chronic hcv infection named occult hepatitis c virus (oci) was described by castillo., 2004. this infection is characterized by absence of anti - hcv antibodies (abs) and hcv rna in serum with elevated liver function tests in the presence of hcv - rna in the liver. furthermore, about 70% of patients with occult hcv infection also have hcv rna in their peripheral blood mononuclear cells (pbmcs) ; the genomic and the antigenomic hcv rna have also been detected in these cells. although detection of genomic hcv - rna strand in liver biopsy specimen is the gold standard and the most accurate method for the diagnosis of occult hcv infection, testing for hcv - rna in pbmcs is an alternative and easy to do when a liver biopsy is not available [8, 9 ]. lymphoproliferative disorders is a term that includes a wide spectrum of pathologies ranging from a minor expansion of a b - cell population (with no clinical significance) to an aggressive high - grade lymphoma. such proliferations of b cells apparently can be triggered as a consequence of a chronic antigenic stimulation resulting from an hcv infection. non - hodgkin lymphoma (nhl) is the hematologic malignancy with the highest prevalence worldwide. incidence rates have grown fast up to the beginning of the new millennium, with an annual percentage increase of nearly 3%, which is faster than that for most cancers. a causative association between hepatotropic viruses, especially hepatitis c virus, and malignant b - cell lymphoproliferative disorders has been demonstrated utilizing epidemiologic data, biologic and molecular investigations, as well as clinical observations. these data indicate that hepatitis c virus may be responsible for the development of some malignant lymphoproliferative disorders [1117 ]. a former study on the malignant complications of chronic hcv infection in egypt and association of hcv with increased risk of b - cell nhl as a whole was reported. recently, another study in egypt proved that hcv is a risk factor for diffuse large b cell, marginal zone, and follicular lymphomas in egypt. nonetheless, assessment of the existence of occult hepatitis c infection in lpd patients has not been addressed in egypt and worldwide till now. the objective of this study was to investigate the occurrence of occult hepatitis c infection in lymphoproliferative disorders patients and to compare its prevalence with that of hcv in those patients. all subjects included in the study were obtained from clinical hematology unit of internal medicine hospital at kasr el - eini school of medicine, cairo university in the period between june 2010 and june 2011. institutional ethical board approval was taken prior to the study, as well as informed consent was taken from all the participants. selection criteria for patients were to being confirm lpd patients, negative for infection with hepatitis b virus (hbv), hiv, epstein - barr virus (ebv), and cytomegalovirus (cmv). out of the recruited 50 patients, there were 20 patients with non - hodgkin lymphoma, 4 patients with hodgkin disease, and 26 patients with chronic lymphocytic leukemia. the other 50 subjects were apparently healthy volunteers who were selected to be negative for hcv ab and serum hcv rna and to be age and sex matched with lpd patients recruited. all the subjects included in the study were subjected for full history taking and clinical examination, complete blood picture (cbc) with differential white cell count, complete liver and kidney functions, and hcv antibodies using the commercially available elisa kits (dia sorin, torino, italy). the patients group was subjected as well to beta 2 microglobulin, abdominal ultrasound for detection of organomegaly and lymphadenopathy, ct abdomen and pelvis for proper diagnosis and staging, immunophenotyping to role out chronic lymphocytic leukemia, lymph node biopsy for diagnosis of non - hodgkin lymphoma and bone marrow biopsy, and immunohistochemistry for staging. serum hcv antibodies were detected by elisa (dia sorin, torino, italy), while hcv genotyping was detected by inno - lipa hcv ii (bayer health care, eragny, france). rna was extracted from serum and pbmc using biozol (bioflux, china, catalogue no. bsj000210001 m80) total rna extraction reagent according to the manufacturer 's protocol. hcv rna plus strand was determined by reverse transcription - polymerase chain reaction (rt - pcr). rna was reverse - transcribed and amplified by one step rt - pcr qiagen kit (catalogue no. 210212, sensitivity 22 viral copies) with appropriate primers (5-cgc gcg act agg aag act tc-3) and (5-ata gag aaa gag caac ca gg-3) as forward and reverse primers, respectively. the lack of contamination in pcr reactions and was assessed by the inclusion of a negative control containing water rather than rna in each assay which did not show any pcr amplification in all experiments. moreover, each sample was done in duplicate to ensure absence of false positive results. thermal cycling conditions were denaturation : for 1 min at 94c, annealing : for 1 min at 55c, extension : for 1 min at 72c for 30 cycles, and final extension for 10 min at 72c. the pcr product (174 bp) was submitted to electrophoresis by using a 1.5 agarose gel and was visualized by ethidium bromide staining under ultraviolet light. hcv rna minus strand was determined by a previously established and described in house rt - pcr assay. reverse transcription was performed in 25 l reaction mixture containing 20 u of amv reverse transcriptase (clontech, usa) with 400 ng (3 l) of total pbmcs rna, 40 u of rnasin (clontech, usa), 0.2 mmol / l from each dntp (promega, madison, wi, usa), and 50 pmol of the forward primer 2ch (for minus strand). amplification of the highly conserved 5-utr sequences was done using two pcr rounds with two pairs of nested primers. first round amplification was done in 50 l reaction mixture, containing 50 pmol from each of 2ch (5-aac tac tgt ctt cac gca gaa-3) forward primer and p2 (5-tgc tca tgg tgc acg gtc ta-3) reverse primer, 0.2 mmol / l from each dntp, 10 l from rt reaction mixture as template, and 2 u of taq dna polymerase (promega, usa) in a 1x buffer supplied with the enzyme. a positive control rna of an hcv patient previously tested and confirmed to harbor that only the negative strand was included. moreover, two types of negative controls were included, a negative rt control having no rna at the reverse transcription step and a pcr negative control having water instead of cdna. the thermal cycling profile was 1 min at 94c, 1 min at 55c, and 1 min at 72c for 30 cycles. the second round amplification was done similar to the first round, except for use of the nested reverse primer d2 (5-act cgg cta gca gtc tcg cg-3) and forward primer f2 (5-gtgcagcctccaggaccc-3) at 50 pmol each. redmond, wa, usa) and spss for windows version 16 (spss inc., chicago, il, usa) software. results were reported as mean standard deviation (sd) or frequency (%) when appropriate. comparison of quantitative variables between the study groups was done using one - way analysis of variance test. correlation was considered large if between 1.0 and 0.5, medium if between 0.5 and 0.3, weak if between 0.3 and 0.1, and no correlation if between 0.1 and 0.0. p value less than 0.05 was considered statistically significant and less than 0.01 was considered statistically highly significant. the demographic and clinical data of the lpd patients group are summarized in table 1, also there were no statistically significant differences between the 2 groups regarding sex (p = 0.0639) and age (p = 0.25). there were highly statistically significant differences between the 2 groups regarding hepatomegaly, splenomegaly, and lymphadenopathy (p < 0.0001). the laboratory data of the patients and controls are shown in table 2, and high statistical significance differences were seen between the 2 studied groups in haemoglobin, platelet count, ast, and alt (p < 0.0001). in lpd patients, hcv ab was detected in 13 out of 50 (26%) patients, and serum hcv rna detection results were identical to hcv ab positivity, so 13 out of 50 (26%) patients were positive. pbmc hcv plus strand was detected in 18 out of 50 (36%) lpd patients (figure 1), and among these, ten (20%) patients were negative for hcv ab and serum hcv rna representing oci patients. pbmc hcv minus strand was checked in the ten oci positive lpd patients only and was undetectable in all of them. in controls group pbmc hcv plus strand was detected in only 2 out of 50 cases (4%) representing oci prevalence in this group. statistical analysis showed significant differences between patients and controls regarding the incidence of occult hcv (p = 0.0001), suggesting that occult hcv is associated with nhl and can be considered as a risk factor for nhl. rt - pcr results for the detection of hcv minus strand in pbmc of oci positive patients were all negative. as shown in table 3, according to pearson correlation, there were no correlations between age, hepatosplenomegaly, sex, tlc, ast, and total bilirubin, while there was positive correlation between hb, platelet count, alt, and positive hcv antibodies lpd patients. in oci positive lpd patients, there were no correlations between age, hepatosplenomegaly, sex, hb, tlc, and alt, while there was positive correlation between platelet count, ast, total bilirubin, and oci positive patients. in hcv negative oci negative lpd patients groups, there was no statistically significant correlation with age, hepatosplenomegaly, sex, tlc, ast, hb, platelet count, alt, and total bilirubin. in both hcv positive and oci positive patients, number of male patients exceeded that of female patients. among ten oci positive patients, six were males, while nine of thirteen hcv patients were males. in order to identify the association between blood transfusion and hcv positivity in lpd patients out of 50 lpd patients, 16 patients received blood transfusion including 5 anti - hcv positive lpd patients, 5 oci positive lpd patients, and 6 lpd patients negative or anti - hcv and oci. there was statistically significant difference between the total number of hcv (hcv + oci) positive lpd and non - hcv positive lpd patients and the blood transfusion, proving positive role of blood transfusion in lpd patients having hcv over those without hcv. multiple reports have described an association between chronic hcv infection and b - cell nhl. one of those concluded that the prevalence of hcv infection in patients with b - cell nhl was 15% much greater than in the general population. in egyptian population, this association was also proved by goldman., 2009. authors have long struggled to prove the existence of occult hepatitis c infection (oci). recently, it has been documented in haemodialysis patients, in chronic hcv patients after svr, in general populations, and in chronic liver disease patients of unknown etiology [2428 ] ; nonetheless, it have never been studied in lpd patients. in egypt, a recent study have shown high incidence of oci in nonalcoholic liver disease (40.7%). to the best of our knowledge, this study is considered the first to investigate the detection of oci in lpd patients. surprisingly, our results showed the detection of oci in 20% of nhl patients compared to 4% oci only in control healthy volunteers. statistical analysis of the differential existence of oci in lpd patients versus healthy controls showed a highly significant p value equal to 0.0312, confirming association of oci with lpd and suggesting its incrimination in lymphomagenesis. it should be noted that detecting oci by testing hcv rna in pbmc is an alternative method when liver biopsy is not available, and that oci is detected in pbmc of 70% only of patients with oci. therefore, it should be expected that the prevalence of oci in lpd patients might exceed that reported in this study if detected in liver biopsies. in this study, 4% of control healthy volunteers were oci positive, which is the first record for the prevalence of oci in healthy individuals in egypt. this is consistent with that recorded by de marco., 2009, which showed evidence that occult hcv infection may occur in 3.3% of population unselected for hepatic disease. in the present study, the patients selection criteria were to be newly diagnosed lpd patient and was blind regarding positivity for hcv antibodies (abs)s in order to compare the prevalence of hcv with that of oci blindly. results showed hcv prevalence of 26%, which is slightly higher than oci (20%). consequently, the total burden of hcv in lpd patients calculated based on the existence of hcv only versus existence of both oci and hcv was raised from 26% to 52%, respectively. the hcv genotype detected in lpd patients positive for either hcv or oci was genotype 4. moreover, in our study, oci was detected in a young male patient 18 years old, reflecting its occurrence in such young age. this, together with the fact that male sex is predominant in oci is accordant with previous studies by and. saad. reported that occult hcv infection seems to induce a mild liver disease, and followup is recommended for the occult hcv patients to monitor progression to overt disease. accordingly, it should be taken into consideration that nhl patients with elevated liver function tests with unknown etiology should be tested for oci and should be carefully followed up. blood transfusion was examined as a risk factor for occurrence of hcv and oci in lpd patients. as shown in the results section, statistical analysis of its role in lpd hcv ab positive patients alone was not significant and similarly for lpd oci alone was not significant. nonetheless a significant correlation was recorded when calculation was based on the total hcv plus oci, raising the inevitable role of oci in studying this risk factor. on the other hand, it should be noted that oci positive nhl patients who did not receive blood transfusion are equal in number with those who received it, indicating that there are two accepted probabilities, either hcv and oci can be acquired in lpd patients as a consequence of blood transfusion of infected blood, or lpd may arise as a disorder due to hcv infection. in conclusion, the present study, although having relatively low number of patients, is the first to demonstrate the occurrence of oci in lpd patients in egypt and worldwide and to highlight the fact that the burden of hcv in lpd is doubled if oci is considered. further studies with larger sample are recommended in order to assess the impact of oci on lpd progression. | background. occult hepatitis c virus infection (oci) was identified as a new form of hepatitis c virus (hcv), characterized by undetectable hcv antibodies and hcv rna in serum, while hcv rna is detectable in liver and peripheral blood cells only. aim. the aim of this study was to investigate the occurrence of oci in egyptian patients with lymphoproliferative disorders (lpds) and to compare its prevalence with that of hcv in those patients. subjects and methods. the current study included 100 subjects, 50 of them were newly diagnosed cases having different lymphoproliferative disorders (patients group), and 50 were apparently healthy volunteers (controls group). hcv antibodies were detected by elisa, hcv rna was detected in serum and peripheral blood mononuclear cells (pbmcs) by reverse transcription polymerase chain reaction(rt - pcr), and hcv genotype was detected by inno - lipa. results. oci was detected in 20% of patients group, compared to only 4% oci in controls group. hcv was detected in 26% of patients group with a slightly higher prevalence. there was a male predominance in both hcv and oci. all hcv positive patients were genotype 4. conclusion. our data revealed occurrence of occult hcv infection in egyptian lpd patients at a prevalence of 20% compared to 26% of hcv. |
epithelial - mesenchymal transition (emt) plays an important role in cancer tumorigenesis. during the emt, epithelial cells change their polarized epithelial phenotype to a spindle - shaped, myofibroblast - like phenotype with high motility, and cancer cells increased cell migration and invasion, which are crucial to cancer prognosis. the partial loss of e - cadherin is generally accepted as a hallmark of the emt, which reduces cell - cell adhesion and destabilizes the epithelial architecture. moreover, vimentin bestows a motile phenotype to cancer cells through changes in cellular architecture and cell - matrix interactions. further, many transcription factors, such as snail, act as repressors of e - cadherin and have been linked to the induction of the emt under different cellular contexts. signal transducer and activator of transcription 3 (stat3) is also involved in emt by regulating the transcriptional regulators of e - cadherin. large studies indicated that the emt was induced by transforming growth factor 1 (tgf-1) in various cancer cells. reported that tgf-1 was overexpressed in anaplastic thyroid cancer, and they found that knockdown of tgf-1 could inhibited cell proliferation and colony formation, and promoted apoptosis in anaplastic thyroid cells. park. found that tgf-1 promotes cancer immune escape, and that it promoted cell proliferation, colony formation, and inhibited apoptosis. tgf- is known to activate akt through pi3k, which in turn activates the mtor complex 1 (mtorc1). the activation of mtorc1 affects cell growth, proliferation, and invasion by modulating protein synthesis through its downstream effector, eukaryotic translation initiation factor p70 s6 kinase. recently, studies reported that sorafenib combined with a mammalian target of rapamycin (mtor) inhibitor was a more effective and tolerable treatment strategy for advanced hcc. inhibition of the mtor pathway reduced migration and invasion ability, and knockdown of mtorc1 induced mesenchymal - epithelial transition. recent studies showed that pkm2 (pyruvate kinase m2) is needed to induce emt. pkm2 is normally not expressed in adult tissues, but is reactivated in tumor tissues. pkm2 controls the final rate - limiting step of glycolysis and is an alternatively spliced variant of the pkm gene. moreover, pkm2 is a crucial glycolytic enzyme in the oncogenic mtor - induced warburg effect, in which hypoxia inducible factor-1 (hif-1) and c - myc - hnrnp cascades are the transducers of mtor regulation of pkm2. in addition, a study reported that loss of snail inhibits cellular growth and metabolism through the mir-128-mediated p70s6k / pkm2 signaling pathway. furthermore, atsushi. found that pkm2 knockdown failed to induce spindle - shaped morphological changes, and hindered e - cadherin reduction and vim increase compared with the control group. we infer that the mtor / p70s6k / pkm2 pathway can regulate the emt state. in this study, we hypothesized that the mtor / p70s6k / pkm2 pathway is involved in regulating tgf-1-induced emt. we investigated how the mtor inhibitor regulated emt, and explored the mechanisms of tumor suppression. our findings suggest that inhibition of mtor / p70s6k / pkm2 signaling promotes cervical tumor suppression. cervical cancer cell lines (hela, siha) (american type culture collection, manassas, va) were grown in dmem, supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 g / ml streptomycin and maintained at 37c with 5% co2. cells were treated with or without 10 ng / ml tgf-1 (peprotech, usa) in a serum - free medium for 48 h. cells were incubated with 0, 25, 50, and 100 nm of rapamycin (cst, danvers, ma). cell viability was assessed by use of a cell counting kit-8 (cck-8) assay. cells were plated in a 96-well plate at 310 cells / well in triplicates and treated with or without rapamycin and tgf-1 for 24, 48, and 72 h. ten l cck-8 was added to each well and incubated for 1 h at 37c. cell viability was calculated by the following formula : cell viability (%) = (od treatment od blank)/(od control odblank) 100%. cells were seeded in 6-well plates at 410 cells / well and then treated with 50 nm rapamycin with or without 10ng / ml tgf-1 for 24 h. apoptotic cells were detected by flow cytometry using an annexin v - fitc kit according to the kit instructions. cells were seeded in 6-well plates, and were cultured with fresh serum - free medium containing tgf-1 with or without the indicated concentration of rapamycin for 24 h. cell monolayers were wounded by scratching with sterile plastic 200 l micropipette tips and photographed using phase - contrast microscopy. the migration distance of each cell was measured after the photographs were converted to photoshop files. scratch distance at 24 h)/scratch distance at 0 h. the cells were harvested by centrifugation and washed with pbs, and then they were lysed in ripa buffer containing protease inhibitors. equal amounts of the protein lysates were electrophoretically separated on 10% sds - page gels and transferred to pvdf membranes. the membranes were blocked with 5% nonfat milk and then incubated overnight at 4c with the primary antibodies : sanil2, stat3, phosphor - p70s6k, e - cadherin, vimentin, and -actin, which were purchased from cst. after incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ecl detection system (bd biosciences). cervical cancer cell lines (hela, siha) (american type culture collection, manassas, va) were grown in dmem, supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 g / ml streptomycin and maintained at 37c with 5% co2. cells were treated with or without 10 ng / ml tgf-1 (peprotech, usa) in a serum - free medium for 48 h. cells were incubated with 0, 25, 50, and 100 nm of rapamycin (cst, danvers, ma). cell viability was assessed by use of a cell counting kit-8 (cck-8) assay. cells were plated in a 96-well plate at 310 cells / well in triplicates and treated with or without rapamycin and tgf-1 for 24, 48, and 72 h. ten l cck-8 was added to each well and incubated for 1 h at 37c. cell viability was calculated by the following formula : cell viability (%) = (od treatment od blank)/(od control odblank) 100%. cells were seeded in 6-well plates at 410 cells / well and then treated with 50 nm rapamycin with or without 10ng / ml tgf-1 for 24 h. apoptotic cells were detected by flow cytometry using an annexin v - fitc kit according to the kit instructions. cells were seeded in 6-well plates, and were cultured with fresh serum - free medium containing tgf-1 with or without the indicated concentration of rapamycin for 24 h. cell monolayers were wounded by scratching with sterile plastic 200 l micropipette tips and photographed using phase - contrast microscopy. the migration distance of each cell was measured after the photographs were converted to photoshop files. the cells were harvested by centrifugation and washed with pbs, and then they were lysed in ripa buffer containing protease inhibitors. equal amounts of the protein lysates were electrophoretically separated on 10% sds - page gels and transferred to pvdf membranes. the membranes were blocked with 5% nonfat milk and then incubated overnight at 4c with the primary antibodies : sanil2, stat3, phosphor - p70s6k, e - cadherin, vimentin, and -actin, which were purchased from cst. after incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ecl detection system (bd biosciences). to detect whether tgf-1 induced emt, we used 10 ng / ml tgf-1 to stimulate hela and siha cells for 48 h. we observed that siha (figure 1b) and hela cells (figure 1c) cells became scattered, acquired a spindle - shaped morphology, and lost cell - cell contacts, which are characteristics of a mesenchymal - like morphology. moreover, e - cadherin expression was abundant in the absence of tgf-1 (pre - emt). when stimulated with 10 ng / ml tgf-1, e in contrast, when stimulated by tgf-1, vimentin increased (post - emt) compared with the pre - emt state. these changes in cell morphology and marker proteins indicate that emt was induced when siha and hela cells were stimulated with 10 ng / ml tgf-1. we evaluated the effect of inhibition of mtor pathway on suppressing proliferation of cervical carcinoma cells. we used cell counting kit-8 (cck-8) assays to determine the effects of rapamycin (an mtor inhibitor) with or without tgf-1 on cell proliferation. cells were treated with 0, 25, 50, and 100 nm rapamycin, and 50 nm rapamycin with or without 10 ng / ml tgf-1. as shown in figure 2, rapamycin inhibited the proliferation of in hela cells in a dose - dependent manner. treatment with tgf-1 significantly increased the proliferation of both cell lines at 48 and 72 h, which was abolished by the addition of rapamycin (figure 2a). cells were treated with 50 nm rapamycin with or without 10 ng / ml tgf-1. tgf-1 significantly increased cell migration in hela cell lines at 24 h, which was abolished by the addition of rapamycin (figure 3a). similar data were obtained from the wound - healing assays of siha cells (figure 3b). to evaluate the effect of inhibition of mtor for antagonizing the anti - apoptosis effect of tgf-1 on cervical carcinoma cells by annexin v - fitc and pi staining, we assessed the effect of rapamycin with or without tgf-1 on the apoptosis of hela (figure 4a) and siha (figure 4b) cells. when cells were treated with tgf-1, the total number of apoptotic cells (early apoptotic+apoptotic) was significantly decreased compared to untreated cells in both cell lines. in cells treated with 50 nm rapamycin, the apoptosis rate significantly increased. furthermore, the addition of rapamycin significantly abolished the tgf-1-induced anti - apoptosis effects in both cell lines. to determine whether the emt condition induces an increase in pkm2, pkm2 expression was detected to compare levels pre- and post - emt state. as showed in figure 1, siha (figure 1b) and hela (figure 1c) cells changed morphology from epithelial to fibroblastic - like and spindle - shaped and lost cell - cell contacts, which are characteristics of a mesenchymal - like morphology. we found that the level of e - cadherin expression was suppressed compared with the pre - emt state. vimentin and snail family zinc finger 2 (snai2) expressions were increased in the post - emt state (figure 5). pkm2 expression was stimulated to increase in the post - emt condition (figure 5). the data show that the induction of emt resulted in a decreased level of e - cadherin, and increased vim and pkm2 expression in hela (figure 5a) and siha cells (figure 5b). moreover, atsushiet. reported that they were able to knock down pkm2 under emt conditions, but pkm2 knockdown failed to induce spindle - shaped morphological changes. meanwhile, pkm2 knockdown hindered e - cadherin loss and vim gain compared with the control. we evaluated the effects of rapamycin (an mtor inhibitor) on pkm2 (a critical glycolytic enzyme), and p70s6k (s6k1, a downstream effector of mtor). to investigate the inhibitory effect on mtor, we used 0, 12.5, 25, 50, and 100 nm rapamycin to treat hela (figure 6a) and siha (figure 6b) cells for 24 h. ribosomal p70 s6 kinase (s6k1) is a main downstream mtor effector. as shown in figure 6, rapamycin, a specific mtor inhibitor, inhibited the phosphorylation of p70s6k in a dose - dependent manner. moreover, we investigated whether mtor / p70s6k signaling decreased pkm2 expression, which is a main downstream p70s6k effector. we found that rapamycin treatment significantly decreased pkm2 in hela (figure 6a) and siha (figure 6b) cells. based on these facts, inhibition of mtor appears to affect cervical cancer through the mtor / p70s6k / pkm2 pathway in cervical cancer cells. to determine whether rapamycin is involved in regulating emt in cervical cancer tgf-1 significantly decreased the expression of e - cadherin and increased the expressions of vimentin, stat3 and snail2 in hela (figure 7a) and siha cells (figure 8a). in addition, at concentration of 50 nm rapamycin reversed tgf-1-induced emt - markers expression by repressing vimentin, stat3 and snail2 expressions and restoring e - cadherin expression in in hela (figure 7a) and siha cells (figure 8a). as shown in figures 7 and 8, we found that tgf-1 significantly increased the phosphorylation of p70s6k, which is the main downstream signaling intermediate of mtor signaling. simultaneously, tgf-1 significantly increased the expression of pkm2 in hela (figure 7a) and siha cells (figure 8a), while rapamycin significantly decreased the expressions of p - p70s6k and pkm2. these results suggest that rapamycin reverses tgf-1-induced emt via the mtor / p70s6k / pkm2 pathway. further, we examined the effect of rapamycin with or without tgf-1 on the morphology of hela and siha cell lines. after stimulation with 10 ng / ml tgf-1 for 48 h, both hela (figure 7b) and siha cells (figure 8b) cells became scattered, acquired a spindle - shaped morphology, and lost cell - cell contact, which are characteristics of a mesenchymal - like morphology. treatment with 50 nm rapamycin for 48 h abolished the tgf-1-induced morphological changes in siha and hela cell lines. to detect whether tgf-1 induced emt, we used 10 ng / ml tgf-1 to stimulate hela and siha cells for 48 h. we observed that siha (figure 1b) and hela cells (figure 1c) cells became scattered, acquired a spindle - shaped morphology, and lost cell - cell contacts, which are characteristics of a mesenchymal - like morphology. moreover, e - cadherin expression was abundant in the absence of tgf-1 (pre - emt). when stimulated with 10 ng / ml tgf-1, e in contrast, when stimulated by tgf-1, vimentin increased (post - emt) compared with the pre - emt state. these changes in cell morphology and marker proteins indicate that emt was induced when siha and hela cells were stimulated with 10 ng / ml tgf-1. we evaluated the effect of inhibition of mtor pathway on suppressing proliferation of cervical carcinoma cells. we used cell counting kit-8 (cck-8) assays to determine the effects of rapamycin (an mtor inhibitor) with or without tgf-1 on cell proliferation. cells were treated with 0, 25, 50, and 100 nm rapamycin, and 50 nm rapamycin with or without 10 ng / ml tgf-1. as shown in figure 2, rapamycin inhibited the proliferation of in hela cells in a dose - dependent manner. treatment with tgf-1 significantly increased the proliferation of both cell lines at 48 and 72 h, which was abolished by the addition of rapamycin (figure 2a). cells were treated with 50 nm rapamycin with or without 10 ng / ml tgf-1. tgf-1 significantly increased cell migration in hela cell lines at 24 h, which was abolished by the addition of rapamycin (figure 3a). similar data were obtained from the wound - healing assays of siha cells (figure 3b). to evaluate the effect of inhibition of mtor for antagonizing the anti - apoptosis effect of tgf-1 on cervical carcinoma cells by annexin v - fitc and pi staining, we assessed the effect of rapamycin with or without tgf-1 on the apoptosis of hela (figure 4a) and siha (figure 4b) cells. when cells were treated with tgf-1, the total number of apoptotic cells (early apoptotic+apoptotic) was significantly decreased compared to untreated cells in both cell lines. in cells treated with 50 nm rapamycin, the apoptosis rate significantly increased. furthermore, the addition of rapamycin significantly abolished the tgf-1-induced anti - apoptosis effects in both cell lines. to determine whether the emt condition induces an increase in pkm2, pkm2 expression was detected to compare levels pre- and post - emt state. cervical cancer cells were stimulated with 10 ng / ml tgf-1. as showed in figure 1, siha (figure 1b) and hela (figure 1c) cells changed morphology from epithelial to fibroblastic - like and spindle - shaped and lost cell - cell contacts, which are characteristics of a mesenchymal - like morphology. we found that the level of e - cadherin expression was suppressed compared with the pre - emt state. vimentin and snail family zinc finger 2 (snai2) expressions were increased in the post - emt state (figure 5). pkm2 expression was stimulated to increase in the post - emt condition (figure 5). the data show that the induction of emt resulted in a decreased level of e - cadherin, and increased vim and pkm2 expression in hela (figure 5a) and siha cells (figure 5b). moreover, atsushiet. reported that they were able to knock down pkm2 under emt conditions, but pkm2 knockdown failed to induce spindle - shaped morphological changes. meanwhile, pkm2 knockdown hindered e - cadherin loss and vim gain compared with the control. we evaluated the effects of rapamycin (an mtor inhibitor) on pkm2 (a critical glycolytic enzyme), and p70s6k (s6k1, a downstream effector of mtor). to investigate the inhibitory effect on mtor, we used 0, 12.5, 25, 50, and 100 nm rapamycin to treat hela (figure 6a) and siha (figure 6b) cells for 24 h. ribosomal p70 s6 kinase (s6k1) is a main downstream mtor effector. as shown in figure 6, rapamycin, a specific mtor inhibitor, inhibited the phosphorylation of p70s6k in a dose - dependent manner. moreover, we investigated whether mtor / p70s6k signaling decreased pkm2 expression, which is a main downstream p70s6k effector. we found that rapamycin treatment significantly decreased pkm2 in hela (figure 6a) and siha (figure 6b) cells. based on these facts, inhibition of mtor appears to affect cervical cancer through the mtor / p70s6k / pkm2 pathway in cervical cancer cells. to determine whether rapamycin is involved in regulating emt in cervical cancer, we examined the expression of emt - related markers using western blot analysis. tgf-1 significantly decreased the expression of e - cadherin and increased the expressions of vimentin, stat3 and snail2 in hela (figure 7a) and siha cells (figure 8a). in addition, at concentration of 50 nm rapamycin reversed tgf-1-induced emt - markers expression by repressing vimentin, stat3 and snail2 expressions and restoring e - cadherin expression in in hela (figure 7a) and siha cells (figure 8a). next, we explored the possible that signaling pathways may be involved. as shown in figures 7 and 8, we found that tgf-1 significantly increased the phosphorylation of p70s6k, which is the main downstream signaling intermediate of mtor signaling. simultaneously, tgf-1 significantly increased the expression of pkm2 in hela (figure 7a) and siha cells (figure 8a), while rapamycin significantly decreased the expressions of p - p70s6k and pkm2. these results suggest that rapamycin reverses tgf-1-induced emt via the mtor / p70s6k / pkm2 pathway. further, we examined the effect of rapamycin with or without tgf-1 on the morphology of hela and siha cell lines. after stimulation with 10 ng / ml tgf-1 for 48 h, both hela (figure 7b) and siha cells (figure 8b) cells became scattered, acquired a spindle - shaped morphology, and lost cell - cell contact, which are characteristics of a mesenchymal - like morphology. treatment with 50 nm rapamycin for 48 h abolished the tgf-1-induced morphological changes in siha and hela cell lines. we investigated whether the mechanisms of the mtor pathway affect tumorigenesis in tgf-1-induced emt in cervical cancer cells. rapamycin, an mtor inhibitor, can inhibit cell proliferation and migration, as well as promoting apoptosis and reversing tgf-1-induced emt via the mtor / p70s6k / pkm2 signaling pathway. moreover, e - cadherin plays a critical role in maintaining the cell surface and mediating normal epithelial tissue functions. overexpression of vimentin in cancer correlates well with accelerated tumor growth and invasion. during emt, cells start to exhibit a mesenchymal phenotype and show increased vimentin expression, with high motility rates. as shown in figure 1, we established an emt model in cervical cancer cells. consistent with these observations, our data indicated that tgf-1-induced cells acquired a striking morphological change and promoted e - cadherin loss and vim gain, as well as promoting proliferation and migration, and inhibiting apoptosis caused by emt. the mtor signaling pathway plays an important role in integrating intracellular and extracellular signals, and is a central regulator of cell metabolism, growth, proliferation, and survival. recent studies reported that using rapamycin derivatives in therapy against liver cancer achieves positive outcomes, leading to the implementation of large clinical trials. pedro. showed that rapamycin (an mtor inhibitor) inhibits tumor growth in pten - negative ishikawa tumor cells. our data also showed that inhibition of mtor significantly decreased cell proliferation, apoptosis, and migration, and reverses emt in cervical carcinoma cells. when cancer cell stayed in the emt state, pkm2 knockdown failed to induce spindle - shaped morphological changes, and hindered e - cadherin reduction and vim increase compared with the control group. our data show that the induction of emt resulted in a decrease level of e - cadherin, and increased vim and pkm2 expression in cervical cancer cells (figure 5), suggesting that pkm2 is involved in the emt state. rps6kb1(p70s6k) is a key downstream target of mtor, and an essential target for regulating translation rates and additional metabolic processes for cell growth. pkm2 is a crucial glycolytic enzyme in the oncogenic mtor - induced warburg effect, in which hypoxia - inducible factor-1 (hif-1) and c - myc - hnrnp cascades are the transducers of mtor regulation of pkm2. sun. reported that mtor upregulation of pyruvate kinase isoenzyme type m2 is critical for aerobic glycolysis and tumor growth. pkm2 level was augmented in mouse kidney tumors and consequent mtor activation, and was reduced by mtor suppression. based on these studies on the interaction of mtor, p70s6k, and pkm2, we investigated whether mtor / p70s6k signaling regulates pkm2 in cells in cervical cancer. the results showed that the phosphorylation level of p70s6k was inhibited and decreased pkm2 expression, which suggests that the mtor / p70s6k / pkm2 pathway is involved in regulation of cervical cancer (figure 6). our data show that tgf-1 significantly increases the expression of p70s6k and pkm2 (figures 7, 8), suggesting the mtor pathway was activated in the process of tgf-1-induced emt, and pkm2 is involved in the process of tgf-1-induced emt. further, we propose that rapamycin exerts its antitumorigenic effects and abolishes tgf-1-induced emt through mtor / p70s6k / pkm2 signaling in cervical carcinoma cells (figure 9). rapamycin, a specific mtor inhibitor, was added to cell cultures to determine whether mtor regulates tgf-1-induced emt. our data showed that rapamycin treatment significantly decreased phosphorylation of p70s6k and pkm2 in siha and hela cells. more importantly, we demonstrated that inhibition of mtor / p70s6k / pkm2 signaling is the therapeutic target of cervical cancer. inhibition of mtor reversed tgf-1-induced emt via the mtor / p70s6k / pkm2 signaling pathway. we believe this is the first study showing that inhibition of mtor reverses tgf-1-induced emt in tumor cells through mtor / p70s6k / pkm2 pathways. our data showed that tgf-1 induced proliferation and emt, and rapamycin inhibits cell proliferation and reverses emt. the mechanism involves suppression of p70s6k activation and reduced pkm2 expression, mediated by inhibiting mtor / p70s6k signaling, which provides novel mechanistic insights into the anti - tumor effects of mtor. | backgroundepithelial - mesenchymal transition (emt) plays an important role in cancer tumorigenesis. transforming growth factor 1 (tgf-1) can induced emt, which could increase tumor migration and invasion. moreover, recent studies have been proven that mammalian target of rapamycin (mtor) is a critical regulator of emt. we investigated the mechanisms of mtor in transforming growth factor 1 (tgf-1)-induced emt in cervical cancer cells.material/methodshela and siha cells were treated with 10 ng / ml tgf-1 to induce emt. then, they were treated with or without rapamycin. cck8 assay was performed to determine cell proliferation. cell migration was detected by wound - healing assay ; apoptosis was analyzed by flow cytometry ; mtor inhibitors inhibited mtor pathway to assess the expression of e - cadherin, vimentin stat3, snail2, p - p70s6k, and pkm2 expression.resultstgf-1 promoted proliferation and migration, and attenuated apoptosis in cervical carcinoma cells. rapamycin abolished tgf-1-induced emt cell proliferation and migration and reversed tgf-1-induced emt. e - cadherin were suppressed, whereas vimentin and pkm2 were increased in hela and siha cells after stimulation with tgf-1. moreover, mtor was activated in the process of tgf-1-induced emt. rapamycin inhibited the phosphorylation of p70s6k. furthermore, inhibition of the mtor pathway decreased pkm2 expression.conclusionsinhibition of the mtor pathway abolished tgf-1-induced emt and reduced mtor / p70s6k signaling, which downregulated pkm2 expression. our results provide novel mechanistic insight into the anti - tumor effects of inhibition of mtor. |
in order to arrive at their conclusion that infanticide is morally permissible, giubilini and minerva espouse a personistic ethic. briefly, this is the view that human beings do not have any inherent dignity in virtue of their humanity. merely being human is not in itself a reason for ascribing value to someone. on the contrary, human beings get their value from their status, understood in technical terms, as persons. accordingly they announce : the moral status of an infant is equivalent to that of a fetus in the sense that both lack those properties that justify the attribution of a right to life to an individual. both a fetus and a newborn certainly are human beings and potential persons, but neither is a person in the sense of subject of a moral right to life. we take person to mean an individual who is capable of attributing to her own existence some (at least) basic value such that being deprived of this existence represents a loss to her. this means that many non - human animals and mentally retarded human individuals are persons, but that all the individuals who are not in the condition of attributing any value to their own existence are not persons. merely being human is not in itself a reason for ascribing someone a right to life. the moral status of an infant is equivalent to that of a fetus in the sense that both lack those properties that justify the attribution of a right to life to an individual. both a fetus and a newborn certainly are human beings and potential persons, but neither is a person in the sense of subject of a moral right to life. we take person to mean an individual who is capable of attributing to her own existence some (at least) basic value such that being deprived of this existence represents a loss to her. this means that many non - human animals and mentally retarded human individuals are persons, but that all the individuals who are not in the condition of attributing any value to their own existence are not persons. merely being human is not in itself a reason for ascribing someone a right to life. the first relates to its explicit dehumanisation of the mentally disabled so that they, as a category, are indistinguishable, morally speaking (though perhaps not physically speaking), from animals, as a category. the second relates to its moral actualism so that only those who are in the condition of attributing any value to their own existence have any right to life. the first feature, the moral conflation of non - human animals and mentally retarded human individuals, is troublesome. it is at odds with the principle that human beings have an intrinsic dignity in virtue of their common humanity so that however disabled, young, old, conscious, awake, ill, diseased, unproductive and irrational we may be, we retain our dignity just by virtue of being human. a human being, notwithstanding illness or inability to exercise higher mental functions, is human and does not thereby degenerate to the level of vegetable or an animal. although our abilities and capacities may fluctuate, we retain our moral worth and our relation to the human family. this is often referred to as the equal dignity principle since it resists the temptation to discriminate morally on grounds of disability or characteristics. it locates value in our common humanity. on this view, human beings are distinctive in part because they are the kind that has moral obligations in a way that other kinds (eg, animals and vegetables) do not. human individuals do not lose that moral distinctiveness however mentally disabled. despite its absence from the authors analysis, the principle is a powerful one embedded both in international law and traditional moral thinking. as a well - known alternative position, it deserves some consideration however anxious the theorist is to arrive at his preferred conclusions. giubilini and minerva assume the truth of personism, leave this question entirely unaddressed and then unsurprisingly conclude that killing newborns is morally permissible because young babies are non - persons and thus more like animals than humans. any plain reading of the universal declaration of human rights and european convention on human rights affirms the same. unjust discrimination on the basis of age, disability or incapacity is among the numerous grounds available to regard personism as seriously dehumanising. part of the reason for the concern to assert the inherent dignity of all human beings is the arbitrariness and discrimination of any system that regards certain members of humanity as right subjects for elimination, as somehow subhuman or morally equivalent to animals. the preamble to the universal declaration of human rights recognises the inherent dignity and... the equal and inalienable rights of all members of the human family [as ] the foundation of freedom, justice and peace in the world. article 2 of the european convention on human rights asserts that everyone 's right to life shall be protected by law. article 14 of the european convention of human rights states that the enjoyment of the rights and freedoms set forth in this convention shall be secured without discrimination. the declaration on the rights of the child proclaims that the child... needs special safeguards and care, including appropriate legal protection, before as well as after birth. given the incentives to dehumanise, abuse and kill vulnerable human beings, in the name of non - therapeutic medical research, the nuremberg code, for example, reminds us that the voluntary consent of the human subject is absolutely essential. this means that the person involved should have legal capacity to give consent. the 20th century is a reminder of how the dehumanisation of certain classes of individual has been at the cost of gross human rights abuse. many of the illicit experiments of that benighted century were performed on children whose parents abandoned them to research, in which they were classified as non - persons or subhuman, for the greater ends of scientific progress and social utility.11 concerns for justice (self - defence, defence against aggressors and, more contentiously, capital punishment, etc), where the idea of an individual 's forfeiting his right to life is at play, need not compromise the principle of equal dignity. the concept of forfeiture is entirely in keeping with the principle because it recognises that, in certain cases, it is necessary to permit intervention in the interests of justice. the principle of self - defence, for example, need not be predicated on arbitrary notions of personhood, moral status, age or ability for that matter. in short, on an immediately appealing, non - discriminatory account, one does not derive one 's rights from one 's technical status as a person. nor need we predicate concerns for justice on fluctuating concepts of personhood and diminishing moral status so that unjust aggressors are somehow lesser persons. on this account we have value in spite of our fluctuating capacities, in spite of our dependence, age and weakness.12 the objection that infanticide ought to be permissible at international law and that plain readings of these conventions should no longer be thought relevant given alterations in attitudes merely begs the question about whether these proposed alterations in western thinking are morally sound. we have seen only too clearly and frequently in the 20th century how alterations in positive law and prevailing attitudes can be fundamentally mistaken and at odds with human dignity. the equal dignity principle notwithstanding, the authors assert without elaboration that however weak the interests of actual people can be, they will always trump the alleged interest of potential people to become actual ones, because this latter interest amounts to zero. this statement highlights another problem with their account and that is their moral actualism. on this view only those demonstrating actual capacities matter morally. we are, after all, at various points in our lives, dependent, asleep, unconscious, young, suckling, aged, intoxicated, disabled and so on. giubilini and minerva nowhere discuss the question of the sedated, sleeping, etc when they assert that we take person to mean an individual who is capable of attributing to her own existence some (at least) basic value such that being deprived of this existence represents a loss to her. but the seriously intoxicated, the sleeping, the sedated and the comatose are not capable of attributing to their own existence some (at least) basic value such that being deprived of this existence represents a loss to them. our propensity to sleep, fall unconscious, go into comas, pass out and so on, demonstrates, in no uncertain terms, that we very often exhibit our status as potential persons and become incapable of attributing value to our own existence. our abilities can and do fluctuate indeed every night when we go to sleep and fail to lay a claim on our lives. potentiality and fluctuating actuality is a feature of the human condition and efforts to predicate value on perceived valuable actual states that persons exhibit is question - begging. manifestly, the authors regard certain kinds of potential persons as having moral status. what is to be made of their account ? here it might be suggested that the authors are concerned only with lack of capacity rather than lack of ability, and that this distinction between types of potentiality affords them a way out of the proposed logical impasse. we may therefore kill babies qua non - persons but not sedated, sleeping etc individuals who are persons. efforts to exclude just those kinds of potential persons who do n't matter morally from the realm of moral status involves them in a circle of the form just those kinds of potential persons who lack moral status lack moral status. why should one 's status as a baby (disabled or otherwise) not matter morally if one 's status as potential person (while sleeping, comatose, sedated or drunk, etc) is so recognised ? efforts to set out criteria like rationality, self - reflective capacity, moral sensibility, and so on are equally problematic. excluding from the world of moral status 1- and 2-month - old babies on the grounds that they lack rationality, self - reflective capacity or moral sense can have the ill - starred effect of excluding also many professors of moral philosophy and 13-year - old boys. these defects alone should not deprive such creatures of their moral status or encourage us to believe that they lack intrinsic dignity. some 15 years ago, drawing on work by jenny teichman,13 i suggested that singer 's personism and actualism were flawed. in innocence and consequentialism : inconsistency, equivocation and contradiction in the philosophy of peter singer i argued that singer was inconsistent in his explanation of a thought experiment. his rejection of baby - farming that is, deliberately creating brain - damaged babies for organ harvesting - implied a logical inconsistency and a contradiction on the face of his own work. at the same time, his views on the permissibility of infanticide were well known and a reason for some of his cachet at the time. baby farming was impermissible on his view because it undermines our attitudes of care and protection for the brain damaged. his infanticide proposal is predicated on his personism. if, however, we can help ourselves to the idea that our attitudes of care and protection matter morally then they must matter also where newborn babies are concerned. accordingly, even if we set aside the equal dignity principle, there are still good utilitarian reasons to reject both infanticide and baby farming as imprudent. in short those who accept the equal dignity principle and traditional morality 's fundamental precepts need not go down this route, since intentionally killing the innocent is always impermissible, but a broader ethical analysis might consider this possibility. let us suppose that undermining attitudes of care and protection for our young by way of abortion (and now infanticide) brings about an erosion of respect for future generations, undermines the caring ethic of the medical profession, undermines patient trust in the medical profession and in the long term adversely affects the birth rates of nations whose dominant ideology would by now be personist in character. one of the well - known charges against utilitarianism is that it can achieve diverse results depending on how the calculus is performed. accordingly, if one chooses a calculus using certain short - term criteria, the theorist can achieve one result. if the theorist opts for a longer - term approach, using another criteria set he can achieve different results. the arbitrariness objection also arises spatially depending on the subjects, preferences, feelings and so on used to calculate best consequences. either way, giubilini and minerva, in their haste to arrive at their radical conclusions, spare us any discussion of the parameters and rationale behind their calculus, and nowhere countenance these broader kinds of factor ignoring any longer - term psychological, demographic, cultural, professional and intergenerational calculations. apart from anything else, given the demographic state of europe, some consideration of these broader implications would seem a sensible place to start. the european green paper confronting demographic change : a new solidarity between the generations and the munich economic summit 2007, for example, highlight the collapse in european birth rates, rising dependency ratios and looming pensions crisis. despite this reality, there is little interest, certainly by these authors and many avowed utilitarians more generally, in addressing the intergenerational conundrum. the question of why western nations, immersed in an ethic that dehumanises their young, are prepared to forego their own future generations altogether remains. at any rate, giubilini and minerva are not in the slightest interested in the longer - term cost of further undermining respect for young human life and blithely proceed to their citation - maximising conclusions. the reader is left wondering what reason other than the circular one of realising their preferred conclusion of permitting the killing of the newborn babe could be given to justify their short - termism. the irrationality of their approach is suggested by the self - serving nature of their preferred calculus. the infanticide proposal is not a new one. citing their own ideological sages, singer, kuhse and hare, and omitting any reference to objectors, giubilini and minerva state : euthanasia in infants has been proposed by philosophers for children with severe abnormalities whose lives can be expected to be not worth living euthanasia in infants has been proposed by philosophers for children with severe abnormalities whose lives can be expected to be not worth living as always, the very language they use is reminiscent of the lebensunwertes leben language favoured by the eugenicist nazi regime. be that as it may, on their own analysis, it is not at all clear quite why the status of a child as disabled should be of relevance given that babies are non - persons in any case. if it is relevant because it would cause displeasure to other actual persons who are the parents, or the hospital authorities, or the officers of the state, any reason is as good as the next for killing the baby. after all it is the displeasure to third parties that is doing the moral work here. yet disability is harped upon throughout the essay by the authors as a reason for killing the child. again, it is worth remembering singer 's mistake. if we are able to refer to our attitudes of care and protection then attitudes of care and protection for the very young and the disabled ought to enter the moral equation. indeed discrimination against the disabled may well display a callousness toward the vulnerable that might be better suppressed by general prohibitions even on singer 's selective criteria. after all, these are likely to have implications for existing disabled and dependent people. these concerns are not even alluded to by giubilini and minerva. in their haste to arrive at their preferred conclusions, they tailor the parameters of their calculus to suit their preferred conclusions. in full swing with their moral actualism, oblivious to the charges of arbitrariness and inconsistency, and unconcerned by charges of discrimination against the vulnerable, the authors insist of a sudden that the psychological pain of giving a child up for adoption should count as a reason in favour of infanticide, because in their view the interests of the actual people involved matter, and among these interests, we also need to consider the interests of the mother who might suffer psychological distress from giving her child up for adoption. the emergence of these feelings of distress out of the blue, as it were, serves to underline once again the arbitrariness and self - serving nature of their account. strangely, the ravaging of the moral sensibilities of the medical professionals involved in the process of using the lethal injection on babies, the confusion and guilt of members of the family who might be swept into this course of action by force of custom or ignorance, the danger of incentivised homicide that would inevitably follow in its wake (what with the demands for organs, medical research, cost minimisation and so on) play no role in the authors reasoning about killing the newborn. the examples of nuremberg (and human rights violations by states even now for example, infanticide and much more in china), are a reminder of how medical research and organ demand can incentivise homicide. the alder hey scandal is testimony to the temptation among medical professionals to use illicit means to achieve perceived progressive ends with long - term loss of public trust. if this is true of organ retention, we can expect the same to be true of infanticide once legalised. cost saving, litigation and payout minimisation, bed clearing, body parts and political malthusianism are all matters that incentivise infanticide and other forms of euthanasia. as i have noted elsewhere,1822 in this environment failures of transparency, (ie, to say, feelings of confusion resulting in wholesale deception, among medical professionals), even in states where euthanasia is legal, becomes both pragmatic and inevitable. belgium is now well known for its failures of transparency with only 52.8% of acts of euthanasia reported to the authorities in flanders.24 these reasons against killing the child, turning carers into killers and fuelling an industry in death, in addition to the longer - term implications mentioned earlier nowhere enter into the authors calculus emphasising the problem of arbitrariness and circularity that characterises the analysis. these pragmatic considerations notwithstanding, it is not at all clear why third - party feelings of distress should trump the life of a newborn baby however disabled. to judge that the treatment involved in his or her care is futile, too expensive or too burdensome to the child is to make a coherent point about the quality of the treatment involved. it need not degenerate into a personistic, actualistic, arbitrary and unjustly discriminatory evaluation of the child 's very life. nor does the analysis leave us blind to the vulnerability of human life, the need we have for the care of others, the importance there is in not incentivising and institutionalising this most serious of offences. as outlined earlier, the infanticide proposal is not new in the academic world where vested medical, pecuniary and political interests define and finance academic debate, but emerging practices, like the self - styled groningen protocol demonstrate that the discussion is far from theoretical. by way of illustration, the dutch experience is useful evidence of the slippery slope from voluntary to involuntary paediatric euthanasia, from euthanasia for the terminally ill to those depressed and lonely people whose elimination is now being proposed by progressive dutch physicians as socially advantageous, from right to die to duty to die. despite being practised by the ancient romans, greeks and spartans, however, infanticide is still largely outlawed in the west, in part, because of judeo - christian prohibitions on the practice. tertullian, for example, notes and abhors the custom in his apology. the practice remains illegal in most jurisdictions in the world. in the new england medical journal25 verhagen and sauer specify that where babies are suffering and parents are questioned by medics and give consent, there should be due diligence with respect to any act of infanticide by lethal injection. aside from highlighting the accuracy of warnings about the logic of voluntary euthanasia naturally implying involuntary euthanasia of those regarded unfit, there are further difficulties with this eliminating suffering by eliminating sufferers approach to medical care.2629 opponents of this new practice note that sound paediatric care does not mean that pain and suffering should go untreated, only that active euthanasia is not the proper treatment. eric kodish, for example, affirms that : high doses of analgesia along with other therapies designed to palliate symptoms are the appropriate alternative to the swift option of infanticide. again the issue of withholding treatment is, all things being equal, distinct from the act of infanticide, particularly when the child is dying of independent causes or where the treatment is over - burdensome or too expensive. as kodish rightly points out : the moral justification for withholding is that the burdens of the technology outweigh the benefit to the infant, a very different premise from the active killing of the infant to end... suffering.... caring for seriously ill infants and children is never compatible with active euthanasia(p. furthermore, the idea that substituted parental consent should determine whether or not the child should be given a lethal injection to end its life is predicated on the idea that parents can not wrong or abuse a child. particularly where parents are ill - informed or mistaken as to the diagnosis or prognosis of the child, the test is manifestly all the more obviously objectionable. for euthanasia to be an expression of the will of the party seeking it, that expression of will should not be substituted by other third parties, however well - meaning or closely related. verhagen and sauer 's final arctic requirement that the act of infanticide be performed with due diligence is another question - begging condition. there is every reason to doubt whether paediatric care should involve the technical skills associated with homicide. indeed, it is this prudential consideration that informs the hippocratic oath in its unadulterated form. the world medical association (resolution on euthanasia adopted general assembly 2002) condemns euthanasia by lethal injection, and urges all national medical associations to refrain from complicity in such practice even if domestic law professes to legalise it. the hippocratic oath, at least in its ancient form : i will give no deadly medicine to any one if asked, nor suggest any such counsel, rejects it. the parliamentary assembly of the council of europe has issued a declaration in which it asserts that [e]uthanasia, in the sense of the intentional killing by act or omission of a dependent human being for his or her alleged benefit, must always be prohibited. these international instruments, the equal dignity principle and principles of traditional morality notwithstanding, the institutionalisation of infanticide and its normalisation among paediatricians involves gravely unacceptable outcomes. giubilini and minerva 's infanticide proposal is predicated on actualism and personism. according to these related ideas, human beings achieve their moral status in virtue of the degree to which they are capable of laying value upon their lives or exhibiting certain qualities, like rationality, self - consciouness and autonomy or being desirable to third parties. actualism and personism are predicated on arbitrary and discriminatory notions of human moral status. our propensity to sleep, become unconscious, pass out and so on, demonstrates that we often exhibit our status as potential persons. dependency on one another is a function of our humanity and involves natural rights, obligations, virtues and necessities. whether or not we are exercising our capacities or abilities, are in pain or desirable to third parties are insufficient grounds on which to judge human moral value. so too are qualities like evincing rationality, capacity for self - reflection or moral sensibility, characteristics that exclude many professors of moral philosophy for a lifetime. even bearing in mind general demands that treatment not be futile, over - burdensome or overexpensive, the equal dignity principle, which affirms the dignity of all human beings however disabled, suggests human dignity does not fluctuate and should not be regarded as fluctuating. again, substituted consent to the euthanasia of third parties, whether unwanted or disabled babies or otherwise, and the implications of institutionalising non - voluntary or involuntary euthanasia in the context of financial, research and political interests in the practice suggest there are broader reasons to reconsider the infanticide proposal. whatever our commitment to the principle of equal dignity, we can predict that eroding the value of human life by normalising infanticide carries with it significant destructive long - term implications for the medical and legal professions, for families and societies, on top of the obvious danger that it presents to the newborn. to elevate some of these implications whilst ignoring others demonstrates the self - serving, arbitrary and irrational nature of efforts to institutionalise euthanasia. | alberto giubilini and francesco minerva 's recent infanticide proposal is predicated on their personism and actualism. according to these related ideas, human beings achieve their moral status in virtue of the degree to which they are capable of laying value upon their lives or exhibiting certain qualities or being desirable to third - party family members. this article challenges these criteria, suggesting that these and related ideas are rely on arbitrary and discriminatory notions of human moral status. our propensity to sleep, fall unconscious, pass out and so on, demonstrates that we often exhibit our status as potential persons who are not in the condition of attributing any value to their own existence. our abilities, age and desirability can and do fluctuate. the equal dignity principle, distinguished in turn from both the excesses of vitalism and consequentialism, is analysed and defended in the context of human rights logic and law. the normalisation of non- and involuntary euthanasia, via such emerging practices as the self - styled groningen protocol, is considered. substituted consent to the euthanasia of babies and others is scrutinised and the implications of institutionalising non - voluntary euthanasia in the context of financial, research and political interests are considered. the impact on the medical and legal professions, carers, families and societies, as well as public attitudes more generally, is discussed. it is suggested that eroding the value of human life carries with it significant destructive long - term implications. to elevate some, often short - term, implications while ignoring others demonstrates the irrational nature of the effort to institutionalise euthanasia. |
a 10-month - old domestic shorthair cat was evaluated for severe esophagitis and protracted vomiting and regurgitation secondary to a sliding (type i) hiatal hernia. the hernia and concurrent upper airway obstruction (nasopharyngeal polyp) were diagnosed with a multi - modality approach, including thoracic and abdominal radiographs, abdominal ultrasound, computed tomography and endoscopy. following unsuccessful attempts at medical management, lower esophageal incompetence was successfully treated by employing a combination of surgical techniques, including herniorrhaphy, esophagopexy and modified (floppy) nissen fundoplication. a multi - modality imaging approach was valuable in completely assessing the extent of this cat s disease. although an untraditional approach, the authors report herein the first clinical description of the use of combined surgical techniques with the floppy nissen fundoplication technique (an antireflux procedure) in a cat. this procedure was used as a first - line surgical technique in this cat with severe lower esophageal incompetence, and may be a viable option for cases non - responsive to other therapeutic interventions. further investigation of this surgical technique is warranted. hiatal hernia, defined as protrusion of abdominal contents through the esophageal hiatus into the thorax, is a complex entity in humans and animals, with a multifactorial etiology and pathophysiology. the latter leads to decreased movement resistance of the gastric cardia from the abdomen to the thorax. intrinsic and extrinsic components of the lower esophageal sphincter prevent regurgitation of gastric contents. disruption or displacement of the lower esophagus or gastroesophageal junction a 10-month - old male intact, 1.4 kg, domestic shorthair cat was evaluated for chronic vomiting and poor body condition despite polyphagia. clinical signs began at 10 weeks of age and included lethargy, fever, stunted growth and abdominal distention occurring within minutes of eating. pyrexia and lethargy resolved with empirical treatment with augmented amoxicillin (14 mg / kg orally q12h). famotidine (0.5 mg / kg orally q24h) and metoclopramide (0.3 mg / kg orally q8h) were added to the treatment plan. results of a serum biochemistry analysis, complete blood count (cbc), serum bile acids, fecal flotation and thyroid testing were normal. in addition, a well - marginated, 2 cm 3 cm, soft tissue opacity, caudal esophageal mass was seen. a mural, extramural or luminal caudal esophageal lesion was suspected, with preliminary differentials including esophageal foreign body, hiatal hernia, congenital megaesophagus and esophageal diverticulum. upper gastrointestinal endoscopy revealed gastroesophageal mucosal hyperemia, distal esophageal dilation and severe esophageal reflux. omeprazole (0.9 mg / kg orally q24h) and small, frequent feedings were added to the treatment protocol. the patient was referred for further diagnostic investigations after 6 months of poor response to medical management. a body condition score of 3/9, stunted growth and a body weight of 1.4 kg were noted on physical examination. repeat serum bile acid concentrations were marginally increased at 15.7 mol / l (reference interval [ri ] 05 mol / l). a cbc demonstrated a mature neutrophilic leukocytosis (42.2 10/l leukocytes, ri 4.215.6 10/l ; 34,943 repeat orthogonal thoracic radiographs showed an intermittently present, large, 5 cm 2 cm 3 cm, well marginated, soft tissue opacity within the caudal esophagus with severe gastric distension (of admixed gas / fluid opacity). during imaging the patient demonstrated signs of respiratory distress (increased respiratory effort, rate and stertor), which resolved with discontinuation of applied manual restraint and supplementation of flow - by oxygenation. the increased respiratory effort noted during thoracic imaging acquisition corresponded to a dynamic change in position of the mid- to caudal sternebral segments on the radiographs (figure 1). thus, combined radiographic and clinical findings raised the suspicion of sliding hiatal herniation secondary to upper airway obstruction. given the propensity toward respiratory decline and the dynamic caudal esophageal mass effect, computed tomographic (ct) imaging was deemed the best - suited modality to evaluate the upper airway, esophagus and stomach. note the presence of gas within the soft tissue mass at the level of the caudal esophagus and the dorsal displacement of the mid - to - caudal sternebral segments due to increased inspiratory effort and respiratory distress. note how the patient s sternebral deformity and the mass at the level of the caudal esophagus resolve with assisted ventilation. dynamic changes in the sternebral segments coupled with increased respiratory effort raised suspicion of an upper airway obstruction, which was likely exacerbated by the stress of restraint for thoracic imaging abdominal ultrasonography revealed a non - motile, fluid - filled stomach and normal pyloroduodenal junction. the portal vein to aortic ratio was normal (0.89 ; ri 0.81.0), making a portosystemic shunt unlikely. although the cat s episode of dyspnea was brief, it prompted a prioritized diagnostic evaluation of the upper airway with contrast - enhanced ct of the head. given the intermittent radiographic mass lesion within the caudal thorax, ct of the thorax was also performed followed by a sedated examination of the oropharynx. premedication, anesthetic induction and maintenance following intubation were maintained with butorphanol (0.1 mg / g i m), midazolam (0.2 mg / kg i m), propofol (2 mg / kg iv) and an isoflurane oxygen admixture, respectively. contrast - enhanced ct demonstrated a non - enhancing, soft tissue density within a thickened and expansile left tympanic bulla (figure 2). an obstructive, 1 cm diameter, similarly minimally enhancing, ovoid mass was identified within the nasopharynx. ct abnormalities were consistent with a nasopharyngeal polyp extending from the left tympanic bulla / eustachian tube to the nasopharynx with secondary hiatal herniation. computed tomography of the head. (a) (b) note the soft tissue density within the expansile left tympanic cavity with thickening of the osseus bulla. note the soft tissue densities within the left tympanic cavity and the nasopharynx computed tomography of the thorax. (a) sagittal, (b) transverse and (c) dorsal, curved planar reconstructed images demonstrating rugal folds within the thoracic esophagus just cranial to the hiatus. note also the diffuse gas distention of the intrathoracic esophagus on all images following imaging, the nasopharyngeal mass (presumed to be a polyp) was extracted via gentle traction and submitted for histopathologic confirmation. the patient recovered without complication and was discharged with buprenorphine (0.015 mg / kg orally q46h), omeprazole (0.9 mg / kg orally q24h) and cisapride (1.8 mg / kg orally q8h). lower esophageal sphincter incompetence and complications secondary to a sliding hiatal hernia were suspected, with consideration given to esophageal hypotonia and reflux esophagitis. surgical correction of the hiatal hernia was recommended given continued poor response to medical management. thirty - three days following polyp removal, the cat was admitted for endoscopy, esophagoscopy, gastroscopy and hiatal hernia repair using a combined approach and employing an antireflux procedure. premedication, anesthetic induction and maintenance following intubation were achieved with diazepam (0.5 mg / kg iv), buprenorphine (0.01 mg / kg i m), propofol (3 mg / kg iv) and an isoflorane oxygen admixture, respectively. lactated ringer s (10 ml / kg / h iv) and a fentanyl continuous rate infusion (0.35 g / kg / min) were administered. endoscopy demonstrated severe esophagitis, scarring and erythema of the lower esophageal mucosa, and grossly normal gastric mucosa (figure 4). (a, b) note the severe esophagitis with irregularly marginated, multifocal erosions. a significant amount of bilious reflux was noted. (d) note the mural twist at the cardia just caudal to the diaphragm. tented margins of the lower esophageal wall ventral midline celiotomy revealed a large, flaccid stomach and marked dilation of the esophageal hiatus. blunt dissection of the esophagus, herniorrhaphy (with 3 - 0 simple interrupted polypropylene simple interrupted diaphragmatic sutures) and diaphragmatic esophagopexy (with 4 - 0 polydiaxione simple interrupted sutures) were performed followed by a floppy nissen fundoplication. the size of the tube was such that it was able to distend the esophagus to some degree but able to be passed easily to the stomach, as per the previously described modified technique. the gastric wall was mobilized 360 degrees dorsomedially around the intra - abdominal esophagus and sutured to itself without tension, using 3 - 0 polypropylene simple interrupted sutures. the cat was kept in the hospital for observation and pain management for 48 h. a fentanyl continuous rate infusion (0.2 g / kg / min) was administered for postoperative pain management. two weeks postoperatively the cat was re - evaluated at the referral center for suture removal. the owner reported three episodes of non - productive retching, but vomiting had resolved. the referring veterinarian provided a 6 week postoperative follow - up report. at that visit the cat weighed 2.1 kg, having gained an additional 0.4 kg, and the owner noted marked improvement in activity levels at home. although the owner reported overall improvement of regurgitation, episodes with retching and production of small amounts of brown, frothy fluid persisted and were noted five times weekly, usually within 3 h of consuming a meal. the cat progressively continued to thrive at home and gain weight, with reports from the owner of once - daily non - productive retching in the 616 week interim following surgery. between this time period, the referring veterinarian provided wellness maintenance with an unchanged regimen of prokinetics and proton pump inhibitors. at the 16 week follow - up at the referral center, the cat s physical examination was normal. this weight gain was attributed to the surgical intervention given that the cat s body weight had nearly doubled in the 16 week postoperative period. thoracic radiographs showed a persistent, mild increase in soft tissue opacity in the region of the caudal esophagus (figure 5). endoscopy revealed resolved esophagitis with mild reflux within the caudal esophagus and no gross esophageal or gastric mucosal abnormalities. irregularity of the lower esophagus was considered related to the surgery (figure 4). note the decreased size of the thoracic cavity compared with figure 1 (a). note the lack of gas within the esophagus and the resolution of the previously described soft tissue mass at the level of the caudal esophagus. mild, normal volume of mixed gas fluid opacity is present within the fundus. note also the normal contour of the ventral thoracic body wall with the more normally positioned sternebral segments due to resolved respiratory distress. note the poorly defined increase in soft tissue opacity just dorsal to the caudal vena cava and cranial to the diaphragm. gas opacity persistent within the fundus and the normal mixed gas fluid opacity within the pylorus. findings are consistent with the surgically induced malposition of the stomach follow - up by email occurred bi - monthly for 24 months postoperatively, then every 6 months up to 48 months postoperatively. over that time the owner reported that the cat had non - productive retching of gradually decreasing frequency (episodes stabilized to 23 times weekly rather than after every meal). reduction of prokinetic therapy (cisapride 1.8 mg / kg) from every 8 h to once daily, as per the referral veterinarian, had no effect on retching frequency. the patient s retching frequency continued to slowly decrease ; by 48 months following surgery no retching episodes were noted and the cat was thriving. a multi - modality imaging approach and combined surgical techniques were used in the juvenile cat of this report in order to diagnose and treat a sliding hiatal hernia. even though respiratory issues were not a primary complaint, restraint for radiographic imaging precipitated a respiratory episode. congenital or acquired hiatal herniations were the primary differentials for the soft tissue opaque mass in the caudodorsal thorax seen on the same study. ct was supportive of this clinical suspicion, revealing a large obstructive mass in the nasopharnynx. upper airway obstruction has been reported in cases of dogs with hiatal hernia, where it is speculated that increased intraesophageal and intrapleural pressure can pull the esophagus and stomach into the thorax. further evaluation of the upper and lower airway is indicated in patients with suspected respiratory disease. in the patient of this report, ct provided details regarding the inappropriate presence of mixed soft tissue and gas densities with rugal fold architecture at the hiatus. the diagnosis of hiatal hernia typically relies heavily on signalment, history, clinical signs and diagnostic imaging. widely available modalities such as radiographs, contrast esophagraphy, fluoroscopy and ct can aid in the diagnosis of the sliding hernia. barium esophagraphy can be especially useful in assessing esophageal motility and caudal esophageal sphincter function. as was the case with the cat of this report, endoscopic evaluation was helpful in evaluating the degree of mucosal changes associated with secondary reflux esophagitis, as well as aiding in medical and presurgical treatment planning. although not a traditional approach, ct was instrumental in a rapid diagnosis for both the cause of the upper airway obstruction and the displaced gastric anatomy through the hiatus. abdominal ultrasonography demonstrated gastric dilation and functionally reduced gastric motility, while ruling out other causes of persistent regurgitation in young cats (such as pyloric stenosis). endoscopy confirmed the presence of esophagitis, gastritis and gastric flaccidity, and also prescreened the patient for the presence of contraindicated disease for the employment of the modified nissen fundoplication technique. repeat endoscopy was valuable in documenting resolved esophagitis. in the private practice setting, this degree of imaging may not be economically or technically feasible. at a minimum, we recommend survey thoracic and abdominal radiographs, and a sedated oropharyngeal exam prior to referral for advanced imaging and surgical intervention of cats with suspected hiatal hernias. in addition, we recommend endoscopy and/or dynamic fluoroscopic evaluation of esophageal motility for cats with evidence of chronic reflux esophagitis and poor response to medical management. removal of the nasopharyngeal polyp was prioritized to alleviate the risk of respiratory decompensation. following a poor response to medical management and protracted episodes of retching and regurgitation, lower esophageal sphincter incompetence due to chronic esophageal reflux was highly suspected and confirmed with presurgical endoscopic evaluation of the lower esophagus. surgical correction of the sliding hiatal hernia with an antireflux procedure, a modified (floppy) nissen fundoplication, was then performed. floppy nissen fundoplication successfully reduced clinical signs (vomiting and weight loss) in this cat in the short term ; in the long term, the cat s clinical signs completely resolved. a sliding hiatal hernia, also called a type i hernia or axial hernia, is described as cranial displacement of the abdominal esophagus, esophageal junction and, sometimes, a portion of the stomach into the thoracic cavity. congenital sliding hiatal hernias in young patients and acquired sliding hiatal hernias in adults are the most common forms seen. when sliding hiatal hernias occur in young animals between the ages of 2 and 4 months, they are usually described as congenital. however, hiatal herniation is a complex entity in humans and animals with an incompletely understood, multifactoral etiology and pathophysiology. given the large obstructive nasopharyngeal mass in the patient of this report, an acquired sliding hiatal hernia was suspected, despite the patient s young age. congenital hiatal herniation was also considered, and coincidental occurrence of the polyp could not be ruled out. patients with sliding (type i) hiatal herniation usually present for emesis and regurgitation. other clinical signs can include ptyalism, nasal discharge, coughing, dysphagia and hematemesis. stunted growth, recurrent gastric dilations, and clinical signs referable to esophagitis and gastroesophageal reflux, such as regurgitation with or without blood, aerophagia, anorexia and weight loss, are often reported. disruption or displacement of the position of the esophagus, gastroesophageal junction and lower esophageal sphincter contribute to sphincter incompetence, leading to gastroesophageal reflux and esophagitis. experimentally, the production of esophageal reflux in cats has been shown to lower gastroesophageal sphincter pressure, resulting in a chronic cycle of reflux and esophagitis. hiatal herniation may be associated with, or exacerbated by, respiratory disease, neuromuscular compromise, megaesophagus, esophageal motility disorders and obesity. as with the cat described in this report, respiratory difficulty and obstruction can be a predisposing factor in the occurrence of hiatal hernia both in dogs and cats. entrapment of gastric fluid orad to the hiatus and loosening of the phrenoesophageal attachment may also contribute to movement of the intrinsic component of the lower esophageal sphincter. patients with subclinical sliding hiatal hernia may not require medical or surgical intervention. in patients with clinical signs, medical management of gastroesophageal reflux should be attempted for a minimum of 30 days when reasonable, but is often unsuccessful. small - volume, frequent feedings of a low - fat diet may aid in gastric emptying and decreased acid production. changing the consistency of the food and feeding from an elevated position may also contribute to esophageal clearing. drug therapy aims to decrease acid production, increase gastric emptying and increase the tone of the esophageal gastric junction. frequently employed pharmaceuticals include sucralfate, antacids, h2 blockers, prokinetic agents and proton pump inhibitors. surgical intervention is recommended if clinical signs (especially regurgitation and high - volume reflux) persist despite medical management. the occurrence and severity of reflux in patients with hiatal hernia is multifactorial. in immature animals, as with the cat of this report, reflux may be significantly increased because of an immature or poorly developed lower esophageal sphincter. reduction of the hiatus alone rarely results in complete resolution of the clinical signs. antireflux procedures are considered superior in patients with gastroesophageal reflux and may obviate the inconvenience and expense of lifelong medical management. as the majority of clinical signs associated with sliding hiatal hernias are related to gastroesophageal reflux, a procedure that is effective in reducing or eliminating reflux has merit. techniques for repairing hiatal hernias in animals vary and there is much debate in the veterinary community regarding which techniques, or combination thereof, are best depending on the type of hernia. described techniques include herniorrhaphy, gastropexy, esophagopexy, antireflux procedures and feeding tubes, or a combination thereof. corrective surgical techniques have been described and include different methods of maintenance of hernia reduction and manipulation of the gastroesophageal sphincter. the nissen fundoplication was developed to treat reflux in humans. in pediatric patients, the modified fundoplication used in conjunction with a gastrostomy tube is commonly used to treat esophageal achalasia or hiatal hernia with severe gastroesophageal reflux. an antireflux procedure combined with herniorrhaphy and esophagopexy were chosen in this patient in an attempt to prevent further reflux, which was causing the clinical signs. in cats, modified nissen fundoplication has experimentally been the most effective procedure in restoring lower esophageal pressure to normal, and the only procedure that helped prevent reflux. the other developed severe necrotizing gastritis in the immediate postoperative period but recovered with medical management. antireflux procedures in small animals have previously been challenged, with opponents arguing that primary lower esophageal sphincter incompetence, the main indication for an antireflux procedure in humans, does not occur in small animals. however, lower esophageal sphincter incompetence is thought to be secondary rather than primary in humans. some authors propose that these techniques are surgically demanding, lend themselves to higher complication rates, and that the rationale for pursuing sphincter enhancement techniques is a failure of response to previous repositioning efforts. given the low number of cases, there is a lack of evidence clearly demonstrating that repositioning techniques lead to irreversible hiatal attenuation and prevention of reflux in the long term. additionally, performance of techniques such as gastropexy could potentially compromise performance of a subsequent antireflux procedure. antireflux procedures have not been reported following failure of repositioning techniques in the veterinary literature. in humans, studies have shown that some patients having no reflux preoperatively developed reflux postoperatively when antireflux procedures were not performed. in these cases, the development of reflux may have been attributable to dissection of the gastroesophageal junction, reduction of the stomach or insufficient crural repair. complete evaluation for a functional esophageal motility disorder was not performed prior to or after surgery with dynamic, fluoroscopic, positive contrast esophagography. in patients with esophageal motility disorders, however, an excellent outcome can be achieved with modified nissen fundoplication, regardless of etiology. the procedure should be avoided in cases with aperistalsis, tight fibrous strictures and short esophagus syndrome. one previous study suggested that the absence of megaesophagus on survey radiography is consistent with a functional esophagus, while other studies emphasize the importance of evaluating for functional motility disorders with a barium esophagram or dynamic fluoroscopic esophagography. additional limitations of this report include lack of esophageal and gastric histopathology and full gastrointestinal panel serum analysis (b12/folate). although this cat was quite young, the possibility of primary inflammatory bowel disease as a contributing factor was considered. advanced imaging was an invaluable tool in the diagnosis, management and employment of combined surgical techniques, which provided an excellent outcome in a young cat with a sliding hiatal hernia, severe lower esophageal incompetence and concurrent reflux esophagitis. although presumed to be secondary to upper airway obstruction, the sliding hiatal herniation may have been congenital in nature. failure of response to medical management, degree of reflux noted on endoscopy, lack of a functional lower esophageal sphincter, suspicion of gastric hypomotility and questionable esophageal functional motility prompted the consideration of combined surgical techniques to include an antireflux procedure. surgical correction consisted of herniorrhaphy, esophagopexy and modified (floppy) nissen fundoplication. regardless of the underlying etiology, advanced imaging and a multi - modality approach can assist in evaluation of candidacy for antireflux procedures. further studies are needed to investigate short- and long - term outcomes of the floppy nissen fundoplication antireflux procedure and combined herniorrhaphy / organopexy in feline and canine cases of sliding hiatal hernia. | case summarya 10-month - old domestic shorthair cat was evaluated for severe esophagitis and protracted vomiting and regurgitation secondary to a sliding (type i) hiatal hernia. the hernia and concurrent upper airway obstruction (nasopharyngeal polyp) were diagnosed with a multi - modality approach, including thoracic and abdominal radiographs, abdominal ultrasound, computed tomography and endoscopy. following unsuccessful attempts at medical management, lower esophageal incompetence was successfully treated by employing a combination of surgical techniques, including herniorrhaphy, esophagopexy and modified (floppy) nissen fundoplication.relevance and novel informationa multi - modality imaging approach was valuable in completely assessing the extent of this cat s disease. although an untraditional approach, the authors report herein the first clinical description of the use of combined surgical techniques with the floppy nissen fundoplication technique (an antireflux procedure) in a cat. this procedure was used as a first - line surgical technique in this cat with severe lower esophageal incompetence, and may be a viable option for cases non - responsive to other therapeutic interventions. further investigation of this surgical technique is warranted. |
the current criteria for diagnosis of amyotrophic lateral sclerosis (als) require neurophysiological evidences of ongoing denervation, defined by fibrillation potentials (fibs) or positive sharp waves, and of chronic partial reinnervation, involving enlarged, unstable motor units with a reduced interference pattern. fibs are detected as long as denervation - reinnervation processes take place, and their assessment is considered a sensitive method to evaluate disease progression and a useful research tool (see, for instance,). at later stages of disease they fade owing to replacement of muscle fibres with fibrous tissue. in mammalian denervated muscle fibres, fibrillations arise because spontaneous activation of voltage - gated na (nav) channels triggers action potentials and contractions. disappearance of fibs has been reported in one als patient receiving fentanyl and propofol during surgery, two anaesthetics that block nav channels [7, 8 ]. riluzole is a well - established blocker of neuronal nav channels, but it also affects other channels in nonneuronal tissues, for instance, muscle acetylcholine receptor channels [912 ]. riluzole also causes a mild block of recombinant and native muscular voltage - gated na current (ina) [10, 13 ], although at concentrations higher than those attained in patients ' plasma. in myotubes given these premises, riluzole might affect fibs in als patients, but this point has received little attention to date, even if riluzole is the only approved drug for als treatment. therefore, we undertook a study of riluzole action on fibs, aimed at filling this gap. human muscle satellite cells can be cultured in vitro in the absence of nerve, and myotubes adequately represent denervated muscle fibres. moreover, satellite cells in vitro preserve traces of their in vivo environment including denervation - induced alterations. satellite cells derived from als patients display a reduced myogenic potential but form multinucleated myotubes (als myotubes ;). as commonly observed for primary human myotubes [1921 ], als myotubes do not contract spontaneously. in the context of nav channels, both denervated muscle fibres and cultured myotubes express nav1.5 channels in addition to nav1.4, the only form present in innervated muscle fibres [22, 23 ]. thus, als myotubes provide a valuable experimental model with functional properties conditioned by donor health status. we therefore first evaluated the effect of riluzole on muscle ina and action potentials in vitro and then verified our conclusions in vivo, in a group of patients. in als myotubes, riluzole affected the amplitude and voltage dependence of ina. as a consequence, spike amplitude and rate of rise were reduced, but action potential firing was not suppressed, suggesting that riluzole would not prevent muscle fibrillations. indeed, fibs were routinely detected during riluzole administration in als patients and brief suspension of the treatment had no univocal effects on fibs frequency. all our data suggest that riluzole, in spite of partially blocking muscle nav channels, does not interfere with muscle fibs. human myotubes were grown from satellite cells derived from muscle biopsies performed at the als centre of policlinico umberto i, sapienza university. all patients gave written informed consent to use part of the biopsy material for the research project entitled study of nicotinic acetylcholine receptor in muscle tissue of als patients, approved by the human subjects ethical committee of policlinico umberto i. satellite cells were obtained immediately after biopsy as described [11, 12 ]. frozen aliquots were thawed for the present study and propagated as previously reported [11, 12 ]. differentiation was induced at 50% confluence by switching to low - serum differentiating medium (dulbecco 's minimum essential medium plus 2% horse serum and penicillin / streptomycin). whole - cell patch - clamp recordings were made at room temperature (2327c) using an axopatch 200b amplifier (molecular devices, union city, ca, usa), driven by pclamp 9 (molecular devices). cells were continuously superfused using a gravity - driven fast exchanger perfusion system (rsc-200, bio - logic, france). external solution contained (mm) 140 nacl, 2.8 kcl, 2 cacl2, 2 mgcl2, 10 hepes - naoh, and 10 glucose, ph 7.3. addition of 4-aminopyridine (2 mm) to block voltage - gated k channels did not affect ina. patch pipettes (25 m) contained (mm) 130 cscl, 10 nacl, 5 bapta, 10 hepes - koh, 2 mg - atp, and 2 mgcl2, ph 7.3. to record action potentials, recordings were considered only if patch series resistance was less than 8 m and compensated by 9599%. riluzole - induced shifts of activation and inactivation curves were unaltered if voltage drop over uncompensated access resistance (ru) was kept into account correcting the test potential (vm) as vcorr = vm ruina and using vcorr in data analysis. current - voltage curves were obtained applying membrane potential steps (5 ms at 1 s interval) in 5 mv increments from 65 mv to + 60 mv, from a steady holding potential of 80 mv. fast inactivation curve was obtained using a two - pulse protocol : prepulse potential (20 ms) was changed from 140 mv to 15 mv in 5 mv increments, followed by a test pulse to 10 mv (5 ms at 1 s interval), as previously done in human muscle fibres. current reversal potential (ena), estimated from plot of peak ina versus vm, was 64 1 mv (n = 30), as predicted by nernst equation (66 mv). conductance was calculated as g = ina/(vm ena) and normalized to the maximal value gmax. values of g / gmax were then fitted with boltzmann equation : (1)ggmax=11+e(vva)/ka, where va is the potential at which conductance is half of maximal value and ka is the slope factor. fast inactivation relationships were constructed plotting current amplitude i, normalized to maximal value imax, versus prepulse potentials and fitting a boltzmann equation to data points : (2)iimax=11+e(vvi)/ki, where vi is the potential at which current is half of maximal value and ki is the slope factor. action potentials were recorded under current clamp conditions using stimuli of increasing amplitude (from 0.2 to 1 na) at 1 s intervals. phase plots, representing changes in membrane potential with time (i.e., the first derivative of membrane potential) versus the value of membrane potential at the corresponding time, were built for each action potential. two data sets were considered statistically different when p 2 on the modified ashworth scale) ; absence of psychiatric disorders or cognitive impairment at first evaluation ; no concomitant therapy with psychoactive drugs. muscle strength of lower limbs was assessed with the medical research council (mrc) score for muscle strength, an ordinal scale ranging from 0 (absence of movement) to 5 (normal contraction against full resistance) that quantifies muscle weakness in isolated muscles or muscle groups. five muscle groups in lower limbs are tested : hip flexors, knee extensors, knee flexors, ankle plantar flexors, and ankle dorsiflexors. an electromyographic instrument (system plus evolution 1.04.0104) employing a one - channel montage (sensitivity 50 v / division ; low frequency filter 20 hz ; high frequency filter 5 khz) was used to detect electrical activity. during the electrophysiological procedures, needle electromyographic signals were recorded from the right anterior tibialis muscle at a site approximating the demarcation between the proximal one - third and distal two - thirds of the muscle. three consecutive needle insertions (10 s each) with the same depth of insertion of needle (1.5 cm) per session were performed in all patients. a reference landmark by indelible pen allowed recording at the same muscle site in the second session. right tibialis anterior compound muscle action potential (cmap) was evoked by peroneal nerve stimulation. the stimulating cathode was placed at the posterior - lateral area of the fibular head. the point for the recording surface electrodes was placed 8 cm from the cathode over the tibialis anterior. the latency of cmap was measured from the stimulus artefact to the onset point, and the amplitude was determined from baseline to the highest negative peak. voltage - gated na current (ina) was recorded in all multinucleated myotubes (38 days differentiation) examined. current density was 0.27 0.03 na / pf (n = 22) with the largest current obtained at test potentials of 20 to 0 mv (figure 1(a)). current was reduced to 28 3% (n = 10) of control value by ttx (1 m, not shown), as expected in myotubes which express a mixture of nav1.4 and nav1.5 channels, with half - inhibitory ttx concentration of 10 nm and 3 m, respectively, thus confirming that recorded signals were bona fide na currents. applying riluzole 1 m, a clinically relevant concentration, 30 s before stimulation, total ina amplitude was 79 2% (n = 22) of untreated value (figure 1(a)). in all cells tested, activation and steady - state fast inactivation curves were modified in the presence of riluzole. the activation curve had a depolarizing shift (figure 1(b)), with a mean increase for va of 2.4 0.3 mv (n = 22, p < 0.0004), and ka was significantly increased by 0.6 0.1 mv (n = 22, p = 0.0004). steady - state fast inactivation curves had a hyperpolarizing shift (figures 1(c) and 1(d)), with vi decreasing by 8.7 1.5 mv (n = 7, p = 0.001) and ki by 3.5 0.5 mv these riluzole - induced shifts in activation and fast inactivation of ina might reverberate on action potential generation, reducing spike amplitude (because of enhanced fast inactivation) and rate of rise (due to reduced current amplitude). we therefore recorded action potentials evoked by depolarizing currents of increasing amplitude, using current clamp mode. in these myotubes, membrane resting potential ranged between 40 and 25 mv, so hyperpolarization (to 89.0 1.4 mv, n = 5) was required to elicit action potentials. depolarizing currents evoked overshooting spikes in all myotubes tested, but amplitude and rate of rise were reduced in the presence of riluzole (figure 2(a)). in the five myotubes tested, spike peak and rate of rise were 29.7 5.1 mv and 119 18 mvms under control conditions and 21.3 5.3 mv and 89 17 mvms, in the presence of riluzole (p 0.002 by paired student 's t - test). action potential peak was delayed by 1.8 0.7 ms (n = 5 ; p = 0.03). furthermore, phase plots showed a small shift in threshold potential, compatible with the depolarizing shift of va measured in each myotube (figure 2(b)). thus, riluzole reduces excitability of myogenic cells but does not prevent action potential generation in response to depolarizing stimuli, suggesting that it might be ineffective on muscle fibrillations. all patients were treated with riluzole (average treatment duration, 13.8 months) and suspended treatment for one week for this study. this time is adequate for riluzole washout, estimated to be complete within about 40 hours of withdrawal. multiple fibs were detected in all patients ' anterior tibialis muscle (figure 3). across the 3 insertions performed, their number had small fluctuations in all but patients 5 and 7, which suggests that variability due to needle position is limited. in the second recording session, one week after riluzole withdrawal, clinical status and cmap amplitude were unchanged for all patients (table 1), indicating that no relevant alteration in muscle innervation took place. fibs continued to be present in all patients, and variability between insertions did not change. when compared to the first recording session, the total number of fibs changed by less than 25% in six patients ; in the other 5, changes ranged between a 55% increase (patient 2) and a 70% decrease (patient 1). overall, there was no statistically significant variation in the number of fibs upon riluzole treatment suspension (p = 0.69) (table 1). the effect of riluzole withdrawal showed no correlation with patients ' age, sex, disease duration, or fibs frequency at baseline (data not shown). in this paper we studied the effect of riluzole on voltage - gated sodium channels in human myotubes and on fibs in als patients. in vitro, we show that riluzole at a therapeutically used concentration reduces ina and shifts the voltage dependence of gating. as a consequence, action potentials evoked by depolarizing currents have lower amplitude and reduced rate of rise. these results clearly indicate that riluzole acts on channels present on muscle fibres. the therapeutic implications of this nonneuronal effect, if any, are presently unknown. for instance, the documented partial block of muscle nav channels may result in dampened spontaneous activations of these channels and limit fibs occurrence in patients ' denervated muscle fibres. here we report that fibs were consistently recorded during riluzole treatment in als patients, a clear indication that riluzole does not suppress muscle fibrillations. we were unable to detect univocal effects on fibs upon riluzole withdrawal in the group of patients considered. the in vitro findings here reported indicate that riluzole has a small effect on action potential firing. data obtained in patients do not disclose even a trend in the change of fibs frequency upon riluzole withdrawal. thus, extension of the in vivo study to a larger number of patients appears of little significance, also considering the discomfort of the procedure and the burden of repeated hospital access for heavily disabled patients. none of the previous studies showing that nav channel blockers can suppress fibs in vivo [5, 6 ] assessed the plasma concentration of the drugs, so it is not possible to correlate disappearance of fibs with the extent of nav channels blockade. in organ cultures of rat muscle, ttx prevented fibs when used at 1 m, which we show here to block a larger fraction of ina than riluzole at the concentration used. thus, it is likely that riluzole - induced block of muscle ina is not sufficient to prevent fibs. the aforementioned studies [46 ] report effects observed within minutes of drug application, whereas our data were collected in patients routinely taking riluzole or after a washout lasting one week, so that it can not be ruled out that adaptations to long - term presence / absence of riluzole contribute to its inability to prevent fibs in the examined patients. a reduced rate of rise of evoked action potentials under riluzole, here reported for cultured myotubes, was also observed in guinea pig cardiac purkinje fibres but not in rat ventricular cardiomyocytes. this study shows that riluzole at a typical clinical concentration partially blocks muscle nav channels and dampens but does not abolish the action potentials evoked by depolarizing stimuli in als myotubes. as suggested by in vitro inability to fully suppress spikes, riluzole does not prevent muscle fibs in als patients, apparently having very limited impact on this symptom. therefore, the pattern of fibs disappearance during disease progression is unlikely to be directly influenced by riluzole - induced block of muscle nav channels. | denervated muscles undergo fibrillations due to spontaneous activation of voltage - gated sodium (na+) channels generating action potentials. fibrillations also occur in patients with amyotrophic lateral sclerosis (als). riluzole, the only approved drug for als treatment, blocks voltage - gated na+ channels, but its effects on muscle na+ channels and fibrillations are yet poorly characterized. using patch - clamp technique, we studied riluzole effect on na+ channels in cultured myotubes from als patients. needle electromyography was used to study fibrillation potentials (fibs) in als patients during riluzole treatment and after one week of suspension. patients were clinically characterized in all recording sessions. in myotubes, riluzole (1 m, a therapeutic concentration) reduced na+ current by 20%. the rate of rise and amplitude of spikes evoked by depolarizing stimuli were also reduced. fibs were detected in all patients tested during riluzole treatment and riluzole washout had no univocal effect. our study indicates that, in human myotubes, riluzole partially blocks na+ currents and affects action potentials but does not prevent firing. in line with this in vitro finding, muscle fibs in als patients appear to be largely unaffected by riluzole. |
a review of the literature indicated an association among high nitrate ingestion, methemoglobinemia, and pathologic changes in bronchi and lung parenchyma. the present study examined a possible correlation among drinking water nitrate concentration, methemoglobin levels, cytochrome b(5) reductase activity, and acute respiratory tract infection with a history of recurrence (rrti). our study was conducted in five village units in the state of rajasthan, india, with nitrate concentrations of 26, 45, 95, 222, and 459 mg no(3) ion / l. we randomly selected 88 children. the children were up to 8 years of age, age matched, and represented 10% of the total population of these areas. we obtained detailed rrti histories and conducted medical examinations. methemoglobin levels and cytochrome b(5) reductase activity were estimated biochemically. the data collected were statistically analyzed using spreadsheet software on a personal computer. we observed strong interdependence between methemoglobin levels and rrti in children up to 8 years of age. methemoglobin levels alone explained 80% of the variation in the rrti cases. this study indicates that methemoglobinemia, secondary to high nitrate ingestion in drinking water, causes rrti. increased production of methemoglobin and free radicals of nitric oxide and oxygen due to nitrate metabolism in the body lead to alveolar damage and mismatching of ventilation and perfusion, which may be the reason for high mortality in children due to rrti.imagesfigure 1 |
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heparina, a highly sulfated glycosaminoglycan, has the highest negative charge density of any known natural biological molecule. heparin has been extensively used as an injectable anticoagulant in many clinical procedures for the prevention of blood clotting, especially during open heart surgery [2, 3 ]. pharmaceutical - grade heparin is derived from mucosal tissues of slaughtered meat animals such as porcine intestine and bovine lung. native heparin is a polymer with a molecular weight ranging from 3,000 to 50,000, and the average molecular weight of most commercially prepared heparin is in the range of 12,000 to 15,000. under physiological conditions, the sulfate groups are deprotonated and attract positively charged counterions (usually sodium) to form a heparin salt. it is in this form that heparin is usually administered as an anticoagulant [5, 6 ]. at the end of the medical procedure, the heparin has to be rendered inactive to prevent possible bleeding problems. therefore, clinicians need to know the exact concentration of heparin remaining when they attempt to neutralize the anticoagulant activity of heparin via precise doses of protamine. the current heparin detection method used in hospital is based on the blood clotting time [7, 8 ] and varies widely due to variation in the blood content of the different individuals. thus, the discovery of accurate, rapid, reliable safe, and low - cost methods of heparin detection remains an important area of sensor research. for example, electrochemical sensors [9, 10 ] and optical sensors [11, 12 ] were intensively studies in the 1990s, and more recent studies have focused on fluorescent spectroscopy [13, 14 ] and uv / vis spectroscopy using gold nanoparticles. luminescence spectroscopy is an analytical method that studies the luminescence emission from a chromophore after their electrons are excited from the ground state to a higher energy level. one of the most attractive features of luminescence spectroscopy as an analytical method is its inherent high sensitivity, wide linear response range, and fast detection speed. however, the luminescence intensity of the chromophore (or excited state complex) can be seriously reduced by collision with other chemical species or enhanced by chemical interactions. through studying the effect of these chemical species on the luminescence intensity of the chromophore, the quantitative determinations of a variety of important inorganic and organic species in trace amounts have been realized by photoluminescence methods. recently, a series of os(ii) carbonyl (co) complexes with two bipyridine or phenanthroline ligands and one pyridyl or triphenylphosphine ligand as the five other ligands have been synthesized in our lab. the osmium centers in these complexes are all in the + 2 oxidation state with an overall + 2 charge of the complex ion, and exhibit high emission intensity in the visible region [17, 18 ]. this research focuses on studying the effect of the heparin polyanion on the photoluminescence of these polypyridyl osmium(ii) complexes, for the purpose of finding potential luminescence markers for the detection of the optically nonactive polyanion heparin. to our surprise, polyanions show unique influences on the luminescence spectra and intensity of these osmium complexes. the 1,10-phenanthroline (phen) complexes were compared with the 2,2-bipyridine (bpy) complexes. for each of these chromophoric ligands, three analogous sets were compared using pyridine (py), triphenylphosphine (pph3), or 4-phenylpyridine (4-phpy) as the sixth ligand. these ligands showed a significant impact on the luminescence characteristics of the osmium complex in the absence as well as in the presence of polyanions. the results of the studies could lead to a new, potential effective detection method for heparin monitoring. the six new os(ii) complexes are [os(phen)2co(pph3)](pf6)2, [os(phen)2co(py)](pf6)2, [os(phen)2co(4-phpy)](pf6)2, [os(bpy)2co(pph3)](pf6)2, [os(bpy)2co(py)](pf6)2, and [os(bpy)2co(4-phpy)](pf6)2. the structure of each complex is shown in figure 1. the synthesis of six osmium complexes was described in [17, 18 ]. heparin sodium sulfate (> 180 usp units / mg), ethanol (99.5+%), reagent or analytical grade naclo4, mgso4, ch3coona, nacl, kbr, nano3, naoh, nano2, kscn, and ki were all obtained from sigma - aldrich. injectable heparin solutions, 100 units / ml and 40 units / ml with 5% dextrose, were purchased from b. braun medical inc. luminescence measurements were made on a perkin elmer luminescence spectrometer ls 50 b equipped with a high - intensity xenon flash lamp. a branson 1210 sonicator was used to help dissolve the osmium complex in solvent when needed. thermo scientific digital pipets were used for the addition of all solutions into the quartz cuvette. the stock solutions of the osmium complexes were prepared by dissolving 0.62.8 mg of each osmium complex in ethanol (ethanol solution) or distilled water (aqueous solution) in a 100 ml volumetric flask. the 1.0 mg / ml heparin aqueous solution was prepared by dissolving 5.0 mg of heparin in 5.00 ml of distilled water. the 0.10 mg / ml and 0.010 mg / ml heparin aqueous solutions were diluted from the 1.0 mg / ml stock solution. for small anions solutions, a 0.100 m solution of each salts was first made and then diluted to 0.0100 m and 0.00100 m. for the luminescence measurements of each osmium complex solution (ethanol, aqueous), 2 ml stock solution was transferred to a quartz cuvette (1 cm 1 cm 4.4 the emission and excitation slits were both set at 10 nm for the luminescence measurements. a prescan was taken first to obtain the wavelengths of the maximum excitation and emission intensities ; then the emission spectrum was recorded by exciting the sample at the appropriate excitation wavelength. in order to study the anion quenching or enhancement of the luminescence of each osmium complex solution, 2.00 ml of the stock solution was placed in the cuvette, and the emission spectrum was measured ; then a small amount (10 l or 20 l) of the anion solution (0.010~1.0 mg / ml for heparin and 0.10 to 0.0010 m for small anions) was added to the cuvette and the emission spectrum was recorded after the solution was well mixed. the anion solution was continuously added to the cuvette, and the emission spectrum was recorded after each addition. the values of the luminescence intensity were taken at a fixed wavelength at or near the emission maximum. first, 10 or 20 l of injectable heparin solution was added to 2 ml of the aqueous [os(phen)2co(4-phpy)](pf6)2 solution. next three portions of 5 l of 1 mg / ml standard heparin solution were added, and the emission spectrum of the solution was measured before and after each addition. the concentrations of the heparin in the injectable solution were calculated using the standard addition curve. the excitation and emission spectra of each complex in their ethanol solution (except for [os(bpy)2co(py)](pf6)2 which could not dissolve in ethanol) and their aqueous solution were first recorded at room temperature under ambient air. all the spectra exhibited broad emission and excitation bands up to 200 nm in width. figure 2 shows the emission spectra in ethanol solution of the two complexes : [os(phen)2co(pph3)](pf6)2, and [os(phen)2co(py)](pf6)2, which exibit the largest difference in luminescence intensity. as shown in table 1 and figure 2, the two os - pph3 complexes have much higher luminescence intensities and shorter emission wavelengths than the two os-(4-phpy) complexes and the two os - py complexes (520 nm versus 560 nm). the shorter wavelength (higher energy) of the emission is caused by the -backbonding ability of the phosphine which stabilizes the homo of the molecule relative to pyridine. the higher luminescence intensities are attributed to the larger steric effect of the pph3 ligand which shields the complex from colliding with the solvent. this slows down the vibrational relaxation to the ground vibrational level of the excited state that must occur prior to emission of a photon of light that occurs when the electron returns to the ground electronic state. all these six complexes exhibit higher luminescence intensities in aqueous solution than in ethanol solution due to the hydrophobicity of the ligands surrounding the osmium center. the spectral changes that occur before and after the addition of small inorganic anion solutions suggest that those anions clo4, so4, ch3coo, cl, br and no3 exhibit very little quenching of the luminescence of the osmium complex with anion concentrations up to 0.01 m, while no2, scn and i anions can dramatically quench the luminescence of the osmium complexes with concentrations at 0.01 mm. however, oh exhibits a small enhancement of the luminescence of the osmium complex ; luminescence intensity slightly increased as the solution ph increased from 6 to 9 in aqueous solution. it was expected that heparin would quench the luminescence of the osmium complexes due to its high negative charges. surprisingly, however, it was found that heparin actually enhanced the luminescence intensity of the osmium complexes in ethanol solutions. the luminescence of each complex in the ethanol and aqueous solution changed quite differently after heparin was added. in ethanol solution, each complex exhibited an increase in luminescence intensity after heparin was added in concentrations greater than 0.5 g / ml. however, the amount of increase varied significantly for each complex. the solutions of [os(bpy)2co(4-phpy)](pf6)2, [os(phen)2co(4-phpy)](pf6)2, and [os(phen)2co(py)](pf6)2 exhibited a very large increase in luminescence intensity while the solutions of [os(phen)2co(pph3)](pf6)2 and [os(bpy)2co(pph3)](pf6)2 exhibited a small luminescence increase. figure 3 shows the luminescence spectra of the ethanol solution of [os(bpy)2co(4-phpy)](pf6)2 in the absence and presence of heparin at different concentrations. the luminescence intensity was increased from a relative emission intensity of 136 in the absence of heparin to 462 by addition of heparin in a concentration range of 5.038.5 g / ml. further, there is an 8 nm blue shift in wavelength of the emission maxima with high heparin concentrations. a plot showing the wider range of heparin concentrations on the enhancement ratio (f / f 0) is shown in figure 4. here, f and f 0 are the luminescence (fluorescence) intensity of the osmium complexes solutions at 560 nm with and without heparin, respectively. the luminescence intensity increased steadily up to a heparin concentration of 56 g / ml. in the lower concentration range (up to 30 g / ml), there is a linear relationship between the f / f 0 ratio and the concentration with an r of 0.99. the heparin enhancement of the [os(phen)2co(4-phpy)](pf6)2 (2.4 mg/100 ml etoh) luminescence has a similar pattern to that seen with [os(bpy)2co(4-phpy)](pf6)2. in this case, the luminescence was enhanced by three and half times when the added heparin concentration was increased to 50 g / ml. once again, there is an 8 nm blue shift of the emission maxima at higher heparin concentrations. in the concentration range of 1 to 40 g / ml, there is a linear relationship between the f / f 0 ratio and the heparin concentration with an r of 0.99. the heparin enhancement of the luminescence of [os(phen)2co(py)](pf6)2 (2.4 mg/100 ml etoh) is smaller than that seen for the 4-phpy complexes. the luminescence intensity was enhanced by two and half times when the added heparin concentration was increased to 2030 g / ml, and then the luminescence started to decrease at heparin concentrations in excess of 30 m / ml. in this case, there is also an 8 nm blue shift of the emission maxima at higher heparin concentrations. the heparin enhancement of the luminescence of the [os(phen)2co(pph3)](pf6)2 (2.4 mg/100 ml etoh) and the [os(bpy)2co(pph3)](pf6)2 (2.1 mg/100 ml etoh) solutions is very small compared to the os-(4-phpy) and os - py complexes. here, the shift in the emission maxima is insignificant (less than 2 nm). figure 5 shows the plot of f / f 0 versus heparin concentration for the two phenanthroline complexes with pyridine and pph3 as the sixth ligand. from figures 4 and 5, it can be seen that the 4-phpy complexes, which have the lowest solution luminescence in the absence of polyanions, showed the highest enhancement in the concentration range of 540 g / ml added heparin concentrations. by contrast, the pph3 complexes showed the smallest enhancement in the same concentration range. the osmium complexes luminescence enhancement by heparin is likely contributed by the close contact of heparin and the complexes, which forms a rigid structure that reduced the solvent collision and quenching. heparin has a long linear structure with anionic sites on its branches when it is dissolved in aqueous solution. when its aqueous solution is added to the ethanol solution of the osmium complex, the anion sites on its branches attract the double positively charged osmium complex cations. as a result, the osmium complex could electrostatically bind to heparin and become surrounded by the heparin molecules (figure 6). in this case, solvent molecules can no longer get close enough to the osmium complex to help relax the excited state, resulting in the emission maximum moving to higher energy. this effect would also explain the increased emission intensity since the rate of nonradiative decay would be greatly reduced in the absence of favorable interaction with solvent molecules. the sixth ligand in these complexes plays an important role in the interaction of these charged sites. the complexes [os(bpy)2co(4-phpy) ], [os(phen)2co(4-phpy) ], or [os(phen)2co(py) ] have small narrow 4-phenylpyridine or pyridine ligand. when heparin is added to their ethanol solution, these osmium complexes could readily approach the heparin polyanion and bind up to two heparin anion sites. these two sites may well form two heparin polyanions branches, thus forming ion pairs between the polyanion and osmium complex. therefore, heparin can cause significant enhancement to the luminescence of the os-(4-phpy) and os - py complexes. further, the luminescence only stops increasing at very high heparin concentrations when all the osmium complex cations are bound to heparin anion sites. by contrast, the [os(phen)2co(pph3) ] and [os(bpy)2co(pph3) ] complexes have a sterically bulky pph3 group, which prevents them from approaching the heparin polyanion closely. therefore, the luminescence intensity of the os - pph3 complexes could not be significantly enhanced by heparin. the effect of heparin on the emission of these osmium complexes in their aqueous solution is quite different from that of their ethanol solution. the aqueous solution of osmium complexes with a pph3 or pyridine group exhibits a decrease in luminescence intensity at low added heparin concentrations ; then the luminescence increases at heparin concentration above 15 to 19 g / ml. however, complexes with the 4-phpy group exhibit a decrease of luminescence at heparin concentrations below 0.5 g / ml then an increase in luminescence intensity is observed with the addition of heparin in the range of 1 to 15 g / ml. this shows that as a polyanion, when not binding to the osmium complexes, heparin is an efficient luminescence quencher in aqueous solutions. this is especially true with the pph3 complexes, where binding only occurs at very high (> 15 g / ml) heparin concentrations. by contrast, the 4-phpy complexes bind more strongly to heparin, and so less of a quenching effect is observed. figure 7 shows the plots of the luminescence enhancement ratio (f / f 0) as a function of heparin concentration for the two os-(4-phpy) complexes in aqueous solution. the high luminescence enhancement ratio of the 1,10-phenanthroline complexes by heparin shown in figure 7 suggests that the [os(phen)2co(4-phpy) ] complex binds more strongly to the heparin polyanion than the analogous 2,2-bipyridine complex complexes. comparing figures 4, 5, and 7, the enhancement of heparin to the [os - co(bpy)2(4-phpy) ] complex is much smaller in aqueous solutions, which indicate a weaker binding to heparin, possibly due to the higher solubility of heparin and the complexes in aqueous solution. in the aqueous solutions of [os(phen)2co(pph3)](pf6)2 or [os(bpy)2co(pph3)](pf6)2, it is even harder for the osmium complex to bind to heparin because of the high solubility of heparin in aqueous solution and the hindrance of the bulky pph3 group. in fact, at low heparin concentration, heparin quenches the luminescence of these two complexes heavily as polyanion. the decrease of the luminescence intensity in the aqueous solution of these two complexes fits the stern - volmer equation (1)f0f=1+ksv[q ]. here f 0 and f are the luminescence signals in the absence and in the presence of a quencher, respectively ; k sv is the stern - volmer quenching constant ; and [q ] is the concentration of the quencher. stern - volmer plot is shown in figure 8 for the [os(phen)2co(pph3) ] complex. the high linearity of the stern - volmer curve, shown at the concentration range of 1 to 12 g / ml in the insert, confirms that heparin exhibits dynamic quenching to the os - pph3 complexes. the nonlinearity at higher concentrations may indicate that osmium complex - heparin ion - pairs have formed. further addition of heparin above 12 g / ml caused the luminescence of these complexes to increase slightly. table 2 summarizes the heparin concentration ranges that induced significant luminescence changes to the complexes studied in ethanol or aqueous solutions. in ethanol solution, [os(bpy)2co(4-phpy)](pf6)2 exhibits the greatest enhancement in the presence of heparin as measured by the highest slope of the f / f 0 versus heparin concentration linear curve. this complex exhibits the longest response range and the highest sensitivity to heparin concentration. in aqueous solutions, the [os(phen)2co(4-phpy)](pf6)2 and both phosphine complexes showed significant luminescence changes at low heparin concentrations. the detection of heparin was then performed with commercial medicinal solutions to evaluate the feasibility. two commercial heparin sodium injection solutions (40 and 100 units / ml in 5% dextrose, in 100 ml plastic bags) from b. braun medical inc. were tested. with the [os(phen)2co(4-phpy) ] complex in aqueous solution, the emission intensities of the osmium solution before and after adding 20 l of these two heparin solutions were measured. the ratios (f / f 0) obtained were 1.30 and 1.97 (n = 5), respectively, showing that the osmium complex indeed had a greater response to higher heparin concentration preparations. however, these medical preparation responses are much higher than the pure heparin solution response. when response curve of heparin in the presence of 5% dextrose was performed, an enhanced osmium complex response to heparin concentration was observed. this enhanced response is likely due to the hydrophobic environment that the polysaccharide, dextrose, created around the osmium complex in aqueous solution, thus reducing the extent of nonradiative decay of the excited states and increasing the luminescence intensity of the osmium complex. to eliminate the background effect, first, 10 or 20 l of the commercial heparin sample solutions was added to the aqueous osmium complex solution ; then three portions of 5 l of the 1.0 mg / ml heparin standard solution were added. the luminescence values of the complex solution after each addition of the sample and standard heparin solutions were measured at 560 nm. with the standard addition curves and using the conversion of 180 units / mg of sodium heparin, the average heparin concentration in the two sample bags was calculated to be 46 3 and 111 the relative standard deviation, rsd for the two corresponding samples are 5.6 (n = 3) and 2.3 (n = 6) respectively, indicating that the precision of the method is acceptable considering the volume added is very small, although they are 11% higher than the labeled value. further tests, with other commercial sources having different backgrounds, are currently under study. the luminescence of osmium carbonyl complexes is stable at room temperature in ethanol and aqueous solutions, though the intensity and wavelength of this emission are heavily dependent on the other five ligands. the effect of heparin on the luminescence of the osmium complexes depends heavily on both the ligand and solvent used. in ethanol solutions, heparin could enhance the luminescence of five osmium complexes at low heparin concentrations, with the greatest enhancement occurring with the os-(4-phpy) complexes. this increased emission intensity may be due to binding of heparin to the double charged complexes which changed the solvent environment of the complexes. in aqueous solutions, heparin quenching the osmium complex emission was more significant as heparin is more soluble in water and behaves as an anion quencher. these osmium complexes are shown to be useful for the fast and sensitive detection of heparin in commercially injectable samples. | the luminescence characteristics of six osmium carbonyl complexes with phenanthroline (phen) or bipyridine (bpy) and pyridine (py), 4-phenylpyridine (4-phpy), or triphenylphosphine (pph3) complexes in the presence of polyanion heparin were studied in both ethanol and aqueous solutions. the influence of heparin on the luminescence of the complexes is heavily dependent on the type of ligands in the complexes and the solvent used. in the ethanol solutions, the heparin solution enhanced the luminescence of the five osmium complexes, with the strongest enhancement to the 4-phenylpyridine complexes ; linear curves were obtained in the luminescence enhancement ratio (f / f 0) versus the heparin concentration range of 140 g / ml. in aqueous solutions, heparin quenching of the complexes was more significant ; a linear quenching curve was obtained with [os(phen)2co(pph3)](pf6)2 in the lower concentration range of 112 g / ml. the interaction of these complexes with heparin in the solutions is discussed. the complexes are shown to be successful in the fast and sensitive detection of heparin in commercial injectable samples. |
functional dyspepsia (fd) is a syndrome characterized by chronic and recurrent gastroduodenal symptoms in the absence of any organic or metabolic disease that is likely to explain the symptoms.1,2 fd is considered to be important to public health, because it is remarkably common, can be disabling, and can pose a major social and economic burden.3 since fd is a highly heterogeneous disorder, numerous pathophysiological mechanisms, such as gastroduodenal motor dysfunction, visceral hypersensitivity, central nervous system dysfunction, helicobacter pylori (h. pylori) infection and psychosocial factors have been suggested to play a role in the development of fd. although numerous epidemiological trials have suggested a higher prevalence of h. pylori infection in fd patients, the results have been conflicting.4 results of a meta - analysis showed that the prevalence of h. pylori infection was greater in patients with dyspepsia than in controls, with an odds ratio of 2.3 (95% ci, 1.9 - 2.7).5 although this result seems to support the role of h. pylori infection in the pathogenesis of dyspepsia, it appears that some of the studies that were included in the analysis were biased by the selection of controls not properly matched for age, socioeconomic status and ethnic background.4 however, recent studies have revealed a subset of patients who developed fd after an episode of gastrointestinal infection. these studies, proposing the concept of post - infectious fd, suggest that an inflammation - immunological circuit also plays an important role in the development of fd.6 it is generally well - recognized that the major cause of gastroduodenal inflammation is h. pylori infection.7 since h. pylori induces activation of a complex and fascinating cytokine and chemokine network in the gastric mucosa,8 it is of little surprise that h. pylori infection has been implicated in the pathogenesis of dyspepsia. for this reason, one of the major research interest is the difference between h. pylori - associated dyspepsia and other functional dyspepsia.9 in this review article, fd in patients with a present or even past history of h. pylori infection is defined as a different disease entity (h. pylori - associated dyspepsia [hpd ]) from fd, especially by focusing on the etiological insight of hpd, and then discusses the therapeutic strategy of hpd. a lot of clinical evidences have been published to investigate whether h. pylori infection is involved in gastric motility disorders and visceral hypersensitivity. however, all of these studies were small - scale studies, and the results were conflicting. thumshirn compared gastric motor and sensory functions in 17 patients with fd and 16 asymptomatic controls, and reported that h. pylori infection did not appear to influence gastric accommodation, but was associated with hypersensitivity in fd patients. on the other hand, some researchers were able to show the association between gastric motility dysfunction and h. pylori infection. mearin investigated the symptomatic pattern in 27 h. pylori - positive and 23 h. pylori - negative patients with fd, and showed that fd patients with h. pylori infection presented no distinctive symptoms in comparison with their h. pylori - negative counterparts, and that h. pylori infection was associated with diminished postprandial antral motility, but did not increase the perception of gastric distension. tucci evaluated the h. pylori infection status, histological features of the gastric mucosa, and the gastric motor and secretory functions in 45 consecutive patients with fd. h. pylori infection was found in 60% of fd patients, as compared with 33% of the 15 healthy controls. no difference was detected in the basal or stimulated gastric acid secretion between the fd patients and healthy controls. gastric emptying was significantly delayed in fd patients as compared with that in healthy controls after adjustments for age and sex. delayed gastric emptying was associated with a low frequency of h. pylori infection, female gender and young age. epigastric pain or burning and postprandial fullness were more severe in patients with h. pylori infection and in those with delayed gastric emptying, respectively. saslow compared 8 h. pylori - positive and 8 h. pylori - negative asymptomatic subjects, and showed that h. pylori infection reduced accommodation, but had no effect on the overall sensation or motor functions of the stomach. however, some studies showed that h. pylori infection did not affect gastric motility or hypersensitivity. leontiadis evaluated 23 fd patients and 17 controls, and showed that although gastric emptying was delayed in fd patients, the gastric emptying rate was not associated with the h. pylori infection status, and was also not affected by eradication of the infection. chang compared 22 h. pylori - negative patients and 38 h. pylori - positive patients with fd, and showed that the h. pylori infection status appeared to have no influence on the incidence of delayed gastric emptying of digestible and indigestible solids. although the results of several clinical studies suggest that h. pylori infection may play a role in the development of fd, the precise pathogenesis of hpd could not be elucidated. since gastric dysmotility and visceral hypersensitivity are induced by a number of confounding factors, such as diet, smoking and psychosocial stress, the association of h. pylori infection with gastric sensation or motor dysfunction might be difficult to be revealed only by clinical studies. a large - scale clinical study controlled for thus, novel biological markers for hpd other than gastric dysmotility and hypersensitivity must be identified. on next section, therefore, the possible pathophysiology of hpd will be reviewed. traditionally, gastric acid hypersecretion induced by h. pylori infection of the gastric antral mucosa has been considered to play a role in the development of dyspepsia. about 10%-15% of patients with h. pylori infection show antral - predominant gastritis, which results in gastric acid hypersecretion.16 in these patients, h. pylori induced a decrease in somatostatin secretion in the antral gland area, leading to an increase in the release of gastrin and subsequently to a rise in acid secretion.17 this mechanism is also considered to underlie the development of duodenal ulcer. these phenomena are reversible, since normal feedback control of gastrin secretion is restored after h. pylori eradication.17,18 however, a few studies investigating the association between the severity of histological gastritis and that of dyspepsia symptoms yielded different results. turkkan reported that dyspepsia symptom scores were higher in patients with mild or moderate chronic inflammation of the corpus and antrum than in those with severe chronic inflammation, although the difference did not reach statistical significance. in studies conducted by joshi and pereira - lima,21 no relationship was found between the severity of histological gastritis and the severity of the dyspeptic symptoms. similarly, van der schaar also found an indirect relationship between the severity of symptoms and the severity of inflammation of the corpus. from these results, we could not reach any definitive conclusion about the association of severity of gastritis or amount of gastric acid secretion with severity of the dyspepsia symptoms. ghrelin, which is produced and secreted by the a - like cells of the oxyntic glands of the stomach, has a well - established role in increasing appetite and food intake and in stimulating gastric emptying and acid secretion.24 - 28 these functions are mediated, at least in part, via vagal nerve pathways.29,30 in gastroduodenal mucosal injury, the levels of plasma ghrelin increased in response to the physiological demand for the purpose of gastroduodenal cytoprotection.31,32 however, in the presence of h. pylori - induced severe gastric mucosal atrophy, the plasma ghrelin concentrations shifted to lower levels.33 - 36 taken together, h. pylori infection may induce gastric motor dysfunction and reduce appetite with suppressed ghrelin secretion. therefore, this peptide may play a role in the onset of fd, especially hpd. in fact, alterations of the plasma ghrelin levels have been reported in fd patients, which frequently correlated with the fd symptom score.37 - 39 some studies showed that plasma ghrelin levels were significantly lower in patients with dysmotility - like fd.28,37 concerning the active ghrelin levels, they were also decreased in patients with postprandial fullness and/or early satiation,40 whereas similar between dysmotility - like fd patients and healthy controls.37 moreover, recent study showed that repeated ghrelin administrations had stimulatory effects on food intake in fd patients.41 however, the opposite results, such as enhanced ghrelin levels in fd patients, were also reported.38,42 leptin is also produced in the stomach, and activates vagal nerve ternimals, reduces appetite and increases mucin secretion.43 leptin may also play a role in the onset of fd, since patients with dysmotility - like dyspepsia have been reported to show higher serum concentrations of leptin.44 on the other hand, serum leptin levels and expression of leptin mrna in the gastric mucosa was enhanced in h. pylori - positive patients,44,45 suggesting that h. pylori infection may reduce appatite with enhanced leptin secretion. the circulatory levels of ghrelin and leptin in hpd patients have not yet been investigated, warranting future research. we recently investigated the role of micrornas (mirnas) in gastric motility disorders associated with h. pylori infection,46 and the results provided a novel insight into the molecular pathogenesis of hpd. histologic examination showed prominent thickening of the muscular layer of the gastric corpus in h. pylori - infected mice. the mirna expression profile revealed that the muscle - specific mirnas, mir-1, mir-133a and mir-133b, were downregulated in the stomach of h. pylori - infected mice. the expression levels of histone deacetylase 4 and serum response factor, which are target genes of mir-1 and mir-133 known to enhance muscular hyperproliferation, were increased. taken together, chronic h. pylori infection downregulates the expressions of muscle - specific mirnas and upregulates the expression of histone deacetylase 4 and serum response factor, which might cause hyperplasia of the muscular layer of the stomach and deregulation of gastric emptying in mice. further human studies will be necessary to validate the association between aberrant expression of muscle - specific mirnas in the muscular layer of the stomach and hpd. recent studies have emerged implicating abnormal motor and autonomic responses in the duodenum perhaps triggering functional responses, including pain and abnormal gastric emptying. increased duodenal acid exposure has been reported in patients with dyspepsia symptoms. at the level of the duodenum, abnormalities may exist in the stimulus intensity, mucosal mrna expression, biosynthesis, release or inactivation of the mucosal mediators, or in the receptor expression on the afferent nerve endings.47 furthermore, talley proposed that changes in the duodenal eosinophil count might be an underlying feature of fd. they also showed that eosinophils were significantly increased in both the bulb and second portion of the duodenum in fd, whereas increase of the mast cells in the second portion of the duodenum was noted in irritable bowel syndrome (ibs).49,50 a link between eosinophils (and other inflammatory cells) and fd would have therapeutic implications. eosinophils are critically dependent on the cytokine il-5 for their maturation in the bone marrow, which also influences eosinophil migration and survival. kindt reported that stimulated lymphocyte expression of il-5 and il-13 was enhanced, whereas stimulated monocytic il-12 and lymphocytic il-10 expression were reduced in both fd and ibs. based on these findings, anti - inflammatory agents, possibly including novel biologics such as anti - il-5 humanized antibodies, could be explored as a possible therapeutic candidates for fd. active duodenitis has been reported to be more common in patients with h. pylori infection.52 genta reported that h. pylori was detected in the gastric metaplastic epithelium of 67.6% of patients with active inflammation of the duodenum. on the other hand, h. pylori infection is well - known to cause eosinophil infiltration of the gastric mucosa.53 taken together, h. pylori might be one of the causes of duodenal eosinophilia, as well as of the onset of dyspepsia symptoms. in addition, gargala reported that the number of intraepithelial lymphocytes in the duodenal mucosa was significantly greater in h. pylori - positive fd patients than in healthy controls, but not different between h. pylori - negative fd patients and healthy controls. the expressions of cd95/fas and hla - dr - expressing cd3 lymphocytes were lower in h. pylori - negative fd patients than in healthy controls. these findings suggest that the phenotypic characteristics of intraepithelial lymphocytes may be different between hpd and h. pylori - negative fd. although a number of clinical trials have assessed the efficacy of h. pylori eradication for the treatment of fd, the studies drew different conclusions. however, it is quite clear that h. pylori eradication treatment is effective in at least a subset of patients with fd.7,55 - 58 according to a meta - analysis of randomized controlled trials to determine the effect of h. pylori eradication on dyspepsia symptoms, h. pylori eradication therapy appears to have a small but statistically significant effect in hpd.59 harvey showed that h. pylori eradication gave cumulative long - term benefit, with a continued reduction in the development of dyspepsia severe enough to require a consultation with a general practitioner up to at least 7 years. the efficacy for patients with hpd in asia would be different from those in western countries, since asian population differs from the western population in many respects, such as prevalent h. pylori strains, including caga gene polymorphisms, levels of acid secretion in the stomach and the severity or pattern of gastritis.58,61 in fact, gwee showed that the patients with fd in asia would have a benefit from treatment for h. pylori infection with as much as a 13-fold increased chance of symptom resolution following its eradication in a double blind, randomized and placebo - controlled trial in singapore - based asian population. there is no evidence of treatment for hpd patients after the successful eradication of h. pylori. at present, a meta - analysis of randomized controlled trials of proton pump inhibitors (ppis) for fd reported that this class of agents was superior to placebo.63 however, much of this benefit may be explained by the presence of concomitant unrecognized gastroesophageal reflux disease (gerd). xiao showed that the prevalence of pathologic esophageal acid reflux without typical reflux symptoms (silent reflux) was 31.7% in fd patients. in addition, ppis were effective in 83.1% of fd patients with silent reflux, and in 54.3% of those without silent reflux. on the other hand, inverse associations are observed between the presence of h. pylori infection and gerd, because of the reduction in gastric acid production by h. pylori colonization of the gastric mucosa.65,66 this suggests that the efficacy of ppis in hpd may be weaker than that in h. pylori - negative fd, which may show strong overlap with gerd. on the other hand, a gastro - protective agent for chronic gastritis would be a therapeutic candidate for hpd. rebamipide, a gastro - protective anti - ulcer drug, has been used for the improvement of dyspepsia symptoms in japan, korea, china and some other countries. rebamipide is known to suppress gastric mucosal inflammation, which is thought to be related to its activity in the inhibition of superoxide anion production from neutrophils and scavenging hydroxyl radicals.67,68 rebamipide administration after h. pylori eradication could promote the restoration of atrophic mucosa in mongolian gerbils.69 chitapanarux reported that rebamipide treatment improved symptom, endoscopic and histologic features of chronic gastritis in patients with dyspepsia symptoms refractory to ppis. talley reported a double - blind, placebo - controlled and multicenter study of rebamipide for the treatment of fd patients with or without h. pylori infection. although a significant improvement of individual symptoms at 8 weeks was not detected, the ratio of patients who requested usage of the study medication again was greater in the rebamipide groups compared with the placebo group in h. pylori - positive patients. during the planning of this study, it was originally projected that a sample size of 100 patients per treatment group would be sufficient to detect a difference in response rate of approximately 20% between the rebamipide treatment group and the placebo treatment group with 80% power at the 0.05 significance level. however, because of the slow patient recruitment and unexpected budget constraints, the trial had stopped prior to completion of enrollment. based on the enrolled population of approximately 50 patients per arm in the h. pylori - negative study and 30 patients per arm in the h. pylori - positive study, the detectable differences would be 30% and 40%, respectively. the 30% superiority over the placebo would be non - realistic hurdle for any medication for fd. miwa also reported a double - blind, placebo - controlled and single - center study of rebamipide for the treatment of fd patients. although the mean changes in overall symptoms after 4 weeks of treatment were not significantly different between the rebamipide and placebo treatment groups, the improvement in symptom score was significantly greater in the rebamipide group for bloating, belching and pain or discomfort that was relieved after a meal. social restriction and pain intensity the ratio of subjects with h. pylori infection were 54.1% in the rebamipide group and 42.4% in the placebo group. however, they did not perform subanalysis by h. pylori status as the number of subjects was rather small. as rebamipide has an anti - inflammatory effect, it might be effective for hpd, but not for fd patients without gastritis. however, there is not enough evidence for the efficacy of rebamipide for dyspepsia symptoms of hpd patients. therefore, the efficacy of all the existing medical treatment, including a gastro - protective agent, for fd should be re - evaluated for hpd and h. pylori - negative fd. therefore, it would be reasonable that the h. pylori " test - and - treat " strategy is not effective in all hpd patients, but is effective in only a subset of hpd patients. h. pylori infection evokes significant inflammatory changes, not only in the gastric mucosa, but also in the gastric muscular layer as well as in the duodenum. we therefore need to conduct further investigation about the true relationship between dyspepsia symptoms and h. pylori infection to determine whether there might be identifiable risk factors for the onset of symptoms. when the rome iii criteria were developed, the role of h. pylori infection in fd was controversial. now, however, the pathophysiology underlying disturbances of gastroduodenal motor or sensory function and dyspepsia symptoms caused by h. pylori infection is gradually being elucidated. therefore, when hpd is considered as an organic disease and as a different disease entity from fd, these conflicting results of previous studies might become more comprehensible. in addition, the differences in the therapeutic strategies between hpd and h. pylori - negative fd are also necessary to be investigated in the future. | advances in basic and clinical research have revealed that helicobacter pylori (h. pylori) infection plays an important role in the development of gastroduodenal dysmotility and hypersensitivity, as also in dyspepsia symptoms. in addition, recent studies have proposed an inflammation - immunological model for the pathogenesis of functional dyspepsia. since h. pylori is the major microbe that provokes a gastroduodenal inflammatory response, it should not be overlooked when considering the pathophysiology of dyspepsia symptoms. in fact, population - based studies have demonstrated that h. pylori is detected more frequently in dyspepsia patients. however, although many clinical studies tried to reveal the association of h. pylori infection with gastric motility dysfunction or hypersensitivity, the results have been conflicting. on the other hand, many etiological features were revealed for the development of h. pylori - associated dyspepsia, such as abnormal ghrelin or leptic secretion, altered expression of muscle - specific micrornas, and duodenal inflammatory cell infiltration. in addition, therapeutic strategy for h. pylori - associated dyspepsia would be different from h. pylori - negative functional dyspepsia. this review focuses the issue of whether h. pylori - associated dyspepsia should be considered as a different disease entity from functional dyspepsia. |
a septic patient is in your intensive care unit and you are concerned that he is behind on his intravascular volume. for a variety of reasons you have decided you would like to give him intravenous colloids. frdrique schortgen and laurent brochard capillary leakage during sepsis is a reason for recommending the use of macromolecules that could preserve the colloid osmotic pressure (cop). the high cost of albumin has facilitated the widespread use of hydroxyethylstarches (he s). outcome studies on sepsis are scarce, and the reasons why we should use he s remain speculative or based on short - term physiological data. we will briefly describe how uncertain are the clinical benefits of these products and, by contrast, how strong is the evidence for numerous adverse effects. both crystalloids and colloids have a similar ability to achieve sufficient volume loading when the volume administered takes into account the capacity of the solution to remain in the intravascular space. to achieve an equivalent plasma volume expansion, a fourfold greater volume of crystalloid may be needed in comparison with 5% albumin. maintaining cop by administration of he one study including septic patients found a higher incidence of pulmonary oedema after crystalloids than after he s. most clinical results have been disappointing, however, and a meta - analysis showed that pulmonary oedema occurrence is similar with colloids or crystalloids. indeed, in the context of a free course of macromolecules across a damaged alveolocapillary membrane, the starling equation indicates that colloidal forces can no longer stop fluid shift. an attractive, although unproven, pharmacological effect of he s comes from experimental studies suggesting that he s could improve microcirculation. boldt and colleagues found a better intramucosal ph in patients receiving he s in comparison with albumin, but two recent studies in septic hypovolaemic patients showed that he s did not improve splanchnic circulation whereas gelatins did. the case for adverse events secondary to administration of he s is much stronger and concerns coagulation disorders, acute renal failure, liver failure and pruritis [8 - 12 ]. initially shown in a situation of ischaemia reperfusion (i.e. renal transplant recipients), the nephrotoxicity of hydroxyethylstarch has been demonstrated in a randomised study during severe sepsis. in comparison with gelatins, he s 200 kda/0.6 induced a twofold higher incidence of acute renal failure. the adverse effects of he s may depend on the molecular weight and the degree of substitution (proportion of hydroxylethyl groups on glucose molecules). the conflicting results coming from coagulation studies cast doubts whether an optimal combination for these two parameters has yet been found. comparing he s 200 kda/0.5 and 130 kda/0.4, jamnicki and colleagues found the same disturbances in in vitro coagulation tests. a recent meta - analysis in cardiac surgical patients showed that postoperative bleeding was more frequent with he s, whatever the molecular weight, than with albumin. concerning acute renal failure, no comparative study supports the hypothesis that the newer compounds are safer. in a prospective randomised study including 150 postoperative patients with sepsis, a 50% increase in the serum creatinine was found at day 3 in the hydroxyethylstarch 200 kda/0.5 group against only a 6% increase with albumin. this difference was not significant, but this result again suggests that doing no harm should be our primary goal. if the only colloids available are he s, the actual equipoise regarding efficacy means that we choose crystalloids to avoid adverse effects on organ function. ellen burnham and greg s martin fluid exchange across the capillary endothelium obeys starling 's law { v = kf [(pc - pi) - (c - i) ] }, which describes the forces governing fluid flux across a semipermeable membrane, such as the human vasculature. hydrostatic pressure and cop () are the primary determinants of fluid flux in this system. when these forces are in balance, homeostasis between the intravascular and extravascular fluid compartments is maintained. the difference in hydrostatic pressure (pcapillaries - pinterstitium) pushes fluid out of the vasculature, while the difference in cop (capillaries - interstitium) draws fluid into the vasculature. the relative effect of oncotic pressure is modulated by the reflection coefficient (), describing the integrity of the capillary wall in preventing translocation of proteins. colloids were developed as a durable alternative to crystalloids and blood products for patients requiring fluid resuscitation., this translates into albumin versus starches, gelatins, dextrans or combination solutions. because of cost differentials, conflicting evidence and the underemphasis of cop in shock states, the solution of choice for resuscitating patients is controversial. the utilisation of a crystalloid solution in volume resuscitation, especially in situations where patients are hypoproteinemic, such as sepsis, may promote extravasation of volume out of the vascular space and into the interstitium, where it is of little help in rectifying hypotension. physiologically, the use of resuscitative fluid containing osmotically active molecules of low molecular weight that are biodegradable with a moderate halflife would be ideal in septic patients, who have greater capillary permeability and, frequently, a low cop. he s solutions contain molecules with a wide range of molecular weights and have an effect on intravascular volume lasting about 24 hours. in the intravascular compartment, he s are progressively hydrolysed into smaller fractions that are ultimately excreted by the kidneys. colloidal agents are more efficacious at restoring plasma volume compared with crystalloids, per unit of fluid given. furthermore, he s continue to provide volume expansion even in states of capillary permeability. investigators have demonstrated that, in hypovolaemic shock, resuscitation with starches or albumin results in a lower incidence of pulmonary oedema, compared with crystalloids. additionally, maintenance of cop may prevent complications of critical illness, including refractory acidosis, acute respiratory distress syndrome, prolonged mechanical ventilation and mortality associated with crystalloid resuscitation. apart from being pure volume expanders, he s have specific pharmacologic properties that may be beneficial in sepsis, such as lowering the circulating levels of adhesion molecules, and thus potentially reducing endothelial activation and damage. in septic patients, endogenous vasopressor production is decreased in patients receiving he s compared with other colloids. additionally, he s may exert useful effects on the microvascular coagulation cascade of these patients by elevating levels of protein c and protein s. the use of he s as a resuscitative fluid in this patient with septic shock makes sound physiologic sense, particularly if cop is already reduced. experimental data have demonstrated the efficacy of he s in the restoration of intravascular volume, and the unique pharmacologic properties of he s may provide additional benefit. finally, frdrique schortgen and laurent brochard in the absence of abnormally high hydrostatic pressure, low cop does not promote lung fluid accumulation. whereas a low cop may induce soft tissue oedema, several effective mechanisms protect against alveolar flooding. low cop is rather a marker of severity for capillary leakage and of the amount of volume needed before acute respiratory distress syndrome onset. the ability of plasma expanders to reverse microvascular damages is not limited to starches, or even to colloids. one might not forget that the best way to reverse low cop and microvascular damages in sepsis remains early and adequate anti - infectious treatment. ellen burnham and greg s martin for improving outcomes in critically ill patients with severe sepsis there is an absence of evidence regarding intravenous solutions. colloids have physiologic advantages over crystalloids, but suffer from higher acquisition costs. in light of recent evidence specifically regarding he s, advocating their use in patients with severe sepsis although he s may be the economic colloids of choice, we must focus our prescribing choices on patient - centred outcomes. association does not indicate causation and, until clinical trials evaluating appropriate clinical outcomes are performed, we will continue to deliver imprecise critical care. intensivists should prescribe intravenous therapy based upon patient - specific factors, recognising that newer starches might obviate the associated risks, which are also absent with natural colloids. | there are few issues in critical care medicine that have a less clearly defined standard of care than the intravenous fluid choice for resuscitation. natural colloids (such as albumin) became popular during the second world war when there was a need to develop a portable, easily stored, blood substitute. early successes led to widespread use and a multibillion dollar industry. it is not surprising given the large demand, high costs and potential adverse effects of natural colloids that synthetic colloids have emerged. in the present article, two groups of clinical investigators remind us of the controversies surrounding the use of synthetic colloids. |
ten healthy volunteers participated in the study (9 females and 1 male, age : 2236). the study was approved by the central denmark region committees on biomedical research ethics (m-20100032). written informed consent was obtained from the participants following oral and written information about the study. the exclusion criteria and the design of the in situ model all participants wore an individually designed acrylic splint in the lower jaw in which four custom - made non - fluorescent glass slabs with a surface roughness of 1,200 grit (menzel, braunschweig, germany) had been inserted into the buccal flanges of each side for biofilm collection (17). the glass slabs were sterilized by autoclaving before being inserted into recessions in the splint with sticky wax (densply, weybridge, uk). the volunteers wore the splint during two 48-h experimental periods, each beginning at 8 am. during the first experimental period, the volunteers removed the splint every 2 h during daytime (eight times a day) and immersed the right - side flange in physiological saline for 2 min (sucrose - free group) and the left - side flange in a 4% sucrose solution for 2 min (sucrose group), the latter to imitate a situation where the individual consumes a diet rich in fermentable carbohydrates. based on studies by imfeld (8), a concentration of 4% sucrose was chosen to achieve ph drops within the optimal ph range of the ph - sensitive probe (ph=4.57) (16). fresh solutions were provided for each day of the experiment. during the second experimental period, the volunteers repeated the procedure described above but immersed the left - side flange in physiological saline (sucrose - free group) and the right - side flange in the sucrose solution (sucrose group). at the end of each experiment, the glass slabs were removed from the splints and stored at room temperature in humid containers until microscopic analysis (< 6 h). for methodological reasons, for each participant (n=10) and for both experimental periods, eight 48-h biofilm specimens were analyzed (four from each side of the splint), yielding a total of 160 biofilms. the order of analysis of the specimens alternated between specimens from the saline immersion side and specimens from the sucrose immersion side. an inverted zeiss lsm 510 meta (carl zeiss, jena, germany) confocal microscope was used for image acquisition. the 543 nm laser line was used and fluorescence emission was monitored simultaneously within 576608 nm (green) and 629661 nm (red) intervals (meta detector). an xl incubator (pecon, erbach, germany) was used to keep the microscope at a temperature of 37c. calibration data of c - snarf-4 for our microscopic set - up and the resulting calibration curve have been published previously (16). ph analysis of the biofilms was performed in pooled salivary solution (18) titrated to ph 7.0, and glucose was added to a concentration of 0.4% (wt / vol). custom - made wells were filled with 100 l of salivary solution mixed with 2 l (50 m) of c - snarf-4 and placed under the confocal microscope. then glass slabs were placed in the custom - made wells with the biofilm facing downward (t=0). for each participant, experimental period and treatment group, ph was monitored for 1 h following the glucose challenge in two replicate biofilms and for 15 min following the glucose challenge in another two replicate biofilms. for 1-h monitoring, six fields of view (fov) per biofilm were imaged at seven consecutive time points., three fov per biofilm were imaged as often as possible (68 times), starting at t=2 min. images were acquired 510 m from the biofilm - glass interface for each microscopic fov. pilot experiments were conducted to exclude the possibility of drift in the measuring system of the microscope. a plaque sample on a glass slab was incubated for 16 h in hepes buffer (ph 5.5) and then placed in a custom - made well with 100 l hepes buffer (ph 5.5) and 2 l of c - snarf-4. two fov were imaged at seven time points during 1 h, as for the experimental biofilms. subsequent image analysis and ratio calculation showed the extracellular ph to be constant over time (data not shown). postprocessing of all images to determine extracellular ph following the glucose challenge was done using the programs daime (digital image analysis in microbial ecology, v. 2) (19) and imagej (www.imagej.nih.gov/ij/, v.1.47) (20). briefly, daime was used to distinguish between bacterial cells and extracellular matrix in the images and to remove the bacterial biomass from all images. image j was used to subtract background fluorescence and to calculate the average ratios between green and red fluorescence in the extracellular matrix of the biofilm images. average ratios for each examined fov and time point were then converted to ph values according to the calibration equation (see equation (1) in schlafer.. the data comprised recordings of extracellular ph and time of recording of this ph as observed in 1) six fovs in each of the four specimens at seven time points over a 1-h period or 2) observed in three fovs in each of the four specimens at 68 time points over a 15-min period, from each of the two experimental periods. the 1-h ph / time data were considered to have a hierarchical structure with fov (n=6) nested in specimens (n=4) nested in individuals (n=10) yielding a total n=240 ; the 15-min ph / time data had a hierarchical structure with fov (n=3) nested in specimens (n=4) nested in individuals (n=10) yielding a total n=120. in the 1-h data, the ph versus time curve for each fov was used to estimate, by interpolation, the ph during five fixed time intervals : 34 min, 67 min, 1213 min, 2930 min, and 5960 min. these ph values correspond to the average ph value in each of these 1-min intervals in the fov. in the 15-min data the ph versus time curve for each fov was used to estimate, by interpolation, the ph value at three fixed time intervals : 34 min, 67 min, and 1213 min. the data were analyzed using mixed - effects linear regression procedure xtmixed of stata 13 (statacorp, college station, tx, usa) to account for the three - level hierarchical structure of the data. this analysis amounts to a variance - components analysis and provides estimates of the average ph, as well as estimates of the between - individuals variance (varsubject), the between - specimen within - individual variance (varspecimenindividual), and the between fov within - specimen within - individual variance (varfovspecimen & individual) of the ph values. analyses were made for each time point and for each immersion solution (saline or sucrose). the significance of the variance components was tested using the likelihood - ratio test, and ph changes were tested using paired - tests between time intervals. ten healthy volunteers participated in the study (9 females and 1 male, age : 2236). the study was approved by the central denmark region committees on biomedical research ethics (m-20100032). written informed consent was obtained from the participants following oral and written information about the study. the exclusion criteria and the design of the in situ model all participants wore an individually designed acrylic splint in the lower jaw in which four custom - made non - fluorescent glass slabs with a surface roughness of 1,200 grit (menzel, braunschweig, germany) had been inserted into the buccal flanges of each side for biofilm collection (17). the glass slabs were sterilized by autoclaving before being inserted into recessions in the splint with sticky wax (densply, weybridge, uk). the volunteers wore the splint during two 48-h experimental periods, each beginning at 8 am. during the first experimental period, the volunteers removed the splint every 2 h during daytime (eight times a day) and immersed the right - side flange in physiological saline for 2 min (sucrose - free group) and the left - side flange in a 4% sucrose solution for 2 min (sucrose group), the latter to imitate a situation where the individual consumes a diet rich in fermentable carbohydrates. based on studies by imfeld (8), a concentration of 4% sucrose was chosen to achieve ph drops within the optimal ph range of the ph - sensitive probe (ph=4.57) (16). fresh solutions were provided for each day of the experiment. during the second experimental period, the volunteers repeated the procedure described above but immersed the left - side flange in physiological saline (sucrose - free group) and the right - side flange in the sucrose solution (sucrose group). at the end of each experiment, the glass slabs were removed from the splints and stored at room temperature in humid containers until microscopic analysis (< 6 h). for methodological reasons, for each participant (n=10) and for both experimental periods, eight 48-h biofilm specimens were analyzed (four from each side of the splint), yielding a total of 160 biofilms. the order of analysis of the specimens alternated between specimens from the saline immersion side and specimens from the sucrose immersion side. an inverted zeiss lsm 510 meta (carl zeiss, jena, germany) confocal microscope was used for image acquisition. the 543 nm laser line was used and fluorescence emission was monitored simultaneously within 576608 nm (green) and 629661 nm (red) intervals (meta detector). an xl incubator (pecon, erbach, germany) was used to keep the microscope at a temperature of 37c. calibration data of c - snarf-4 for our microscopic set - up and the resulting calibration curve have been published previously (16). ph analysis of the biofilms was performed in pooled salivary solution (18) titrated to ph 7.0, and glucose was added to a concentration of 0.4% (wt / vol). custom - made wells were filled with 100 l of salivary solution mixed with 2 l (50 m) of c - snarf-4 and placed under the confocal microscope. then glass slabs were placed in the custom - made wells with the biofilm facing downward (t=0). for each participant, experimental period and treatment group, ph was monitored for 1 h following the glucose challenge in two replicate biofilms and for 15 min following the glucose challenge in another two replicate biofilms. for 1-h monitoring, six fields of view (fov) per biofilm were imaged at seven consecutive time points., three fov per biofilm were imaged as often as possible (68 times), starting at t=2 min. images were acquired 510 m from the biofilm - glass interface for each microscopic fov. pilot experiments were conducted to exclude the possibility of drift in the measuring system of the microscope. a plaque sample on a glass slab was incubated for 16 h in hepes buffer (ph 5.5) and then placed in a custom - made well with 100 l hepes buffer (ph 5.5) and 2 l of c - snarf-4. two fov were imaged at seven time points during 1 h, as for the experimental biofilms. subsequent image analysis and ratio calculation showed the extracellular ph to be constant over time (data not shown). postprocessing of all images to determine extracellular ph following the glucose challenge was done using the programs daime (digital image analysis in microbial ecology, v. 2) (19) and imagej (www.imagej.nih.gov/ij/, v.1.47) (20). briefly, daime was used to distinguish between bacterial cells and extracellular matrix in the images and to remove the bacterial biomass from all images. image j was used to subtract background fluorescence and to calculate the average ratios between green and red fluorescence in the extracellular matrix of the biofilm images. average ratios for each examined fov and time point were then converted to ph values according to the calibration equation (see equation (1) in schlafer. (16)). the data comprised recordings of extracellular ph and time of recording of this ph as observed in 1) six fovs in each of the four specimens at seven time points over a 1-h period or 2) observed in three fovs in each of the four specimens at 68 time points over a 15-min period, from each of the two experimental periods. the 1-h ph / time data were considered to have a hierarchical structure with fov (n=6) nested in specimens (n=4) nested in individuals (n=10) yielding a total n=240 ; the 15-min ph / time data had a hierarchical structure with fov (n=3) nested in specimens (n=4) nested in individuals (n=10) yielding a total n=120. in the 1-h data, the ph versus time curve for each fov was used to estimate, by interpolation, the ph during five fixed time intervals : 34 min, 67 min, 1213 min, 2930 min, and 5960 min. these ph values correspond to the average ph value in each of these 1-min intervals in the fov. in the 15-min data the ph versus time curve for each fov was used to estimate, by interpolation, the ph value at three fixed time intervals : 34 min, 67 min, and 1213 min. the data were analyzed using mixed - effects linear regression procedure xtmixed of stata 13 (statacorp, college station, tx, usa) to account for the three - level hierarchical structure of the data. this analysis amounts to a variance - components analysis and provides estimates of the average ph, as well as estimates of the between - individuals variance (varsubject), the between - specimen within - individual variance (varspecimenindividual), and the between fov within - specimen within - individual variance (varfovspecimen & individual) of the ph values. analyses were made for each time point and for each immersion solution (saline or sucrose). the significance of the variance components was tested using the likelihood - ratio test, and ph changes were tested using paired - tests between time intervals. 2 show representative examples of the extracellular ph curves from three individuals during the 15- and 60-min experiments in the absence and presence of sucrose, respectively. for both experimental groups, the observed ph drops differed considerably between different fovs from one biofilm, between different specimens from the same individual, and between individuals (figs. 1 and 2, and supplementary fig. these differences were usually maintained until the end of the observation period (60 min). ph curves from three individuals with different patterns of ph drops (sucrose - free group). (a)(c) show data from the 15-min analysis and (d)(f) from the 60-min experiment. (a)(c) and (d)(f) show two different fields of view (fov) in one biofilm from the sucrose - free group. (a) and (d) show overlays of the red and green fluorescent emissions of a c - snarf-4-stained biofilm after exposure to 0.4% glucose. the dye concentration is the highest inside bacterial cells, which allows removal of the bacterial biomass from the images via digital image postprocessing. (b) and (c) show the fov in (a) after removal of the bacteria and ratiometric ph calculation, 8 and 63 min after exposure to glucose, respectively. for visualization, the average ph dropped from 6.48 (in b) to 6.04 (in c). (e) and (f) show the fov in (d) after removal of the bacteria and ratiometric ph calculation, 5 and 60 min after exposure to glucose, respectively. the average ph dropped from 5.90 (in e) to 5.12 (in f). bars=20 m. the variance components analyses showed significant variation between individuals (varindividual), between specimens within individuals (varspecimenindividual), and between fov within specimens (varfovspecimen & individual) at every time interval (table 1 ; for examples, see fig. 1 and supplementary fig. 2). while varspecimenindividual tended to be the greater source of variation, the two other variance components were similar, being only slightly lower than varspecimenindividual. moreover, the analysis indicated similar values of the three variance components for biofilms from the sucrose - free group and the sucrose group (table 1). variance of biofilm ph between individuals (varindividual), between specimens within individuals (varspecimenindividual), and between field of view (fov) within specimens within individuals (varfovspecimen & individual) at different time intervals in the sucrose - free group and the sucrose group. 15-min experiment : n=10 individuals4 glass slabs3 fov=120 ; 60-min experiment : n=10 individuals4 glass slabs6 fov=240 ; in a few instances, extracellular ph could not be determined owing to the microscopic field being entirely covered by bacterial cells. extracellular ph dropped in all biofilms from the sucrose - free group after addition of saliva containing 0.4% glucose (fig. the mean ph for all fovs of this group dropped to 6.39 at the 34 min interval, and to 5.63 at the 5960 min interval (fig. all fovs taken together, half of the final h+ ion concentration (at 60 min) was reached after 16.7 min. in most fovs, nevertheless, ph continued to fall throughout the experimental period (figs. 1 and 3). mean extracellular ph values of all examined fields of view in five fixed time intervals. the first three time intervals (34 min, 67 min, 1213 min) are based on the 15 min data and the last two intervals (2930 min, and 5960 min) on the 60-min data. black data points show the mean phse for the sucrose - free group and white data points show the mean phse for the sucrose group. biofilms in the sucrose group showed similar extracellular ph drops as biofilms in the sucrose - free group. the mean ph value in the extracellular space decreased from 6.38 at the 34 min interval to 5.59 at the 5960 min interval (fig. 3 (white datapoints)). as in the biofilms grown without sucrose, an initially rapid ph drop was observed, with half of the final h+ ion concentration being reached after 18.6 min. no statistically significant differences were observed in mean ph between the two treatment groups during any of the analyzed time intervals (34 min, 67 min, 1213 min, 2930 min, or 5960 min). the present study applies ratiometric imaging to systematically record the extracellular ph in young dental biofilms from several individuals for time periods up to 60 min. we demonstrate that the ph drops differ between sites from different individuals, between sites from different specimens, and within single biofilms grown on one glass slab. it is well known that large inter - individual differences exist in biofilm formation rate, architecture and microbial composition (2124), as well as host - specific differences in saliva flow and chemistry (25, 26). consequently, differences in the ph drops between individuals were expected (8, 10, 27). however, it was surprising that the variation in the ph drops was not more pronounced between individuals than between specimens from the same individual, and even between fovs from the same specimen in the same individual. these results shed new light on dental biofilm metabolism, providing evidence for a pronounced chemical heterogeneity within thin developing dental biofilms. we assume that five key processes influence ph in the biofilms in this study : 1) supply of glucose and buffering ions, 2) oxygen depletion, 3) bacterial acid production, 4) bacterial acid consumption, and 5) diffusion of acids within and out of the biofilms. glucose limitation is not likely to account for the observed differences in ph within the same biofilm, because it is delivered in excess and diffuses readily in the thin (1030 m) biofilms (28). (29) have previously shown rapid ph drops in deeper layers of five - species laboratory biofilms (up to 70 m) upon exposure to 0.4% glucose. likewise, buffering ions deriving from the salivary solution should penetrate quickly through the thin biofilms. the observed differences in ph within the same biofilm typically arose within short time (< 5 min ; see fig. 1 and supplementary fig. 2), indicating that oxygen depletion and acid production occur at different rates in closely juxtaposed areas. as soon as differences in ph are present within one biofilm, diffusion of h ions starts, thereby counteracting the chemical gradient. still this is particularly interesting, as the reaction chamber represents a closed system without fresh medium supply and clearance of acids. diffusion was not able to compensate for the continuously higher production of h ions in what we might call acidogenic hot spots in the biofilms. first, differences in the bacterial composition within a biofilm might result in different metabolic activities. studies have documented the existence of distinct microcolonies of bacterial species or genera in both in - situ - grown dental biofilms (21, 30) and in biofilms on natural teeth (14, 15), which could reflect an altered ecological milieu in some areas compared with others. second, different metabolic activities might derive from bacteria being in different physiological states, depending on the area of the biofilm. it has been shown that bacterial vitality varies across dental biofilms (31), and differential access to nutrients and metabolites, as well as the presence of water channels (32), might potentially impact acid production and diffusion. finally, the role of the extracellular matrix for the conservation of acidic microenvironments must be appraised. recently, the spatial distribution of ph in a mixed - species biofilm model consisting of streptococcus mutans, actinomyces naeslundii, and streptococcus oralis was recorded, and the authors demonstrated low - ph areas inside complexes of exopolysaccharide, arguing that the extracellular matrix might act as a barrier against diffusion of metabolites between microenvironments (33). importantly, the presence of acidogenic hot spots in close spatial relation to biofilm areas with low acid production may contribute to explain the observation of adjacent areas with demineralization and remineralization on tooth surfaces. progressive demineralization of the dental hard tissues is likely to occur underneath acidogenic hot spots in the biofilm, as shown by the multifocal pattern of demineralization in microradiographs of root caries lesions (34). the initial ph drop found in this study is in accordance with previous findings and resembles the initial phase of a classical stephan curve (6). we assume that oxygen depletion in the reaction chamber by the biofilm bacteria occurs quickly in our experimental model due to the small size of the custom - made wells. fermentation of glucose is enhanced as soon as anoxic conditions are reached in certain areas of the biofilm, and the ph begins to drop (35). after some time, acid production slows down, possibly because of the toxic effect of the fermentation acids on the acidogenic bacteria (36, 37). this was also seen in the present study, where half of the final h ion concentration was reached after less than 20 min in both experimental groups. it should be emphasized that biofilm ph in this study was recorded ex vivo in small custom - made wells without flow and clearance of metabolites. restoration of near - neutral ph, as seen in a classical stephan response in vivo, could therefore not occur. interestingly, even though the rate of the ph drop decreased with time, ph continued to fall throughout the experimental period, supporting a continued acid production. compared with other studies investigating biofilm ph upon exposure to carbohydrates, ph drops observed in the present study were moderate (10, 12). the fact that we studied thin, young biofilms from caries - free individuals may serve as an explanation. xiao (33) observed observed a positive correlation between biofilm thickness and ph in deep layers of the biofilm. furthermore, smaller ph drops in 2-day - old biofilms than in older biofilms have been documented previously (7, 8). finally, regardless of other factors caries - free individuals may elicit a reduced ph - response to sugar challenge compared with caries - active individuals (6, 38). it was an unexpected finding that the ph drops did not differ between biofilms exposed to sucrose and biofilms grown without repetitive sucrose supply. while other studies have shown an increased acid production in biofilms exposed to sucrose during growth, these results were obtained for older biofilms (5, 39). in studies using ph telemetry (7, 8), ph drops below 55.5 as our biofilms were collected from healthy, caries - free individuals with an early microflora that is unlikely to comprise high numbers of aciduric microorganisms (40), we conclude that it takes more than 2 days with regular sucrose exposure to modulate species composition and biofilm metabolism. the absence of flow and consequently the lack of acid clearance obviously impact the study results. different ph profiles might be observed under flow conditions. on the one hand, constant supply of fresh saliva with neutral ph and the removal of protons on the other hand, removal of protons from the biofilm may reduce diffusion of protons between acidogenic hot spots and sites with low acid production leading to a more pronounced difference in ph between these sites. as biofilms were incubated with saliva titrated to ph 7 at the beginning of an experiment, the baseline ph value was not actually calculated. such calculations would be difficult as ph drops during sample handling before the first image acquisition. however, control biofilms incubated with glucose - free sterile saliva showed a constant ph of 7 (data not shown). a clear drawback of microscopy - based ph recordings is that monitoring of multiple fov reduces the number of time points that can be collected for each individual fov. comparison of the 15-min data (three fov per biofilm) with the 60-min data (six fov per biofilm) showed that the initial rapid ph drop in the biofilms was underestimated in the 60-min experiments. other technical difficulties regarding image acquisition and image analysis have been discussed elsewhere (16, 41). in conclusion, the present study is the first of its kind to apply the combination of ph ratiometry and digital image analysis to systematically record extracellular ph in intact dental biofilms from 10 individuals for up to 1 h. we revealed heterogeneous ph landscapes within the three - dimensional biofilm architecture, including the presence of acidogenic hot spots. the data suggest that sucrose supply during growth does not affect ph development in young dental biofilms. | background and objectiveph in dental biofilms is of central importance for the development of caries. we used the ratiometric ph - sensitive dye c - snarf-4 in combination with digital image analysis to monitor extracellular ph in dental biofilms grown in situ with and without sucrose supply.designdental biofilms (48 h) from 10 individuals were collected on glass slabs mounted on intra - oral appliances. during growth, appliances were immersed extra - orally in either physiological saline or 4% sucrose for 2 min, eight times per day. fluorescence emissions of c - snarf-4 in deep layers of the biofilms were recorded ex vivo with confocal microscopy for 15 min or for 1 h after exposure to 0.4% glucose. extracellular ph was determined ratiometrically using digital image analysis.resultsextracellular ph dropped rapidly in most examined sites after addition of glucose. distinct ph microenvironments were observed within single biofilms. the variation in ph was similar between sites within the same biofilm and sites from different individuals. ph drop patterns did not differ between biofilms exposed to sucrose - free and sucrose - rich environments.conclusionthe present study is the first of its kind to apply the combination of ph ratiometry and digital image analysis to systematically record extracellular ph in intact dental biofilms from several individuals for up to 1 h. we observed highly heterogeneous ph landscapes and the presence of acidogenic microenvironments acidogenic hotspots within the biofilms. the data suggest that ph drops in young (48 h) dental biofilms are independent of the sucrose supply during growth. |
delivery is a natural and physiologic process, but a painful and exhausting phenomenon both for mother and those who care her. this process constitutes a very important physiologic event of woman 's life with significant physical, mental, and emotional effects. delivery is accompanied by pain, mental stress, probable physical injuries, and rarely, death. midwives are responsible to provide care and support for delivery of mothers in non - complicated deliveries. quality and the way of providing midwifery cares are among the factors affecting childbirth outcome. midwife 's functions and actions during this critical stage of woman life may lead to different outcomes ranging from life to death and from heath to physical injuries, with significant effects on the mental and emotional health of mother and child. delivery care is a process that aims to maintain the health of mothers and children and facilitates their normal growth and development later in life. according to yanger these indications are introduced as five valuable cs including communication, condolence, continue, commitment, and courage. in conventional delivery cares within delivery departments, generally a midwife is responsible for providing care for several delivering women and she mostly needs to do clinical care including monitoring of fetal heart rate and progress of delivery, prescribing drugs, recording delivery data, etc. precise observational studies in several hospitals have shown that in most of the cases, the women are left alone during delivery and the midwives only spend one - fourth of their time in the bedside of women in delivery room (this time is very short for establishing a continuing and sympathetic relationship with delivering woman), while being alone during delivery causes fear, anxiety, and forlornness in the delivering women. commission of health care in 2007 announced based on a review that 46% of women are left alone during labor, which causes worry in them. in the aforementioned conditions, the woman fears from being alone and also fears from pain, distress, contempt, being naked, and losing control on their behavior, their child 's health and death. facing these fears, anxieties, and tension resulting from predicting negative events is an experimental turning point in delivery, and the delivering woman shall benefit from continuing support and companionship for confronting with them. the policy of providing continuing midwifery care in england 's midwifery centers includes providing personal support to women during labor and delivery through one - to - one care (one midwife to one delivering woman) at the first and second stages of delivery. the benefit of this policy is a continuous, persuasive, and responsive support for woman during delivery. on the other hand, fear is the most important factor in causing severe pains and influences normal progression of delivery. one of the preventive methods in difficult and prolonged deliveries is providing proper support and precise care of woman during delivery. stress causes muscle contraction and, consequently, increases the severity of pain and the pain increases mother 's stress and anxiety. this may lead to slowing fetal heart rate and prolonging the second phase of delivery by causing a defective cycle. the presence of a caring person (preferably midwife) at the bedside of parturition (delivering) woman can facilitate the contractile activities and uterine bloodstream by decreasing mother 's anxiety. the results of hodnett 's study in 2007 showed that the childbirth outcomes of women receiving continuous care during labor were improved significantly compared to the conventional care group. meanwhile, continuing attendance of midwife, and so, continuing of care in all phases of delivery reinforces woman 's body ability in producing endogenous analgesics or endorphins. unnecessary intervention of midwifery such as induction, premature amniotomy, and perineal laceration are amongst the important factors that decrease satisfaction, and may deteriorate the midwifery care outcomes and cause improper experiences for mothers and midwives. but continuing care may lead to less delivery stimulation and induction, shorter duration of labor, decrease in midwifery surgical operations, lower episiotomy and cesarean, and less need to apply labor 's pain - reliving drugs, and thus, to a more physiologic delivery. other benefits of continuing care are increasing vaginal delivery, decreasing infection in mother and child, more satisfaction of mother and midwifery, and increasing breast feeding. this research was planned to study the effect of continuing delivery care on several childbirth outcomes such as type of delivery, perineal laceration, using oxytocin for stimulating delivery, and duration of delivery stages. this research was a quasi - experimental study and was conducted on parturition (delivering) women referring to tabriz 29 bahman hospital in 2009. according to a primary study, 84 samples were defined, and finally, the number was increased to 100. those parturition (delivering) women, who referred for vaginal delivery and satisfied the requirements for the study, were selected. the inclusion criteria included willingness for participating in the study, not having indications of abnormal delivery (e.g. not having multiple pregnancies, risky pregnancy, placenta or amniotic fluid problems), not having cesarean record, having normal pelvic diameters, height over 145 cm, not having mental diseases or problems in which the mother can not communicate with others (such as deafness and blindness), not using any unauthorized drugs, being in active phase of delivery, estimated weight of fetus likely to be less than 4000 g, live and full - term fetus, not having premature laceration in fetal membranes for more than 12 h, not having any unnatural bleeding from the vagina, number of pregnancies should be less than 5, mother 's age ranging 18 - 35 years, not having internal surgical diseases, and not having any risk for fetus (such as me conium defecation or fetal distress). the exclusion criteria included mother 's refusal of receiving continuing care and any emergency cases of mother and fetus, caring for which was beyond the responsibility of midwife. according to pre - recorded information in admission office of tabriz 29 bahman maternity hospital (research location), from a total of about 400 women who are admitted monthly in the delivery unit, at least 100 the inclusion criteria for the current study. based on the allocated data collection duration for this study (3 months), two series of 50 random numbers based on the serial number of admission in the delivery unit from among 300 delivering women who were predicted to be admitted in next 3 months were selected. the first series of women were allocated to routine care group and the second series to continuing care group randomly. according to the pre - planned arrangements during the data collection stage when a delivering woman from the experimental group was enrolled (at any time), the researcher was contacted to attend the delivery unit as soon as possible, and therefore, she provided the before, during, and after delivery cares by continuing attendance on the patient 's bed and initializing a one - to - one care. in the routine care group, when the delivering woman was enrolled in the study, she received the necessary cares by a group of unknown midwives and the midwives were not obliged to attend on a specific patient 's bedside. conventionally, in the labor room, four to five midwives are responsible to provide care for the delivering woman, and the delivery may be supported by one midwife and episiotomy excision repair done by another midwife. in general, the total number of midwives who provide care during delivery may reach five to six persons. while in the experimental group, only one midwife provided all cares and attended on the delivering woman 's bedside continuously. allocating samples in the two groups was completely random and all contextual factors that seemed to have an effect on results [such as mothers age, rank of pregnancy, type of delivery (full term vs. pre term), abortion, gestational age, etc. ] the checklist included three sections : section 1 included personal and social information, section 2 was related to history of earlier pregnancies (number of pregnancies, previous delivery and abortion, pregnancy age at the time of delivery, and mother 's age), and the third section was related to the outcome of the current delivery. content validity of the prepared checklist was assessed based on the viewpoints of informants, and cronbach 's alpha coefficient was used to test its reliability. the data analysis was done by spss version 13 ; t - test was used for comparing the amount of applied oxytocin during labor and mann whitney u test for comparing the type of delivery and laceration of delivery canal in the two groups. it shall be reminded that there was no limitation regarding use or non - use of oxytocin, its dose, and doing or not doing episiotomy in the two groups, and all of these were based on the delivering mothers needs and their health conditions, and after receiving confirmation from on - call physicians and midwives. since all research samples had the requirements for vaginal delivery and none of them were previously willing to have cesarean, choosing the type of delivery was done based on the needs and conditions of the delivering woman and on - call physicians diagnosis. this research project has been ethically and scientifically approved by research deputy of tabriz university of medical sciences. the social characteristics of the individual research subjects showed that majority of the patients were in the age range of 21 - 27 years (54%). most of patients were housewives (95%) and there was no statistically significant difference between the two groups (p = 0.64) regarding the mothers occupation. also, 91% of the patients husbands were self - employed and the rate was not statistically different in the two groups (p = 0.29). the levels of patients education were different in the two groups (p = 0.001). the educational level of the patients husbands was mostly in the secondary level and 3% were illiterate. the educational levels of patients husbands were same in both the groups (p = 0.075). comparing the types of delivery between the two groups (n = 100) comparing the level of perineal laceration in the two groups (n = 93) comparing the amount of oxytocin used in labor between the two groups (n = 100) reproductive profile of the research subjects was as follows. considering the number of pregnancies, 78% in the experimental group and 72% in the control group had referred with first pregnancy. considering the number of deliveries, 80 % in the experimental group and 82% in the control group did not have delivery record. also, 88% in the experimental group and 84% in the control group did not have abortion record. considering the pregnancy age, the average of pregnancy age in the experimental group was 39.54 0.81 and in the control group was 39.63 0.9. regarding the mothers age, the average of mothers age in the experimental group was 23.8 49.79 and in the control group was 24.6 33.22. the majority of women (i.e. 98%) in the experimental group and 88% in the control group had vaginal delivery. the results of mann whitney u test showed that although the number of cesareans in continuing care group was less, this difference was not significant statistically between the groups (p = 0.051). the results of independent t - test showed that the amount of oxytocin used in the first phase in both groups had a statistically significant difference (p = 0.001), such that in the continuing care group, the average dose was 0.94 1.7unit and in the conventional care group, it was 3.45 0.6 unit. meanwhile, the applied oxytocin in the second phase was significantly different in both groups (0.001), such that in the continuing care group, the average dose was 0.04 0.17unit and in the conventional care group, it was 0.48 0.8 unit. the laceration type in the experimental group was mostly of level 1, while in the control group, it was mostly done in level 2. in the experimental group, there was no level 4 laceration, but it was done in one case in the control group. the results of mann whitney u test showed that the level of perineal laceration had significant difference between the two groups (0.001). this study showed that childbirth outcomes may improve by providing continuing delivery cares by midwives and this causes less injury in the perineal area of the delivering women. the study findings showed that almost in all delivering women who had received continuing care, the performed perineal laceration was at level 2 or less, while the laceration was in this level only among two - thirds of the women in the conventional care group and the remaining had deeper lacerations. similar studies had been conducted investigating the level of perineal laceration, and the results of saultz and albedaiwi (2004) showed that perineal laceration in the conventional care group had a statistically significant difference with the continuing care group. it also must be noted that episiotomy incision in the continuing care group was less than in the conventional care group. hodnett 's study in 2008 which was done on two groups of conventional and continuing care showed that in the continuing care group, the dosage of drug for reduction of delivery pain, need for neonatal resuscitation, and need to do episiotomy were less, but the vaginal or perineal laceration was high. even though the continuing attendance of midwife in continuing care in all delivery phases strengthens the ability of woman 's body in producing endocrine or endorphin analgesics and the endorphins cause comfort, drowsiness, and increase euphoria, these studies showed that the effects of other factors such as non - accurate monitoring of the perinea, or ritgen maneuver, large size of fetuses head, race of the studied samples, or other reasons in increasing the perineal laceration should not be ruled out completely. the current study showed that less oxytocin is required during delivery in continuing midwifery care. that indicated less oxytocin use for delivery induction in the continuing care group compared to the conventional care group. not interfering in different phases of natural delivery (such as using oxytocin for induction and stimulating natural birth) the midwives sometimes face with conditions in which they are obliged to interfere potentially, while midwifery 's function and occupation is based on natural pregnancy and delivery. these interventions with accepted and recognized complications are only proposed when their advantages outweigh their disadvantages. although the type of delivery in the two groups of continuing and conventional care was not significantly different, the number of cesarean deliveries in the continuing care group was less than in the conventional care group. the results of page. and campbell showed that the type of delivery in the two groups of continuing and conventional care was the same, which is consistent with the results of the present study (3 and 22). (2000) showed that the type of delivery in the two groups of conventional care and delivery care with less caregiver (continuing care) had significant difference, which is inconsistent with the results of the present study. such an observation may result from the selected retrospective design or the sample size of the study. several factors are involved in determining the delivery type ; fear is the most important factor in creating severe pains and causing non - progression of natural delivery. so, it seems essential to provide sufficient care by a midwife during labor to decrease the rate of cesarean delivery and surgical complications for the mother and newborn. although based on the findings of this study continuing care did not decrease the rate of cesarean delivery significantly, it is worth to at least consider its efficacy in future complementary studies. in a systematic review by johantgen. from 1990 to 2008 in america which was entitled comparison of delivery and labor care provided by certified nurse - midwives and physicians, the results showed that care processes such as using epidural anesthesia, stimulating delivery, and doing episiotomy were applied less by nurses and midwives than physicians. the perineal laceration among mothers who were cared by the first group was less and their breast feeding was higher, but the childbirth outcomes based on the type of birth attendants were not statistically significant. the results showed that providing one - to - one delivery care and continuous attendance of the midwife on the bedside of delivering woman had positive effect on improvement of birth outcomes. so, providing the choice of one - to - one care for women in delivery rooms must be considered where it is logistically possible. | background : continuation of delivery care by a midwife, and establishing a relationship between the midwife and the delivering woman, is so important for women, and preserving such relationship increases woman 's calmness and self - confidence. the current research aims at studying the effect of midwifery continuing care during delivery on delivery outcomes.materials and methods : this study was a quasi - experimental research conducted on childbearing women referring to tabriz 29 bahman hospital. one hundred women were randomly assigned to either experimental (n = 50) or control (n = 50) group. in the experimental group, the women were cared exclusively with a midwife from the active phase continuously, while in the control group, women were cared with several midwifes conventionally. the birth outcomes were recorded in both valid and reliable groups (checklists). data were analyzed using spss version 13.0.results:type of delivery was the same in both the groups (p = 0.051). in the experimental group, grade of the perineal lacerations was lower (p = 0.001) ; also, in this group, less oxytocin was used in the labor stage (p = 0.001).conclusions : the results showed that providing one - to - one delivery care and continuous attendance of the midwife on the bedside of delivering woman had positive effect on improvement of birth outcomes. so, providing the choice of one - to - one care for women in delivery rooms must be considered where it is logistically possible. |
injection use is one of the most important medical procedures used globally, mostly for administration of drugs and immunization. in a developing country like india unsafe injection practices put patients and healthcare providers at risk of infectious and noninfectious adverse events and have been associated with a wide variety of procedures and settings. an injection is said to be safe if it does not harm the recipient, does not expose the provider to avoidable risk, and does not result in wastes that is dangerous for the community. injuries caused by needle sticks and sharps due to unsafe injection practices are the most common occupational hazard amongst health care personnel worldwide. the health care workers include doctors, nurses, laboratory technicians, dentists, phlebotomists, and refuse workers. the national institute of occupational safety and health (niosh) defines needle stick injury (nsi) as injuries that are caused by needles such as hypodermic needles, blood collection needles, intravenous (iv) stylets and needles used to connect part of iv delivery systems. the world health organization estimates that about 3 million needle stick injuries occur every year with 90% occurring in developing countries. these needle stick injuries pose a serious health problem particularly for transmission of blood borne viruses like hiv, hepatitis b, and hepatitis c. the risk of hiv transmission without prophylaxis is 0.3% from percutaneous injury. for unvaccinated healthcare workers, the risk from single needle sticks or cut exposure to hbv - infected blood ranges from 6 to 30%, depending on the hepatitis b antigen status of the source individual. who estimates that annually 21 million hepatitis b infections, 2 million hepatitis c infections, and 260,000 hiv / aids cases may be caused by reuse of syringes and needles without sterilization accounting for 40%, 32%, and 5%, respectively. an effective vaccine is available against hepatitis b infection but none is available against hiv or hepatitis c. hence it becomes all the more pertinent to reenforce safe injection practices amongst the medical personnel. medical interns who begin their clinical career after completing their graduation are exposed to blood and blood products in various settings like immunization sessions, drawing blood samples, assistance during surgery, conducting deliveries, and so forth. it is important that they are fully aware of safe injection practices and related biomedical waste management. even before starting their internship, medical students face similar type of exposures during their clinical postings in the departments of obstetrics and gynecology, medicine, surgery, accident and emergency, orthopedics, pediatrics, and so forth, thus making them vulnerable to deadly infections. thus, safe injection practice is critically important and its lack poses a major occupational health hazard for health care professionals. we chose to conduct this study on medical interns because they have less clinical experience and are more at risk of unsafe injection practices. there is a dearth of intervention based published studies on knowledge of interns on injection safety in india. with this background, we planned this study with the following objectives. objectives the aim is to determine the existing knowledge of medical interns regarding safe injection practices and related biomedical waste management.the aim is to measure the change in the level of awareness following an iec package regarding safe injection practices and related biomedical waste management amongst interns.the aim is to determine the status of hepatitis b vaccination among the interns. the aim is to determine the existing knowledge of medical interns regarding safe injection practices and related biomedical waste management. the aim is to measure the change in the level of awareness following an iec package regarding safe injection practices and related biomedical waste management amongst interns. study design. we conducted a follow - up study. study period. the study area was a tertiary care teaching hospital and medical college, new delhi, india, which is situated in central part of delhi. this medical college had an intake of 130 students for the course of mbbs of four and half years ' duration. study population and sample size. in india, the bachelor of medicine and bachelor of surgery (m.b.b.s) professional course consists of a four and half years ' degree course consisting of four professional examinations followed by one year of compulsory rotatory internship in all major disciplines in which their bedside clinical skills are enhanced. after successful completion of internship, so, the study population comprised of all the medical interns (106) who were to begin their compulsory rotatory internship for a period of one year from january 1, 2012 to december 31, 2012. a total of 106 interns were enrolled for the study. out of them a predesigned semistructured questionnaire was developed using previous published reports on injection safety [610 ] and pretested in the outgoing batch of interns in november 2011. iec package in the form of hands, on workshop, power point presentation, video - films, charts on injection safety, and biomedical waste management was used. the areas covered in all the above iec package included the three criteria of safe injection by who which are as follows : no harm to the recipient, no harm to the provider, no harm to the community, diseases transmitted by unsafe injection, vaccines to prevent blood borne infections, knowledge about autodisable (ad syringes), exposure to needle stick injuries, steps for prevention of needle stick injuries, postexposure prophylaxis against hiv, hepatitis b vaccination, disposal of injection related biomedical waste, and so forth. methodology. as a part of skill development of interns, theory classes and practical supportive supervision with respect to safe injection practices and biomedical waste management are done on a regular basis in their two months posting of community medicine every year. we tried to analyze this regular activity. a common briefing is planned for all the interns every year before the start of their internship training which was held for this batch on december 31, 2011. the purpose of the study was explained and informed consent of the entire group was obtained. they were administered a predesigned, standardized, self - administered pretested questionnaire for the baseline assessment regarding their awareness of safe injection practices. the questionnaire consisted of questions related to all the topics covered in the iec package as mentioned in the paragraph above under study instruments. a batch of 1820 interns was posted in the department of community medicine for a period of two months starting from january 1, 2012. in this way, six batches (a to f) rotated in the department in a period of one year from january 1, 2012. we also conducted a one - day workshop in first week of january 2012 for the entire batch of interns after the pretest to sensitize them regarding safe injection practices and biomedical waste management. subsequently, in the first week of their posting in community medicine, each batch (a to f) was given an intervention package consisting of a power point presentation and a video - film on safe injection practices and biomedical waste management. along with it charts and posters were shown by a single investigator during their period of posting in the department throughout the year. they also got hands - on experience in safe injection practices and biomedical waste management during their postings in health centers and during immunization sessions. in this way, they were exposed to repeated iec to reinforce their knowledge and skills required for injection safety and biomedical waste management. in the last week of their two months posting in the department of community medicine, they were again given the same self - administered questionnaire as a posttest and their change in the level of knowledge was assessed. bivariate analysis using mcnemar 's chi - square was used to examine the association between each dependent variable at pretest and posttest. out of the total 106 pretest and posttest questionnaires, 101 were complete and were analyzed with a response rate of 95.28%. almost all the interns (98%) had good knowledge in the preintervention assessment, about diseases which can be contracted by unsafe injections, namely hiv, hepatitis b and hepatitis c. majority (92.1%) knew about hepatitis b vaccine to prevent infection with this virus. all the interns (100%) knew about the three most commonly used routes for giving injection. most of the interns (93.1%) had good knowledge about life - saving drugs present in an emergency tray which is extremely important for management of emergencies (like anaphylaxis) following administration of injections. almost two - thirds knew correctly about the postexposure prophylaxis (pep) against hiv (74.3%) and the time period after which pep is not effective (76.2%). only 59.4% interns had knowledge about not using spirit swab before administration of any live vaccine (bcg, measles) (table 1). however, the knowledge was poor for some aspects of injection safety. only 23% interns knew correctly about all the three criteria for a safe injection and only 10% knew correctly the correct method of opening a glass ampoule and correct handling of syringe after giving injection. only 24% interns were aware of the do not 's after a needle stick injury. nearly one in four (26.7%) of them knew correct method to dispose gloves. tables 1 and 2 depict the comparison of the pre- and postintervention assessment of knowledge and skills ' regarding injection safety and related biomedical waste management, respectively. a highly significant (p < 0.001) improvement in the knowledge of interns was observed after intervention with respect to the three criteria of a safe injection, use of autodisabled syringe, cleaning of injection site, not to use spirit swab before administering any live vaccine, procedure to open a glass ampoule, precautions and do 's and do not 's after nsis, reporting of nsi, ideal time for initiating postexposure prophylaxis (pep), handling needle and syringe after giving injection, and disposal of injection related waste. other aspects in which significant improvement in knowledge of interns was observed after iec intervention were knowledge about common complications after giving injection (p = 0.03), time after which pep is not effective (p = 0.01) and disposal of needle cap (p = 0.04). however, there was no significant improvement in knowledge regarding postexposure prophylaxis after nsi (p = 0.21) and disposal of broken ampoule (p = 0.28). baseline (pretest) information regarding the immunization status of interns against hepatitis b was collected. it was observed that 67 (66.3%) interns had completed the three - dose hbv vaccination schedule, 22 (21.8%) were partially immunized with either one or two doses, and remaining 12 (11.9%) were not immunized against hepatitis b. after the intervention, there was significant (p < 0.001) improvement in their immunization status. as many as 78 (78.2%) interns had completed the 3-dose hbv vaccination schedule, 20 (19.8) were partially immunized, and only three interns remained unimmunized. at the baseline stage, information was collected on history of needle stick injuries in the past, that is, during the four and half years of m.b.b.s. six interns (5.9%) reported needle stick injury in the past before the start of their internship ; four of them had one needle stick injury while the other two had inflicted it twice. information was also collected about needle stick injuries which occurred after the start of internship. twenty - eight (27.7%) interns reported nsi with 22 interns having one exposure, 5 interns had two exposures, and one intern had injury thrice during the period of their internship. as a medical student in the m.b.b.s curriculum, students are taught about safe injection practices and biomedical waste management. it was seen that nearly all (98%) medical interns had good knowledge about the diseases transmitted by unsafe injections, namely, hiv, hepatitis b, and hepatitis c. all the interns knew about the three most common routes of giving injection viz intramuscular, intravenous, and intradermal. while giving any injections, be it for therapeutic or preventive purpose, an emergency tray should always be at hand and interns should be aware of the life - saving drugs in that tray such as adrenaline, antihistamine, and hydrocortisone. this aspect is often neglected and it is very important for management of emergencies following administration of injections. hence health professionals should have adequate knowledge regarding the emergency drugs and their usage so that any anaphylactic situations, if arose, can be managed effectively. this also shows that our m.b.b.s curriculum is quite robust in educating them with adequate knowledge about various issues of injection safety. however, the study reveals that some practical aspects of injection safety were not covered during their clinical postings making them vulnerable to risk carried by unsafe injection practices. the present study showed that the iec intervention package was effective in significantly improving the knowledge and practical aspects of injection safety and related biomedical waste management. a highly statistically significant improvement was observed in many variables related to knowledge about injection safety as evident in table 1. this shows that the intervention package administered to the interns after the baseline collection of information (preintervention) in the form of workshop, lectures, charts, and hands on training was helpful in improving the knowledge and practices of interns regarding various aspects of injection safety. this implies that interns who are inexperienced at the beginning of their medical career need regular reenforcement of basic knowledge and practices of universal precautions including injection safety. this should specially be emphasized in the clinical departments where chances of nsi are higher. they also need regular hands on training when they are posted in immunization clinics, wards, labour room, laboratories, accident and emergency department, and wherever they are exposed to blood and blood products. after intervention, there was improvement in immunization status against hepatitis b. but there were some interns who, even after intervention, remained partially immunized or unimmunized. this raises a question, whether we should make hepatitis b vaccination mandatory for those who are partially immunized or unimmunized at the beginning of internship, when risk of exposure to need stick injury is higher. similar result of hepatitis b immunization (66.5% and 87.5%) was reported from vellore and tehran, respectively. it was surprising to note that 6 (5.9%) interns had suffered an nsi earlier as medical students while pursuing their m.b.b.s course. this means that they were exposed to blood and blood products during their clinical, laboratory, and labour room postings during m.b.b.s. however, this problem was quite low as compared to results of other studies done on medical students in malaysia (14.1% and 38.3%) [9, 10 ]. these observations suggest that safe injection practices and sound knowledge about biomedical waste management should get due attention during m.b.b.s. all the interns who had a nsi exposure received counselling and postexposure prophylaxis and also were encouraged to report all nsi. internship is a period where practical knowledge is imparted and opportunity is given to acquire clinical skills. at the same time, there is need to safeguard interns against avoidable occupational hazards including risk of acquiring blood borne infections like hiv, hepatitis b, and hepatitis c. universal precautions, injection safety, and biowaste management need to be not only covered during graduate courses (mbbs) but also reinforced during internship. the limitation of our study was that we did not interview all the interns together at the end of their one - year internship, that is, on december 31, 2012. so the information regarding the actual status of hepatitis b immunization and nsi history may be underreported. this is because the postintervention assessment was carried out at the end of their two months posting in community medicine department. hence it was carried out at different times for different batches of interns which may have introduced bias. we conducted a single workshop for the entire batch of interns at the beginning of the study. we should have conducted a workshop for every batch of interns at the beginning of their posting in community medicine for the results to be more reliable. however, the rest of iec package (power point presentations, videos, and charts) was given to each batch of interns. recall bias, particularly exposure and history of nsi during mbbs course, is another limitation of this study. the baseline knowledge of interns was good in certain aspects of injection safety, namely, diseases transmitted by unsafe injections and their prevention, common routes of giving injections, and life - saving drugs in emergency tray. their knowledge was satisfactory regarding pep while it was poor in some other aspects like who criteria for safe injections, opening of glass ampoule, do not 's after needle stick injury, and biomedical waste management. we conclude that there was a change in the level of knowledge as the iec intervention package was effective in significantly improving the interns ' knowledge regarding safe injection practices, prevention of nsis, and injection related waste management. periodic reenforcement of the interns with hands on training and iec intervention will significantly protect them from nsis and prevent the spread of blood borne pathogens. | injuries caused by needle sticks and sharps due to unsafe injection practices are the most common occupational hazard amongst health care personnel. the objectives of our study were to determine the existing knowledge and practices of interns and change in their level following an information education and communication (iec) package regarding safe injection practices and related biomedical waste management and to determine the status of hepatitis b vaccination. we conducted a follow - up study among all (106) interns in a tertiary care teaching hospital, delhi. a predesigned semistructured questionnaire was used. iec package in the form of hands - on workshop and power point presentation was used. a highly significant (p < 0.001) improvement in the knowledge of interns was observed after intervention with respect to the three criteria of a safe injection and cleaning of injection site. thus, the baseline knowledge of interns was good in certain aspects of injection safety, namely, diseases transmitted by unsafe injections and their prevention. we conclude that iec intervention package was effective in significantly improving the interns ' knowledge regarding safe injection practices and biomedical waste management. almost two - thirds of interns were immunised against hepatitis b before the intervention and this proportion rose significantly after the intervention. |
it implies tolerance to the semiallogeneic fetus, which possesses half maternal genes and half paternal genes. the more genetically distinct the fetus is, the greater the immunological tolerance becomes during pregnancy. this occurs during pregnancies by egg donation (ed), when the fetus is allogenic. such tolerance also intervenes in modulating pregnancy - related pathologies, including preeclampsia. as it complicates up to 8% of pregnancies, preeclampsia is the leading cause of maternal and perinatal mortality and morbidity. adverse perinatal outcomes, such as prematurity and intrauterine growth restriction, are related to this condition. preeclampsia is a pregnancy - specific disease characterized by the development of both hypertension and proteinuria.. moreover, african - american and filipino women and a low socioeconomic status are associated with increased risk. although the precise etiology of the disorder is still unknown, deficient early placentation is particularly associated with early onset preeclampsia. in fact, abnormal placentation is thought to be immunologically mediated [2, 3 ]. as prevention and prediction of preeclampsia are still not possible, symptomatic clinical management should focus on preventing maternal morbidity (e.g., generalised seizures of eclampsia) and mortality. from the epidemiological viewpoint, there is a higher incidence of pregnancy - induced hypertension in pregnancy by ed, which oscillates between 16% and 40% of all cases. compared to pregnancy by autologous in vitro fertilization (ivf), the prevalence of hypertensive complications is 2637% as opposed to 8%, with an odds ratio (or) of 7.1 for the ed group. furthermore, the incidence of pregnancy - induced hypertension is higher in ed - pregnant women, who are not related to the donor and have not been previously exposed to the donor 's sperm [35 ]. the aim of this review is to analyze the maternofetal immunological tolerance phenomenon and its possible relation with preeclampsia onset, because this could justify the higher prevalence of this pathology in ed pregnancies as a result of an inadequate maternal immunological response to the allogeneic fetus. numerous fetal, maternal, and placenta - based mechanisms protect the fetus against the maternal immune system. the trophoblast hardly expresses the molecules of the main histocompatibility complex (mhc) or the human leukocyte antigen (hla), which contribute variability within the same species. this and other immunoregulatory mechanisms endeavor to avoid fetal cells from being innately rejected upon their arrival [6, 7 ]. even before implantation, the receptive maternal setting for the host is reflected in the uterus. scarce cytotoxic activity to foreign agents and the outstanding capacity to segregate cytokines of uterine natural killer (unk) cells in the maternofetal interphase intervene in the extensive hemodynamic remodeling that the pregnant uterus undergoes [6, 7 ]. a systemic maternal response is prepared even before the zygote reaches the uterus, which is based on the expression of the cytokine profile that is characteristic of t helper lymphocytes (th) type 2 (th2) [6, 7 ]. during human placentation, first, the endometrium is differentiated in the dense cell matrix known as the decidua. third, a subtype of these cells, fetal extravillous cytotrophoblast (evt), penetrates maternal vessels, which alters and replaces the endothelium and part of the muscle layer. in this way, the maternal uterine arteries are transformed into wide low - resistance vessels due to the destruction of their muscle layer, which leads to increased maternal flow to the placenta [8, 9 ]. placental villosities, composed of the cytotrophoblast and covered by syncytiotrophoblast, are immersed in the circulating maternal blood that comes into contact with its constituent immune cells. the exact mechanisms involved in maternal immunological tolerance which allow for a semiallogeneic or allogeneic pregnancy in ed [1012 ] are still unknown. for example, some authors postulate that the well - known maternal inflammation associated with preeclampsia may arise from a high concentration of the syncytiotrophoblast microparticles circulating in the mother 's blood. these would overactivate the response of maternal monocytes through their toll - like receptors (tlr-) 1 [1316 ]. these already begin in the secretory phase of the female menstrual cycle, and they adapt to the immune response from a preconceptional stage. among the cell components, the immunoregulatory and proangiogenic functions of unk cells and antigen - presenter cells (macrophages and dendritic cells (dcs)) are highlighted. the four main populations of decidual leukocytes present in early - stage pregnancy are unk cells, macrophages, dcs, and t - cells. of these, the most abundant are unk, macrophages, and t cd3 + cells (cd8 + and rarely cd4 +). nk cells are characterized by the expression of surface markers cd56 and cd16 and are subdivided into two populations based on the density of marker cd56 (bright - strong or dim - medium). of the nk cells circulating in peripheral blood, 9095% of them are highly cytotoxic and belong to the cd56 dim cd16 + phenotype. the rest of them possess cd56 bright cd16- and are highly efficient at secreting cytokines. in the decidua, the majority of nk cells possess a cd56 bright cd16- phenotype. thus, unk cells differ phenotypically from nk cells in peripheral blood and are characterized by poor toxicity and a good capacity to secrete cytokines and angiogenic mediators. they rapidly proliferate during the late secretory phase of the menstrual cycle and drop in number after the halfway point of human gestation. the fact that unk cells are present before implantation, and even in the decidua of an ectopic pregnancy, suggests that they are induced by signals regulated by stromal endocrine factors rather than by embryonic tissue. unk cells regulate trophoblast invasion through the secretion of angiogenic growth factors, cytokines, and chemokines [20, 21 ]. moreover, the ability of unk cells to kill semiallogeneic fetal cells or allogeneic cells in ed pregnancies is limited. the close contact between the evt and decidual leukocytes suggests the existence of paracrine interactions between maternal leukocytes and fetal cells. cytokines produced by unk cells at the human fetal - maternal interface include interleukin (il) 8, interferon - inducible - protein-10 (ip-10), and the most synthesized cytokine by unk, regulated upon activation normal t - cell expressed and secreted (rantes), triggers the migration of the invasive trophoblast. angiogenic factors of unk include vascular endothelial growth factor (vegf) and placental growth factor (plgf), as well as the most abundant, nkg5. the genuine interest in the role that immunity plays in vascular remodeling emerges from the study of hanna., who demonstrated in vitro and in vivo that unk cells participate in uterine spiral artery remodeling by promoting angiogenesis at embryonic implantation sites by means of a gradient of cytokines and vasoactive mediators [21, 22 ]. subsequent evidence has shown that during angiogenic activation, hormone factors and the hypoxic setting are also capable of regulating the production of angiogenic factors such as vegf and their interaction with endothelial cells. these cells possess activator or inhibitor receptors which belong to three main families : the type - c lectin family (cd94/nkg), the killer immunoglobulin - like receptor (kir), and immunoglobulin - like transcripts (ilt or the leukocyte immunoglobulin - like receptor). the effector functions of nk cells depend on fine tuning between these inhibitor and activator receptors, and they are considered activated when kir receptors are constitutively expressed. it has been demonstrated that extravillous trophoblast cells express maternal and paternal hla - c. the hla - c ligands for maternal kir receptors are divided into two groups, c1 and c2, which are defined by a dimorphism at position 80 of the 1 domain. the interaction between the trophoblast hla molecules and the kir receptors of the unk cells of the maternal endometrium inhibits cytotoxic activity and modulates cytokine production and growth factors by unk cells to favor trophoblast growth, endometrium invasion, and vascular remodeling. so the kir2d receptors (containing two immunoglobulin - like domains) of unk cells are better capable of recognizing trophoblastic hla - c than the kir of the nk in peripheral blood. depending on the combination of haplotypes, kir2d can act more like an activator or more like an inhibitor [25, 26 ]. the genomic kir region contains a family of highly polymorphic and homologous genes localized in chromosome 19q13.4 inside the leukocyte receptors complex. according to populational studies, the order of the kir genes along the chromosome has mainly determined two different haplotypes : a (lacks activator receptors) and b (possesses activator and inhibitor receptors) [25, 27 ]. the maternal kir genotype can be aa (inhibitor), ab, or bb. the combination of the maternal kir aa genotype with a fetal hlac2 (hlac) increases the risk of preeclampsia. as this interaction gives rise to a strong inhibitor signal, it is considered that the inhibition, and not the activation of unk cells, predisposes to preeclampsia. unk cells would be unable to participate in uterine arterial remodeling because they are inhibited. therefore, the presence of activator receptors in unk cells to protect against preeclampsia has been proposed (figure 1). hence unk cells ' functionality during pregnancy depends on the combination of two polymorphic genes. these are the maternal genotype for kir (aa, ab, or bb) and fetal hla - c haplotypes [c1 (hlac) or c2 ]. during pregnancy, the frequency of the maternal kir aa genotype increases with pathologies related to defective placentation (preeclampsia, intrauterine growth restriction, and recurrent spontaneous abortions), but only when the fetus possesses more c2 genes than the mother (e.g., maternal c1/c1 with fetal c1/c2 and maternal c1/c2 with fetal c2/c2) or when the only c2 that the fetus possesses is of paternal origin. therefore, a deleterious effect of paternal allogeneic c2 and the early - stage role in pregnancy of these receptor / ligand pairs in reproductive failure pathogenesis have been postulated. it is known that the telomeric b region of haplotype kir b protects against these alterations in pregnancy, especially when the fetus possesses the c2 gene. in general terms, different human populations present a reciprocal relation between the frequency of aa and that of hla - c2, which suggests a selection against this combination. in normal pregnancies, recognition of fetal hla - c by receptor kir - bb of unk triggers the release of cytokines by unk cells. these include transforming growth factor - beta (tgf-), whose participation in immunoregulation and angiogenesis has been well - established, and angiogenic factors placenta growth factor (pigf), and vascular endothelial growth factor (vegf). conversely in preeclampsia, when kir - aa of maternal unk cells recognize the hla - c of the extravillous trophoblast, unk cells display a poorer expression of these mediators, as well as an overexpression of antiangiogenic factors like soluble endoglin (seng) and soluble fms - like tyrosine (sflt1) kinase-1 factor. seng inhibits tgf-1 from binding to the surface of its receptors and diminishes nitric oxide - mediated endothelial signaling. interestingly, significantly lower pigf levels, but with higher sflt1 and seng concentrations, have been demonstrated before gestation week 30 in the serum or plasma of pregnant women who have developed preeclampsia if compared with pregnant women who have not develop this disease. serum levels of granulysin, a cytotoxic granule protein of nk cells and cytotoxic t lymphocytes, are significantly high in preeclamptic patients as compared with women with normal pregnancies [31, 32 ]. indeed, the proportion of granulysin - producing cytotoxic t - cells notably increases in the peripheral blood of preeclamptic patients in comparison to healthy pregnant women. preeclamptic women do not show significantly different serum levels of rantes, a cytokine produced by unk cells at the human fetal - maternal interface, if compared with healthy pregnant women. nevertheless, the placental gene expression of rantes has been found to be upregulated in severe early onset preeclampsia from gestational weeks 25 to 27 when compared with placental samples of uncomplicated pregnancies in similar gestational weeks. further studies are required to elucidate the exact contribution of rantes in inducing a tolerogenic maternal immune response to allow for trophoblast survival, migration, and invasion. these studies would provide a better understanding of its role in pregnancy complications, such as recurrent spontaneous abortions or preeclampsia. several research lines have demonstrated the key role played by antigen - presenter cells (apc) in the maternofetal interphase during pregnancy. dcs, which are the most powerful apc, are required to initiate and modulate immune responses and to induce immunological tolerance [3537 ]. in humans, the density of endometrial immature dcs (cd1a+) is significantly greater than that of mature dcs (cd83 +) throughout the menstrual cycle. indeed the total number of cd1a+ dcs is much larger in the basal layer of the endometrium than in the functional layer during the secretory phase. cd1a, a highly specific and sensitive marker of immature dcs, mediates a hla - independent antigen presentation pathway. during the first trimester of pregnancy, most dcs express dc - specific adhesion receptor dc - sign (dendritic cell - specific icam - grabbing nonintegrin, classified as cd209). in fact, the dc - sign expression at the maternal - fetal interface in the rhesus macaque has been reported as an early response by the primate maternal immune system to the implanting embryo. uterine dcs direct maternal receptivity by regulating decidual tissue remodeling and angiogenesis in mice. indeed, uterine dcs play a key role in embryo implantation, when they show an immature phenotype. during intrauterine and extrauterine pregnancies, the immature dc status prevails, which has been related to the interaction with unk. the mature dc status has been associated with implantation failure [17, 38, 41 ], whereas the majority of decidual immature dc - sign+ dcs are in close contact with unk ; cd83 + mature dcs relate to cd3 + t - cells. regarding the adaptive response, dcs participate in tolerance induction as they are crucial for agonist - induced t regulatory cells (treg) differentiation. heme oxygenase-1 (ho-1) is a microsomal enzyme with anti - inflammatory, antiapoptotic, and antiproliferative properties. ho-1 reduction is related to murine pregnancy complications, such as abortion. in murine pregnancies blockage of ho-1 renders dcs a mature state, which promotes the action of effector t - cells. indeed both ho-1 and its metabolite carbon monoxide promote implantation and placentation [44, 45 ]. pregnancy disorders such as preeclampsia and intrauterine growth restriction are associated with ho-1 lessening and the impaired remodeling of maternal spiral arteries. interestingly, carbon monoxide induces the proliferation of unk cells and the remodeling of spiral arteries in pregnant hypertensive ho-1 mutant mice. accordingly in a clinical mice model of intrauterine growth restriction, carbon monoxide prevented fetal death by reducing free haem levels in circulation. dcs and macrophages, present in the human endometrium, play a role in decidualization and implantation. after unk, macrophages are the second most abundant population in the maternofetal interphase in both implantation and early pregnancy development [48, 49 ]. macrophages congregate around spiral arteries, while the placenta develops and supports vascular remodeling by releasing proangiogenic factors, such as vegf and mmps and by removing apoptotic cells. they also generate a wide range of cytokines, mainly for their function as apc, and they specifically produce high levels of il-10, a well - known anti - inflammatory mediator. hence they inhibit t - cell responses through e2 prostaglandin production, and they also produce tryptophan metabolites that can abolish t - cell proliferation. functional macrophage maturation leads to a macrophage effector phenotype, either m1 or m2 [18, 50 ]. in contrast, m2 macrophages are polarized by being exposed to an environment containing the cytokines of th2 (il-4, il-10) and glucocorticoids. m1-type macrophages, or classically activated monocytes, participate in the progression of inflammation by segregating tumor necrosis factor (tnf) and il-12, and they play a role in tissue destruction [50, 51 ]. m2 macrophages, or alternatively activated monocytes, generate an enhanced production of anti - inflammatory cytokines, il-1 receptor antagonist, il-10, and transforming growth factor (tgf-) beta. furthermore, m2 macrophages express the macrophage mannose receptor, which mediates host defense and the elimination of the substances produced in inflammatory processes [48, 49 ]. the polarization of decidual macrophages toward m2, found in normal pregnancies, indicates that their immunosuppressive activities are critical for maintaining immunological homeostasis during pregnancy [18, 50 ]. the innate immune response of macrophages is regulated by signaling mediated by pattern recognition receptors, that is, toll - like receptors (tlrs). recognition of microbes by tlrs on macrophages is the primary host defense mechanism in the decidua [18, 48, 50 ]. at the end of pregnancy, macrophages with an inflammatory phenotype participate in cervical ripening and in onset of labor. paradoxically in patients who have undergone ivf, the inflammatory environment generated by performing an endometrial biopsy before implantation entails a higher implantation rate. a study of tissue samples from spontaneous abortions and elective terminations of pregnancy has shown an increased population of decidual macrophages in spontaneous abortions. the fas - ligand (fas - l) overexpression of these decidual macrophages during spontaneous abortions has also been demonstrated. fas - l is a transmembrane protein that binds to the fas receptor and triggers apoptosis to fas - expressing cells. therefore, it has been hypothesized that the fas - l expression by decidual macrophages forms part of m2-like polarization. the fas - l expression by decidual macrophages could induce apoptosis to fas - bearing activated t - cells to potentially diminish deleterious maternal immune responses against the semiallogeneic or allogeneic embryo in ed pregnancies. the decidual differentiation of macrophages and dcs is regulated by the granulocyte - macrophage colony - stimulating factor (gm - csf). in the nonpregnant human endometrium, luminal and glandular a peak in the gm - csf mrna levels has been observed during the window of implantation. the mrna for the gm - csf receptor has been localized in endothelial cells of the spiral artery. gm - csf levels in decidual cells are higher in patients with preeclampsia if compared with gestational - age matched controls. besides, the cytokines involved in preeclampsia tnf and il-1 upregulate gm - csf mrna in cultured first - trimester human decidual cells. in line with this, gm - csf levels in blood and gm - csf / total protein levels in the placenta are significantly higher in gestations with preeclampsia than in normal pregnancies. therefore, it is feasible to hypothesize that increased gm - csf in patients with preeclampsia might contribute to dc maturity and the decidual macrophage polarization to m1. these mediate the clearance of pathogens, apoptotic cells, and immune complexes by forming a membrane attack complex (mac), which leads to cell lysis [54, 55 ]. the complement system can be activated in three pathways : the classic pathway is triggered by antigen - antibody complexes ; the alternative pathway is spontaneously and continuously activated ; the lectin pathway is triggered by the binding of mannan - binding lectin to mannose residues on the surface of microorganisms [54, 55 ]. irrespectively of the mechanism of activation, all the pathways converge to generate c3 convertases, which transform c3 into its active components, c3a and c3b. c3b is a main effector of the complement that tags nonself cells for destruction by phagocytes. c3b also binds to c3 covertases to form c5 convertase, which transforms c5 into c5b and powerful proinflammatory mediator c5a. c5b associates not only with c6, c7, c8, but also with many units of c9, to form a lytic pore that inserts into cell membranes, known as the mac (c5b-9). c3a and c5a, commonly known as anaphylatoxins given their role in anaphylactic shock, facilitate pathogen clearance by increasing vascular permeability, inducing inflammatory cell chemotaxis, and releasing cytokines. c3a and c5a exert these proinflammatory effects by binding to their respective receptors, c3a receptor (c3ar), and the two receptors for c5, c5a receptor (c5ar ; cd88) and c5a receptor - like 2 receptor (c5l2). syncytiotrophoblasts, villous cytotrophoblasts, and evt express the three regulatory proteins of the complement, these being decay - accelerating factor (daf), membrane cofactor protein (mcp), and cd59 which avoids the formation of the mac and subsequent cell lysis. thus, excessive complement activation is prevented in successful human pregnancies thanks to the presence of these three regulatory proteins in trophoblast membranes. the component of complement c1q plays a very important role in trophoblast migration, spiral arteries remodeling, and normal placentation. the decidual endothelial cells (decs) covering spiral arteries acquire the ability to synthesize c1q. this protein is bound to the cell surface and acts as a physical link between endovascular trophoblasts and dec to favor the vascular remodeling process. in line with this, c1q - deficient pregnant [c1q (/) ] rats present the main findings of human preeclampsia : hypertension, albuminuria, endotheliosis, diminished placental vegf, and elevated levels of soluble vegf receptor 1 (sflt-1), with high fetal mortality. mannose - binding lectin (mbl) activates the lectin pathway of the complement. the level of mbl in the vaginal cavity changes during the menstrual cycle, which is produced locally by vaginal cells. mbl apparently plays a key role in embryonic implantation because an analysis of uterine aspirates obtained upon oocyte capture for ivf has revealed a high level of mbl in patients whose infertility was of unknown etiology as compared to patients who underwent ivf / icsi for male factor or tubal infertility. although the serum levels of mbl rise during pregnancy, its function is still to be clarified. patients with preeclampsia show higher median plasma mbl concentrations when compared to women with uncomplicated pregnancies. likewise the association of a genetically related mbl polymorphism with mbl diminished the functional activity that protects against preeclampsia. interestingly, the serum obtained from preeclamptic women has been found to prevent the interaction between evt and decs, which avoids the endovascular invasion of trophoblastic cells. increased serum mbl in women with preeclampsia inhibits the interaction of evt with c1q, which interferes with the process of evt adhesion to and migration through decs. the lectin pathway of complement is also activated by ficolins, which mediate a primitive opsonophagocytosis. they are soluble molecules of the innate immune system that recognize carbohydrate molecules on microbial pathogens, apoptotic, and necrotic cells. these reduced plasma ficolin-2 concentrations in preeclampsia might contribute to the development of the maternal syndrome of the disease by the impaired removal of the trophoblast - derived material released into the maternal circulation by the hypoxic and oxidatively stressed preeclamptic placenta. although the complement components are normally high during pregnancy, excessive complement activation, particularly enhanced c5a synthesis, is associated with pregnancy complications such as recurrent abortion, preterm birth, and preeclampsia. sflt-1 sequesters vegf and pigf, which are crucial growth factors for normal placental development and for successful pregnancy. therefore, it has been postulated that c5a can harm angiogenesis by contributing to abnormal placentation, which allows fetal loss in early pregnancy stages or preeclampsia in later stages. alternatively, c5a can cause preterm birth by inducing cervical ripening and by releasing a large number of birth - prompting mediators. preeclamptic patients have significantly higher c4d, c3a, and c5b9 levels and substantially lower c3 concentrations than healthy pregnant women. indeed higher plasmatic levels of c5b9, or excessive terminal complement activation, have been found in preeclamptic patients with intrauterine growth restriction as compared with those presenting normal intrauterine growth. in human pregnancies at between gestation weeks 10 and 15, the plasma levels of activation product bb (derived from factors b which initiate c3b activation through the alternative pathway initiation complex), activated c3 (c3a), c5 - 9, and the serum levels of angiogenic factors pigf, sflt-1, and seng, have been quantified. surprisingly, multiparous women who changed their partner presented higher bb levels and were 5 times more likely to develop preeclampsia as compared to women who were still with their same partner since their last pregnancy. women with preeclampsia present significantly higher plasma levels of c5a than women with uncomplicated pregnancy. before gestation week 20, women who later developed any hypertensive disease related to pregnancy or gestational hypertension showed higher plasma levels of c3a when compared with those who did not develop these diseases. in the placentas of human severe early - onset preeclampsia, a low c3ar expression has been found as compared to women with preterm nonpreeclamptic pregnancies. the activation of complement c3ar through autoantibodies has been revealed to contribute to preeclampsia pathogenesis. the human maternal angiotensin ii type 1 receptor agonistic autoantibody stimulates the deposition of complement c3 in placentas and kidneys of pregnant mice through the activation of angiotensin ii type 1 receptor. interference with c3a signaling through a complement c3ar - specific antagonist significantly decreases hypertension and proteinuria in angiotensin ii type 1 receptor agonistic autoantibody - injected pregnant mice. complement c3ar antagonist significantly not only inhibits autoantibody - induced circulating sflt-1, a well - known antiangiogenic protein related to preeclampsia, but also reduces small placental size with damaged angiogenesis and intrauterine growth restriction. in humans, it has been demonstrated that the placentas of preeclamptic patients present a significantly higher c3 deposition than normotensive controls. in cultured human villous explants, its complement c3ar activation has been seen as an important mechanism that underlies autoantibody - induced sflt-1 secretion and decreased angiogenesis. nevertheless, the low c3ar expression in the placentas of women with preeclampsia indicates that further studies are required to evaluate the usefulness of the postulated therapy. cells of the innate immune system respond to infectious microorganisms by pattern recognition receptors, such as tlrs. these recognize the sequences expressed by microbes named pathogen - associated molecular patterns (pamps), such as bacterial lipopolysaccharide (lps) or viral dsrna. since both first trimester and term placentas show tlrs, the placenta may recognize pathogens through these receptors and could induce a subsequent immune response. it is known that human first - trimester trophoblasts constitutively secrete chemokines like growth - related oncogene, growth - related oncogene, il-8, and monocyte chemotactic protein-1 (mcp-1). the ligation of tlr-3 by viral poly (i : c), or tlr-4 by bacterial lps, significantly increases the trophoblast secretion of chemokines.. moreover, tlr-3 stimulation induces rantes secretion by trophoblast cells, which is chemotactic for monocytes. a significant increase in the tlr-4 protein expression is observed in placental trophoblasts of preeclamptic patients as compared to normotensive pregnant women [66, 67 ]. surprisingly, the expression of tlr-2 and tlr-4 in maternal neutrophils has been found to diminish in preeclampsia when compared with normal pregnant controls of similar gestational ages. further studies are required to explain the discordant tlr-4 expression between placental trophoblasts and maternal neutrophils in preeclamptic patients. as a response to the paracrine signals from the trophoblast, the proinflammatory cytokines, chemokines, and angiogenic factors in decidual stromal cells are significantly induced. in line with this, a study was carried out with human endometrial stromal cells decidualized with progesterone, which were treated with either conditional media from human trophoblasts (tcm) or control - conditioned media (ccm) from nondecidualized stromal cells. it revealed that the most overexpressed genes at 12 hours of treatment were chemokines cxcl1 (gro), il8, c - x - c chemokine receptor type 4 (cxcr-4), and other genes implicated in the immune response, such as pentraxin 3 (ptx3), il6, and tnf-induced protein 6 (tnfaip6), or metaloproteinases (mmp1, mmp10, and mmp14). the downregulated genes were growth factors, such as igf1, fgf1, and the genes involved in wnt signaling. paracrine interactions between evt and the maternal decidua are therefore essential for successful embryonic implantation, which occurs in an enriched cytokine / chemokine environment where stromal cells ' mitotic activity is limited in the invasive implantation phase. a prior in vitro study also revealed that the most overexpressed genes by endometrial stromal cells during implantation, due to the effect of the trophoblast, were those involved in inflammatory response, immune response and chemotaxis (ptx3, il8, il1 receptors, il18 receptor, and tnfaip6), cell growth regulators (igf - binding proteins 1 and 2), and signal transduction. downregulated genes were those involved in proteolysis (mmp11) and cell death, transcription factors, and the genes involved in the humoral immune response (cd24 antigen). the main secretory product of a pregnant woman 's decidualized endometrium is insulin - like growth factor binding protein-1 (igfbp1). its interaction with the 51 integrin of the evt cell surface triggers its migration in an igf - independent manner. whereas decidual igfbp1 production increases progressively during the first and second trimesters of uncomplicated pregnancies, women destined to develop preeclampsia present low serum levels of igfbp1, which may indicate decidual dysfunction. the vast majority of the trophoblast that comes into contact with maternal tissue does not possess the antigenic determinants required for maternal t - cell activation ; indeed it prevents the potential maternal antifetal rejection. the syncytiotrophoblast that is the main trophoblast to come into contact with the maternal immune system lacks classic class i and ii hla antigens. evt has an invasive phenotype and forms columns of cells that invade the maternal decidua and replace the endothelium of spiral arteries. this evt expresses a single class i hla expression pattern with nonpolymorphic molecules, which include hla - e, -f, -g, and -c. hla - g is crucial in maternal tolerance to the semiallogeneic or allogeneic fetus in ed pregnancies. the hla - g expression in evt has been found throughout pregnancy. with fas - l, soluble hla - g induces cd8 + t cell apoptosis [24, 29 ]. hla - g expressed in evt inhibits not only cytotoxic t lymphocyte responses, but also nk cell functions. a leader peptide of hla - g forms a complex with hla - e on the trophoblast cell surface and binds to the cd94/nkg2 receptor in nk cells. hla - e, which is found in all the cells expressing hla - c or hla - f, is localized mainly on the evt surface that invades the maternal decidua. like hla - f, it can promote fetal growth as its expression coincides with the rapid fetal growth period. although polymorphic hla - c is also present in evt, it is not as highly polymorphic as hla - a and hla - b. of all the hla class i molecules expressed by evt, only hla - c displays the variability required to constitute a fetal alloantigen, and it is recognized by maternal unk cells through their kir receptors. fas - l from fetal evt or maternal decidual cells, coupled with soluble hla - g from evt, induces cd8 + t - cell apoptosis [23, 28 ], thus increasing maternofetal immune tolerance. isolated first - trimester trophoblast cells have been described to not show fasl on their membrane but to also express a cytoplasmic form. after the disruption of these microvesicles, the secreted fasl induces t - cell apoptosis through the activation of the fas pathway. this knowledge has been supported by a subsequent in vivo study, which has only found fasl production and storage in first - trimester human syncytiotrophoblast, but not in the cytotrophoblast. on the other hand, it has been recently reported that the fas - l a-670 g polymorphism is associated with increased risk of preeclampsia. therefore, the desire to gain a better understanding of the fetal fasl expression and its contribution to maternofetal tolerance may inspire further studies. it is expressed in both evt and villous trophoblasts in humans, where it may inhibit maternal t - cell activation through the deprivation of tryptophan t - cells. the pharmacological inhibition of ido activity in murine pregnancy has been demonstrated to induce maternal t - cell - mediated rejection of the allogenic, but not the syngenic concept. nevertheless, the genetic deletion of ido in mice results in normal litter size as compared to ido - sufficient control mice. it is worth noting that lack of ido in mammals can be compensated by the tryptophan dioxygenase enzyme, which induces tryptophan catabolism. optimal maternal t - cell activation requires the connection between the t - cell receptor (tcr) and the hla antigenic peptide of the antigen - presenting cell (apc). in order to provoke efficient t - cell activation, a positive costimulatory signal is required, which is mediated by the interaction between cd28, which is constitutively expressed in most mature t - cells, and molecules b7 - 1 and b7 - 2 exposed by apc. interestingly, blockade of b7 - 1 and b7 - 2 at the time of murine implantation has been reported to induce the inhibition of maternal fetus rejection in abortion - prone cba / jxdba/2 matings. moreover, the frequency of b7 - 1 and b7 - 2 expressing activated monocytes in peripheral blood of preeclamptic patients is lower than in normal pregnant woman. b7 - 1 and b7 - 2 also bind in another receptor in t - cells, cytotoxic lymphocyte antigen-4 (ctla-4). their union provides an inhibitory signal that plays a key role in the negative regulation of the immune system. susceptibility to recurrent spontaneous abortion mediated by a polymorphism in the ctla4 gene has been suggested. another costimulatory pathway that plays a role in peripheral tolerance is defined by the programmed death-1 receptor and its ligands, pdl1 and pdl2. in pregnancy, while pdl1 or b7-h1 is expressed by all the trophoblast populations, pdl2 or b7-dc is present in the syncytiotrophoblast in early pregnancy. it is known that pdl1 is essential to maintain maternofetal tolerance, and its blockade or deficiency results in poor fetal survival and a shift toward th1 placental cytokines. initially, it was suggested that the human fetus is not rejected by the maternal immune system due to the prevalent cytokine production of th2 cells. the th2 cytokines produced at the maternal - fetal interface would inhibit th1 responses, leading to fetal survival. yet while th2 cells predominate in early pregnancy decidua, th1 cells prevail in the nonpregnant endometrium, particularly in the proliferative phase. thus, the th1/th2 ratio peaks in the proliferative endometrium and significantly decreases in the secretory phase and reaches its lowest level in the early pregnancy decidua. similarly, the th2 cytokine expression, specifically il-6 and il-10, is 10-fold higher in the early pregnancy decidua as compared to the nonpregnant endometrium. besides, progesterone stimulates a th2-type response, decreases inflammatory cytokines, and restrains allogeneic responses to allow fetal survival. the characteristic cytokines of th2 cells are il-4, il-5, il-6, il-9, il-10, and il-13. although these cells participate in the development of humoral immunity against extracellular pathogens, they also repress the functions of phagocytic cells. th1 cells not only synthesize interferon - g (ifn - g), il-2, and tumor necrosis factor - a (tnf-), but also trigger cell - mediated immunity and phagocyte - dependent inflammation. a tendency for immune th1 responses has been found in human pregnancy - related complications, such as recurrent spontaneous abortions. significantly higher serum levels of th2 cytokines, il-6, and il-10 and considerably lower levels of the th1 cytokine, ifn - g, have been reported in normal pregnancy as compared to unexplained recurrent pregnancy losses. accordingly, patients reporting recurrent pregnancy losses and infertile women with multiple implantation failures after ivf present increased t helper 1 cytokine responses by circulating t - cells. the injection of each th1 cytokine, like ifn - g, tnf-, and il-2, or the coadministration of these, in pregnant mice significantly increased fetal resorption. however, the function of a major chemokine in the th1 response, rantes, may prove essential for modulating the responses of specific t - cells for alloantigens during normal pregnancy. indeed, successful pregnancies are accompanied by increased serum levels of rantes, which are lower in patients who suffer recurrent abortions. it has been demonstrated in vitro that rantes specifically suppresses alloactivated maternal t - cells. so the high levels of progesterone present during normal pregnancy, particularly on the maternofetal surface, can be predictors of rantes production at levels required to induce a tolerogenic immune response locally. rantes (also known as ccl5) is a proinflammatory chemokine that can act as a modulator of alloantigen - specific t - cell responses in healthy pregnancy. whereas in successful pregnancies the serum levels of rantes are high, they are low in recurrent spontaneous abortions. thus, rantes might cooperate in the maternal tolerogenic immune response to allow trophoblast cell survival and migration. it is known that in peripheral blood in preeclampsia, the percentage of th1 cells and the th1/th2 ratio are significantly higher, while the percentage of th2 cells is significantly lower than in the third trimester of healthy pregnancy. a change to th1-type immunity is expressed in the serum of preeclamptic patients by an increase in the il2/il4 and ifng / il4 ratios. in addition, preeclampsia is associated with a proinflammatory systemic environment due to the elevated circulating levels of proinflammatory cytokines il-6 and tnf - alpha, chemokines il-8, ip-10, and mcp-1, and adhesion molecules intercellular adhesion molecule 1 (icam-1) and vascular cell adhesion protein 1 (vcam-1) as compared to normal pregnancy. surprisingly, the increased ip-10, mcp-1, icam-1, and vcam-1 concentrations in preeclamptic patients correlate significantly with blood pressure values and liver and renal function parameters. in line with this, the peripheral blood mononuclear cell production of il-12, which induces th1 responses, diminishes in normal pregnant women but increases in preeclamptic patients. in the maternal immune response against the fetus, which may be considered a semiallograft or an allograft in pregnancies by ed, the role of cd4+cd25+foxp3 + treg cells is particularly relevant. the transcriptional regulatory protein forkhead box p3 (foxp3) is a transcriptional repressor required for the development and function of treg cells. it has been identified in deciduas cd4 + t - cells expressing foxp3 with high levels of cd25 (cd4+cd25foxp3 +) or low levels of cd25 (cd4+cd25). whereas decidual cd4+cd25 treg cells are involved in the regulation of immune responses in humans, decidual cd4+cd25 t - cells display an activated phenotype by expressing raised levels of cd69 and low levels of foxp3 and cytotoxic t lymphocyte antigen (ctla)-4. decidual cd4+cd25 treg cells contribute to the maternal immune tolerance of fetal antigens, since deciduas in early human pregnancy contain abundant cd4+cd25 treg cells that express ctla-4 at high levels. these cells prevent the proliferation of autologous cd4+cd25 t - cells. moreover, the proportion of decidual cd4+cd25 t - cells is substantially lower in spontaneous abortion as compared to induced abortions. treg cells are essential in the induction and maintenance of mhc class ii antigen - specific tolerance. although hla ii is not expressed in villous or evt, the trophoblastic cell debris containing the intracellular fetal hla - dr antigen circulates in maternal blood. it has been postulated that immature dcs, acting as apcs, catch these debris and induce peripheral tolerance through the induction of treg cells. the immune regulation of cd4 + t - cells is carried out mainly by treg cells. the apoptosis of cd8 + t - cells is induced by the soluble hla - g and fas ligand expressed in evt. the regulation of both cd4 + and cd8 + t - cells results in maternofetal tolerance. treg cells also enhance the maternal tolerance of the fetus through the expression of ctla-4 on their surface. the ligation of ctla-4 by transmembrane protein b7 of apcs results in an increased ido expression on decidual and peripheral blood dcs and monocytes / macrophages. circulating treg cells increase during early pregnancy, reach a higher level during the second trimester, and decline postpartum. estrogen has been suggested to promote maternofetal tolerance by increasing treg cells since the treatment of nave mice with e2 increases both the cd25 + cell number and the foxp3 expression level. in addition, estrogen treatment and pregnancy induce a similar foxp3 protein expression. in line with this, in vivo and in vitro elegant mice models have provided evidence that progesterone increases the proportion of cd4+cd25 + treg cells and il-10 expression and enhances their suppressive function. additionally at equivalent physiological doses to midterm pregnancy, progesterone, but not estradiol, converts tcd4+cd25 t - cells into cd4+cd25 + treg cells. it has therefore been suggested that progesterone extends treg cell populations by means of nuclear progesterone receptors. besides, ru 486 significantly decreases the amount and function of treg cells at the maternofetal interface before the onset of induced abortion. the significantly reduced foxp3 expression has been reported to be accompanied by a significant increase in proinflammatory factors. a subpopulation of tcd4 + effector cells, th17, differs from th1, th2, and treg cells. th17 cells secrete il-17 and express cc chemokine receptor type 6 (ccr6). whereas the prevalence of tregs lowered, that of th17 cells increased in both the peripheral blood and decidua of patients with unexplained recurrent miscarriage as compared to healthy early pregnant women. patients with unexplained recurrent miscarriage display diminished suppressive activity of tregs in th17 cells when compared with healthy women who underwent early elective abortion. nowadays, it is believed that unexplained recurrent spontaneous abortions could be an alloimmune disease associated with defective maternofetal tolerance in which treg cells play a key role. as foxp3 is a crucial regulatory factor for the development and function of treg cells ; foxp3 gene deficiency suppresses the regulatory function of treg cells. accordingly, a significant association has been found between foxp3 gene polymorphisms rs3761548a / c and rs2232365a / g and unexplained recurrent abortions in a chinese female population. tregs are essential for pregnancy maintenance, and low levels have been found in pregnancy complications. thus not only women with unexplained recurrent spontaneous abortions, but also patients with preeclampsia display low levels of tregs in both maternal blood and placenta. in fact in preeclamptic patients, the percentage of cd25 cells in the cd4 + t cell population in peripheral blood mononuclear cells is considerably lower than in women with normal pregnancies and nonpregnant healthy controls. moreover, placental samples from preeclamptic patients show a low percentage of foxp3 + cells in cd3 + t - cells as compared to those reported in normal pregnancy subjects. it has been suggested that cytotoxic t - cells increase at the decidua basalis in preeclampsia since the cd8 + t / cd3 + t - cells ratio in placental preeclamptic samples was much higher than in the samples taken from healthy pregnancies. the frequency of conventional cd4 + cd25high foxp3 + tregs and that of nonconventional cd4 + cd25 foxp3 + tregs diminish in peripheral blood in preeclamptic patients as compared to healthy pregnant women. in addition, the prevalence of th17 cells and the th17/treg ratio increases in peripheral blood in preeclampsia as compared with normal pregnancy. since the complete fetal genome is allogeneic to the mother in ed pregnancies, maternofetal immune tolerance is particularly essential for pregnancy success. the substantially large number of t cd4 + and nk cells in the basal plaque of placentas from ed pregnancies, if compared with those from nondonor ivf pregnancies, may reveal that maternal immune tolerance against the fetal allograft is enhanced. strangely enough, it has been recently suggested in maternal tolerance to the semi- or allogeneic fetus in ed pregnancies that peripheral or extrathymic treg cells are vital as they block the immune response to foreign antigens. conversely, thymic treg cells suppress autoimmunity [100, 101 ]. semen exposure for the induction of t regulatory cells. besides exposure to trophoblastic cell debris, exposure to sperm hla - dr antigens expressed on sperm might induce hla ii antigen - specific tolerance. murine models have shown that treg cells are activated by male antigens. as a matter of fact, seminal fluid expands the pool of treg cells in the para - aortic lymph nodes draining the uterus and induces the accumulation of treg cells in the uterus prior to embryo implantation. treg cells accumulate in the mouse uterus in the receptive phase of the estrus cycle, and seminal fluid further promotes treg expansion. on the other hand, soluble hla class i in seminal fluid may induce hla i - specific tolerance.. such exposure may increase maternal immune tolerance to paternal hla class i and ii antigens before pregnancy. in pregnancies achieved by donated spermatozoa, women have not been previously exposed to semen and the fetus is a semiallograft to the mother. since the risk of preeclampsia in donated spermatozoa is very high (18.2%), semen exposure would reduce the risk of preeclampsia. along these lines, risk of preeclampsia has been studied with intracytoplasmatic sperm injection (icsi) using either ejaculated sperm or surgically obtained sperm, and both cases involve exposure to seminal fluid. whereas in icsi with ejaculated sperm sperm exposure exists, exposure is absent in icsi with surgically obtained sperm. the risk of preeclampsia in icsi with ejaculated sperm is the same as that for ivf cases, 4%. oddly enough, the risk of preeclampsia in icsi using surgically obtained sperm is 11%, which is significantly higher than in icsi with ejaculated sperm. in addition, as the risk of preeclampsia in ed pregnancies with former exposure to the partner 's semen is high (16.00%), the allogeneic fetus may constitute a risk factor of preeclampsia. in donated embryo transfer cases, the fetus is allogeneic to the mother, and no former semen exposure is involved. in such cases, onset of preeclampsia may be related to the gradual decrease of treg cells, which induce paternal antigen - specific tolerance during the third trimester of pregnancy [29, 91 ]. in addition, a protective effect of multiparity in preeclampsia has been described. despite the possibility of memory t - cells decreasing after delivery, seminal thus in a second pregnancy with the same partner, the number of memory t - cells may rapidly increase. this protective multiparity effect in preeclampsia it is also noteworthy that the longer the interval between second and third deliveries with the same partner, the higher the risk of preeclampsia. this finding may be explained by the progressive decrease in memory t - cells after delivery in the second or third pregnancy. memory t - cell levels reach their lowest levels at more than 10 years after the last delivery, and seminal priming maintains these tolerance - inducing t - cells at a low level. therefore, in a subsequent pregnancy, some of these women may not achieve adequate tolerance which, in turn, raises the risk of preeclampsia. regarding the adaptive maternal humoral immune response during pregnancy, paternal anti - hla antibodies similarly, fetal hla - specific b - cells have been detected in murine pregnancies. fetal antigens induce an adaptive maternal humoral immune system response. accordingly, maternal antibodies against fetal hla can be generated, which are especially prone to increase when the hla mismatches between the mother and fetus are high. since ed pregnancies may be associated with a larger number of hla mismatches than spontaneously conceived pregnancies, women with ed pregnancies might produce higher levels of antibodies. it remains unknown whether adverse clinical consequences occur as a result of the maternal humoral immune response to fetal antigens. in fact, antipaternal hla antibodies and antifetal t - cells are present in many normal pregnancies. in preeclamptic patients, the autoantibody against angiotensin ii type i receptor (at1-aa) has been found. it binds to the at1 receptor, which is highly expressed in the placenta and triggers the activation of an intracellular cascade, which results in the production of antiangiogenic factors sflt1 and endoglin. whereas b1 cells develop during fetal and perinatal life, b2 cells are produced during postnatal life. b1 cells may be subdivided into b1a and b1b cells based on the expression of cellular marker cd5 by b1a cells, but not by b1b cells. b2 and b1b cells produce adaptative antibodies upon antigen stimulation, while b1a cells synthesize natural antibodies in the absence of antigenic stimuli. it is known that aat1-aa autoantibodies are produced by the cd19+cd5+b1a cells, but not by cd19+cd5b2 cells, obtained from peripheral blood of nonpregnant women and stimulated in vitro with serum from preeclamptic women. during human pregnancies, proportions of cd5 b1a cells thus, it has been suggested that the reduction of circulating b1a cells during pregnancy may contribute to maternofetal immune tolerance since these cells are the main producers of poly - reactive antibodies. in pregnant women with uncomplicated pregnancies, cd19+cd5 + levels are significantly lower toward the third trimester, while cd19+cd5 + levels remain high in preeclamptic patients. fetal hla arises from the donor 's ovule and from the biological father of the future newborn child. in spontaneous pregnancies, it has been shown that hyperactivation of th1 and th2 by an allogeneic fetus is specific for ed pregnancy in the first trimester of pregnancy if compared with ivf pregnancies and pregnancies by natural conception. another regulatory counteractive mechanism in ed pregnancies is reflected by the preferable activation of th2 and the relative suppression of the th1 chemokine expression. the larger number of mismatches in the five most immunogenic hla antigens (hla - a, -b, -c, -dr, and -dq) in ed pregnancies may have clinical consequences. indeed, the healthy uncomplicated term pregnancies containing a hla - c mismatched child induce a higher percentage of cd4+cd25 activated - t cells in decidua parietalis and contain functional cd4+cd25 regulatory t - cells in decidual tissue when compared with hla - c - matched pregnancies. moreover, a significant correlation between the total number of hla - a, hla - b, hla - c, hla - dr, and hla - dq mismatches and the percentage of activated cd4+cd25 t - cells in decidua parietalis has been described. therefore, further activation by fetal hla - a, hla - b, hla - dr, and hla - dq may occur in pregnancy. as trophoblast cells do not express these hla molecules, the microchimeric fetal cells that express hla antigens before entering the decidual tissue may activate a number of decidual t - cells in the periphery. activation in the decidua might occur by hla - c, which explains the prevailing effect of an hla - c match on the functional faculties of decidual t - cells. a meta - analysis revealed that the or for pregnancy - induced hypertension after ed, as compared to conventional assisted reproductive techniques, was 2.57 (95%ci, 1.913.47). moreover, the or for pregnancy - induced hypertension after ed, if compared to the control naturally conceived pregnancy group, was 6.60 (95%ci, 4.559.57). a subsequent retrospective study reported that the incidence of both gestational hypertension and preeclampsia was significantly higher in ed pregnancies than in pregnancies by autologous ivf (24.7% versus 7.4%, and 16.9% versus 4.9%, resp. although the literature describes higher maternal morbility in ed pregnancies (pregnancy - induced hypertension, preeclampsia, bleeding complications during the first trimester), a higher rate of complications (intrauterine growth restriction, congenital anomalies) for the fetus or newborn has not been demonstrated. nonetheless, ed pregnancies are more likely to end in preterm birth than pregnancies by autologous ivf (34% versus 19%). it is known in spontaneous preterm births that maternal anti - hla class i seropositivity is significantly higher than in term births. ed pregnancies (fetal allograft) may be associated with higher maternal anti - hla i seropositivity than pregnancies by autologous ivf or those spontaneously conceived (fetal semi - allograft). therefore, the higher levels of maternal anti - fetal hla i antibodies in ed pregnancies may be the cause of the higher incidence of preterm birth in these pregnancies when compared with autologous ivf or spontaneous pregnancies. typification of donors ' and recipients ' hla to select haploidentical combinations can be considered in ed pregnancies in order to make them more immunologically comparable to spontaneous pregnancies. during pregnancy, the maternal immunological response allows maternal tolerance to the semiallogeneic or allogeneic fetus in ed pregnancies. a defective maternofetal immune response may contribute to the development of pregnancy - related complications, such as bleeding complications during the first trimester, pregnancy - induced hypertension, preeclampsia, or preterm birth. therefore, suitable knowledge of the maternal immune response during pregnancy will enable us to understand the etiopathogeny to elucidate prevention and to improve the treatment of these pathologies. | maternofetal immune tolerance is essential to maintain pregnancy. the maternal immunological tolerance to the semiallogeneic fetus becomes greater in egg donation pregnancies with unrelated donors as the complete fetal genome is allogeneic to the mother. instead of being rejected, the allogeneic fetus is tolerated by the pregnant woman in egg donation pregnancies. it has been reported that maternal morbidity during egg donation pregnancies is higher as compared with spontaneous or in vitro fertilization pregnancies. particularly, egg donation pregnancies are associated with a higher incidence of pregnancy - induced hypertension and placental pathology. preeclampsia, a pregnancy - specific disease characterized by the development of both hypertension and proteinuria, remains the leading cause of maternal and perinatal mortality and morbidity. the aim of this review is to characterize and relate the maternofetal immunological tolerance phenomenon during pregnancies with a semiallogenic fetus, which are the spontaneously conceived pregnancies and in vitro fertilization pregnancies, and those with an allogeneic fetus or egg donation pregnancies. maternofetal immune tolerance in uncomplicated pregnancies and pathological pregnancies, such as those with preeclampsia, has also been assessed. moreover, whether an inadequate maternal immunological response to the allogenic fetus could lead to a higher prevalence of preeclampsia in egg donation pregnancies has been addressed. |
ultrasound is routinely used to evaluate small breast masses. a large proportion of these small masses are benign but unfortunately many go to biopsy or excision despite benign imaging characteristics. utilizing our case of adult male pilomatrixoma, we discuss the work - up of small breast masses, focusing on ultrasound characteristics, and stress conservative management if the typical benign sonographic features are present. despite a very low propensity for malignant degeneration, surgical excision is the treatment of choice for pilomatrixomas, mainly for symptomatic and cosmetic reasons in a pediatric population. given the age, location, and low chance of malignant degeneration, conservative management may be considered in rare case of pilomatrixoma of the adult breast. a 53-year - old male presented with a painless hard palpable pea - sized lump, in the right breast, near the nipple. a referral for mammography was made as the man had a strong family history of breast cancer with two sisters diagnosed at ages 42 and 50. diagnostic mammography showed a well - circumscribed, oval - shaped mass of the medial right breast, with smooth borders, and multiple small punctuate calcifications [figure 1 ]. gray - scale ultrasound examination revealed a small superficial isoechoic well - marginated oval mass parallel to the skin, with smooth borders, and multiple hyperechoic foci corresponding to calcifications seen on mammogram [figure 2 ]. the lesion appeared benign on ultrasound examination, but given the patient 's concern and a strong family history of breast cancer, a core biopsy was performed. the patient remained anxious about malignancy despite this benign diagnosis, so the mass was excised two months later with an unchanged pathologic diagnosis. mediolateral oblique view of mammogram demonstrates a small well - circumscribed oval mass just underneath a radiopaque skin marker in the medial right breast near the nipple. a gray - scale ultrasound image of the lesion shows a well - defined isoechoic oval mass, parallel with the skin measuring 7 mm 3.5 mm, containing the small hyperechoic foci of calcification observed on the mammogram. pilomatrixoma, originally named calcifying epithelioma of malherbe, was initially described in 1880 by dr. chenantals malherbe as a calcified epithelioma of sebaceous glands. developing in the subcutaneous tissue and arising from the matrix cells of the hair bulb, these benign tumors usually develop during the first two decades of life and have been very rarely described to degenerate to pilomatrixcarcinoma. the tumors are commonly found on the head with the cheek being a frequent location, followed by the neck and upper extremities in decreasing frequency. pilomatrixomas are more seldom found on the trunk and lower extremities and are very rarely found in the breast. despite a very low chance for malignant degeneration surgical excision is the treatment of choice as these tumors do not regress and occur in cosmetically unpleasant locations, usually the head and neck in a pediatric population., there have been only five reports of pilomatrixoma of the adult male breast in the english literature. the tumors were removed in two cases without undergoing medical imaging, most likely due to the size of the tumor. two other reports described typical benign features of the tumor ; one with mammography and sonography and the other with mammography alone. sonography is routinely used to evaluate palpable masses as well as incidental masses found on screening mamography. traditionally, ultrasound was used for differentiation of cystic from solid masses but its role has evolved to serve as a guide in characterization of masses into benign, indeterminate, or malignant categories. the negative predictive value for small breast masses with benign ultrasound features has been quoted to be greater than 99% in several studies.[710 ] features that have been described to most reliably characterize masses as benign are a round or oval shape, circumscribed margins, and width - to - anteroposterior dimension ratio greater than 1.4., if agreed upon by three different readers, these three features predicted benignity with 100% accuracy. in fact, pilomatrixomas on imaging are commonly well - defined subcutaneous nodules. also, most are heterogeneously hyper- to isoechoic with acoustic shadowing and hyperechoic calcifications, as seen in our case. in general, a differential diagnosis of a superficial breast lesion includes seborrheic keratosis, dermal nevus, epidermal inclusion cyst, and basal cell carcinoma. although the majority of superficial tumors are benign, management is frequently based on results of the triple test (physical exam, imaging, and biopsy) and biopsy is performed despite benign imaging characteristics. we suggest a conservative approach of interval follow - up for any small superficial breast mass with benign imaging characteristics. such masses can be assigned into a bi - rads 3 category without a pathologic diagnosis [figure 4 ] or following a benign biopsy result to ensure stability over time. also, conservative management can be considered in cases of pilomatrixoma of the adult breast. although, most advise surgical excision of the tumor, especially in a pediatric population, an asymptomatic pilomatrixoma cosmetically acceptable to the patient seems safe to monitor with imaging as there is very low chance of malignant degeneration. given the location and lack of symptoms, conservative management was discussed with our patient but excisional biopsy was ultimately performed due to anxiety resulting from a strong family history of breast cancer. a well - circumscribed breast lesion on gray - scale ultrasound parallel to the skin surface has not been biopsied but is likely to be benign given its stability on sonography for nearly two years. | pilomatrixomas are uncommon benign skin neoplasms arising from the hair follicle matrix. they occur more commonly in children than adults. most originate on the head, neck, or upper extremities, less commonly on the trunk or lower extremities, and very infrequently in the breast. we present a rare case of pilomatrixoma of the breast in an adult male. as the patient had a strong family history of breast cancer, a full work - up of the breast mass was performed. ultimately, an excisional biopsy was carried out for patient reassurance. |
endovascular therapy for cerebral vascular diseases has expanded in both the variety and number of devices. conventionally, an animal vascular model is used for initial testing of new devices.1 2 to date, swine and dog models have been reported.3 however, animal experiments are labor intensive and expensive. fresh human placenta on the other hand is relatively easy to obtain from hospitals with obstetrics service. although the placenta has been widely used in biomedical research,4 5 its application in neurointerventional research has not been found in the literature. we now report on the preliminary applications of human placenta as an ex vivo vascular model in this rapidly expanding research field. written consent for donation of placenta for research was obtained from the mother before the baby was delivered. eighteen placentas were obtained during the period 20112013 for various vascular interventions. after cleansing with normal sterile saline solution to remove any blood adhered to the placenta surface, the amnion sac was peeled off from the chorion and removed by sharp dissection at the cord insertion, thus exposing the chorionic plate vessels on the chorion surface. the umbilical cord was shortened to about 10 cm to allow easy catheterization of the umbilical arteries and vein. a 5 french gauge straight tip diagnostic catheter or size 16 venous puncture catheter was used to catheterize the umbilical arteries. a pressurized intra - arterial infusion bag was used to delivery heparinized saline solution into the placental arteries (figure 1a). a motorized pump was used to simulate pulsation of the vessels. a solution of heparin in normal saline was used to irrigate the specimen until the vessels were free of blood clots. by taking advantage of the semitransparent nature of the placental vessels, direct observations of changes within the blood vessels and the behavior of catheters, clot and devices under a leica oh5 neurosurgical microscope (wetzlar, germany) with high magnification, were made. results were documented by high definition digital recording and photography. by erecting the placenta with a styrofoam board support on a tray, digital subtraction angiography (dsa) was performed (figure 1b). this set - up was to allow escape of fluid and contrast material from the placenta to the tray by gravity, thus keeping the radiological field clear (figure 1c). (2) simulation of stent assisted coiling and flow diversion on an aneurysm model. (3) simulation of intra - arterial thrombolysis. (4) simulation of embolization of arteriovenous malformation (avm) with glues. (5) simulation of mechanical thrombolysis and comparison of different devices. (6) vascular model for training of neurointerventionalists. all 18 placentas were examined macroscopically and the diameters of the first order chorionic plate artery (cpa) and vein were recorded. the presence of any anastomosis between the two umbilical arteries near the placental insertion was noted. nos 1, 2, 3, 4, and 5, a neurosurgical microscope was used for microdissection, visualization of flow inside the vessel, and high definition photography. in experimental study no 3, ex vivo intra - arterial thrombolysis was performed with 500 000 units of urokinase. with the use of a pulsatile pump, the placenta was continuously perfused with ringer 's solution via a 6 french gauge catheter to simulate a normal circulation (figure 1a). a 5 mm in length with 4 mm diameter clot specimen was loaded into the junction of the first and second order arteries through the guiding catheter. under high magnification, the lytic change of the clot sample was observed through the almost transparent cpa. in experimental study no 5, different thrombectomy devices were tested for ease of deployment and effectiveness in clot retrieval en masse. after single passage of the device, the labeled vessel was fixed in situ by vascular perfusion of 4% formaldehyde in normal saline for 60 min under a perfusion pressure of 50 mm hg and a draining pressure of 0 mm hg at the placental capillaries region. the vascular samples were dissected and prepared for paraffin sections and en face endothelium examination. eosin stain to visualize the vessel morphology and cluster of differentiation 31 (cd31) immunohistological stain to confirm the existence of endothelium, and were then examined by microscopy. for en face examination, the tissue samples were incubated with a mixture of 5 unit / ml alexa fluor 488 phalloidin (invitrogen, camarillo, california, usa), 1% bovine serum albumin (santa cruz biotechnology, santa cruz, california, usa), 0.5% triton x-100 (invitrogen), and physiological buffered saline for 1 h at 37c. after rinsing three times in physiological buffered saline for 5 min each time, the samples were stained with a 1:500 dilution of to - pro-3 (invitrogen) for 20 min at room temperature. the fluorescence stained samples were placed on superfrost plus gold slides with the endothelium facing upward, and then mounted with pro - long gold antifade reagent (invitrogen). after covering with a micro glass and the edges sealed, the slides were examined with a lsm 7 duo confocal microscope (carl zeiss, jena, germany). the placenta was perfused with dextrose non - ionic solution before the application of n - butylcyanoacrylate (nbca)/ethiodized oil (lipiodol) mixture ; 18% nbca was used to visualize the angiographic appearance and polymerization time. ethylene vinyl alcohol copolymer in dimethyl sulfoxide (onyx) was tested for the degree of penetration and reflux. the cerebral cortical veins on the brain surface always cross over the arteries (figure 2a) while the cpas always cross over veins when observed from the fetal surface (figure 2b). similarities were found in vascular branch patterns and histological cross sections of the human cerebral artery (figure 2c, e) and the cpa (figure 2d, f). (a) brain surface at the region of the sylvian fissure where the sylvian vein crosses the temporal branch of the middle cerebral artery (white arrow). (b) chorionic surface after removal of the amnion membrane showing the chorionic plate artery (black arrow) crossing the vein. (c) digital subtraction angiography (dsa) of the left internal carotid artery (ica) in a human subject. (e) hematoxylin eosin (h&e) staining of a cross section of the human ica showing the presence of the tunica elastica (black arrow). (f) h&e staining of a cross section of the chorionic plate artery showing the absence of the tunica elastica. intracranial stents such as pipeline, neuroform, and wingspan stents (covidien, usa) were performed with the use of a delivery catheter. one of the main side branches of the placenta vessel was ligated to simulate a wide neck aneurysm (figure 3a). in one model, flow diversion was observed with examination of the interface between the pipeline device and the aneurysm (figure 3b). similarly, deployment of coil through the stent strut into the aneurysm was done. under magnification, (a) simulation of flow diversion of a wide neck aneurysm by the pipeline device (pd). a side branch of the chorionic plate artery (cpa) was ligation to form an aneurysm - like sac. (b) the vessel was opened to inspect the surface of the pd and clot formation. (c) simulation of intra - arterial thrombolysis with clot sample (black arrow) was loaded into the bifurcation of a cpa. (f) digital subtraction angiography appearance of the embolized vessel (white arrow). degree of adhesiveness of onyx with the detachable tip of the perfusion catheter was observed. the dose of lytic agent was diluted to 10 ml, and the time to complete lysis of the clot was 23 min on average (figure 3c, d). the dsa appearance resembled embolization of the avm feeding vessel (figure 3e, f). the perforating branches of the placenta vessel are very similar to the en passage configuration of avm. the polymerization times for both glues were similar to the clinical situation even when the placentas were perfused with dextrose solution instead of fresh blood. deployment of devices such as the penumbra separator 3d (penumbra europe gmbh, germany), solitaire fr stent (covidien), and similar stent - like constructs were performed in a preselected branch of the placenta artery under continuous ringer 's solution perfusion (figure 4a, b). after applying single passage with a thrombectomy device the change in endothelium lining after thrombectomy was compared with the normal control. the hematoxylin since the endothelium of cpa was positive for the cd31 stain (figure 4d), the change in endothelial lining could be compared for two different devices after thrombectomy at the same placenta. the change was confirmed by confocal microscopy and quantitative measurement of cell loss (figure 4e, f). this initial study was affirmative of endothelium loss after the use of the thrombectomy device. no concrete conclusion could be drawn from these findings as the number of vessels examined was too small for statistical analysis. (a) image tracing of the chorionic plate artery (cpa) on the surface of the placenta. (b) mapping of the cpa with designation of control and testing segments for comparing embolectomy devices. (c, d) cross sectional images of a cpa obtained by light microscopy. the tissue samples were stained with hematoxylin eosin and cd31 for endothelium, respectively. (e) optical section two - dimensional images at 1 m showing endothelial cell nuclei. (f) a three - dimensional image was constructed with a series of optical section two - dimensional images along the thickness of the vessel wall (z axis). three neurosurgical trainees participated in this study and performed dsa by catheterizing the umbilical artery or vein at the loose end of the umbilical cord. various modes of angiographic examination were performed, such as hand injection of contrast or glue, or deployment of stent or thrombectomy devices. the placental model was well suited for training of pushing nbca glue and withdrawal of a delivery catheter, which require multiple practices to achieve ideal results in that split second manipulation. the cerebral cortical veins on the brain surface always cross over the arteries (figure 2a) while the cpas always cross over veins when observed from the fetal surface (figure 2b). similarities were found in vascular branch patterns and histological cross sections of the human cerebral artery (figure 2c, e) and the cpa (figure 2d, f). (a) brain surface at the region of the sylvian fissure where the sylvian vein crosses the temporal branch of the middle cerebral artery (white arrow). (b) chorionic surface after removal of the amnion membrane showing the chorionic plate artery (black arrow) crossing the vein. (c) digital subtraction angiography (dsa) of the left internal carotid artery (ica) in a human subject. (e) hematoxylin eosin (h&e) staining of a cross section of the human ica showing the presence of the tunica elastica (black arrow). (f) h&e staining of a cross section of the chorionic plate artery showing the absence of the tunica elastica. intracranial stents such as pipeline, neuroform, and wingspan stents (covidien, usa) were performed with the use of a delivery catheter. one of the main side branches of the placenta vessel was ligated to simulate a wide neck aneurysm (figure 3a). in one model, flow diversion was observed with examination of the interface between the pipeline device and the aneurysm (figure 3b). similarly, deployment of coil through the stent strut into the aneurysm was done. under magnification, (a) simulation of flow diversion of a wide neck aneurysm by the pipeline device (pd). a side branch of the chorionic plate artery (cpa) was ligation to form an aneurysm - like sac. a heparinized human blood sample was perfused into the vessel for 30 min. (b) the vessel was opened to inspect the surface of the pd and clot formation. (c) simulation of intra - arterial thrombolysis with clot sample (black arrow) was loaded into the bifurcation of a cpa. (f) digital subtraction angiography appearance of the embolized vessel (white arrow). degree of adhesiveness of onyx with the detachable tip of the perfusion catheter was observed. the dose of lytic agent was diluted to 10 ml, and the time to complete lysis of the clot was 23 min on average (figure 3c, d). the dsa appearance resembled embolization of the avm feeding vessel (figure 3e, f). the perforating branches of the placenta vessel are very similar to the en passage configuration of avm. the polymerization times for both glues were similar to the clinical situation even when the placentas were perfused with dextrose solution instead of fresh blood. deployment of devices such as the penumbra separator 3d (penumbra europe gmbh, germany), solitaire fr stent (covidien), and similar stent - like constructs were performed in a preselected branch of the placenta artery under continuous ringer 's solution perfusion (figure 4a, b). after applying single passage with a thrombectomy device the hematoxylin eosin staining method demonstrated the vessel wall layers (figure 4c). since the endothelium of cpa was positive for the cd31 stain (figure 4d), the change in endothelial lining could be compared for two different devices after thrombectomy at the same placenta. the change was confirmed by confocal microscopy and quantitative measurement of cell loss (figure 4e, f). this initial study was affirmative of endothelium loss after the use of the thrombectomy device. no concrete conclusion could be drawn from these findings as the number of vessels examined was too small for statistical analysis. (a) image tracing of the chorionic plate artery (cpa) on the surface of the placenta. (b) mapping of the cpa with designation of control and testing segments for comparing embolectomy devices. (c, d) cross sectional images of a cpa obtained by light microscopy. the tissue samples were stained with hematoxylin eosin and cd31 for endothelium, respectively. (e) optical section two - dimensional images at 1 m showing endothelial cell nuclei. (f) a three - dimensional image was constructed with a series of optical section two - dimensional images along the thickness of the vessel wall (z axis). three neurosurgical trainees participated in this study and performed dsa by catheterizing the umbilical artery or vein at the loose end of the umbilical cord. various modes of angiographic examination were performed, such as hand injection of contrast or glue, or deployment of stent or thrombectomy devices. the placental model was well suited for training of pushing nbca glue and withdrawal of a delivery catheter, which require multiple practices to achieve ideal results in that split second manipulation. although the placenta shows a wide variation in its anatomy between individuals, in the majority of the cases it is perfused with blood by two umbilical arteries and one vein. the blood vessels spread and divide in the two - dimensional plane between the amnion and the chorion on the fetal side of the placenta, constituting the cpas. the cpas form a disperse pattern where they divide dichotomously, in two equal branches, with gradually diminishing diameter, giving off small branches along their course. the primary branches of the umbilical vasculature that course through the chorionic plate periodically dive beneath this stratum to establish the circulation of primary vascular ramifications ending in the terminal villi.6 the two dichotomized branches are extremely useful in adapting them for comparison of two devices or using one as control on the same placenta. resembling the cortical blood vessels of the human brain, the cpas and their branches are end arteries.7 the veins roughly follow the course of the arteries, showing a similar branching pattern. firstly, the blood pressure difference between the cerebral artery and vein is more than 100 mm hg while between the chorionic artery and vein it is only 30 mm hg under normal physiological conditions. due to different pressure requirements, the properties and thickness of the vessel wall between the cerebral artery and vein are distinctly different while the chorionic vessels show less disparity. as a result, both placental artery and venous systems can be used to simulate the cerebral artery whereas the animal model is limited to one. secondly, the chorionic artery is superficial to the vein while the cerebral cortical artery is the reverse. as the chorionic artery tree is superficial, movement of materials inside the vessel lumen can be visualized throughout the entire length without disruption. there are four extra - embryonic membranes that are part of the placenta, including the yolk sac, amnion, allantois, and chorion. for the brain, there are also four layers of membrane coverings the fibrous dura, meningeal dura, arachnoid, and pia matter. in most placentas there is a single anastomosis, first described by hyrtl in 1870, between the umbilical arteries near the cord insertion.8 the anastomosis is promising for simulation of communication between the left and right cerebral vascular territories. further experiment is required to validate the usefulness of this model for cross flow study. cooling and irrigation with iso - osmotic fluid such a ringer 's solution can preserve the tissues. we found that the cells within the blood vessels of the human placenta in particular were still viable to take up vital staining for histological studies within 6 h after placenta delivery (figure 4). cd31 is a constituent of the endothelial intercellular junction, also designated as pecam-1 (platelet endothelial cell adhesion molecule 1).9 immunohistological cd31 stain has been applied to routinely processed tissues containing endothelial cells.10 phalloidin binds f - actin with high selectivity, and has been used to visualize and quantify f - actin in tissue sections and cell cultures.11 the to - pro-3 stain is a sensitive detector of double stranded nuclei acids.12 it may be worthwhile exploring the applications of molecular markers in the ex vivo model. most of the placentas demonstrated various degrees of chorionic plate vessel spasm soon after delivery. the smooth muscle constriction may be triggered by the release of angiotensin ii from the placenta.13 in our observation, the pressurized infusion of ringer 's solution for 1015 min can also restore the normal luminal size of the vessels. after the application of heparin and urokinase infusion, the existing clot within the arteries can be dissolved and passed to the venous system of the placenta. the only difference is the presence of the autonomic nervous supply to the smooth muscle layer in the cerebral blood vessel where the placenta is deprived of any nerve supply. with general features closely resembling the brain vessel in terms of diameter, tortuosity, elasticity, and bifurcation pattern at the chorion surface, the human placenta is well suited as an ex vivo vascular model to simulate various clinical situations. interventional procedures are well known to carry a high risk of procedure related complications, such as inadvertent perforation of a vessel, kinking of a catheter or guidewire, fracture of catheters, and overshooting of embolization materials. for trainees, apart from computer simulation training, the placental model can provide start up practice in the dsa room before they embark on performing interventional procedures in real clinical situations. the experimental studies in this paper suggest that it is feasible to use human placenta as an ex vivo vascular model in neurointerventional surgery research and training purposes due to its vessels resembling the brain vasculature. the model opens doors for investigation of new endovascular devices during the development phase in the future. | backgroundhuman placenta is a convenient resource for biomedical research, and has not yet been used for neurointerventional surgery research.objectiveour objective was to explore the feasibility of using human placenta to test various endovascular interventions and for training.design18 placentas soon after delivery were prepared for six pilot studies. (1) study on anatomical similarity to human cerebral vessel. (2) simulation of stent assisted coiling and flow diversion on an aneurysm model. (3) simulation of intra - arterial thrombolysis. (4) simulation of embolization of arteriovenous malformation with glues. (5) simulation of mechanical thrombolysis and comparison of different devices. (6) vascular model for training of neurointerventionalists.resultswhen the chorionic plate vessels were compared with the cerebral cortical vessels, similarities were found in vascular branch patterns, histological cross sections, and angiographic appearances. due to the semitransparency of its vessel wall, performance of flow diverter and stent assisted coiling of an aneurysm could be visualized under direct microscopic observation. similarly, timing of clot lysis and glue polymerization could be estimated. endothelial change after thrombectomy could be assessed by histological methods. from these pilot studies, the placenta model could be adopted to simulate various clinical situations. it is also ideal for interventional radiology training.conclusionsit is feasible to adopt the human placenta as an ex vivo vascular model in neurointerventional surgery research due to the fact that its vessels resemble the brain vasculature. |
one of every five children aged less than 5 years in low - income, developing countries is malnourished. globally, undernutrition is associated with more than one - third of all deaths in this age group. acute malnutrition defined by weight - for - height z - score (whz) 1 year) (adjusted or (aor) : 3.1, p = 0.004), have an undernourished mother (body mass index (bmi) < 18.5), (aor : 2.8, p = 0.017), have a father with no or a low - paying job (aor : 5.8, p < 0.001), come from a family having a monthly income of less than 10,000 taka, (1 us$ = 80 taka) (aor : 2.9, p = 0.008), and have shorter period of predominant breastfeeding (aor : 2.7, p = 0.013) (table 3). the aim of this study was to identify risk factors associated with acute malnutrition (whz < 2, which includes both moderate and severe wasting) in our population of 659-month - old children. our study shows that the major associated / risk factors for acute malnutrition among these children were older age of the child, undernourished mother, jobless father or father with a low - paying job, low total family income, and poorer breastfeeding practices. some of these factors may operate in synergy to increase the risk of acute malnutrition. older age as a risk / associated factor for acute malnutrition of children in our study might reflect a selection bias. however, jeyaseelan and lakshman from tamil nadu, india, and kikafunda. from uganda also observed older age of a child to be significantly associated with malnutrition. proper nutrition and child care are essential to begin in early childhood to prevent malnutrition and ensure maximum potential for normal psychomotor development as children grow older. similar to other studies from bangladesh [811 ] and africa, the current study observed maternal malnutrition (bmi less than 18.5) as an independent risk factor for children 's wasting / acute malnutrition. undernourished mothers often deliver low - birth - weight (lbw) infants, which is an attributable risk factor for increased childhood malnutrition, morbidity, and mortality. the capacity to adequately breastfeed may also be compromised [1416 ] in maternal malnutrition and such mothers might have less energy to appropriately nurture their children. the fathers of most (84%) of the wasted children in our study were rickshaw pullers or day laborers. likewise, a study from south india also found that children of fathers who were day laborers were ~3 times more likely to be severely underweight. moreover, they often result in erratic or insecure incomes, at times yielding little or no earnings on a particular day. income insecurity leads to food insecurity forcing family members to consume poor food quality and/or amount. low family income as a possible risk factor of acute malnutrition, as found in this study, was also reported by ahmed. in wasted children under 2 years old, by nahar. in severely underweight under-5 children in bangladesh, and by jeyaseelan and lakshman in undernourished children in tamil nadu, india. our finding of improper / inadequate breastfeeding as an associated factor with acute malnutrition is in accordance with the findings of several other studies [10, 19, 20 ] from bangladesh and elsewhere, in which early supplementation with infant formula or cow 's milk, early introduction of semisolid complementary foods, and inadequate breastfeeding were important risk factors for malnutrition in children. during the critical period of early infancy, the hygienic and nutritional risks associated with bottle - feeding and artificial milk are well known [2123 ], and previous studies also found that breastfeeding had a significant and substantial impact on overall survival of undernourished children [24, 25 ]. it is also possible that the association with a shorter duration of predominant breastfeeding could be an example of reverse causality, whereby children who were ill and undernourished stopped breastfeeding or were provided with other foods. one of the possible limitations of the present study is that the same personnel who obtained the anthropometric measurements of the children and mothers also conducted the interviews, so interviewer biases could be there. however, most of the variables identified as risk / associated factors for acute malnutrition in our study were objective in type. the other limitation was the cross - sectional nature of the present study, which did not allow us to state the identified associated factors as definite causally related risk factors. it is worthwhile to mention that icddr, b has developed ready - to - use foods using locally available food ingredients. these foods can be used to prevent and to treat moderate wasting in children living in food - insecure communities. moreover, the associated factors identified for acute malnutrition in this study can be incorporated into the design and targeting of preventive interventions. interventions that motivate behaviours more consistent with recommended infant and young child feeding practices would be expected to have a positive impact. certain factors and possible causes of acute malnutrition are complex and involve societal and broad - based preventive programs. | to assess the risk factors for acute malnutrition (weight - for - height z - score (whz) 1 year) (adjusted or (aor) : 3.1, p = 0.004) ; have an undernourished mother (body mass index < 18.5), (aor : 2.8, p = 0.017) ; have a father with no or a low - paying job (aor : 5.8, p < 0.001) ; come from a family having a monthly income of < 10,000 taka, (1 us$ = 80 taka) (aor : 2.9, p = 0.008) ; and often have stopped predominant breastfeeding before 4 months of age (aor : 2.7, p = 0.013). improved understanding of these characteristics enables the design and targeting of preventive - intervention programs of childhood acute malnutrition. |
transplants of the retina are among the new strategies being used in the treatment of genetic and degenerative macular diseases. moreover, literature dated from 2004 to 2011 was comprehensively examined via medline and pubmed searches for the following terms : auto-, homo-, heterologous transplantation, retina, stem cells, cultivated cells. tissue and cell therapy of retinal diseases are reviewed, including full - thickness retina / retinal pigment epithelium (rpe)/choroid graft ; full and partial thickness rpe / choroid complex grafts ; rpe / bruch membrane complex graft ; and rpe, iris pigment epithelium and stem cell grafts. recommendations for transplants, as well as the benefits and weaknesses of specific techniques in retina transplants, are discussed. auto- and allogenic transplants of a full or partial thickness retina / rpe / bruch membrane / choroid complex represent an alternative treatment offered to patients with some macular diseases. stem cell transplantation to reconstruct and regenerate the macula requires further biomolecular and animal research studies. genetic and degenerative retinal diseases currently present scientific and ophthalmological challenges. in the united states, approximately 6 million people have retinal diseases, in particular from retinopathies such as age - related macular degeneration (amd), diabetic retinopathy and retinitis pigmentosa [13 ]. amd accounts for 75% of cases of legal blindness in people over the age of 50 years in all industrialized countries. photodynamic therapy or intravitreal anti - vascular endothelial growth factor (vegf) injections represent the first line of wet amd treatment, but are of limited efficacy [48 ]. implantation of electronic chips to stimulate the brain, as well as transplantation of donor tissue and cells, are new therapeutic strategies currently being explored to reconstruct the photoreceptor / retinal pigment epithelium (rpe) complex and to restore central vision in different retinopathies [1114 ]. an animal model of retinal degeneration demonstrated the importance of the survival and integration of transplanted tissues. an evolutionary phenomenon that prevents immunogenic inflammation, macular destruction and blindness [12,13,1618 ]. blood - tissue barriers that limit the ingress of blood - borne immunogenic factors ; immunosuppressive properties of pigment epithelial cells ; and the expression of cd95 ligand on ocular parenchymal cells, which triggers the apoptosis of effector t cells, all contribute to the immune tolerance of the eye. human rpe cells have the particular ability to suppress t - cell activity by direct contact (toll - like receptors serve as the first line of defense [17,9,10 ]) and via secretion into the aqueous humor and vitreous body of soluble immunomodulatory / suppressive factors, such as cytotoxic t lymphocyte - associated antigen-2 programmed cell death 1 ligand, thrombospondin-1 and transforming growth factor [19,2125 ]. these overlapping mechanisms enable acceptance of foreign tissue grafts for extended intervals unlike conventional body sites that summarily reject such grafts and delay symptoms of retinal rejection (ie, shrinkage, architecture loss and glial transformation accompanied by low - grade inflammation). full - thickness retina / rpe / choroid complex allogenic grafts were performed to treat patients with retinitis pigmentosa and geographic atrophy in dry amd. during a 5-year study, graft samples were harvested from the enucleated eyes of human embryos after abortion in the 10 to 15 week of pregnancy. it was then placed in a special applicator (allowing gentle lifting and correct direction of the tissue) and grafted under the central retina by means of an upper nasal retinotomy. the procedure was finalized with the use of a laser around the retinotomy and performance of a perfluorocarbon - oil exchange. after the retina transplantation, synaptic junction stimulation at the host / donor interface was required, which was performed in this group of patients by showing them various colorful moving images on video. seventy percent of the patients (n=10) showed improved visual acuity scores 6 months after the transplantation. the nutritious effect of the graft on the photoreceptors or the stimulation of mller cells, leading to new synaptic junctions in the host / donor interface, may have contributed to better visual acuity. the new immunization was not found in human leukocyte antigens ; embryonic tissues were less immunogenic and the blood - brain barrier stopped the infiltration of the graft s antigens. five years after surgery, no graft rejection symptoms, such as macular edema or encapsulation, were found clinically or by optical coherence tomography. however, in the graft area, pigment loss occurred in 80% of patients after 36 months, which was not interpreted as rejection because of the improvement in visual acuity. the investigators also suggested that the improvement could result not only from the graft, but also from the lensectomy and vitrectomy, which had been performed before transplantation during the same procedure. the administration of neutrofins (an indirect result) rather than the replacement of the degenerated tissue could also have contributed to the good results. it is doubtful, however, whether this type of graft would be commonly accepted (because of ethical and religious dilemmas). autotransplantation (autotranslocation) of rpe / choroid complex graft is called a full - thickness autologous patch or a patch graft (pg). such grafts were performed in patients with wet amd (choroidal neovascularization in amd [cnv / amd ]) [3135 ], rpe rip and dry amd (geographic atrophy in amd). a pg consists of the rpe, bruch membrane, and choriocapillaris, as well as medium and large choroidal vessel translocation. the differences between pg methods depend on the manner in which the neovascular tissue is removed and the graft is prepared, as well as on the tissue translocation technique used (see below). one method involved the removal of cnv by means of perimacular retinotomy and harvesting of the graft from the superior temporal equatorial retina. it was first encircled with continuous laser impacts and then excised as deep as the sclera with vertical scissors. the retina was peeled from the sheet and pg was implanted under the fovea through a central retinotomy. the procedure was finalized by applying the laser around the retinotomy and performing a perfluorocarbon - oil exchange. one study compared the results of this pg technique with those of full macular translocation (fmt) surgery (360). the results after 1 year were comparable, but after 4 years the results of fmt surpassed those of pg (the deterioration of visual acuity from 0.87 to 1.38 logmar in pg and from 0.9 to 0.69 logmar in fmt ; more extensive rpe damage and inferior fixation occurred in the pg group). three reasons for the inferior results in the pg group have been suggested : (1) iatrogenic detachment of the retina caused by the fluid in fmt does not damage the natural, strong adhesion of the photoreceptors and rpe to the extent that peeling does ; (2) in pg, the tissue is harvested from the equatorial retina and rpe, which is less specific and less sensitive than paramacular rpe, on which the retina is placed and rotated by 45 in fmt ; and (3) the perfusion disorder can last for several days after pg and the translocation of the full choroid section does not prevent ischemia and does not contribute to the metabolic balance of the retina. in another study the investigators compared results of autologous rpe / choroid pg with an rpe cell - suspension graft. three or more lines in best corrected visual acuity at 24 months follow - up was found in 28.5% of patients in the rpe patch group and in 14.2% of patients in the rpe cell - suspension group ; a loss of 3 or more lines occurred in 14.2% in each group (n=7 in each group). examination by fluorescein and indocyanine green angiography showed revascularization of all pgs and normal choroidal vasculature in the area of rpe atrophy in all patients of the rpe cell - suspension group. however, part of the successfully transplanted patch grafts showed intrastructural changes on spectral - domain optical coherence tomography (ie, irregularities in the cell layers ; scarring or gliosis and an absence of photoreceptors and outer nuclear layer). this may explain the limited visual gain of rpe pgs, which should theoretically be a better option than rpe suspension grafts (loose, unevenly distributed rpe cells without their own basal lamina are worse support for photoreceptors than a functional monolayer in sheet grafts). in another method for pg surgery, a temporal 180 retinotomy was performed and the mobilized retina placed on the nasal area. the vast retinal area bared in this way enabled less extensive cnv removal. with special scissors, the graft was excised from the central circumference of the fundus in the superior temporal quadrant and was not encircled by the laser. in cases of hemorrhage, the nutrient vessel supporting the graft was coagulated with diathermia and/or the intraocular pressure was raised. moreover, perfluorocarbon injected into the intrinsic surface reversed the retina and stabilized the graft. the procedure was finalized by applying a laser along the retinotomy and by performing a perfluorocarbon - oil exchange. between 4 and 20 months after surgery, visual acuity improved in 60% of eyes, stabilized in 30% and deteriorated in 10% (n=13). this allows for delicate cnv excision and maximum preservation of residual, healthy rpe and subretinal vessels ; in addition, it enables and accelerates revascularization of the graft. folding and stretching of the tissue during implantation was not required (it took place in the graft through paramacular retinotomy). the laser was not used prior to tissue resection to avoid the shrinkage and infolding of the graft s edges. it is vital for the graft to be large enough to entirely cover the area of the recipient s damaged rpe and choroid. the tissue junction contributes to the nutrient bridge vessel at the host / donor interface and to early graft revascularization. in indocyanine green angiography, the graft s nutrient vessels did not run radially, but in parallel as ladder rungs. pvr occurs in 2050% of patients after a 360 retinotomy and in 31% of patients after paramacular retinotomy ; a large incision area and good perfusion of the central retina lead to pvr. lack of pvr in the above - mentioned group is ascribed to limited (180) retinotomy in the poorly vascularized circumferential area of the retina and routine performance of phacoemulsification with intraocular lens implantation. the right access allows for maximum excision of the vitreous base, which prevents shrinkage and pvr. in some patients, extensive tissue thickness makes penetration of nutrients and reperfusion difficult, thereby leading to graft fibrosis beginning just 3060 days after autotranslocation. animal research has shown that a partial graft, with no medium and large choroid vessels, is easily integrated with the recipient s bed. in 63% of the animals, the partial graft was found to be viable (ie, revascularization and monolayered rpe cells were present). autologous rpe / bruch membrane complex grafts were performed in patients with hemorrhagic cnv in wet amd. a temporal 180 retinotomy was performed with cnv removal on the external side of the retina and laser around the retinotomy with a perfluorocarbon - oil exchange. the 4 mm graft was excised with a microvitreoretinal knife on the cauterized area from the central circumference in the superior temporal quadrant. the rpe / bruch membrane complex was separated from the choroid vessels with a special s - shaped spatula and scissors. patients showed improved early treatment of diabetic retinopathy study visual acuity scores that averaged 28.6 to 47.7 after surgery, central fixation occurred in 30% of eyes, and the appropriate graft morphology without depigmentation was found in 82% of eyes (n=21). visual acuity scores were reported to be comparable to the results of full - thickness grafts. however, the thinness of the partial graft, without the retina and the choroids, is perceived as an advantage in this graft it easily settles after perfluorocarbon, does not require curling, enables penetration of nutrients from the choroid to the retina, and its edges do not tend to curl, which prevents the formation of subretinal membranes. according to various authors [4144 ], the reported long - term results of rpe / bruch membrane pgs are not satisfactory because the achieved overall visual gain is about 1 line of best corrected visual acuity, and proliferative vitreoretinopathy complications occur in up to 45% of eyes. full or partial tissue transplantation is one of the current strategies for macular pathology treatment, but most grafts fail to integrate into the recipient s bed, lose their architecture and do not function [4547 ]. moreover, allotransplants of fetal or postmortem tissue lead to rejection and the need to apply immunosuppressants and, at the same time, they raise moral and ethical concerns. autologous rpe cell grafts were performed in patients during the surgical removal of cnv in exudative amd (cnv / amd) or cnv in retinal angiomatous proliferations (cnv / rap) and can be a therapeutic option for anti - vegf non - responders or where anti - vegf fails to improve visual acuity (rpe tear, massive subretinal hemorrhage). after pars plana vitrectomy, nasally from the optic nerve, flat detachment of the retina was created and rpe cells were mobilized in the iatrogenic bubble by means of fluid injection. the cells were aspirated, removed by centrifugation and then counted and prepared for injection of suspension. the cnv was removed by a second paramacular retinotomy, and the suspension of rpe cells was injected under the fovea. the procedure was finalized by applying a laser to the retinotomy and performing a perfluorocarbon - oil exchange. in a 1-year follow - up period, visual acuity improved or stabilization occurred in 80% of patients with wet amd and in 68% of patients with rap. however, the long - term results of autologous rpe cells grafts are not promising. the technique is not viable because the grafts do not contribute to functional monolayer formation and the scattered cells atrophy. unevenly distributed, multilayered rpe cells without their own basal lamina and extracellular matrix are unable to grow well on an aged or diseased host s bruch membrane, which is a crucial factor in the survival of transplanted rpe cells, their adherence and differentiation, and their ability to phagocytose photoreceptor outer segments [5052 ]. the fovea requires much more stimuli than just new rpe cells in order to function properly. a biostable synthetic membrane (ie, expanded polytetrafluoroethylene (eptfe) polymer modified by ammonia gas - plasma treatment) was tested experimentally as a bruch membrane prosthesis a scaffold for rpe cell growth and differentiation. defluorination of the fluoropolymer increases surface hydrophilicity, incorporates surface polar groups and facilitates cell attachment. arpe-19 cells (a non - immortalized human rpe cell line that mimics the human situation), seeded on a modified eptfe scaffold at a density of 1500 cells / mm and cultured in the presence of retinoic acid (to promote rpe differentiation), formed a differentiated and functional monolayer capable of phagocytosing photoreceptor outer segments. the scaffold serves as a carrier substrate, protected from the hostile influences of an aged and diseased bruch membrane ; facilitates cell interaction and flow of nutrients (vitamin a, glucose, fatty acid) ; and represents a possible treatment to repair retinal degeneration and restore vision in affected patients. according to krishna., the biostable, biocompatible eptfe membrane provides stable replacement of the natural bruch membrane and long - term support for transplanted rpe cells. the membrane seems to be a better option than a synthetic biodegradable membrane or tissue - based material [50,5357 ]. despite advances in microfabrication technology, the functional results of various rpe cell transplantation techniques in humans are worse than anti - vegf treatment and are associated with a high rate of serious complications (eg, massive hemorrhage, pvr). autotransplantation of iris pigment epithelium (ipe) cells is not performed in humans because of the potential for significant damage to the iris. non - modified ipe allotransplantation is also not performed in humans, because it does not significantly improve visual acuity. however, ipe cells have become a focus of interest because in vivo they have been shown to express mrna for proteins involved in the metabolism of retinol. they also phagocytize the external segments of photoreceptors and accumulate lipofuscin ; thus, they significantly impact visual acuity. subretinal human ipe xenotransplantation was performed in rats (the royal college of surgeons) with iatrogenic macular dystrophy (genetically engineered gene change for the photoreceptors of tyrozine kinosis, leading to inhibition of phagocytosis of the external segments of photoreceptors through rpe and lipofuscin accumulation, as well as to subsequent atrophy of photoreceptors). a human iris (from a cadaver) the human ipe cell xenotransplant was shown in vivo to protect photoreceptors without any significant adverse effects ; it also produced protective neurotrophic growth agents and stimulated the regeneration of photoreceptors. in future, ipe cells may turn out to be useful for transferring genetic information to photoreceptors and rpe. specific retinal cells, similar to other neurons, have limited capability for proliferating and regenerating spontaneously. stem cells, as well as engineered retina (ie, retinal aggregates that are laboratory - generated from tissue - specific progenitor stem cells [pscs ]), are new therapeutic strategies for the replacement of degenerative photoreceptors and rpe cells in patients [45,57,6179 ]. the multipotent stem cells differentiate into multiple, specific cell types of 1 lineage (meso- or endo- or exo - dermy), known as pscs. multipotent stem cells can be harvested from different tissues, such as embryo, neuron, bone marrow, and eye (including the ciliary body, iris, and retina). retinal pscs, formed from exodermy, have the ability to differentiate into photoreceptors, rpe, mller glial cells and bipolar cells in a favorable environment that includes growth factors and/or chemical modulators. at present, research on retinal pscs has been conducted in vitro and with animals [45,60,78,8284 ], and the source of optimum retinal pscs has not yet been determined. embryonic human stem cells can differentiate into retinal neurons that are similar to adult retinal stem cells, but this manner of obtaining adult stem cells in humans has raised serious moral and ethical concerns and dilemmas. neural stem cells differentiate to photoreceptors and glia in rats, but when implanted into an animal retina, often become immature and fail to express rhodopsin. a human neural progenitor xenograft was performed in 21-day - old rats with iatrogenic macular degeneration and pigment dystrophy. adult human stem cells found in the retina, mller cells or rpe are considered to be the optimum source of retinal pscs [45,6165 ]. the concept of grafting both 3-dimensional (3d) cultures and differentiated retinal pscs seems to be promising. 3d cultures are much better suited than monolayer cultures because the graft involves the 3d structure of the recipient s bed. cultured aggregates of retinal progenitors appear on approximately the 10th day of culturing. as a result of cellular bioengineering, the cultured 3d structured cells were differentiated in such a way as to create several cell lines specific for the particular tissue. photoreceptor differentiation was performed by upregulation of rhodopsin and aanat enzyme (implicated in melatonin synthesis). this differentiation was confirmed by reverse transcription - polymerase chain reaction in reference to the following factors : rod transcription factor nrl, nr2e3, interstitial retinal binding protein and rhodopsin kinase. differentiation toward other cell lineages was demonstrated by the presence of tyrosine hydroxylase in amacrine and ganglion cells, as well as by the presence of calbindin and gnb3 in cone cells. the resulting 3d cultured and differentiated psc aggregates are able to blend into the degenerated bed in the recipient and are capable of producing neurotrophic factors, leading to regeneration of the photoreceptor / rpe complex and reconstruction of the subretinal environment in retinitis pigmentosa and amd in humans. the development of microfabrication technology improved the delivery aspect of 3d aggregates for transplantation in an experimental study. a novel biodegradeable, 3d thin - film cell encapsulation scaffold served as the carrier platform for aggregates. the transplantation vehicle was constructed from thin - film 22.5-d polycaprolactone (pcl) layers (< 10 m) thermally bonded to form the 3-d cell encapsulation scaffold (< 30 m) with varying protrusions, cavities, pores and particles. the 3d scaffold was permeable and promoted the delivery of retinal progenitor cells and their appropriate retention in subretinal space. in another study, mouse embryonic stem cells cultured with the extracellular matrix proteins added to the medium, over a period of 9 days formed 3d spherical aggregates called embryoid bodies. subsequent invagination of its apical surface formed an optic cup - like structure with a 2-walled structure consisting of retinal pigment epithelium and neural retina, with synaptic activities. the formation of the optic cup occurred in a self - directed way (was not caused by a lens or surface ectodermal tissues). according to several studies, self - formation of fully stratified 3d neural retina tissue from stem cells opens up a new strategy for the transplantation of the artificial retinal tissue sheets, rather than simple cell grafting. this optic - cup structure can be further induced to form an entire eye structure (lens, iris, cornea, and sclera), and can serve as the cell - culture models for drug screening and characterization of the disease phenotype. such models may be obtained with retinal pscs, which derived from the persons with some specific mutant genotypes associated with the diseases, such as macular degeneration or retinitis pigmentosa. natural retinal barriers inhibitory extracellular matrix and cell adhesion molecules (cd44, neurocan), as well as the outer limiting membrane (olm) that form a natural barrier between the subretinal space and the outer nuclear layer, weaken cellular host - donor integration. degradation of these molecules and transient chemical disruption of the olm facilitates host - donor integration in animals. the combined transplantation of retinal pscs with biodegradable microspheres of poly lactic - co - glycolic acid, which deliver active matrix metalloproteinase 2, stimulated the removal of the matrix inhibitory barrier and enhanced cellular host - donor integration in mice. targeted disruption of olm junctional proteins, crumbs homologue 1(crb1) and zona occludens (zo-1), as well as intravitreal injection of the glial toxin dl - alpha - aminoadipic acid 72 h prior to transplantation, induced transient chemical disruption of the olm (maximal at 72 h, recovered by 2 weeks) and led to a significant increase in the number of transplanted photoreceptor precursors integrated within the outer nuclear layer in degenerated mouse retinas. this study showed that targeted disruption of an undesirable physical barrier in the retina may be an effective and practical strategy for retinal repair. the usefulness of research on stem cells has also been confirmed by autologous non - myeloablative hematopoietic stem cell transplantation (hsct). hsct was shown to be effective in the therapy of autoimmune - related retinopathy and optic neuropathy syndrome (arron) that was resistant to conventional therapies (prednisone, methotrexate, cyclophosphamide, plasmapheresis, intravenous immunoglobulin). after hsct, the clinical manifestations (visual acuity, visual field, electroretinography, and antibody activity against pig retina and pig optic nerve) of arron appeared to stabilize. the techniques presented for auto- or allogenic retina transplants, both full and partial, represent the currently available surgical alternatives in the treatment of neovascular and atrophic macular diseases. however, retina transplant requires complicated vitreoretinal surgery and in some cases might be followed by delayed reperfusion of the graft. the difficulty of retina transplantation lies in the highly specific nature of the complex and demanding neuronal tissue. stem cell transplantation in retinal diseases is of increasing importance, but further laboratory tests in genetic engineering and animal testing are required to find an optimal source of pscs for rpe cells and photoreceptors. stem cell transplantation also raises new ethical dilemmas that require further consideration. | summarybackgroundtransplants of the retina are among the new strategies being used in the treatment of genetic and degenerative macular diseases. moreover, various cell cultures are being tested to treat retinal disorders.material/methodsliterature dated from 2004 to 2011 was comprehensively examined via medline and pubmed searches for the following terms : auto-, homo-, heterologous transplantation, retina, stem cells, cultivated cells.resultstissue and cell therapy of retinal diseases are reviewed, including full - thickness retina / retinal pigment epithelium (rpe)/choroid graft ; full and partial thickness rpe / choroid complex grafts ; rpe / bruch membrane complex graft ; and rpe, iris pigment epithelium and stem cell grafts. recommendations for transplants, as well as the benefits and weaknesses of specific techniques in retina transplants, are discussed.conclusionsauto- and allogenic transplants of a full or partial thickness retina / rpe / bruch membrane / choroid complex represent an alternative treatment offered to patients with some macular diseases. stem cell transplantation to reconstruct and regenerate the macula requires further biomolecular and animal research studies. |
pulmonary cryptococcosis is a type of fungal infection, initiated by inhalation of the organism, cryptococcus neoformans, from an environmental source (1). it most commonly occurs in immunocompromised hosts, such as human immunodeficiency virus (hiv)-infected or cancer patients, and rarely involves the immunocompetent hosts (1, 2). imaging features, associated with pulmonary cryptococcosis, which mimic hematogeneous metastases, are rare in immunocompetent patients. to our knowledge, this is a rare presentation of pulmonary cryptococcosis with computed tomography (ct) characteristics of hematogeneous lung metastases, and it 's valuable to show the pathologic correlation with positron emission tomography (pet) scan. a 49-year - old man was admitted with a complaint of dry cough over the last two weeks, and pulmonary nodules were incidentally found on his chest radiography (fig. 1a) at our outpatient department. due to the pulmonary nodules of unknown cause, ct of the chest was arranged. multiple well - defined nodules (number : more than 10) were observed in both lungs (fig. each nodule differed in size, ranging from 4 mm to 12 mm ; moreover, the nodules were randomly distributed in every lobe, accompanied by minimal pleural effusions. no recent travel history was noted. the physical examination and all the laboratory tests presented no remarkable findings. however, tumor markers, such as carcinoembryonic antigen, squamous cell carcinoma antigen, and prostate - specific antigen were all detected to be in the normal range. the gastroscopy and colonoscopy also showed within normal findings. after failing to find any possible primary malignancy, we arranged a whole body 18f - fluorodeoxyglucose (fdg) pet scan to evaluate the possible primary malignancy our preoperative diagnosis of hematogeneous lung metastases was based on the presence of multiple pulmonary nodules of varying sizes, which distributed randomly throughout the lung ; however, the findings of the whole - body fdg pet scan indicated that the nodules were possibly benign. two nodules each from the right middle and lower lobe were surgically removed ; histopathology revealed a fibrotic nodule with mild peripheral granulomatous inflammation and many cryptococcal spores (fig. a series of examinations were performed for greater detail, such as immunoglobulin levels, hiv by enzyme - linked immunosorbent assay, serum and cerebrospinal fluid cryptococcal antigens, but tested negative. the patient was then administered intravenous fluconazole, shifting to oral drugs 400 mg per day, for three months, after discharge. the nodules remain stable in size and number, during a one year follow up. the pulmonary manifestations in patients with cryptococcal infection can present with a variety of appearances, showing from air - space consolidation to ill- or well - defined nodules / masses (2 - 7). in some cases, mixed associated findings, such as mediastinal lymphadenopathy, pleural effusions or cavitation, may be also noted. the number of nodules was often less in immunocompetent patients than in the immunocompromised ones (6, 7) in our patient, ct scans revealed multiple nodules, demonstrating relatively aggressive characteristics. the varying sizes of the pulmonary nodules and the random bilateral distribution mimicked the presentation of hematogeneous pulmonary metastases. further, the nodular margins were well - defined without other associated findings, which was difficult to link to an infectious process. on reviewing recent case studies and articles (table 1), we noted that most multiple cryptococcal nodules have a tendency to cluster in one or few lobes in the immunocompetent patients, but cryptococcosis may also rarely show scattered nodules, such as in our case. besides the aggressive nodular pattern, the totally absence of fdg uptake is also rare. there are only few studies combining the ct pattern showing pulmonary nodules with the discussion of pet signals. the standardized uptake value (suv) of the cryptococcosis in these reports ranged from 0.93 to 11.6 (7, 9). some lesions may be interpreted as benign by showing low suvs. but most cases are interpreted as likely malignant lesions before biopsy because of the high suv (9). in our patient, a suv of 2.5 has been traditionally used as a cut - off value for differentiating malignancy (7), but in pulmonary cryptococcosis, the suv may vary widely, from mild to marked uptake (7, 9). in our patient, the intensity of fdg uptake was virtually absent for each lung lesion, which may indicate a low probability of malignancy (10). but fdg uptake generally depends on the size of the nodule. the small size (4 - 12 mm) of nodules, in this case, might also contribute to the absence of fdg uptake ; hence, we decided to perform an open lung biopsy. histopathologic results explained the cause of absence of fdg uptake : the nodule mainly composed of fibrotic and necrotic changes, with mild inflammatory cell infiltration. the lack of active inflammatory process told us the reason for absence of fdg pet signals. in conclusion, we report an image presentation of pulmonary cryptococosis, which mimics hematogeneous metastases. pet did not demonstrate fdg uptake, suggesting a low probability for malignancy, and it correlated well with the pathologic finding : an infectious nodule composed of fibrosis and necrosis. it might be a crucial piece of information for clinicians when making management decisions, in cases of multiple lung nodules mimicking hematogeneous metastasis. | the radiologic appearance of multiple discrete pulmonary nodules in immunocompetent patients, with cryptococcal infection, has been rarely described. we describe a case of pulmonary cryptococcosis, presenting with bilaterally and randomly distributed nodules on a computed tomography, mimicking hematogeneous metastases. positron emission tomography does not demonstrate 18f - fluorodeoxyglucose (fdg) uptake, suggesting a low probability for malignancy, which is a crucial piece of information for clinicians when making a management decision. we find the absence of fdg uptake correlates with the pathologic finding of an infectious nodule, composed of fibrosis and necrosis. |
congestive heart failure is a leading cause of morbidity and mortality in developed countries, with an estimated prevalence of 1% to 2% in the general population, reaching 7% to 8% in individuals aged > 75 years.1 elevated blood pressure appears to play an important role in the pathogenesis of both left ventricular (lv) systolic and diastolic dysfunction, at least partly by increasing cardiac afterload.2 this has led to the therapeutic concept of reducing cardiac afterload, which has proven highly beneficial in heart failure with reduced ejection fraction but not in heart failure with a preserved ejection fraction.3 thus, broadening the understanding of the role of central hemodynamics in the pathogenesis of systolic and diastolic lv dysfunction is critical to inform the development of novel therapeutic approaches. blood pressure is routinely measured at the brachial artery, whereas the actual cardiac pressure load is determined by the hemodynamics in the proximal aorta. this is a relevant distinction, as central pulse pressure may differ from brachial pulse pressure, particularly in younger adults, due to agerelated differences in central pressure augmentation. the extent to which central pressure augmentation is explained by peripheral wave reflection or by the windkessel function of the proximal aorta is the subject of ongoing debate.4, 5 limited data suggest that central pressure may be more closely related to cardiovascular disease than peripheral blood pressure.6 furthermore, variable relations between aortic stiffness (which increases early systolic load on the heart) and wave reflection (which increases late systolic load) may have differing implications for systolic and diastolic lv structure and function.7 in contrast, the steady component of blood pressure, ie, the mean arterial pressure, is fairly constant throughout large arteries.8 in this context, the relations of central hemodynamics with cardiac structure and function have not been fully delineated. we hypothesized that the pulsatile component (and its determinants aortic stiffness and pressure augmentation) and the steady component of central blood pressure may have different relations with lv structure and function. thus, we investigated the crosssectional relations of central hemodynamics and aortic stiffness to echocardiographic measures of lv structure and systolic and diastolic function in a large communitybased sample. individuals were derived from the framingham offspring and the framingham third generation cohorts, which have been described.9, 10 the framingham offspring cohort was recruited in 19711974 and includes individuals who are children of the framingham original cohort or the children 's spouses. the framingham third generation cohort, recruited in 20022005, comprises children of the framingham offspring cohort. participants of both cohorts are evaluated approximately every 4 to 8 years in our study clinic during a visit that includes a medical history, physical examination, and phlebotomy. the present investigation is based on examination cycle 8 of the framingham offspring cohort (20052008) and examination cycle 1 of the third generation cohort (20022005). of 3021 participants who attended offspring examination cycle 8 and 4095 participants recruited into the third generation cohort, we excluded 252 individuals with atrial fibrillation, 703 for missing echocardiographic measurements, 334 for missing tonometry, and 28 for missing covariates ; that left 5799 (2184 offspring, 3615 third generation) individuals for this investigation. excluded individuals were generally older and had a higher morbidity (higher prevalence of diabetes, hypertension, and prevalent cardiovascular disease) than those included in the analyses. the study protocols were approved by the boston university medical center institutional review board, and participants signed informed consent. supine brachial systolic and diastolic blood pressures were obtained using an auscultatory device.11 arterial tonometry measurements were performed as previously described.11, 12, 13 briefly, arterial tonometry (using a standard applanation tonometry device) with simultaneous ecg was performed on the brachial, femoral, and carotid arteries. transit distances were assessed by body surface measurements from the suprasternal notch to the pulserecording site. mean arterial pressure (map) was derived from integration of the brachial waveform calibrated with bp at the time of tonometry. direct measurement of carotid pressure, as compared to transfer function based estimates, is associated with a smaller difference between central and peripheral pulse pressure.14 details of signal analyses and data processing have been published elsewhere.11, 12, 13 we primarily assessed 4 measures of arterial stiffness and central hemodynamics : (1) carotidfemoral pulse wave velocity (cfpwv), the current reference standard for aortic stiffness, (2) central pulse pressure, ie, the blood pressure amplitude in the proximal aorta, (3) augmentation index, ie, the fraction of central pulse pressure attributable to late systolic pressure augmentation (expressed as percentage), and (4) mean arterial pressure. secondary analyses assessed additional measures of central pulse wave form and peripheral reflection, in particular forward wave amplitude, reflected wave amplitude, and the global reflection coefficient. all echocardiograms were evaluated by an experienced sonographer or cardiologist based on a standardized reading protocol. cardiac dimensions were quantified using the leadingedge technique as recommended by the american society of echocardiography (ase). lv mass was calculated according to ase guidelines, applying the method of devereux.15 the sum of the diastolic thicknesses of the septum and posterior wall was used as an estimate of lv wall thickness. early systolic mitral annulus velocity (e) was measured at the lateral mitral annulus using tissue doppler imaging and transmitral doppler flow velocities recorded using a standardized protocol. repeated analysis of diastolic function measures (mitral e and a peak velocity, tissue doppler e and a peak velocity) yielded interobserver correlation coefficients of > 0.97. lv dimension, lv mass, and left atrial (la) diameter distributions were skewed and therefore natural logarithmically transformed for all analyses. our primary analyses focused on cfpwv, central pulse pressure, mean arterial pressure, and augmentation index. analyses of lv dimensions (lv mass, lv wall thickness, and diastolic dimension) were performed in 3 stages : (1) adjusting only for age, age, sex, height, and study cohort (offspring vs third generation) ; (2) additionally adjusting for clinical risk factors (excluding blood pressure) and antihypertensive medication, ie, weight, heart rate, diabetes, serum total cholesterol, highdensity lipoprotein cholesterol (hdlc), triglycerides, fasting glucose, prevalent cardiovascular disease, current smoking, intake of angiotensinconverting enzyme inhibitors, angiotensin receptor blockers, blockers, diuretics, calcium channel blockers (binary variable for each class) ; and (3) additionally adjusting for brachial map. analyses evaluating lv systolic and diastolic function (fractional shortening, e, e / e) were constructed similarly in a staged design : (1) adjusting only for age, age, sex, height, and study cohort ; (2) additionally adjusting for clinical risk factors (excluding blood pressure) and antihypertensive medication, ie, weight, heart rate, diabetes, total cholesterol, hdlc, triglycerides, fasting glucose, prevalent cardiovascular disease, current smoking, intake of angiotensinconverting enzyme inhibitors, angiotensin receptor blockers, blockers, diuretics, calcium channel blockers ; (3) additionally adjusting for lv mass ; and (4) further adjusting for either map (in models investigating pulsatile blood pressure traits) or central pulse pressure (in models investigating map). for figure construction we also estimated leastsquares means based on regression models with lv structure or function traits as dependent variable and tertiles of cfpwv as predictor variable, adjusting for the covariates of stage 2. logarithmically transformed lv diastolic diameter was then backtransformed to original scale ; thus, means represent geometric means. given that our primary analyses assessed the relation of 4 tonometry traits (central pulse pressure, map, cfpwv, augmentation index) to 3 lv structure traits (lv mass, lv diastolic diameter, lv wall thickness) in 3 statistical models, as well as the relation of the same 4 tonometry traits to 3 lv function traits (lv fractional shortening, e, e / e) in 4 statistical models, we introduced a bonferroni correction for (433)+(434)=84 statistical tests and regarded a pvalue of 0.0006 (0.05/84) as statistically significant. individuals were derived from the framingham offspring and the framingham third generation cohorts, which have been described.9, 10 the framingham offspring cohort was recruited in 19711974 and includes individuals who are children of the framingham original cohort or the children 's spouses. the framingham third generation cohort, recruited in 20022005, comprises children of the framingham offspring cohort. participants of both cohorts are evaluated approximately every 4 to 8 years in our study clinic during a visit that includes a medical history, physical examination, and phlebotomy. the present investigation is based on examination cycle 8 of the framingham offspring cohort (20052008) and examination cycle 1 of the third generation cohort (20022005). of 3021 participants who attended offspring examination cycle 8 and 4095 participants recruited into the third generation cohort, we excluded 252 individuals with atrial fibrillation, 703 for missing echocardiographic measurements, 334 for missing tonometry, and 28 for missing covariates ; that left 5799 (2184 offspring, 3615 third generation) individuals for this investigation. excluded individuals were generally older and had a higher morbidity (higher prevalence of diabetes, hypertension, and prevalent cardiovascular disease) than those included in the analyses. the study protocols were approved by the boston university medical center institutional review board, and participants signed informed consent. supine brachial systolic and diastolic blood pressures were obtained using an auscultatory device.11 arterial tonometry measurements were performed as previously described.11, 12, 13 briefly, arterial tonometry (using a standard applanation tonometry device) with simultaneous ecg was performed on the brachial, femoral, and carotid arteries. transit distances were assessed by body surface measurements from the suprasternal notch to the pulserecording site. mean arterial pressure (map) was derived from integration of the brachial waveform calibrated with bp at the time of tonometry. diastolic blood pressure and integrated map were used to calibrate carotid pressure tracings. calibrated carotid pressure direct measurement of carotid pressure, as compared to transfer function based estimates, is associated with a smaller difference between central and peripheral pulse pressure.14 details of signal analyses and data processing have been published elsewhere.11, 12, 13 we primarily assessed 4 measures of arterial stiffness and central hemodynamics : (1) carotidfemoral pulse wave velocity (cfpwv), the current reference standard for aortic stiffness, (2) central pulse pressure, ie, the blood pressure amplitude in the proximal aorta, (3) augmentation index, ie, the fraction of central pulse pressure attributable to late systolic pressure augmentation (expressed as percentage), and (4) mean arterial pressure. secondary analyses assessed additional measures of central pulse wave form and peripheral reflection, in particular forward wave amplitude, reflected wave amplitude, and the global reflection coefficient. all echocardiograms were evaluated by an experienced sonographer or cardiologist based on a standardized reading protocol. cardiac dimensions were quantified using the leadingedge technique as recommended by the american society of echocardiography (ase). lv mass was calculated according to ase guidelines, applying the method of devereux.15 the sum of the diastolic thicknesses of the septum and posterior wall was used as an estimate of lv wall thickness. early systolic mitral annulus velocity (e) was measured at the lateral mitral annulus using tissue doppler imaging and transmitral doppler flow velocities recorded using a standardized protocol. repeated analysis of diastolic function measures (mitral e and a peak velocity, tissue doppler e and a peak velocity) yielded interobserver correlation coefficients of > 0.97. lv dimension, lv mass, and left atrial (la) diameter distributions were skewed and therefore natural logarithmically transformed for all analyses. our primary analyses focused on cfpwv, central pulse pressure, mean arterial pressure, and augmentation index. analyses of lv dimensions (lv mass, lv wall thickness, and diastolic dimension) were performed in 3 stages : (1) adjusting only for age, age, sex, height, and study cohort (offspring vs third generation) ; (2) additionally adjusting for clinical risk factors (excluding blood pressure) and antihypertensive medication, ie, weight, heart rate, diabetes, serum total cholesterol, highdensity lipoprotein cholesterol (hdlc), triglycerides, fasting glucose, prevalent cardiovascular disease, current smoking, intake of angiotensinconverting enzyme inhibitors, angiotensin receptor blockers, blockers, diuretics, calcium channel blockers (binary variable for each class) ; and (3) additionally adjusting for brachial map. analyses evaluating lv systolic and diastolic function (fractional shortening, e, e / e) were constructed similarly in a staged design : (1) adjusting only for age, age, sex, height, and study cohort ; (2) additionally adjusting for clinical risk factors (excluding blood pressure) and antihypertensive medication, ie, weight, heart rate, diabetes, total cholesterol, hdlc, triglycerides, fasting glucose, prevalent cardiovascular disease, current smoking, intake of angiotensinconverting enzyme inhibitors, angiotensin receptor blockers, blockers, diuretics, calcium channel blockers ; (3) additionally adjusting for lv mass ; and (4) further adjusting for either map (in models investigating pulsatile blood pressure traits) or central pulse pressure (in models investigating map). for figure construction we also estimated leastsquares means based on regression models with lv structure or function traits as dependent variable and tertiles of cfpwv as predictor variable, adjusting for the covariates of stage 2. logarithmically transformed lv diastolic diameter was then backtransformed to original scale ; thus, means represent geometric means. given that our primary analyses assessed the relation of 4 tonometry traits (central pulse pressure, map, cfpwv, augmentation index) to 3 lv structure traits (lv mass, lv diastolic diameter, lv wall thickness) in 3 statistical models, as well as the relation of the same 4 tonometry traits to 3 lv function traits (lv fractional shortening, e, e / e) in 4 statistical models, we introduced a bonferroni correction for (433)+(434)=84 statistical tests and regarded a pvalue of 0.0006 (0.05/84) as statistically significant. characteristics of the entire framingham offspring and third generation cohort are given in the left part of table 1 ; the characteristics of the final study sample after exclusions (see methods for details) are shown in the right part of table 1. lv hypertrophy was present in 1574 individuals (27%) of the study sample ; impaired systolic lv function was present in 46 individuals (1%). the unadjusted pairwise correlations between our primary tonometry and blood pressure traits are provided in table 2. clinical, echocardiographic, and hemodynamic characteristics available n values were as follows : n=7032, n= 6995, n=7096, n=7115, n=6986, n=6987, n=6984, n=7116, n=7094, n=6976, n=6497, n=6500, n=6751, n=6494, n=6823, n=6761, n=6587, n= 6918, n=6878, n=6797. acei indicates angiotensinconverting enzyme inhibitor ; arb, angiotensin receptor blocker ; e, carotidfemoral pulse wave velocity ; e, peak early diastolic mitral inflow velocity ; e, peak early diastolic mitral annulus velocity ; lv, left ventricular. ai indicates augmentation index ; cfpwv, carotidfemoral pulse wave velocity ; dbp, diastolic blood pressure ; map, mean arterial pressure ; pp, pulse pressure ; sbp, systolic blood pressure. unadjusted correlations between the tonometry measures and echocardiographic traits reflecting cardiac dimensions are presented in table 3. adjusted relations of central hemodynamics and aortic stiffness with lv dimensions are displayed in table 4. mean arterial pressure was positively associated with lv mass, even after multivariable adjustment (p0.003). in secondary analyses, we related additional subphenotypes of the central pressure waveform, ie, forward wave, reflected wave, and reflection factor, to lv structure (table 5). in multivariable (including map)adjusted analyses, forward and reflected waves generally showed similar associations with lv mass, lv diastolic diameter, and lv wall thickness (r=0.0530.089, p0.05 in all models). in contrast, higher map was associated with lower e and higher e / e (all p0.0001), and the association and directionality persisted with multivariable adjustment. in secondary analyses (see table 8), forward wave amplitude correlated with fractional shortening (r=0.086, p0.05). secondary analyses : relations of central pressure waveform components with left ventricular function data are partial pearson correlation coefficients, adjusted for age, age, sex, height, study cohort, weight, heart rate, diabetes, total cholesterol, hdlc, triglycerides, fasting glucose, prevalent cardiovascular disease, current smoking, intake of angiotensinconverting enzyme inhibitors, angiotensin receptor blockers, blockers, diuretics, calcium channel blockers, mean arterial pressure and left ventricular mass. e, maximum early diastolic mitral annulus velocity ; e, maximum early diastolic mitral inflow velocity. additionally adjusted for characteristics of the entire framingham offspring and third generation cohort are given in the left part of table 1 ; the characteristics of the final study sample after exclusions (see methods for details) are shown in the right part of table 1. lv hypertrophy was present in 1574 individuals (27%) of the study sample ; impaired systolic lv function was present in 46 individuals (1%). the unadjusted pairwise correlations between our primary tonometry and blood pressure traits are provided in table 2. clinical, echocardiographic, and hemodynamic characteristics available n values were as follows : n=7032, n= 6995, n=7096, n=7115, n=6986, n=6987, n=6984, n=7116, n=7094, n=6976, n=6497, n=6500, n=6751, n=6494, n=6823, n=6761, n=6587, n= 6918, n=6878, n=6797. acei indicates angiotensinconverting enzyme inhibitor ; arb, angiotensin receptor blocker ; e, carotidfemoral pulse wave velocity ; e, peak early diastolic mitral inflow velocity ; e, peak early diastolic mitral annulus velocity ; lv, left ventricular. ai indicates augmentation index ; cfpwv, carotidfemoral pulse wave velocity ; dbp, diastolic blood pressure ; map, mean arterial pressure ; pp, pulse pressure ; sbp, systolic blood pressure. unadjusted correlations between the tonometry measures and echocardiographic traits reflecting cardiac dimensions are presented in table 3. adjusted relations of central hemodynamics and aortic stiffness with lv dimensions are displayed in table 4. mean arterial pressure was positively associated with lv mass, even after multivariable adjustment (p0.003). in secondary analyses, we related additional subphenotypes of the central pressure waveform, ie, forward wave, reflected wave, and reflection factor, to lv structure (table 5). in multivariable (including map)adjusted analyses, forward and reflected waves generally showed similar associations with lv mass, lv diastolic diameter, and lv wall thickness (r=0.0530.089, p0.05 in all models). in contrast, higher map was associated with lower e and higher e / e (all p0.0001), and the association and directionality persisted with multivariable adjustment. in secondary analyses (see table 8), forward wave amplitude correlated with fractional shortening (r=0.086, p0.05). secondary analyses : relations of central pressure waveform components with left ventricular function data are partial pearson correlation coefficients, adjusted for age, age, sex, height, study cohort, weight, heart rate, diabetes, total cholesterol, hdlc, triglycerides, fasting glucose, prevalent cardiovascular disease, current smoking, intake of angiotensinconverting enzyme inhibitors, angiotensin receptor blockers, blockers, diuretics, calcium channel blockers, mean arterial pressure and left ventricular mass. e, maximum early diastolic mitral annulus velocity ; e, maximum early diastolic mitral inflow velocity. additionally adjusted for in the present investigation we examined differing associations of pulsatile and steady components of central blood pressure and aortic stiffness with left ventricular structure and function. first, both steady (ie, map) and pulsatile (ie, central pulse pressure) components of central blood pressure were correlated positively with lv mass. however, whereas higher map was primarily associated with higher lv wall thickness, central pulse pressure was positively associated with both lv diameter and lv wall thickness. second, higher map and greater aortic stiffness were correlated inversely with lv diastolic function, and these correlations at least partly persisted in models adjusting of lv mass. third, aortic stiffness, as assessed by cfpwv, was associated inversely with lv diastolic function but showed no independent correlation with lv dimensions or circumferential lv systolic function. whereas the relation between peripheral blood pressure and cardiac structure has been well documented,16 few studies have assessed relations among cardiac structure, central hemodynamics, and vascular stiffness using detailed tonometry measures. these prior studies were limited by small sample size17 or an indirect assessment of central hemodynamics,18 restricted to noneuropean ancestry individuals18, 19, 20 or relied on electrocardiographic lv hypertrophy.21 in a sample of 1272 chinese indiviudals, wang reported that central pulse pressure was associated with lv mass.19 however, their study did not separately assess associations with lv wall thickness versus lv dimensions. an independent association of proximal (ascending) aortic stiffness with lv mass was recently reported in 347 elderly participants of the age, gene / environment susceptibility (ages)reykjavik study cohort.22 that study investigated the hypothesis of a direct coupling between lv and a stiffened proximal aorta as a novel type of mechanical, rather than hemodynamic, load on the left ventricle and thus did not evaluate relations with cfpwv. to our knowledge, the present analysis is the largest investigation of the relation of central hemodynamics and cfpwv (the reference measure of aortic stiffness) with cardiac structure and function in a sample of european ancestry. we observed that higher central pulse pressure is associated with both higher lv diameter and greater wall thickness, whereas higher map (steady component of central pressure) is primarily associated with higher lv wall thickness (rather than with lv diameter). thus, the steady and pulsatile components of central blood pressure may conjointly yet variably contribute to differences in lv mass. of note the fact that neither aortic stiffness nor wave reflection is independently associated with lv mass in our analyses suggests that the relation between lv mass and pulse pressure may be largely attributable to mismatch between ventricular outflow and the ability of the aorta to accommodate that flow, as recently described by torjesen.23 existing literature on the relations among central hemodynamics, vascular stiffness, and cardiac function is sparse, and most studies were of limited size,24, 25, 26, 27, 28 restricted to certain patient populations,27 or did not include tissue doppler assessment of lv diastolic function.20 abhayaratna investigated the relation of arterial stiffness to lv diastolic dysfunction in a sample of 188 elderly individuals and observed a significant correlation between central pulse pressure and severity of diastolic dysfunction.29 however, cfpwv was not associated with diastolic dysfunction in their study. russo reported in 983 individuals that several tonometryderived measures of central hemodynamics, greater arterial stiffness, and more wave reflection were all associated with worse lv diastolic function.30 however, after multivariable adjustment, only the ratio of central pulse pressure to stroke volume index (a measure of global arterial stiffness) remained associated with lv diastolic dysfunction. kang measured brachialankle pulse wave velocity in 1929 individuals in shanghai and reported an association with diastolic heart failure.31 notably, cfpwv was not measured in the latter studies. our considerably larger analysis demonstrates that higher mean arterial pressure and aortic stiffness are associated inversely with measures of lv diastolic function. the fact that these associations persisted after adjustment for lv mass is consistent with the notion that diastolic dysfunction may be only partly dependent on lv hypertrophy.32 aortic and cardiac stiffness (hence lv diastolic dysfunction) may share etiologic mechanisms such as excessive tissue fibrosis. the extent to which aortic stiffness may contribute to lv diastolic dysfunction or that common pathophysiological mechanisms may contribute to parallel increases in both aortic and cardiac stiffness if greater aortic stiffness is indeed shown to contribute to lv diastolic dysfunction in additional studies, therapeutic interventions aimed at decreasing macrovascular stiffness may be a promising tool for the prevention and mitigation of diastolic dysfunction, a premise that warrants further study. the relations of central pulse pressure and augmentation index to lv diastolic dysfunction were not straightforward in our analyses. whereas we observed a consistent association of higher central pulse pressure with higher e / e (a surrogate measure of elevated lv filling pressure), the association of central pulse pressure with the fillingphase measure of diastolic relaxation (ie, e) was weak and changed directionality after adjustment for potential confounding by map. hence, central pulse pressure and augmentation index appear to be primarily associated with lv filling pressures rather than with lv diastolic relaxation. interestingly, we observed a positive correlation between higher central pulse pressure and lv systolic function, which may seem surprising. the most likely explanation for this finding is that a greater fractional shortening corresponds to higher stroke volume and peak flow rate in the proximal aorta and thus may be a cause rather than a consequence of higher central pulse pressure. similarly, the lack of an association between map and lv systolic function in our crosssectional analysis may possibly be explained by various opposing effects of map on lv function. an acute increase in map reduces lv systolic function (inverse relation) but may promote lv hypertrophy and remodeling, which restore lv wall stress and systolic function to normal levels (thereby resulting in a null relation). first, our study sample is community based and predominantly comprised of middleaged adults of european ancestry. the applicability of our findings to younger individuals, to other ethnicities or certain patient groups, remains to be explored in future studies. second, our study is crosssectional and observational ; thus, causal inferences can not be drawn. also, we would like to emphasize that our study was focused on the physiological relations of central hemodynamics with cardiac structure and function. we did not investigate whether central blood pressure may be more strongly related to certain echocardiography traits than to arm blood pressure. in fact, brachial and central pulse pressure were highly correlated (r=0.93) in our data. consequently, the findings for brachial pulse pressure were similar to those observed for central pulse pressure (see table 9).14 similarly, we did not study aggregate measures of pulsatile and steady pressure components, eg, central systolic blood pressure. brachial versus central pressure in relation to echocardiographic traits e indicates maximum early diastolic mitral inflow velocity ; e ', maximum early diastolic mitral annulus velocity ; lv, left ventricular.data are partial pearson correlations, adjusted for age, age, sex, height and study cohort. however, we would like to underscore that our main analyses were adjusted for multiple potential confounders. last, we have performed multiple statistical tests, potentially inflating the type 1 error rate. however, we accounted for multiple testing using a bonferroni correction. of note, most of our findings were highly statistically significant (p<0.0001) and hence likely to be true associations. whereas the relation between peripheral blood pressure and cardiac structure has been well documented,16 few studies have assessed relations among cardiac structure, central hemodynamics, and vascular stiffness using detailed tonometry measures. these prior studies were limited by small sample size17 or an indirect assessment of central hemodynamics,18 restricted to noneuropean ancestry individuals18, 19, 20 or relied on electrocardiographic lv hypertrophy.21 in a sample of 1272 chinese indiviudals, wang reported that central pulse pressure was associated with lv mass.19 however, their study did not separately assess associations with lv wall thickness versus lv dimensions. an independent association of proximal (ascending) aortic stiffness with lv mass was recently reported in 347 elderly participants of the age, gene / environment susceptibility (ages)reykjavik study cohort.22 that study investigated the hypothesis of a direct coupling between lv and a stiffened proximal aorta as a novel type of mechanical, rather than hemodynamic, load on the left ventricle and thus did not evaluate relations with cfpwv. to our knowledge, the present analysis is the largest investigation of the relation of central hemodynamics and cfpwv (the reference measure of aortic stiffness) with cardiac structure and function in a sample of european ancestry. we observed that higher central pulse pressure is associated with both higher lv diameter and greater wall thickness, whereas higher map (steady component of central pressure) is primarily associated with higher lv wall thickness (rather than with lv diameter). thus, the steady and pulsatile components of central blood pressure may conjointly yet variably contribute to differences in lv mass. of note, however, the relation between lv mass and pulse pressure is likely bidirectional. the fact that neither aortic stiffness nor wave reflection is independently associated with lv mass in our analyses suggests that the relation between lv mass and pulse pressure may be largely attributable to mismatch between ventricular outflow and the ability of the aorta to accommodate that flow, as recently described by torjesen.23 existing literature on the relations among central hemodynamics, vascular stiffness, and cardiac function is sparse, and most studies were of limited size,24, 25, 26, 27, 28 restricted to certain patient populations,27 or did not include tissue doppler assessment of lv diastolic function.20 abhayaratna investigated the relation of arterial stiffness to lv diastolic dysfunction in a sample of 188 elderly individuals and observed a significant correlation between central pulse pressure and severity of diastolic dysfunction.29 however, cfpwv was not associated with diastolic dysfunction in their study. russo reported in 983 individuals that several tonometryderived measures of central hemodynamics, greater arterial stiffness, and more wave reflection were all associated with worse lv diastolic function.30 however, after multivariable adjustment, only the ratio of central pulse pressure to stroke volume index (a measure of global arterial stiffness) remained associated with lv diastolic dysfunction. kang measured brachialankle pulse wave velocity in 1929 individuals in shanghai and reported an association with diastolic heart failure.31 notably, cfpwv was not measured in the latter studies. our considerably larger analysis demonstrates that higher mean arterial pressure and aortic stiffness are associated inversely with measures of lv diastolic function. the fact that these associations persisted after adjustment for lv mass is consistent with the notion that diastolic dysfunction may be only partly dependent on lv hypertrophy.32 aortic and cardiac stiffness (hence lv diastolic dysfunction) may share etiologic mechanisms such as excessive tissue fibrosis. the extent to which aortic stiffness may contribute to lv diastolic dysfunction or that common pathophysiological mechanisms may contribute to parallel increases in both aortic and cardiac stiffness can not be assessed in our crosssectional analyses and therefore remains to be elucidated. if greater aortic stiffness is indeed shown to contribute to lv diastolic dysfunction in additional studies, therapeutic interventions aimed at decreasing macrovascular stiffness may be a promising tool for the prevention and mitigation of diastolic dysfunction, a premise that warrants further study. the relations of central pulse pressure and augmentation index to lv diastolic dysfunction were not straightforward in our analyses. whereas we observed a consistent association of higher central pulse pressure with higher e / e (a surrogate measure of elevated lv filling pressure), the association of central pulse pressure with the fillingphase measure of diastolic relaxation (ie, e) was weak and changed directionality after adjustment for potential confounding by map. hence, central pulse pressure and augmentation index appear to be primarily associated with lv filling pressures rather than with lv diastolic relaxation. interestingly, we observed a positive correlation between higher central pulse pressure and lv systolic function, which may seem surprising. the most likely explanation for this finding is that a greater fractional shortening corresponds to higher stroke volume and peak flow rate in the proximal aorta and thus may be a cause rather than a consequence of higher central pulse pressure. similarly, the lack of an association between map and lv systolic function in our crosssectional analysis may possibly be explained by various opposing effects of map on lv function. an acute increase in map reduces lv systolic function (inverse relation) but may promote lv hypertrophy and remodeling, which restore lv wall stress and systolic function to normal levels (thereby resulting in a null relation). first, our study sample is community based and predominantly comprised of middleaged adults of european ancestry. the applicability of our findings to younger individuals, to other ethnicities or certain patient groups, remains to be explored in future studies. second, our study is crosssectional and observational ; thus, causal inferences can not be drawn. this is particularly relevant as some of the observed correlations are likely bidirectional. in addition, the potential for residual confounding can not be eliminated. also, we would like to emphasize that our study was focused on the physiological relations of central hemodynamics with cardiac structure and function. we did not investigate whether central blood pressure may be more strongly related to certain echocardiography traits than to arm blood pressure. in fact, brachial and central pulse pressure were highly correlated (r=0.93) in our data. consequently, the findings for brachial pulse pressure were similar to those observed for central pulse pressure (see table 9).14 similarly, we did not study aggregate measures of pulsatile and steady pressure components, eg, central systolic blood pressure. brachial versus central pressure in relation to echocardiographic traits e indicates maximum early diastolic mitral inflow velocity ; e ', maximum early diastolic mitral annulus velocity ; lv, left ventricular.data are partial pearson correlations, adjusted for age, age, sex, height and study cohort. however, we would like to underscore that our main analyses were adjusted for multiple potential confounders. in unadjusted analyses, last, we have performed multiple statistical tests, potentially inflating the type 1 error rate. however, we accounted for multiple testing using a bonferroni correction. of note, most of our findings were highly statistically significant (p<0.0001) and hence likely to be true associations. in the present investigation, we assessed relations of steady and pulsatile components of central hemodynamics and aortic stiffness to cardiac structure and lv systolic and diastolic function in a large communitybased sample with a broad age range. steady and pulsatile components of central blood pressure were jointly and variably related to components of lv structure. aortic stiffness and map were associated inversely with measures of lv diastolic function but not with lv systolic function. this work was supported by the nhlbi, framingham heart study (nhlbi / nih contracts n01hc25195 and hhsn268201500001i), the boston university school of medicine, and by hl076784, g028321, hl070100, hl060040, hl080124, hl071039, hl077447, hl107385, 2k24hl04334, and r01hl126136. dr mitchell is owner of cardiovascular engineering inc (a company that develops and manufactures devices to measure vascular stiffness) and serves as a consultant to novartis, merck, and servier. | backgroundthe differing relations of steady and pulsatile components of central hemodynamics and aortic stiffness with cardiac dimensions and function have not been fully elucidated.methods and resultscentral hemodynamics and carotidfemoral pulse wave velocity (cfpwv, a measure of aortic stiffness) were measured by arterial tonometry in 5799 participants of the framingham heart study (mean age 51 years, 54% women) and related to echocardiographic left ventricular (lv) dimensions and systolic and diastolic function using multivariableadjusted partial pearson correlations. mean arterial pressure (map, steady component of central blood pressure) was associated positively with lv wall thickness (r=0.168 ; p<0.0001) but showed only a weak direct association with lv diastolic dimension (r=0.035, p=0.006). central pulse pressure (pulsatile component of central blood pressure) showed a direct correlation with both lv diastolic dimension and lv wall thickness (r=0.08 and 0.044, both p<0.0001 in multivariable models that included map). cfpwv was not associated with lv structure (all p0.27) in mapadjusted models). both map and cfpwv were associated inversely with lv diastolic function (e ; r=0.140 and 0.153, respectively ; both p<0.0001), and these associations persisted after additional adjustment for lv mass and central pulse pressure (r=0.142 and 0.108, both p<0.0001). map and cfpwv were not associated with lv fractional shortening (p0.10), whereas central pulse pressure was positively related (r=0.064, p<0.0001).conclusionspulsatile and steady components of central pressure are conjointly yet variably related to lv structure. cfpwv is related to lv diastolic function but not to systolic function. additional studies are warranted to confirm these observations. |
measurement of disease activity is critical for longitudinal assessments in both observational studies and clinical trials. in the field of rheumatology, more than 250 assessment tools have been developed and validated to evaluate pathology, symptoms, function, and health status of patients with rheumatic diseases. such instruments should be compatible with regulatory requirements of the food and drug administration (fda) and generally require prospective studies for completion of the validation process. igg4-related disease (igg4-rd) is an increasingly recognized immune - mediated disease that is characterized by a lymphoplasmacytic infiltrate enriched with igg4-positive plasma cells and a distinctive storiform fibrosis of affected organs. commonly involved organs include the pancreas, biliary tree, orbits, salivary glands, and retroperitoneum, among many others. the serum igg4 level is often but not always elevated. because of the novelty of igg4-rd, little effort to date has been devoted to the development of outcome measures for this newly recognized condition. a disease responder index is a tool designed to detect any changes in disease activity and identify improvement and worsening in the same and/or different organ systems. a responder index permits objective quantification of the treatment response by providing standardized outcome measures. assessing clinical response and not simply serologic response is increasingly important to establish endpoints in randomized control trials. glucocorticoids are the standard first - line treatment for igg4-rd and patients whose disease has not reached an advanced stage of fibrosis generally respond well to this treatment, at least initially. the first is the complex, multiorgan system nature of this disease, which makes it difficult to summarize the state of disease activity across all organs. the second is the fact that the stage of disease activity can differ across organs, such that a patient can have active inflammation likely to respond to immunosuppression in one organ and advanced fibrosis (less likely to respond to treatment) in another. we have developed an igg4-rd responder index (igg4-rd ri) for use as an outcome measure in an ongoing pilot trial of rituximab in this condition. we intend that this instrument will measure not only disease activity but will also incorporate features that capture the need for urgent treatment and catalogue disease - related damage. this paper is designed to provide information on the development and implementation of the igg4-rd ri. we report the philosophy behind the development of the igg4-rd ri to date, the steps taken to create the instrument through the enlistment of assistance of international experts in this condition, and the plans for completion of the igg4-rd ri validation process. the igg4-rd ri was designed to assess disease activity from visit to visit using clinician - generated assessments of both objective and subjective measures. the igg4-rd ri uses a scoring system from 04 for each organ system or site and asks the clinician to rate the extent of disease activity and damage at the time of the clinical encounter. the igg4-rd ri was revised by the organizing committee of the international igg4-related disease symposium, held in boston in october, 2011 [http://www2.massgeneral.org/pathology/symposium/igg4_related_systemic_dis.asp ]. this group was comprised of 39 experts from 9 countries, with subspecialty expertise in rheumatology, gastroenterology, allergy / immunology, nephrology, surgery, pathology, and radiology. further revisions were made following a simulation exercise involving six paper case descriptions of real patients, completed by igg4-rd symposium participants. finally, both the igg4-rd ri that emerged from these development steps and a physician global assessment (pga) were used to assess the disease retrospectively in terms of disease activity and damage. the pearson 's correlation coefficient was then calculated to compare the igg4-rd ri and pga responses. the igg4-rd ri was modeled on the birmingham vasculitis activity score for wegener 's granulomatosis (bvas / wg). the bvas / wg is a formally validated and widely used instrument for the measurement of disease activity in granulomatosis with polyangiitis (formerly wegener 's granulomatosis) and microscopic polyangiitis, a pair of distinct but overlapping conditions often termed antineutrophil cytoplasmic antibody (anca)-associated vasculitides (aavs). the bvas / wg is a clinician - scored instrument in which each disease activity in each organ system is graded persistent, worse, or none at each clinic visit. the number of items of persistent or worse for each organ system is totaled and used to quantify the states of disease flare, persistent disease, or remission. the bvas / wg was selected because of the experience of one of the authors (j. h. stone) as a lead developer of this instrument ; similarities between anca - associated vasculitis and igg4-rd, including the propensities for multi - organ system involvement ; the broad range of disease activity between flare and remission ; the high frequency of disease - related damage (which must be distinguished from active disease) ; the absence of reliable biomarkers that necessitates reliance upon clinical indices for longitudinal assessments. the igg4-rd ri, designed to emphasize ease of use, includes specific reminders to consider activity within all organs involved commonly in igg4-rd (figure 1). physicians enter a score from 04 for each organ / site affected, indicating whether the organ / site is normal, improved, new or recurrent, or worse on treatment. the physician also provides yes / no answers for each organ site to the questions of whether the disease is symptomatic ; whether the disease activity requires treatment urgently ; whether the organ dysfunction observed is related to damage rather than (or in addition to) active disease. at the end of this table, the serum igg4 concentration in milligrams per deciliter is entered along with a score of 04, indicating whether the igg4 concentration has improved, become newly or recurrently elevated, or increased despite treatment since the last visit. the scoring scheme for serum igg4 concentration, therefore, parallels the schemes for individual organ system activity assessment. the cumulative glucocorticoid dose (in prednisone equivalents) since the last visit and total igg4-rd ri score the numbers for each organ score refer to disease activity, distinguished from organ dysfunction related to damage : 0 signifies the absence of active disease in that organ. a score of 0 is appropriate when the organ system has never been affected by active igg4-rd, or when previously evident disease within that organ has resolved ; 1 indicates that disease activity within an organ has improved but still persists to some degree ; 2 indicates that the disease within that organ has remained persistent and unchanged since the last visit ; 3 indicates the presence of new or recurrent disease activity ; 4 refers to disease that has worsened despite treatment. the organ sites were selected for inclusion in the igg4-rd ri based on a review of the existing literature (table 1). for ease of conceptualization, the sites of potential organ involvement are listed from head to toe. this structure is similar to that of the bvas / wg scoring sheet, on which disease activity is scored by organ system, and each disease site is assigned a designation of normal, persistent disease activity, and new / worse disease activity, with numerical scores corresponding to each state. the igg4-rd ri category of other organ / site involvement is important because the protean nature of this disease makes it impossible to capture all potential sites of disease. in addition, we anticipate that new clinical manifestations of this disease and possible even new sites of organ involvement will be described as the clinical phenotype of this disease is understood more fully. lists of the most common symptoms and signs within a given organ system are included in the igg4-rd ri instructions (see the appendix), principally as a reminder to the clinician of the possible disease manifestations to consider when scoring disease activity and damage. the physician simply denotes on the form the presence or absence of symptoms for a given igg4-rd site. good clinical judgment and a thorough knowledge of the disease manifestations of this condition are essential, as with any clinical responder index. some disease manifestations of igg4-rd require the immediate institution of treatment to prevent permanent organ damage. for example, igg4-related sclerosing cholangitis can lead to cirrhosis within several months of diagnosis and requires the prompt initiation of therapy. in contrast, the lymphadenopathy of igg4-rd remains unchanged for prolonged periods in many patients and may never require treatment. the urgent column in the igg4-rd ri is designed to capture aspects of the disease that require the immediate start of immunosuppression in order to preserve organ function. the score for an organ site is doubled when the need to initiate treatment for active igg4-rd at a particular or organ / site is considered urgent. for example, if a patient has new igg4-related sclerosing cholangitis, the total score for that organ / site would be 6 instead of 3. similarly, if the patient 's biliary status has worsened despite therapy since the time of the last visit and an urgent escalation of therapy is required to treat the igg4-related sclerosing cholangitis, then that organ score would be 8 rather than 4. only the score of the individual organ site is doubled in this setting, not the total igg4-rd ri score. organ damage results from active disease and in some cases both active disease and damage can be present in the same organ system simultaneously. in other cases, immunosuppression must be targeted to active igg4-rd, not to damage resulting from previously active therapy. the most appropriate use of immunosuppression is to control active disease and prevent disease - related damage. it is particularly ideal to employ immunosuppression at a stage of disease when the histopathology is characterized by a lymphoplasmacytic infiltrate rather than a predominance of acellular fibrosis. radiographic studies such as computed tomography (ct) and positron emission tomography with ct (pet - ct) can aid the clinician in determining which organs have been damaged. for example, even conceding that active disease might be present simultaneously with damage within the pancreas, the finding of atrophic changes by ct scan within that organ would be considered to be the result of damage. in such a case, both active disease and disease - related damage should be scored. the serum igg4 level may become elevated in a patient experiencing an active flare [6, 10 ]. however, not every patient with igg4-rd has an elevated serum igg4 level at baseline, even before treatment. it is well established that classic igg4-rd can be active in the absence of elevated serum igg4 concentrations. the igg4-rd serum level in the igg4-rd ri is scored according to normal, improved, persistent, new, recurrent, or worsened despite treatment. the sum of the disease activity in all of the organ sites plus the serum igg4 concentration score (also graded on a 04 scale) yields the total activity score. an individual active organ site is doubled for urgency and added to the other organ sites. this number can be compared between visits to assess the disease activity over time as well as being used for a clinical trial endpoint. the longitudinal recording of damage, though not included in the overall disease activity score, is essential to the formulation of the patient 's overall outcome. glucocorticoids are the cornerstone of igg4-rd treatment, and most patients respond promptly to this treatment, at least initially. thus, careful recording of the dose of prednisone (or prednisone equivalent) in the interval between the current visit and the preceding one is essential to a full understanding of the degree of disease activity. the igg4-rd ri went through several development stages and iterations before arriving at its current format. a simulation exercise using paper case descriptions of six real patients was sent to attendees of the international igg4-rd symposium (held in boston, ma, usa october 2011). the participants received written instructions on how to apply the igg4-rd ri but did not attend a training session (the appendix). twenty - one individuals participated in this exercise, providing valuable feedback from a cross - section of investigators interested in igg4-rd. the physicians who completed the exercises included a variety of subspecialists, particularly rheumatologists and pathologists (figure 2). the simulation exercises were presented as clinical vignettes, including data from histories, physical examinations, laboratory results, and radiologic findings for each case. accompanying each clinical vignette was at least one clinical photograph, radiology study, or histologic image to illustrate the case effectively. the physicians then used all of the information presented in the simulation exercise to complete a separate igg4-rd ri scoring sheet for each case. the results were scored for each participant against standardized answers prepared by consensus of the four authors. the purpose of this exercise was to solicit feedback on the igg4-rd ri from physicians who were experts in the evaluation of patients with this disorder from different perspectives. these included scoring organ involvement in which clinical symptoms and signs had resolved entirely as improved but persistent (i.e., 1) rather than resolved (i.e., 0). another scoring discrepancy resulted from incorrectly scoring patients off treatment who were recurrent (3) as if they were receiving treatment (4). a third error was failing to double the organ / site score, when disease requiring treatment urgently was present. comments from the participants in this exercise contributed substantially to important revisions of the draft instrument. we reformatted the scoring sheet in order to address the common scoring differences from the simulation case exercises. the next step in the development of the igg4-rd was the retrospective use of the instrument for fifteen individual clinic and in - patient evaluations among patients in the massachusetts general hospital igg4-rd registry. two blinded rheumatology experts scored an igg4-rd patient visit using either the igg4-rd ri or the physician 's global assessment scale (pga). as the field of igg4-rd is poised to move beyond the descriptive phase of the disease, validated outcome measures are required to advance the understanding of this condition and the assessment of new treatment approaches. the current iteration of the igg4-rd ri marks an important step toward the availability of useful outcome measures in this disease. we anticipate that additional validation steps for this instrument will be required, but this paper describes accurately the philosophy and goals behind the igg4-rd ri. the development efforts to date have created a one - page instrument supported by the instruction manual shown in the appendix. data included on this single page include indications of disease activity across a full spectrum of potential organ involvement ; the serum igg4 concentration ; assessments of the need for treatment on an urgent basis ; the recording of damage in organ systems ; the sum of recent glucocorticoid use. expertise with the use of the igg4-rd ri may, therefore, become a concise and important tool for clinical trials and other investigations related to this disorder. although the developers of the igg4-rd ri have relied significantly upon the bvas / wg in creating this instrument, the igg4-rd ri differs in important ways from the bvas / wg. the urgent column in the igg4-rd ri highlights features of the disease that require the prompt institution of treatment and is, therefore, analogous to the major designations given to some organ system manifestations in the bvas / wg. however, the bvas / wg does not record disease damage on the same page. rather, clinical trials in aav have generally used a separate instrument, the vasculitis damage index, for this purpose. although it is critical that the concepts of disease activity and damage be kept separate and recorded appropriately during clinical assessments, it may be useful to have an indication of damage on the same one - page case report form even if damage does not contribute to the overall disease activity score. this model matches more closely the decision - making process that clinicians undertake on a daily basis in encounters with patients : are the signs of organ dysfunction due to active disease, or are they more accurately a reflection of damage rather than a process that requires more intensive immunosuppression ? the igg4-rd ri will find its greatest use in the research setting, either in the context of clinical trials or in other types of investigations that require the careful longitudinal assessments of patients ' clinical status. because consistency of its application from visit to visit is critical, it will be most useful to ensure whenever possible that the same investigators complete the igg4-rd ri for the same patient across all visits. significant debate now exists within the community of igg4-rd investigators about the utility of serum igg4 concentration measurements in the diagnosis and management of this disorder. inclusion of the serum igg4 concentration in the igg4-rd ri at this point permits an analysis of the value of this measurement in the context of other organ disease assessments. we hypothesize that further analysis of these data will confirm the utility of serial measurements, at least in a subset of patients. this hypothesis, however, requires confirmation through studies of larger numbers of patients in a variety of states of disease activity. the simulation case exercises illustrated some shortcomings in early iterations of the igg4-rd ri that led to appropriate revisions of the original index. the experience with the simulation exercise highlighted the importance of adequate training with the instrument prior to its use in the research setting. the igg4-rd ri is simpler than many clinical assessment tools for multiorgan diseases, but both a thorough understanding of the clinical breadth of igg4-rd itself and a high degree of familiarity with the index are required in order to employ it effectively. we anticipate that a focused period of instruction for investigators in the context of a formal training course will be required before this tool can be used in the context of a clinical trial. in conclusion, progress in igg4-rd will be contingent upon the ability to assess patients rigorously in a longitudinal manner, using validated outcome measures. the igg4-rd ri described in this paper represents a broad effort at the development of a disease activity and responder index that can be employed in clinical trials and other investigations of patients with this emerging immune - mediated condition. the next steps in validation will include a multicenter study of patients recruited from a core group of sites with extensive experience in the diagnosis and management of this disease. | igg4-related disease (igg4-rd) is a multiorgan inflammatory disease in which diverse organ manifestations are linked by common histopathological and immunohistochemical features. prospective studies of igg4-rd patients are required to clarify the natural history, long - term prognosis, and treatment approaches in this recently recognized condition. patients with igg4-rd have different organ manifestations and are followed by multiple specialties. divergent approaches to the assessment of patients can complicate the interpretation of studies, emphasizing the critical need for validated outcome measures, particularly assessments of disease activity and response to treatment. we developed a prototype igg4-rd responder index (igg4-rd ri) based on the approach used in the development of the birmingham vasculitis activity score for wegener 's granulomatosis (bvas / wg). the igg4-rd ri was refined by members of the international igg4-rd symposium organizing committee in a paper case exercise. the revised instrument was applied retrospectively to fifteen igg4-rd patients at our institution. those scores were compared to physician 's global assessment scale for the same visits. this paper describes the philosophy and goals of the igg4-rd ri, the steps in the development of this instrument to date, and future plans for validation of this instrument as an outcome measure. |
the cell morphodynamics underlying the processes that control the organized spatial distribution of cells during the embryonic development are nowadays largely unknown. a single fertilized egg - cell (zygote) undergoes cellular divisions, differentiations, and interactions giving rise to specific forms of tissues and organs. the reconstruction of the nuclei shape and the identification of their number and position during embryogenesis are an essential tool for an integrated understanding of such morphogenetic processes. this kind of information is relevant to estimate the cell growth and identify cell divisions and apoptosis. it is a helpful tool to extract parameters such as the cell proliferation rate in time and space. it is relevant for investigating stem cells populations and early steps of cancerogenesis, opening the way for the preclinical evaluation of anticancer drug effects in vivo. we apply in this paper a region - based segmentation algorithm to segment the nuclei in a live zebrafish embryo. since during the acquisition the embryo is growing up, the cell number strongly increases. furthermore, since prior and during mitosis the nuclei condense and lengthen along the future cell division plane, the segmentation is a helpful tool to identify cell divisions. we consider and compare two different forms of the equation originally proposed by chan and vese. comparison is performed through qualitative and quantitative analysis of results on both phantoms and real data. we then apply to our dataset the formulation that ensures the best performances. in the rest of the paper we introduce the active contour model without edges and both tested formulations. in section 4 the zebrafish (danio rerio) is a model organism extensively studied to explore the gene function and vertebrate development [27 ] and might soon become a major model organism for preclinical drug testing by pharmaceutical industries. although distant from humans, nevertheless it has comparable organs and tissues and may supplement higher vertebrate models. compared with mice, zebrafish embryo develops rapidly and externally to the mother, favoring its manipulation. its transparency allows the visual inspection of the internal anatomy. due to its ability to regenerate fins, skin, the heart, and the brain, it is an object of study to understand healing / repair mechanisms in vertebrates. to reconstruct the shape and the position of every nucleus in a live zebrafish embryo is necessary to stain all the cells and use an acquisition technique with micrometrical resolution. this is a very challenging approach requiring specific methodologies and tools in embryos engineering and microscopy imaging. recent advances in imaging strategies open the way to in 4d imaging of live animals with a resolution at the cellular level and enough contrast to allow segmentation of individual cells. through the ubiquitous expression of fluorescent proteins in the zebrafish embryo, it is possible to label all the cells and perform time - lapse clsm (confocal laser scanning microscopy) imaging throughout embryonic development. the embryo used in this paper to design and validate our strategy has been stained through injection at the one cell stage of rnas encoding farnesylated mcherry fluorescent protein and histone h2b / egfp fusion protein. this allows the simultaneous acquisition of two different signals, membranes and nuclei, respectively. in this paper the images have been acquired using a confocal microscope leica sp2 aobs with a 40x/0.8 na water objective and 488 nm and 561 nm laser light excitation. we used the best compromise in terms of embryo survival and spatial and temporal resolution, as suggested in. the data has been acquired for four hours (25c under the microscope), starting at 3.5-hour postfertilization (development at 28c) from the animal pole. due to the lower temperature, during the acquisition the normal embryo development is slowed down and by the end it reaches the shield stage (see figure 1). images are acquired parallel to the focal plane xy, at regularly spaced z depth to build a z stack. the procedure has been repeated every 5 minutes to generate a temporal sequence of 49 volumes. the dimension of each voxel is 0.58 0.58 1.04. in the entire period of development, the whole embryo looks like a sphere with a diameter of 740. as the 3d images have a physical dimension of 300 300 31, they cover only the top part of the embryo, as depicted in figure 1. observing figure 2(a) is possible to note how the typology of staining influences the features of original data. as the histone h2b protein is a component of the chromatin, nuclei in mitosis conversely cell nuclei in interphase show a nonuniform image intensity and boundaries often not clearly defined. we explain in next section how this peculiarity leads to the choice of the segmentation method. one of the most popular approaches for image segmentation is the active contour model or snake, based on the evolution of a curve attracted by image boundaries in order to detect objects. implemented such models using level set methods, introduced first by osher and sethian and extensively used to track the evolution of fronts in a variety of applications. with the level set methods the desired curve of active contour models is embedded as zero level set of an implicit function, overcoming the drawbacks of the original snakes models as the difficulties related to topological transformations. however, the above - mentioned active contour models are edge - based ; that is, a curve is evolved with a speed function depending on a precomputed edge indicator defined by the image gradient. dealing with image derivatives, a preprocessing step is required to remove the noise and smooth the image. if the objects are not well contrasted, the denoising can disrupt image information and preclude the correct identification of objects surface. lately a region - based level - set method has been proposed by chan and vese to segment objects whose boundaries are not defined by a gradient. the model does not require preprocessing and is based on the informations provided directly by the image intensity. the chan vese approach is then well adapted to situations in which images are noisy, and it is difficult to extract the boundary of the desired object. let us consider a 3d image i as a real positive function defined in a rectangular domain. the chan vese model moves an arbitrary surface s for decomposing the image i into two regions of approximately piecewise - constant intensities : the objects to be detected and the background. such decomposition is obtained by minimizing the following energy functional : (1)e(s, si, so)=(area(s)) + iinside(s)|i(x, y, z)si|2dx dy dz + ooutside(s)|i(x, y, z)so|2dx dy dz, where si and so are, respectively, the averages of i inside and outside s, and 0, i > 0, and o > 0 are fixed parameters. while the first term of the right side of (1) controls its smoothness, the surface s separates the image into distinct regions depending on their mean values. the parameter plays an important role in the segmentation process, as it acts like a scale parameter. let, s is represented as a zero level set of an implicit function : such that (2)s=={(x, y, z):(x, y, z)=0},inside(s)=={(x, y, z):(x, y, z)>0},outside(s)=={(x, y, z):(x, y, z) 0 and the function thus constructed is then smoothed with few scale steps of the heat equation. this initialization provides an initial solution close to the final one and does not depend on a previous segmentation. actually an alternative could be to use the result of segmentation at time t as initial solution for the segmentation at time t + 1. but this choice does not consider that the number of nuclei changes between subsequent volumes. indeed some nuclei divide and others are close to the bottom of the acquired volume and can go out of the imaged part of the embryo. moreover, our solution allows the simultaneous segmentation, for example, on different processors, of all the volumes in the time lapse series. we keep i = o = 1, while the scale parameters have been chosen by our previous analysis and set to 0.1. we use in every case a time step of 0.1 and the steady state is reached after about 1500 iterations. from the isosurface corresponding to the zero level set of the final solution are then extracted the connected components, whose number provides the counting of nuclei in the processed volume. both for extracting the isosurface zero and its connected components we make use of the open - source vtk (visualization toolkit) library, which provides widely used filters for image visualization and analysis. in the first a cutting plane of last volume is compared with a cut of segmented surfaces. the white outline well reproduces the nuclei boundaries even if they are not clearly defined, and small regions are correctly detected. in figure 7 is visualized a segmentation (red) together with three orthoslices of original data. observing the nuclei cut by planes it is possible to see the white contour line of original data. as previously introduced, this number can be quantified by counting the connected components extracted from the segmented surfaces. we show in figure 9 an example of connected components extraction on last volume of the time lapse series. the number of nuclei detected in our dataset during the embryo development is instead plotted as graph in figure 10. at the beginning we detected 333 nuclei, nevertheless we would like to point out that the number of detected cells is not strictly increasing but shows small oscillations around an exponential trend. this behavior occurs because the cells are moving and the imaging includes only the top part of the embryo. cells close to the bottom of the imaged volume can then be located either on the inside or on the outside. in order to estimate the ability of our method of correctly extracting the number of cells, we then compared the number of nuclei identified by a manual computation on the first volume of the time lapse series (340 nuclei) with the number automatically detected. by our evaluation the correct cells number is then slightly underestimated, but our error is less than 3%. the main source of error is given by nuclei very close, if not overlapped, in original data. this drawback could be overcome by combining signal of both nuclei and membranes, by refining the segmentation locally, or through the multiphase level - set approach. we applied the active contour model without edges to time lapse series of confocal images representing cells nuclei in a live zebrafish embryo. as the nuclei in our dataset are varying in shape and size, we considered two forms of the originally proposed model equation. the maximum and total errors have been quantified as the hausdorff distance and the mean hausdorff distance between the data and a gold standard obtained through manual segmentation. we proved that our suggestion of using = 1 is the formulation with the best performances. this form of the active contour model without edges has then be applied to a dataset which represents the development of a zebrafish embryo between the shield and the sphere stage. from the results of segmentation have been afterwards extracted the connected components, whose counting allowed to quantify the number of cells. the corresponding error has been estimated through a comparison between the number of cells detected by the algorithm and the number of cells detected by a manual computation in a selected volume. | this paper is devoted to the segmentation of cell nuclei from time lapse confocal microscopy images, taken throughout early zebrafish embryogenesis. the segmentation allows to identify and quantify the number of cells in the animal model. this kind of information is relevant to estimate important biological parameters such as the cell proliferation rate in time and space. our approach is based on the active contour model without edges. we compare two different formulations of the model equation and evaluate their performances in segmenting nuclei of different shapes and sizes. qualitative and quantitative comparisons are performed on both synthetic and real data, by means of suitable gold standard. the best approach is then applied on a number of time lapses for the segmentation and counting of cells during the development of a zebrafish embryo between the sphere and the shield stage. |
avian influenza (ai) is a highly contagious disease caused by type a influenza viruses, a member of the family orthomyxoviridae. influenza a viruses have antigenically related nucleocapsid and matrix proteins but are classified into subtypes on the basis of their haemagglutinin (h) and neuraminidase (n) antigens. although fouchier. reported the presence of 16 h and 9 n subtypes of influenza a viruses, recent studies [3, 4 ] have identified additional h17n10 and h18n11 subtypes in bats. many species of domestic and wild birds worldwide have been shown to be susceptible to infection with avian influenza viruses (aivs), with aquatic birds constituting a major reservoir of these viruses which have particularly been reported to occur in poultry in either the highly pathogenic or low pathogenic forms ; the overwhelming majority of isolates are of low pathogenicity for chickens and turkeys [5, 6 ]. specifically, turkeys are susceptible to a wide range of influenza a viruses and, as a major exception to the host range restriction rule, are routinely infected with swine - like influenza viruses. infections in turkeys range from asymptomatic to severe disease including respiratory tract disease, depression, drop in egg production, and high mortality [7, 8 ]. the segmented nature of the influenza virus genome allows for reassortment of genes when a susceptible host is coinfected with different influenza virus subtypes, which may be from different species. this interspecies transmission of influenza viruses has been reported by several authors [912 ]. influenza viruses with novel combinations of gene segments from different influenza - susceptible species have been isolated from turkeys [12, 13 ], which have been indicated to be more susceptible to ai infections than chickens. although h3- and h5-subtype influenza viruses are known to infect avian and mammalian species, including humans [15, 16 ], the h3-subtype viruses are usually not the subject of conventional surveillance, which is biased towards detection of highly pathogenic avian strains, therefore leading to fewer h3 virus isolations from poultry flocks. since 2005, ai has spread from southeast asia to over 60 different countries, resulting in the direct death or slaughter of over 250 million poultry. recently, an outbreak of highly pathogenic avian influenza (hpai) h5n2 that affected mostly turkey flocks was reported in several counties in minnesota, usa [18, 19 ]. in nigeria which has a poultry industry of about 160 million birds estimated at us$ 250 million, ai has been reported in several domestic and wild birds such as chickens, ducks, waterfowls, and spur - winged geese or whistling ducks. although these studies emphasize the importance of surveillance for aiv infections in the natural hosts, there has been little or no report of surveillance for the disease in turkeys, which form almost 2% of the total poultry population, in nigeria. according to the oie, in serologic surveillance programs, the test to detect the anti - nucleoprotein antibody is the method commonly used because it detects antibodies to a cross - reactive antigen shared by all influenza a viruses. some authors [26, 27 ] have suggested that competitive enzyme - linked immunosorbent assay (celisa) is effective for large - scale surveillance of aiv in avian flocks or herds of other species and this test has been used to detect antibodies against different aiv strains, including h3n8 and h5n2, in chickens, ducks, turkeys, and other avian species. these authors reported that since the celisa is high in sensitivity but relatively low in specificity in turkeys, quails, and pheasants, its use for surveillance purposes should be followed by a conventional standard test (e.g., haemagglutination inhibition assay) when specific species need to be tested [27, 28 ]. therefore, as part of on - going surveillance for ai in poultry, we carried out a serologic survey to investigate the prevalence of avian h3- and h5-subtype influenza virus antibodies in turkey flocks in three states of southwest nigeria. a total of 520 (203 males and 317 females) apparently healthy turkeys sampled from 22 different flocks located in oyo, osun, and ondo states, southwest nigeria (figure 1), were used for this study. while 315 samples were collected from 13 flocks in oyo state, 95 were collected from six flocks in osun state and 110 samples were collected from only three flocks in ondo state. additionally, the turkeys comprised 331 local and 189 exotic varieties. the southwest region plays a leading role in poultry production since an estimated 65% of nigeria 's commercial poultry population is concentrated there [25, 29 ]. about 2 ml of blood was aseptically collected from the brachial vein of each turkey using sterile syringes and needles. the blood was allowed to clot at room temperature while sera were separated and stored at 20c until tested. the farmers were interviewed on issues such as ai vaccination of their flocks and type of poultry they preferred to rear while the practices they employed in rearing the turkeys were observed. additionally, the sex and breed of the turkeys as well as management system (free - range, semi - intensive, or intensive) were recorded. the turkeys were grouped based on age into growers (018 weeks) and adults (> 18 weeks). a competitive elisa kit (bionote inc., korea) for the quantitative detection of anti - nucleoprotein antibodies to aiv was used to screen the sera. according to the manufacturer the test was performed following the kit protocol and results were read at 450 nm using a microplate elisa reader (optic ivymen system, model 2100c, biotech sl, madrid, spain). the percentage inhibition (pi) for each sample was calculated from the absorbance values obtained. positive samples (pi value 85) were screened by haemagglutination inhibition (hi) test for aiv subtype - specific antibodies using a panel of reference antigens comprising low pathogenic avian influenza (lpai) h3n8 and h5n2 viruses and 4 haemagglutinating units of each antigen according to standard protocol. data obtained were analysed with column statistics and fisher 's exact test (two - tailed) using graph pad prism version 5.0 (graph pad software, san diego, ca, usa) and p values < 0.05 were considered significant. interviews conducted with the farmers revealed that the sampled turkeys were not vaccinated against ai and rearing of turkeys with chickens was a common practice on some of the farms (figure 2). it was also observed in two farms (one each in oyo and osun states) that the turkeys were kept in close proximity to pig pens while the farmers interviewed in ondo state indicated their preference for rearing chickens instead of turkeys. based on the elisa, prevalence of anti - aiv antibodies in the tested turkey sera was 6.0% (19/315), 4.2% (4/95), and 0% (0/110) for oyo, osun, and ondo states, respectively, with overall seroprevalence of 4.4% (23/520). compared to the intensively raised turkeys, those reared on free - range system had significantly higher aiv antibody prevalence (table 1), with p value of 0.034 and odds ratio (or) of 9.4 (95% ci : 1.751). however, there was no significant difference in seropositivity based on state, breed, age, and sex of the birds although a greater proportion of the hi test - positive birds were intensively reared adult, female local turkeys from oyo state (table 2). the hi antibody titres ranged from 1 : 8 to 1 : 2048 and 1 : 64 to 1 : 2048 for lpaiv h3n8 and h5n2 subtypes, respectively. of the tested sera, 18 were positive for anti - aiv h3n8 antibodies only and four were positive for both anti - aiv h3n8 and h5n2 antibodies, while five did not contain antibodies to either of the two aiv subtypes. mean hi antibody titres of 5.4 0.6 log2 (95% ci : 4.16.8) and 9.8 1.3 log2 (95% ci : 5.813.7) were obtained for the h3n8- and h5n2-positive sera, respectively. the current global influenza situation is characterized by a number of trends that must be closely monitored. these include an increase in the variety of animal influenza viruses cocirculating and exchanging genetic material, giving rise to novel strains. specifically, avian influenza viruses continue to be a problem worldwide because they are potentially highly infectious and can rapidly spread and cause disease in domestic poultry, and some may also infect other animal hosts, including humans. moreover, clark and hall noted that the first sign of lpai infection in domestic poultry is often seroconversion, which may be the only evidence of infection with some lpai subtypes (i.e., no clinical signs present). these observations, coupled with previous reports of aiv infections in nigeria [2124, 32, 33 ], underscore the need for continuous surveillance for these infections in nigerian poultry. the detection of antibodies to lpaiv h3n8 and h5n2 in apparently healthy turkeys in this study indicates that lpai h3n8 and h5n2 virus strains presently circulate in exotic and local turkey flocks in oyo and osun states, southwest nigeria. since vaccination against ai is not currently officially permitted in nigeria and all the farmers interviewed in this study claimed that they did not vaccinate their birds against avian influenza, the antibodies detected in these birds could only have resulted from seroconversion following natural infection with the viruses. thus, the birds could serve as reservoirs shedding the viruses into the environment, thereby playing a crucial role in the epidemiology of the disease. this finding is consistent with previous reports of infection with lpaiv h3 and h5 subtypes in poultry elsewhere [27, 34, 35 ] and corroborates the observation of clark and hall that the first sign of lpai infection in domestic poultry is often seroconversion, which may be the only evidence of infection. the nondetection of anti - aiv antibodies in turkey sera from ondo state could be due to the fact that the samples were collected from only three flocks where the farmers interviewed indicated their preference for rearing chickens instead of turkeys. importantly, the detection of antibodies to both h3n8 and h5n2 lpai viruses in some turkeys in this study is of public health concern because coinfection with different influenza viruses might provide the opportunity for reassortment, leading to the emergence of novel reassortant strains with zoonotic potential. this finding is similar to that of song. who also detected antibodies against h3n8 and h5n2 aiv strains in chickens, turkeys and other avian species in korea. two of the farms (one each in oyo and osun states) from which seropositive birds were detected in this study also kept pigs in pens close to the turkey houses. according to kapczynski., coproduction of swine and turkeys on the same farm may increase the opportunity for reassortment between different ai and mammalian influenza viruses due to close contact between these species. additionally, it has been reported that if a field virus is a low pathogenic type, there is the risk of it mutating to a highly pathogenic form after circulating in susceptible poultry. thus, the practice of rearing different animal species in the same pen or in close proximity, as observed in this study, may play a vital role in interspecies transmission of aivs in nigeria. it is interesting to note that five of the elisa - positive sera did not contain antibodies to either h3n8 or h5n2 lpai virus. this suggests the possibility of other aiv subtypes circulating among turkeys in the study area. there is therefore a need to further investigate aiv strains present in turkey flocks in southwest nigeria using a larger panel of reference aiv subtypes as well as virus isolation and molecular identification techniques such as reverse transcriptase - polymerase chain reaction. the detection of higher level of seropositivity to lpai h3 virus in this study compared to the h5 subtype is significant because, unlike aquatic birds, poultry are not natural hosts of h3-subtype influenza viruses and, generally, h3-infected poultry are asymptomatic. furthermore, although the global focus remains on h5, h7, and h9 viruses as emerging disease threats for avian and mammalian species, the genetic and pathogenic changes found among h3 viruses suggest a need to determine the current status of h3 aiv infections in poultry, especially among chickens and turkeys, which constitute the largest poultry species in nigeria. moreover, it is known that normal asymptomatic infection of avian species can silently maintain and transmit h3 influenza viruses provided there is an opportunity for genetic reassortment with other prevalent strains (e.g., h5 or h9) in avian populations, and this may be promoted where different avian species are kept together. thus, mixed farming of turkeys and chickens, observed to be practiced on some farms in this study, is a critical management risk that should be discouraged. since turkeys, like swine, have been implicated as another mixing vessel for generating influenza reassortants of human and avian origin in the field, additional studies are advocated to determine their potential role in the zoonotic transmission of aiv strains in nigeria. in this study, it was observed that the odds of detecting anti - aiv antibodies were 9.4 times higher in free - range than in intensively managed turkeys. this could be due to the fact that birds (and other animals) on free - range are often neglected and allowed to scavenge for food, thus exposing them to infectious agents. according to alexander, most evidence obtained on the prevalence of influenza in different types of poultry and from different geographical locations supports the view that the primary introduction is from feral birds. therefore, influenza viruses are most likely to infect poultry reared in a way that allows contact with feral birds, such as on free - range. based on this, we recommend that free - range rearing of turkeys in the study area be discouraged as it has the potential to favour spread of ai infections between poultry populations. additionally, in order to better understand the epidemiology of ai in southwest nigeria, we propose the fact that future surveillance for the disease should be done in combination with innovative tools such as social network analysis and poultry market chain analysis, both of which have successfully been used elsewhere [3841 ], for the control of within - country and transboundary livestock disease outbreaks. these innovations, which should involve stakeholders like the commercial and small - holder poultry farmers, poultry sellers and butchers, and live - bird market operators, have the benefits of being able to provide a network - based approach that offers new insights into disease transmission dynamics as well as an analytical framework that allows characterization of an entire poultry industry and interlinkages among various actors in the industry [38, 42, 43 ]. ultimately, this approach will serve as a basis for developing more effective strategies aimed at mitigating the risk of contracting and transmitting ai among poultry farms in nigeria, thus reducing the socioeconomic impact of the disease on the poultry value chain. the findings of this study not only reveal that lpai h3n8 and h5n2 viruses presently circulate in exotic and local turkey flocks in oyo and osun states, southwest nigeria, but also emphasize the importance of routine surveillance for aivs in different avian species as part of an early warning plan for the prevention of ai outbreaks in nigeria. we advocate that farmers be educated on the dangers inherent in the practice of free - range rearing of birds as well as raising of different avian species together or in close proximity with other animals. this knowledge will help eliminate farm practices that could otherwise have enhanced the spread or interspecies transmission of aivs in the country. lastly, the use of social network and market chain analyses for future ai surveillance investigations is proposed as a basis for developing more effective ai control strategies. | since the first outbreak of avian influenza (ai) in nigeria in 2006, there has been continuous monitoring of the disease in chickens with little attention given to turkeys. as part of on - going surveillance for ai in southwest nigeria, we used a competitive elisa to detect anti - ai virus antibodies in 520 turkey sera obtained from poultry farms in oyo, osun, and ondo states while haemagglutination inhibiting antibodies against low pathogenic ai viruses (lpaivs) were detected using h3n8 and h5n2 subtype - specific antigens. the overall seroprevalence obtained by elisa was 4.4% (23/520). of the 23 elisa - positive samples, 18 were positive for anti - aiv h3n8 antibodies only and four were positive for both anti - aiv h3n8 and h5n2 antibodies indicating a mixed infection, while five were negative for antibodies to either of the two aiv subtypes. considering that turkeys have been implicated as a mixing vessel for generating influenza virus reassortants of human and avian origin, the detection of antibodies to lpaiv h3n8 and h5n2 in these turkeys is of public health concern. we advocate further studies to determine the potential role of turkeys in the zoonotic transmission of aivs in nigeria. additionally, the practice of rearing turkeys with chickens should be discouraged. |
bioflavonoids are a group of polyphenolic compounds that are common throughout the plant kingdom. they are widely studied and have been found to promote healthy living in epidemiologic studies. much of the attention that bioflavonoids have attracted is due mainly to the french paradox. this is the dietary anomaly in which people in the mediterranean culture have a higher fat intake but a lower incidence of cardiovascular disease and increased longevity. one of the most widely studied bioflavonoids is, epigallocatechin-3-gallate (egcg), the most abundant polyphenol in green tea. it has been studied extensively during the past decade for its therapeutic potential in various cancers, alzheimer s disease, obesity, and diabetes. despite these promising therapeutic uses, the plasma concentrations of egcg that are required are prohibitively high. thus, novel methods for improving the oral bioavailability of egcg are desirable. in a previous study, we demonstrated that proprietary nanolipidic particles could be used to improve the bioactivity of egcg for reducing amyloid beta production in a cell model of alzheimer s disease while also more than doubling the oral bioavailability. although this could potentially overcome the problems thwarting the clinical translation of egcg, the technology is limited because it requires an alcohol suspension. until now there have been no reports of any of the crystalline forms of egcg in the cambridge structural database (csd) including the crystal structure of pure egcg which makes it worth studying from a structural perspective. therefore, we undertook the synthesis of cocrystals of egcg and also crystallized its pure form. this was accomplished by taking advantage of crystal engineering concepts and several crystallization techniques. cocrystal formers (ccfs), has emerged over the past decade as a materials science approach to generate novel solid forms of active pharmaceutical ingredients with improved physicochemical properties. we recently reported four new cocrystal forms of another flavonoid, quercetin, which improved solubility by up to 14-fold and oral bioavailability by up to 10-fold.(27) unlike quercetin, egcg is highly soluble in water. previous pharmacokinetics experiments have shown that egcg is absorbed rapidly in the gut following oral administration. thus, egcg would likely fall into the biopharmaceutics classification system (bcs) 3 : high solubility, low permeability. we hypothesized that new cocrystals of egcg with reduced water solubility might exhibit pharmacokinetic profiles that are more desirable for drug development. moreover, we hypothesized that reducing the solubility would decrease the rate of dissolution and slow the absorption of egcg in vivo. we report herein the results of our structural, solubility, and pharmacokinetic studies. green tea - derived egcg (> 95% purity by hplc) was purchased from http://www.herbs-tech.com. isonicotinamide (inm, > 99% purity), isonicotinic acid (ina, > 99% purity), nicotinamide (nic, > 99% purity), and nicotinic acid (nac, 98% pure) were purchased from sigma - aldrich corporation (st. louis, mo, usa). all crystallization and cocrystallization experiments were either conducted at room temperature or at 4 c. egcg (65.0 mg, 0.14 mmol) was dissolved in 1 ml of acetonitrile (99% pure). the resulting solution was layered on 2.5 ml of dichloromethane and was allowed to stand in the refrigerator. egcg (200.0 mg, 0.436 mmol) was dissolved in 2.5 ml of acetonitrile (99% pure). the solution was layered onto 6 ml of dichloromethane and 2 ml of nitrobenzene and then allowed to stand in a refrigerator. yellow crystalline needles were harvested after 24 h. the pure crystal line form of egcg, form iv, was obtained by heating forms ii or iii at 120 for 2025 min. form iv crystals can also be reproduced by dissolving egcg (45.0 mg 0.098 mmol) in 1 ml of acetonitrile (99% pure). the solution was layered on 2.5 ml of dichloromethane and seeded with form iv and was allowed to stand in the refrigerator. egcg (45.80 mg, 0.099 mmol) and iso - nicotinamide (99% pure, used as received, 12.2 mg, 0.0998 mmol) were dissolved in 5 ml of water. colorless block - like crystals of egcginm5h2o were harvested in less than 5 min. however to further grow better quality crystals, 1:1 water / methanol (v / v %) solvent was used, and the crystals were obtained in one day. egcg (45.80 mg, 0.099 mmol) and nicotinamide (99% pure, used as received, 12.2 mg, 0.0998 mmol) were dissolved in 2 ml of water. looking carefully under the microscope a colorless block like single crystal was separated from the cluster and was used for single crystal x - ray diffraction analysis. egcg (45.80 mg, 0.099 mmol) and iso - nicotinic acid (99% pure, used as received, 12.3 mg, 0.0998 mmol) were dissolved in 5 ml of water. egcg (458.0 mg, 0.99 mmol) and nicotinic acid (99% pure, used as received, 123.0 mg, 0.998 mmol) were slurried in 2 ml of water overnight in a vial. the vial was left in the hood undisturbed, and after two days a cluster of block shaped crystals was obtained. open alumina crucibles were used for analysis from 30 to 300 c at 5 c / min heating rate under nitrogen purge. thermal analysis was carried out employing a ta instruments dsc 2920 differential scanning calorimeter. an empty pan, sealed in the same way as the sample, was used as a reference pan. a typical sample (28 mg) was heated in a dsc from 30 to 300 c at a 5 c / min heating rate. the experimental data were analyzed using commercially available software (ta universal analysis 2000 ; ta instruments). all of the forms of egcg were characterized by infrared spectroscopy using a nicolet avatar 320 ft - ir instrument. the samples were placed on the sample holder with the help of vacuum grease and exposed, at room temperature, to cu k radiation (= 1.54056 ; 40 kv 30 ma, bruker axs d8 advance). for all the samples, the angular range was 30 2, with a step size of 0.05 2. intensity counts were accumulated for 0.5 s at each step. single crystals of forms ii, iii, and iv and cocrystals egcginm5h2o, egcgnic9h2o, egcgina3h2o, and egcgnacxh2o were examined under a microscope, and suitable crystals were selected for single crystal x - ray crystallography. single crystal x - ray diffraction data on forms iii and iv were collected on a bruker - axs smart apex ccd diffractometer with monochromatized mo k radiation ((= 0.71073), whereas for form ii and cocrystals egcginm5h2o, egcgnic9h2o, egcgina3h2o, and egcgnacxh2o, data were collected on a bruker - axs smart apex 2 ccd diffractometer with monochromatized cu k radiation (= 1.54178). both of the diffractometers are connected to a kryo - flex low temperature device. the structure was solved using shelxs-97 (direct methods) and refined using shelxl-97 (full - matrix least - squares on f2) contained in apex2 and wingx v1.70.01 programs packages. hydrogen atoms of ch and ch2 groups were placed in geometrically calculated positions and included in the refinement process using a riding model with isotropic thermal parameters : uiso(h) = 1.2ueq (ch2, ch). hydrogen atoms of oh, nh groups and water molecules have been found from difference fourier map and refined using dfix (d(o h) = 0.84 for hydroxyl groups, dfix 2.2 antibumping restraints for two hh distances) and sadi restraints for water molecules (o h and hh distances) with uiso(h) = 1.5ueq(oh). some of water molecules were observed to be disordered in the structure. hydrogen atoms of ch and ch2 groups were placed in geometrically calculated positions and included in the refinement process using riding model with isotropic thermal parameters : uiso(h) = 1.2ueq(ch2, ch). hydrogen atoms of oh and nh groups were placed in geometrically calculated position and refined using afix 147 and afix 93 correspondingly. positions of hydrogen atoms h4, h15, and h25a were further refined using a distance restraint (ho{h - bond acceptor }) of 1.865(8) as found from a csd analysis. some of the water molecules were observed to be disordered, and it was not possible to locate hydrogen atoms for those molecules. hydrogen atoms of ch and ch2 groups were placed in geometrically calculated positions and included in the refinement process using a riding model with isotropic thermal parameters : uiso(h) = 1.2ueq(ch2, ch). hydrogen atoms of oh groups were placed in geometrically calculated position and refined using afix 147 or afix 83. the observed residual electron density 1.258 el/ can probably be attributed to the presence of small satellite crystals and/or disordered chains of ina. the cocrystals egcginm5h2o, egcgnic9h2o, egcgina3h2o, and egcgina were made in bulk for dissolution studies using the slurry method. egcgnacxh2o was not included in the dissolution study due to difficulties in reproducing it in bulk powder form. slurry experiments to reproduce egcgnacxh2o resulted in a glue - like material rather than a powder. for the remaining cocrystals, stoichiometric amounts of the starting materials were stirred overnight in 23 ml of water with the help of a magnetic stir bar on a stir plate that produced the cocrystals with 100% yield. the purity of the bulk material was tested by powder x - ray diffractometry (pxrd) and differential scanning calorimetry (dsc). a uniform particle size (between 53 and 75 m) for the bulk powder was obtained for all the cocrystals and egcg by sieving using standard astm sieves. solubility studies were performed on egcg, egcginm5h2o, egcgnic9h2o, egcgina3h2o, and egcgina using uv / vis / nir spectrophotometry in water at room temperature. the wavelength used for the determination of egcg was 305 nm since there is no interference with the ccfs at this wavelength. the dissolution studies were conducted by taking approximately 3 g of the cocrystal in 50 ml of water and stirring with a magnetic stir bar at ca. 125 rpm for 4 h. aliquots were drawn from the slurry at regular time intervals (5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 120, 150, 180, and 240 min) and filtered using a 0.45 m nylon filter. the filtrates were diluted appropriately and analyzed to measure the concentration of egcg (at 305 nm) by using a uv / vis / nir spectrometer. the remaining undissolved solid was analyzed by pxrd and dsc to confirm phase stability. all animal studies were conducted in accordance with a university of south florida iacuc - approved protocol. dawley rats (n = 3 per group) weighing 200250 g were purchased from harlan laboratories (indianapolis, in). the rounded tip catheters were surgically implanted into the jugular vein of the rats making multiple, precise blood draws painless to the animal. the rats were food (not water) deprived for 18 h prior to the start of the experiment. corn oil was selected as the gavage vehicle because all crystal forms were observed to be insoluble in it. all egcg forms were sieved to attain a particle size between 53 and 75 m prior to suspending in corn oil at 20 mg of egcg per ml. the egcg formulations were delivered via oral gavage at a dosage of 100 mg egcg per kg body weight. blood was collected at the following time points : 0, 5, 10, 30, 60, 120, 240, and 480 min. because heparin was kept in the catheter lines to prevent clotting, a small amount of blood was drawn and discarded before collecting each sample. approximately 300 l of blood was collected in edta tubes for each time point. the samples were kept on ice to preserve their integrity and then centrifuged at 4000 rpm for 10 min, after which the plasma was transferred to sterile centrifuge tubes. a preservative solution was added to each plasma sample at 10% (v / v) concentration to ensure the integrity of the egcg during storage. this preservative was comprised of 20% ascorbic acid (to prevent oxidation) and 0.1% edta (to scavenge any metal contaminants). the samples were stored at 80 c until they were analyzed for egcg content. to accurately quantify the concentration of egcg in the plasma, a previously described method was employed using liquid chromatography with tandem mass spectrometry. the standard spiking solutions were prepared by diluting the stock solution to 1000 and 100 g / ml using acetonitrile water (1:1, v : v). both solutions were protected from light using amber vials, and all solutions were stored at 20 c. for this analysis two standard curves were prepared : one with a higher (100.100 g / ml) dynamic range, and the other a lower range (100010 ng / ml). the results indicated that the standard curve performance was within acceptable range for bioanalytical method acceptance (r > 0.99). mean plasma egcg concentrations and the standard error in the mean (sem) were graphed using graphpad prism software (graphpad software, inc.). phoenix winnonlin version 6.3 (pharsight corporation, mountain view, ca) was used to conduct a noncompartmental analysis of the pharmacokinetic data and generate the pharmacokinetic parameters. the reported pharmacokinetic parameters included cmax, tmax, area under curve (auc), relative bioavailability (frel), and apparent terminal half - life (hl_lambda_z). relative bioavailability was determined by dividing the mean auc of each egcg formulation by the control. two - tailed t - tests were used to assess the statistical significance at each time point for the pharmacokinetic curves. the ccfs were selected based on the supramolecular synthon approach. according to this approach the ccfs were identified based on the functional groups present on egcg and analyzing the frequency for the occurrence of supramolecular synthons (homo and hetero) with other functional moieties. this analysis was carried out via the csd, an archive of over 600 000 organic crystal structures. the csd also offers a software platform that facilitates statistical analysis of packing motifs, thereby providing empirical information on common functional groups and how they associate at the molecular level. several fragments of egcg were identified, and a csd analysis was conducted to determine if these fragments and/or the whole molecule (egcg) are susceptible to form cocrystals with carboxylic acids, alcohols, or weak bases. furthermore, the selected ccfs should be suitable for use in drug products (e.g., fda approved, gras or eafus listed). several potential ccfs were selected based on the above - mentioned criteria. however, we were unable to obtain cocrystals with any other ccfs except those presented herein. in addition, efforts to obtain pure crystalline forms of egcg via different crystallization techniques led to the isolation of two solvates which upon desolvation resulted in a pure crystalline form of egcg. interestingly, all of the isolated cocrystals are in the form of hydrates. egcg is a large and flexible molecule containing a number of hydrogen bond donors and acceptors. furthermore, the raw material used is egcg monohydrate, and dehydration of the starting material is reversible. except for egcgina3h2o, all cocrystals became amorphous following dehydration. egcgina3h2o upon dehydration yields an anhydrous cocrystal of egcg and ina. we believe that water plays a pivotal role in crystallizing these cocrystals, but it is difficult to say whether the water molecules are playing a space - filling role in channels or they are an integral part of the crystal lattice. form ii crystallizes in the monoclinic c2 space group and is a high z structure consisting of two egcg, three acetonitrile, three water, and a disordered dichloromethane molecule in the asymmetric unit. the two symmetry - independent egcg molecules form sheets with voids that accommodate the solvent molecules, as shown in figure 2a and b. (a) egcg molecules in form ii forming sheets with channels. (b) a typical channel being occupied by solvent molecules in form ii. form iii, a solvated form of egcg with nitrobenzene and water, crystallized in the orthorhombic p21 space group with one molecule of each component in the asymmetric unit. in the crystal structure the egcg and water molecules form sheets. (a) illustration of a representative sheet formed by egcg and water molecules in form iii. (b) nitrobenzene molecule sandwiched between sheets of egcg and water molecules in form iii. pure egcg, form iv, crystallizes in monoclinic p21 space group with one egcg molecule in asymmetric unit. the voids are interpenetrated with other sheets to exhibit 2-fold interpenetration as shown in figure 4a and b. (a) crystal packing of form iv ; crinkled sheets with cavities were created via several o the single crystal x - ray structure analysis reveals that egcginm5h2o is a pentahydrate of the 1:1 cocrystal of egcg and inm. egcg molecules and inm molecules interact through one point hydrogen bonds (o hn, on : 2.756 (2)) between a hydroxyl group of egcg molecules and the aromatic nitrogen of inm molecules. ho (oo : 2.662 (4)) hydrogen bonds formed between hydroxyl moieties of egcg and carbonyl moieties of inm molecules and thereby form zigzag chains as illustrated in figure 5. the crystallization of egcg and nic in water results in the formation of a 1:1 cocrystal nonahydrate. it crystallizes in p1 space group with two molecules each of egcg and nic molecules and nine water molecules in the asymmetric unit (figure 6a). the n h functionality of the nic molecules interact with one of the o h moieties present of ring a of egcg with an no bond distance of 2.570 (2) (figure 6b). the cocrystallization of egcg and ina resulted in the formation of a cocrystal trihydrate which crystallizes in the orthorhombic space group p21p21p21. the ina and egcg molecules interact with each other through water molecules (figure 7a). one of the three water molecules interacts with two egcg and one ina molecule through the following h - bonds : (a) the o h of the water molecule interacts with the c o moiety of the ina molecule with a bond distance of 2.739 (3) ; (b) the o h of the water molecule interacts with two neighboring egcg molecules with h - bond distances of 2.704 (5) and 2.752 (3). the other water molecule forms a trifurcated bond with three different egcg molecules as presented in figure 7b through o ho bonds of 2.667 (3), 2690 (2), and 2.712 (2). ho bifurcated hydrogen bonds (2.540 (2) and 2.986 (3)) (figure 7a). the 1:1 cocrystal of egcg and nac crystallized as a hydrate in monoclinic space group c2. unlike the other cocrystals the stiochiometry of water in the egcg - nicotinic acid (nac) is indefinite. we were unable to determine the number of water molecules in the crystal lattice of egcg - nac cocrystals due to poor quality crystals and single crystal data (see table 1). from the crystal structure c bond angle is 122.76, and the c o bond distances are 1.237 and 1.252. each of the nac zwitterions interacts two neighboring egcg molecules via the following h - bonds : (a) an h - bond formed between one of the o h groups on ring b with one of the carboxylates of the nac molecules at a distance of 2.588 (3) ; (b) an h - bond between the n h of the nac molecule and one of the o h functionalities present on ring a of another egcg molecule (n ho : 2.902 (3)). the intermolecular h - bonding between egcg and nac zwitterions is illustrated in figure 8. illustration hydrogen bonding between egcg and nac molecules in egcgnacxh2o cocrystal (water molecules are not shown in the figure for clarity). egcg is relatively soluble in water compared to many other flavonoids such as quercetin. we determined the solubility profiles of the four new egcg cocrystals reported herein and observed that all of the cocrystals exhibit reduced solubility. the solubility studies were conducted for 4 h because after 1 h it was observed that the solubilities remained almost constant and did not change for the next 3 h. this suggests that the solubilities had reached equilibrium after 1 h. figure 9 shows the dissolution profiles of egcg and the egcg cocrystals. the maximum solubility of egcg determined experimentally was approximately 23.6 mg / ml during the 4 h dissolution experiment. the egcg cocrystals were found to exhibit far lower aqueous solubilities when compared to egcg as illustrated in figure 9. the cocrystal that exhibited the highest aqueous solubility was egcgnic9h2o (2.95 0.11 mg / ml). egcgina (anhydrous), egcginm5h2o, and egcgina3h2o were observed to exhibit solubilities of 1.227 0.036, 1.401 0.122, and 0.97 0.07 mg / ml, respectively. after the dissolution experiment has concluded (4 h), the identity of the powders was confirmed by pxrd and dsc. egcgina anhydrous had converted to the hydrate form, egcgina3h2o, while the remaining cocrystals were found to be stable up to at least 4 h.. dissolution profiles of (a) egcg and its cocrystals and (b) the cocrystals alone in water. the pharmacokinetic curves for egcg and the egcg cocrystals are presented in figure 10. each cocrystal is shown separately (solid line) against the egcg control (dashed line). egcg peaked rapidly at 7.50 2.74 min (tmax) due to its high solubility and consequent high rate of dissolution. the cocrystals peaked in the plasma in the following order : egcgnic9h2o, egcginm5h2o and egcgina3h2o, egcgina. surprisingly, only two of the cocrystal forms resulted in improved bioavailability : egcgina3h2o and egcgina. these cocrystals had modest improvements in relative bioavailability at frel of 1.37 and 1.05, respectively. egcg peaked more rapidly than all of the cocrystal forms and exhibited the shortest apparent terminal half - life, 249.35 60.73 min. this was most apparent with the egcgina3h2o and egcgina cocrystals, which exhibited drastic changes in the time to reach maximal plasma concentrations (tmax) at 30.00 24.49 and 160 61.97 min, respectively. in some cases, these pharmacokinetic profiles might be advantageous to the rapid peak and elimination profile of free egcg. < 0.05. due to an insufficient number of data points in the elimination phase of the pharmacokinetic profile, egcg research increased drastically when researchers reported that epidemiological evidence indicated lower rates of certain cancers in populations that consumed the most green tea. since then, numerous molecular mechanisms for these beneficial properties have been discovered. not surprisingly, egcg has become one of the most popular nutraceutical ingredients in the world. egcg is highly susceptible to oxidation, first pass metabolism, and rapid efflux. numerous groups have evaluated delivery systems to overcome these limitations with egcg, but none have tried cocrystallization. cocrystallization has been used extensively to improve the solubility and/or bioavailability of poorly soluble apis. this study is the first report on the bioavailability effects of reducing the solubility of a highly soluble api. we evaluated the structure, solubility, and pharmacokinetics of four new cocrystals of egcg, all of which were found to exhibit reduced water solubility. we hypothesized that these changes in solubility would change the pharmacokinetics of egcg, perhaps improving its bioavailability. when interpreting our data, it is important to note that we used a nonconventional gavage vehicle (corn oil) to prevent presolubilizing of the sieved crystalline forms. our egcg control peaked approximately 16.5 min more rapidly than previously published pharmacokinetic data at the same dose in rats using a saline gavage vehicle. furthermore, we previously assessed the pharmacokinetics of an egcg nanolipidic particle suspension in 10% ethanol. for an accurate comparison this resulted in lower levels of free egcg measured in the plasma (cmax = 116.57 ng / ml). nonetheless, the results presented in this paper should be considered a stand - alone comparison of egcg and egcg cocrystals in a common vehicle. at first glance, it seems that cocrystallization might not be the most useful method for improving the bioavailability of egcg, since only two cocrystals exhibited improved relative bioavailability and the increases were very modest (table 3). although we did not monitor the metabolic profile of the circulating egcg, it is likely that the blunted bioavailability increases were due to the well - known metabolism issues with egcg, which is left unchecked in these cocrystal forms. an additional contributor to the observed relative bioavailability for this experiment is apparent when considering the variability for the egcg control (table 3). one of the experimental rats achieved three times higher plasma levels of free egcg than observed in the other rats. however, if this animal had been excluded as an outlier, the relative bioavailability for the cocrystal forms would have increased. this decision was driven by our desire to minimize the use of animals and based on previous pharmacokinetic studies performed in our lab. however, the pharmacokinetic study suffered from unexpected levels of variability, which should be considered when drawing conclusions from this data. the pharmacokinetic parameters presented in table 3 include the means with standard deviations. for the egcgina, an asterisk marks the hl_lambda_z, because there were insufficient data points in the elimination phase such that the cmax had to be included in the noncompartmental analysis in winnonlin. this only occurred with the egcgina cocrystal because we did not expect such a robust change in the tmax. even though our new forms of egcg did not lead to large increases in oral bioavailability, this does not mean that cocrystallization can not benefit the commercialization of egcg. the ability to have multiple solid forms of egcg with different physicochemical properties and pharmacokinetic profiles could be very useful. further examination of figure 10 indicates that some of the egcg cocrystals exhibited different pharmacokinetic profiles. the egcgina3h2o and egcgina cocrystals not only had the highest relative bioavailabilities of the cocrystals that we evaluated but also resulted in higher concentrations of free egcg after 60 and 120 min, respectively. this plateau effect could be useful for achieving more sustained levels of egcg in the blood. furthermore, because pharmaceutical and nutraceutical products often contain combinations of ingredients with synergistic effects, cocrystallization could be used to select forms with similar pharmacokinetics as the other ingredients. our results generally support our hypothesis that reducing the solubility reduces the rate of dissolution and delays the time at which maximal plasma levels are reached (tmax). egcgnic9h2o was found to be the most water - soluble cocrystal form and had the most rapid tmax after free egcg. egcginm5h2o and egcgina3h2o however, egcgina peaked at 160 min (table 3) and was found to exhibit similar solubility to that of egcginm5h2o and egcgina3h2o (figure 9). normally, it might be reasonable to assume that other pharmacokinetic parameters are being changed, like elimination rate or metabolism. however, this can not be the case for egcgina and egcgina3h2o since they have the same ccf. as expected, the hydrate exhibited slightly lower water solubility and peaked later in comparison to the other more soluble egcg forms. because anhydrous and hydrous forms were evaluated, we were able to conclude that the presence of ina does not explain the discrepancy observed with the egcgina cocrystal. nonetheless, it is very clear that cocrystallization can be used to generate novel forms of egcg with modulated dissolution and pharmacokinetic profiles (see also supporting information). in conclusion, the present study demonstrates how crystal engineering concepts can be utilized for isolating new cocrystals of egcg and that with the aid of several crystallization techniques we were able isolate the first pure (i.e., nonsolvated, nonhydrated) crystal form of egcg. however, cocrystallization might not be the best option for improving the bioavailability of bcs 3 compounds like egcg, but it could be very useful when absorption kinetics is critical such as it is in synergistic and/or combination products. for example, selection of a cocrystal form that exhibits a dissolution rate - limited pharmacokinetic profile could achieve sustained plasma levels of free active ingredient similar to other sustained release preformulation techniques like transdermal delivery. sustained plasma levels are preferred so that the therapeutic bioactivity of the active ingredient is maintained for a longer duration. even though transdermal delivery can also accomplish sustained plasma levels, it has numerous disadvantages to oral formulation such as complicated production, increased cost, and reduced patient compliance due to common side effects like skin irritation at the adhesion site. this study therefore lends credence to the concept of using cocrystallization to modulate the pharmacokinetics of highly water - soluble compounds. in this study, we evaluated four new solid forms of egcg with different solubility properties and consequent pharmacokinetic profiles. although we confirmed that a single cocrystal with reduced solubility relative to a highly soluble pure form is unlikely to produce improved bioavailability, the ability to modulate the pharmacokinetic curve using crystal engineering could be very useful in the clinical translation of active ingredients. in theory | the most abundant polyphenol in green tea, epigallocatechin-3-gallate (egcg), has recently received considerable attention due to the discovery of numerous health - promoting bioactivities. despite reports of its poor oral bioavailability, egcg has been included in many dietary supplement formulations. conventional preformulation methods have been employed to improve the bioavailability of egcg. however, these methods have limitations that hinder the development of egcg as an effective therapeutic agent. in this study, we have utilized the basic concepts of crystal engineering and several crystallization techniques to screen for various solid crystalline forms of egcg and evaluated the efficacy of crystal engineering for modulating the pharmacokinetics of egcg. we synthesized and characterized seven previously undescribed crystal forms of egcg including the pure crystal structure of egcg. the aqueous solubility profiles of four new egcg cocrystals were determined. these cocrystals were subsequently dosed at 100 mg egcg per kg body weight in rats, and the plasma levels were monitored over the course of eight hours following the single oral dose. two of the egcg cocrystals were found to exhibit modest improvements in relative bioavailability. further, cocrystallization resulted in marked effects on pharmacokinetic parameters including cmax, tmax, area under curve, relative bioavailability, and apparent terminal half - life. our findings suggest that modulation of the pharmacokinetic profile of egcg is possible using cocrystallization and that it offers certain opportunities that could be useful during its development as a therapeutic agent. |
ancient infection of germline cells with exogenous retroviruses established a genome - wide random embedment of proviruses, called endogenous retroviruses (ervs), and mendelian genetics governs their inheritance to the offsprings. ervs are reported to exist in the genome of all vertebrates and constitute approximately 8% of the human genome and 10% of the mouse genome [24 ]. the majority of ervs identified so far are reported to be defective primarily based on their inability to encode intact polypeptides for gag (group specific antigen), pol (reverse transcriptase), and env (envelope) genes, which are essential for the retroviral life cycle. however, recent studies identified a number of ervs, which retain intact coding potentials for gag, pol, and/or env genes, and some of them are reported to be associated with a range of normal physiology (e.g., placental morphogenesis) as well as pathogenic processes (e.g., multiple sclerosis, schizophrenia, injury, and chronic fatigue syndrome) [610 ]. on the other hand, biology of porcine ervs (pervs) has been studied extensively because of the potential transmission of pervs to humans as an adverse side effect of xenotransplantation. the env glycoproteins of certain human ervs (hervs) have been implicated in diverse disease processes [1216 ]. for instance, the env glycoproteins of herv - k, herv - e, and erv-3 were characterized as tumor - associated antigens in different types of cancer [1518 ]. the herv - w env glycoprotein, called syncytin-1, is highly expressed in glial cells within central nervous system of multiple sclerosis, an autoimmune disease, patients. it is proposed that potent proinflammatory properties of syncytin-1 contribute to neuronal inflammation and resultant damage to oligodendrocytes during the progression of multiple sclerosis. on the other hand, syncytin-1 and herv - frd env glycoprotein, called syncytin-2, are reported to play an essential role during embryonic development by controlling formation of placental syncytiotrophoblasts primarily through their highly fusogenic properties [1922 ]. additional env glycoproteins have been identified and characterized from murine ervs (syncytin - a and syncytin - b) and endogenous jaagsiekte sheep retrovirus (enjsrv), and their roles in placenta morphogenesis are similar to syncytin-1 and syncytin-2 [7, 23, 24 ]. the findings from recent studies provide evidence suggesting that env glycoproteins of certain ervs play a critical role in biological processes of normal physiology as well as diseases. during a survey of expression profile of mulv - erv subgenomic env transcripts in various normal tissues of c57bl/6j mice, two putative full - length env transcripts were identified in the ovary. in this study, the biological characteristics of these two mulv - erv env genes, named envov1 and envov2, were investigated by examining a selective set of pathophysiologic parameters. female c57bl/6j mice (approximately 12 weeks old) were purchased from the jackson laboratory (bar harbor, me) and housed according to the guidelines of the national institutes of health. the animal use and care administrative advisory committee of the university of california, davis, approved the experimental protocol. three mice were sacrificed by cervical dislocation for tissue collection without any pretreatment, and tissue samples were snap - frozen. rna isolation and cdna synthesis were performed primarily according to the relevant protocols provided by the kit manufacturer. briefly, total rnas were extracted using an rneasy kit (qiagen, valencia, calif) and cdnas were synthesized using 100 ng of total rna from each sample (tissue or cell) and the sensiscript reverse transcriptase (qiagen). the primers capable of amplifying the full length as well as subgenomic mulv - erv transcripts were designed based on the maids (murine acquired immunodeficiency virus) virus - related provirus (genbank no. s80082) : forward, 5-cat ttg gag gtc cca ccg aga-3 (mv1k) and reverse, 5-ctc agt ctg tcg gag gac tg-3 (mv2d). the following are the primer sets used for inflammatory mediators : cox-2 (forward, 5-aca cag tgc act aca tcc tga c-3 and reverse, 5-atc atc tct acc tga gtg tc-3), icam-1 (forward, 5-agc tgt ttg agc tga gcg aga-3 and reverse, 5-ctg tcg aac tcc tca gtc a-3), il-1 (forward, 5-gac agt gat gag aat gac ctg-3 and reverse, 5-gaa ctc tgc aga ctc aaa ctc ca-3), il-6 (forward, 5-gcc ttc cct act tca caa gtc cg-3 and reverse, 5-cac tag gtt tgc cga gta gat ctc-3), inos (forward, 5-aca agc tgc atg tga cat cga-3 and reverse, 5-cag agc ctg aag tca tgt ttg c-3), and tnf- (forward, 5-gca tga tcc gcg acg tgg aa-3 and reverse, 5-aga tcc atg ccg ttg gcc ag-3). in addition, -actin (forward, 5-cca act ggg acg aca tgg ag-3 and reverse, 5-gta gat ggg cac agt gtg gg-3) was used as an internal expression control. the density of amplified products (applied only for inflammatory mediators) was measured using kodak molecular imaging software ver. 4.5 (carestream health, rochester, ny), and it was normalized to -actin control. the rt - pcr products of the mulv - erv subgenomic transcripts (~2.9 kb) were cloned into the pgem - t easy vector (promega, madison, wis) followed by plasmid dna preparation using a kit from qiagen, and sequencing analysis at davis sequencing inc (davis, calif) or molecular cloning laboratory (south san francisco, calif). 10 (invitrogen, carlsbad, calif) or editseq and megalign program within dnastar ver. the coding regions of the envov1 and envov2 were amplified by pcr from their respective original cdna clones using a set of primers embedded with restriction enzyme sites for cloning into the pcdna4/hismax (invitrogen) : forward with noti, 5-cgc ggc ggc cgc atg gaa ggt cca gcg ttc tc-3, envov1-reverse with xhoi, 5-ggc tcg agt tat tca cgt gat tcc act ttt tct gg-3, and envov2-reverse with xhoi, 5-ggc tcg agt tat tca cgt gat tcc act tct tct gg-3. the amplified coding sequences after 10 pcr cycles were cloned into the pgemt - easy vector (promega) followed by digestion with noti and xhoi and subsequently cloned into pcdna4/hismax (invitrogen). the gp2 - 293 packaging cells (purchased from clontech, mountain view, calif), tsa201 cells (a derivative of hek293 cells), cos-7 cells, and cos-1 cells were maintained in dulbecco 's modified eagle medium (dmem, invitrogen) supplemented with 10% fetal bovine serum, streptomycin, and penicillin g. five other cell lines (hela, neuro-2a, mdck, hct 116, and nih3t3) were cultured according to the protocols recommended by the american type culture collection (manassas, va). the gp2 - 293 cells, which were seeded onto a 6-well plate at a concentration of 5 10 cells per well, were cotransfected with pqclin (clontech, mountain view, calif) and pcdna4/hismax - envov1 or pcdna4/hismax - envov2 plasmid using lipofectamine 2000 (invitrogen). the following env proteins were used for tropism and infectivity controls : ecotropic (peco), 4070a amphotropic (pampho), 10a1 amphotropic (p10a1) with a broader host range than 4070a, and g glycoprotein of the vesicular stomatitis virus (vsv - g) (clontech). culture supernatants containing pseudotype viral particles were passed through a 0.45 m filter (fisher scientific, pittsburgh, pa). transfection efficiency was estimated by counting the stained cells under the microscope after x - gal staining. for each cell line (a total of 8 cell lines) employed for tropism and infection analysis, 5 10 cells / well were seeded onto a 24-well plate and incubated overnight in preparation of viral transduction. subsequently, the medium was replaced with 0.5 ml of serial dilutions of culture supernatants containing pseudotype lacz - mulv virions, in which polybrene (sigma, milwaukee, wis) was added (8 g / ml), followed by washing after 4 hours and incubation with 0.5 ml of fresh media for 2 days. the infected cells were treated with fixing solution (2% formaldehyde and 0.2% glutaraldehyde in pbs) and stained with x - gal solution. cells stained blue were counted under the microscope as an infection unit. to confirm expression of the pcdna4/hismax - envov1 or pcdna4/hismax - envov2 construct, western blot analysis was performed following transfection into tsa201 cells using fugene 6 reagent (roche, mannheim, germany). at 2 days after transfection briefly, the membrane, blocked in 5% nonfat dry milk (nfdm), was incubated with a goat antibody specific for gp69/71 of rauscher mulv (1 : 2000 dilution with 5% nfdm in tbst [tris - buffered saline with tween 20 ]) obtained from viromed biosafety laboratories (camden, nj) followed by an anti - goat - hrp antibody (1 : 5000, santa cruz biotechnology, santa cruz, calif). the protein signal was visualized using ecl reagents (ge healthcare, pittsburgh, pa). a similar protocol was used to detect env glycoprotein from supernatants of the gp2 - 293 cells producing pseudotype lacz - mulv virions. hela cells, which were transfected with the pcdna4/hismax - envov1 or pcdna4/hismax - envov2 construct, were harvested and transferred into 0.1% poly - l - lysine coated coverslips and incubated for 1 day. cells were then immunostained with a goat antibody specific for gp69/71 (1 : 200 diluted in culture medium, viromed biosafety laboratories) and fixed with both 4% paraformaldehyde. fixed cells were incubated with a texas - red - conjugated antigoat igg secondary antibody (1 : 200 diluted in pbs, vector laboratories, burlingame, calif) and stained cells were visualized by a zeiss microscope using axiovison software version 4.5 (carl zeiss, jena, germany). hela cells, which were transfected with the pcdna4/hismax - envov1 or pcdna4/hismax - envov2 construct, were subjected to cytotoxicity assay using a cytotoxicity detection kit (roche, south san francisco, calif) according to the protocol recommended by the manufacturer. absorbance was measured at 490 nm with a reference at 600 nm using a reader from molecular devices (sunnyvale, calif). cell proliferation rate was measured from these cells using the colorimetric mtt (3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (sigma, milwaukee, wis) assay as described previously. absorbance was read at 560 nm with a reference at 600 nm using a reader (molecular devices). all experiments were performed at least in triplicate, and 4 independent experiments were repeated. raw264.7 cells, which were transfected with the pcdna4/hismax - envov1 or pcdna4/hismax - envov2 construct, were harvested at 1 day after transfection, and they were examined for expression of a set of inflammatory mediators at mrna levels by rt - pcr, and the relevant protocols and reagents are described in section 2.2 above. statistical analysis was performed using two - tailed student 's t - test and statistical significance was determined as p <.05 and p <.01. the expression profiles of mulv - erv env genes in various normal tissues (liver, lung, salivary gland, adrenal gland, brain, skin, ovary, and uterus) of c57bl/6j mice were investigated. a number of putative subgenomic transcripts with varying sizes, ranging from ~1 kb to ~5 kb, which may be generated by splicing and/or deletion, were differentially expressed in each tissue. among them were ~2.9 kb bands presumed to be amplified from full - length mulv - erv env transcripts, and their expression was evident in the ovary and uterus as well as other tissues (figure 1(a)). sequencing analysis revealed that the two 2,892 bp transcripts were env mrnas, which were generated by a single splicing using the well - characterized donor and acceptor signals. a subsequent open reading frame analysis revealed that the two full - length mulv - erv env genes, named envov1 and envov2, retain intact coding potential for env glycoproteins of 641 amino acids. while the nucleotide and polypeptide sequences of the envov2 was identical to an env gene of an polytropic murine leukemia virus (mulv)-related retroviral sequence from nfs / n mice, the envov1 has not been reported yet. prior to the functional characterization of the envov1 and envov2, they were aligned with four different reference env polypeptides displaying different host tropisms : ecotropic, xenotropic, polytropic, and modified polytropic (figure 1(b)). it turned out that both envov1 and envov2 had a higher level of sequence similarity to the polytropic / modified polytropic env polypeptides compared to the others. both the envov1 and envov2 share the identical sequence in the variable region a and proline rich region, while one amino acid residue was different in the variable region b and r peptide, respectively. to identify the putative mulv - ervs encoding the envov1 and envov2, respectively, the c57bl/6j genome sequence (build 37.1) from the national center for biotechnology information (ncbi) was surveyed with the respective env nucleotide sequences using the blast program. the putative proviruses presumed to encode the envov1 and envov2 were mapped to ideogram data of chromosome 8 and ideogram data of chromosome 18, respectively. both mulv - ervs retained the coding potential for env polypeptide, and envov2 also had the coding potential for pol polypeptide (figure 1(c)). to examine whether the envov1 and envov2 are able to produce full - length env polypeptides, they were overexpressed in a human cell line followed by western blot detection using an anti - gp69/71 (env) antibody. a protein band of ~75 kda, which was about the size of mulv - erv env polypeptides, moreover, the subcellular distribution of these env polypeptides was examined by transient transfection followed by immunocytochemistry using the same antibody used for the western blot analysis. both the envov1 and envov2 proteins were evidently expressed on the cell membrane as was expected from the retroviral env polypeptides (figure 2(b)). two relevant characteristics, tropism and infectivity, of the envov1 and envov2 polypeptides were determined using a retroviral packaging system and compared to reference env proteins with known host tropisms : ecotropic, amphotropic, and pantropic. prior to the analyses of infectivity and tropism traits, the packaging potential of the envov1 and envov2 polypeptides and release of pseudotype lacz - mulv virions were confirmed by detection of ~75 kda bands in the culture supernatants collected after transfection (figure 3). interestingly, a markedly higher level of env protein was detected in the supernatants presumed to contain envov2-packaged pseudotype virions compared to the envov1 samples. this finding may suggest that the envov2 polypeptide is more efficiently produced and/or packaged during the course of virion assembly compared to the envov1. the infectivity and tropism traits of the envov1 and envov2 polypeptides were then examined by infection of various cell types derived from human, nonhuman primate, mouse, and dog. it revealed that the pseudotype lacz - mulv virions packaged with either envov1 or envov2 were capable of infecting both mouse as well as nonmouse cells suggesting their polytropic tropism trait, which is consistent with the alignment data presented in figure 1 (table 1). while the pseudotype envov2-lacz - mulv virions demonstrated infectivity that is very similar to the amphotropic and pantropic controls, the envov1-lacz - mulv virions had substantially low infection titers compared to the controls, probably due to low expression level and/or inefficient packaging potential during virion assembly. in this experiment, the cytopathic effects of the envov1 and envov2 polypeptides were examined by overexpression followed by measurement of cytotoxicity and inhibition of cell proliferation. cytotoxic property of the envov2 polypeptide was clearly demonstrated by both colorimetric quantitative assay and microscopic examination of morphological characteristics, including adherence to culture plate (figures 4(a) and 4(b)). in contrast, no significant cytotoxic effects were observed in the cells overexpressed with the envov1 polypeptide compared to negative controls. on the other hand, the overexpression of the envov2 polypeptide, but not envov1 polypeptide, evidently inhibited cell proliferation, which was measured by colorimetric quantitation of cell growth (figure 4(c)). it is likely that inhibition of cell proliferation by the envov2 polypeptide is linked to its cytotoxic effect, and it is unclear how its high infection titer correlates with the cytopathic characteristics. to investigate whether the envov1 and envov2 play a role in inflammation, changes in mrna expression of a set of inflammatory mediators the set include proinflammatory mediators of cox-2 (cyclooxygenase-2), icam-1 (intercellular adhesion molecule 1), inos (inducible nitric oxide synthase), il-1, il-6, and tnf-. the expression of four proinflammatory mediators, cox-2, inos, il-1, and il-6, was significantly increased by overexpression of envov2 polypeptide but not envov1 (figures 5(a) and 5(b)). in contrast, no significant changes in icam-1 and tnf- levels were detected following the overexpression of either the envov1 or the envov2 polypeptide. the findings from this study suggest that the envov1 and envov2 polypeptides differentially participate in certain signaling events controlling the production of inflammatory mediators. two mulv - erv env genes with intact coding potential, named envov1 and envov2, were isolated from the ovary of normal c57bl/6j mice and their biological properties were characterized. although the sequence of one (envov2) of the two env genes has been reported previously, its biological functions have not been characterized. the findings from this study suggest that both the envov1 and envov2 polypeptides, which were determined to confer polytropic tropism, participate in a range of biological processes, such as retroviral packaging, cell death, proliferation, and inflammation. the results from this study suggest that putative mulv - ervs, or unidentified exogenous retroviruses, which are packaged with either envov1 or envov2 polypeptide, are capable of infecting cells of mice as well as other species, such as humans, nonhuman primates, and dogs. de novo as well as stress - related activation of the mulv - ervs, which are packaged with these env polypeptides, may be followed by infection of specific cells of local as well as distant. in addition to the potential cytopathic effects examined in this study, the genomic random integration of the proviral dnas may be directly linked to various pathogenic outcomes following infection. further in vivo studies are needed to determine infectivity of the mulv - ervs packaged with these env polypeptides in mice as well as other species. ervs have been associated with a range of diseases, such as sepsis, multiple sclerosis, and injury whose central pathology includes inflammatory conditions [12, 3337 ]. some reports provided evidence that certain erv env gene products, but not the relevant virus particles, play a role in the inflammatory processes associated with various pathologic phenotypes [38, 39 ]. the herv - w syncytin-1 exerted its inflammatory effects by induction of proinflammatory mediators, such as il-1, il-6, il-12, inos, and tnf-, leading to neuron inflammation in multiple sclerosis patients [12, 40 ]. the findings from this study that the envov2 polypeptide is capable of modulating inflammatory mediators suggest its potential roles in immunologic homeostasis as well as in various diseases involving inflammatory conditions, such as sepsis [41, 42 ]. a markedly higher level of packaging and subsequent release of pseudotype lacz - mulv virions was predicted with the envov2 compared to the envov1, based on the detection of abundant env polypeptide in the culture supernatants of envov2 samples. it is consistent with the finding that the envov2-packaged virions (pseudotype envov2-lacz - mulvs) had higher infection titers compared to the envov1-packaged virions (pseudotype envov1-lacz - mulvs). it is possible that the putative high packaging rate with the envov2 polypeptide is directly linked to its efficient transcription and/or translation as well as stability. on the other hand, abundant presence of the envov2 polypeptide in the cytoplasm may explain, at least in part, the characteristics of cytotoxicity and inhibition of proliferation compared to the envov1. throughout the entire coding sequences, nine amino acid residues (v22i, r24 g, r158 g, q161r, r362 g, g518r, g528r, d608, and k640e) were different between the envov1 and envov2 polypeptides. further investigation may be needed to learn the roles of these polymorphic residues in stability as well as pathogenic characteristics, including infectivity, of the envov1 and envov2 polypeptides. the findings from this study indicate that certain mulv - erv env polypeptides, such as envov2, may participate in a range of pathophysiologic processes as an envelope of mulv - erv virions and/or independently. | envelope (env) proteins of certain endogenous retroviruses (ervs) participate in various pathophysiological processes. in this study, we characterized pathophysiologic properties of two murine leukemia virus - type erv (mulv - erv) env genes cloned from the ovary of c57bl/6j mice. the two env genes (named envov1 and envov2), with 1,926 bp coding region, originated from two mulv - erv loci on chromosomes 8 and 18, respectively. envov1 and envov2 were ~75 kda and predominantly expressed on the cell membrane. they were capable of producing pseudotype murine leukemia virus virions. tropism trait and infectivity of envov2 were similar to the polytropic env ; however, envov1 had very low level of infectivity. overexpression of envov2, but not envov1, exerted cytotoxic effects and induced expression of cox-2, il-1, il-6, and inos. these findings suggest that the envov1 and envov2 are capable of serving as an env protein for virion assembly, and they exert differential cytotoxicity and modulation of inflammatory mediators. |
fcs was used to determine the relative affinity of -syn100 for large unilamellar vesicles (luvs) composed of 100% popg or 1:1 (mol / mol) pg : pc under dilute conditions. binding of fluorescently labeled protein to unlabeled vesicles results in a shift in the autocorrelation curves to the right (longer diffusion times) (figure 1a) ; the autocorrelation curves can then be fit to extract the fraction of bound protein and to determine an apparent binding affinity, kd (see materials and methods in the si for details). these measurements found that -syn100 binds to 100% popg vesicles with 60 times greater affinity than to 1:1 pg : pc vesicles (kd = 2.25 and 136.9 m, respectively) under our buffer conditions. this result agrees with previous studies showing that -syn100 binding to lipids is driven primarily by electrostatic interactions between anionic lipid headgroups and positively charged lysine residues in the membrane - binding region of the protein, although the complex roles of hydrophobic lipid protein interactions and entropy can not be ruled out. (a) fcs traces for -syn100 in the absence (black) or presence of equal concentrations of 1:1 pg : pc (blue) or 100% popg (red) luvs. the greater shift to the right in the 100% popg curve reflects a larger fraction of -syn100 bound relative to the 1:1 pg : pc curve. (b) -syn100 tubulation capacities for 1:1 pg : pc and 100% popg at equal bound density, compared with buffer. the inset shows the corresponding absorbance traces for systems with 1:1 pg : pc or 100% popg and buffer or -syn100. in order to test the effect of headgroup charge on -syn100-induced tubulation at equal bound - protein density, we adjusted the added protein concentration on the basis of the measured kd value. again, because of our previous measurements under saturating conditions, this ensured equal density of bound protein. the ability of -syn100 to tubulate liposomes was assayed by monitoring the change in the amount of scattered light from a liposome solution in the presence of -syn100. we quantified -syn100 s tubulation capacity by determining the ratio of the initial scattering intensity before addition of protein to the near - final scattering intensity (t = 2400 to 2500 s) for each absorbance trace. figure 1b shows the dramatic loss of -syn100-induced tubulation in the 1:1 pg : pc mixture compared with the 100% popg vesicles. while -syn100 causes a rapid decrease in the amount of light scattered by 100% popg vesicles (figure 1b inset), the signal change for the 1:1 pg : pc vesicles is equivalent to that of the control. thus, we have confirmed that previous reports of reduced tubulation in 1:1 mixtures hold under conditions of equally bound protein. as we will explore in depth below, this does not necessarily mean that the difference in binding energy does not dominate the biophysics of tubulation. in order to begin to understand the remodeling phenomena that take place in 100% popg time - averaged height surfaces, h(x, y), reflect the spontaneous curvature of a system by removing long - wavelength temporal fluctuations of the membrane. h(x, y) was determined using the method of our previous -syn study (see the si). figure 2a presents h(x, y) for -syn100 bound to pure popg and 1:1 pg : pc bilayers determined over the last 5 s of the simulations. we quantified h(x, y) by determining the percent excess area for each system as (1 ah(x, y)/axy) 100%, where ah(x, y) is the area along the height surface and axy is the projected area (see the si). there is a significant increase in the percent excess area (figure 2b, blue) in the popg system (0.51%) compared with the 1:1 mixture (0.30%) (see supplemental table 1). on a per - protein basis, the magnitude of the induced spontaneous curvature appears to be small on the scale of global membrane remodeling. however, the cumulative effect imparted by multiple proteins organizing in a localized membrane region could induce enough curvature stress to breach the energy threshold required for membrane remodeling. as will be discussed below, recent work by the voth group shed light on an important phenomenon where linear aggregation of n - bar domain proteins occurs prior to macroscopic membrane remodeling. a similar mechanism for protein aggregation / alignment may occur for -syn100, acting as local nucleation points for tubule formation. an additional set of simulations presented below will expand on this point. (a) time - averaged height surfaces, h(x, y), for 1:1 pg : pc (left) and popg (right), determined over the last 5 s of the simulations (b) percent excess area per protein (blue) and integrated total lipid order (green) for 1:1 pg : pc and popg. pg : pc (top) and popg (bottom) (solvent, gray ; headgroup, cyan ; carbonyl - glycerol, green ; acyl - chain, magenta ; -syn100, black line). (d) local total order parameter sz(x, y) for the membrane near the protein (top) and opposite the protein (bottom) in 1:1 pg : pc (left) and popg (right) (warm colors = more ordered, cool colors = more disordered). the insets correspond to pure lipid sz(x, y) for each lipid composition. the location of the n - terminus of -syn100 is indicated with (the protein itself is not shown). it has been proposed that subtle changes in the depth of partitioning of an ah into the hydrophobic acyl - chain region can dramatically alter the induced (local) curvature and that this curvature is dependent on the membrane s hydrophobic thickness. figure 2c shows that -syn100 partitions slightly deeper in the pg : pc mixture when the depth is measured relative to the lipid component density (zpep) (results for all of the other 1600:1 -syn100 systems are presented in supplemental figure 2). however, zpep is not the most relevant measure of partition depth in the context of curvature induction. we define the hydrophobic thickness of the membrane, 2dc, as the z distance between the points with equal probability for acyl - chain or solvent densities. there are two structural parameters that dictate the magnitude and sign of the induced spontaneous curvature : (1) the monolayer hydrophobic thickness dc and (2) the extent to which the protein partitions into dc. interestingly, for a given protein density (1600:1 or 400:1), regardless of the pc mole percent, the protein partitions to the same depth relative to dc (see supplemental table 1). however, what does change with pc mole percent is the hydrophobic thickness (both in pure and protein systems), which increases by 0.16 nm in popg relative to the 1:1 pg : pc mixture (see supplemental table 1). although this shift is quite small below the resolution of the martini cg beads current theory on ah curvature suggests that with equal relative partition depth, changes in hydrophobic thickness of 0.2 nm are capable of doubling the spontaneous curvature. thus, these data suggest a strong correlation between hydrophobic thickness and curvature induction. a shift in the membrane s hydrophobic thickness modulates how other structural features of the membrane respond to -syn100 s relative partition depth, particularly the acyl - chain order parameter. we characterized the acyl - chain conformations around -syn100 using a local total lipid order parameter, sz(x, y) (see the si for details of the method). figure 2d illustrates sz(x, y) for the region of the monolayer near the protein (top) and opposite the protein (bottom) for the systems with 1:1 pg : pc (left) and popg (right), with the corresponding data for protein - free bilayers given in the insets (see supplemental figures 2 and 3 for complete results for other 1600:1 -syn100 systems). the lipids near the protein are equally disordered for both systems, but the change relative to the bulk is much greater for popg. when we quantified sz(x, y) as a function of distance from the protein, we observed an asymmetry across the bilayer, sz = sz, opposite sz, protein. this asymmetry extends out to distances of 5 nm (supplemental figure 3e, f), with the largest asymmetry developing in the popg system. when only the first shell of lipids is considered (by integration out to 1 nm from the protein), the order asymmetry shows a strong correlation with the excess area, where the popg system again experiences twice the effect relative to the 1:1 mixture (figure 2b, green bars). we speculate that because of the increased dc in popg, there is a greater volume beneath the protein that must be accommodated by the neighboring lipid s acyl chains, leading to more disordering of the lipids and giving rise to increased order asymmetry. the simulation data comparing -syn100 in 100% pg and the 1:1 pg : pc mixture suggest, but in no way prove, a direct correlation between the depth of partition relative to the hydrophobic thickness, the order parameter asymmetry across the leaflets, the induced positive curvature, and tubulation. however, this correlation does not take into account the difference between the binding affinities for 100% popg and the 1:1 pg : pc mixture. one explanation for the role of the affinity difference relates to the electrostatic repulsion between pg headgroups, which is lessened in the mixture. as a result, the 100% popg membrane is under greater lateral pressure than that of the mixture. binding of -syn can relieve this pressure by screening lipid lipid interactions through lys although current theory focuses on the importance of protein partition depth relative to hydrophobic thickness, it is possible that the protein inserts at the optimal zpep position (figure 2c) for tubulation in the 100% pg bilayer. parsing the relative contributions of these driving forces binding energy, partition depth, hydrophobic thickness, and order perturbations is far from trivial. in an effort to isolate the binding - energy component, we engineered a minimally altered variant of -syn100 that would partition to the same depth in pure popg bilayers (maintaining a constant dc and local curvature induction) but have a reduced kd. s membrane binding domain comprises seven imperfect heptad repeats with consensus sequence xktkegvxxxx (x = any residue). we replaced the hydrophobic nac domain (the sixth heptad) with a replicate of the fifth heptad (gavvtgvtava ektkeqvtnvg). the anticipated effect on kd and the depth was uncertain, as the alteration reduces the hydrophobicity while adding extra charged residues (zero change in net charge). because lys residues in -syn associate strongly with pg headgroups, we suggest that any reduction in measured binding affinity should be attributed to the loss in hydrophobicity. we first tested the depth of partition and the order parameter asymmetry of this nac - null protein using simulations, and figure 3a shows that indeed the engineered variant has nearly identical binding depth and order parameter asymmetry as -syn100. the nac - null variant actually partitions slightly less deeply, and this is manifested in a slightly greater curvature field (supplemental table 1 and supplemental figure 4a c). experimentally, we found that the binding affinity of nac - null was reduced 6-fold, which is considerably less of an effect than the lipid headgroup charge with the native -syn100 sequence but significant nonetheless. we also found that at equal bound protein density, the nac - null variant induced an approximately 50% reduction in tubulation (figure 3b and supplemental figure 4d), also a smaller but significant effect. thus, on the basis of the similar partition depths and order asymmetry along with the reduced kd, the results for the engineered nac - null construct strongly suggest that while there may be a range of partition depths over which tubulation is possible, the binding energy is a major player in dictating whether tubulation occurs. (a) comparison of protein partition depths and integrated order parameter asymmetries for -syn100 (left) and nac - null (right) proteins. (b) experimental tubulation capacities at equal bound protein density for popg + buffer (black), popg + -syn100 (blue), and popg + nac - null (red). (c) comparison of excess areas per protein for the low - density (1600:1, blue) and high - density (400:1, green) systems for -syn100 (left) and nac - null (middle) in popg and -syn100 in 1:3 pg : pc (right). (d) time - averaged height surfaces for high - density (400:1) -syn100 (left) and nac - null (middle) systems in popg and -syn100 in 1:3 pg : pc (right). the average protein position is indicated with white spheres, and the n - terminus of the protein is indicated with. because the nac - null variant shows decreased affinity relative to -syn100 in popg despite the presence of extra lys residues, it is likely that binding of the native protein (and possibly the stability of the bound protein furthermore, given the 60-fold reduction in affinity of -syn100 for 1:1 pg : pc versus popg, an effect that has previously been shown to be electrostatically driven these findings strongly suggest at least a two - stage binding process : (1) electrostatically driven adsorption of the unfolded protein and (2) a combination of electrostatic and hydrophobic stabilization of the -helical bound state. we note that whereas our experiments capture the full - binding process, the simulations probe only the second stage. binding energy not only dictates the equilibrium distribution of bound and unbound protein but also reflects the strength (stability) of the interaction between the protein and the lipids in its solvation shell. a tighter coupling be it between charged (lys / pg) or hydrophobic groups should be manifested as a larger, more stable complex whose diffusion in the membrane will be slowed by size. this may increase the likelihood of stable, nucleating assemblies of proteins. in an attempt to more deeply understand the binding energy difference that dictates the reduced tubulation in the nac - null variant, we simulated the two proteins (-syn100 and nac - null) at high density on pure pg bilayers (for details, see materials and methods in the si). figure 3d shows the calculated curvature fields for -syn100 (left) and nac - null (middle). the -syn100 system shows a broad area of positive spontaneous curvature spanning well beyond the local region of a single protein (it should be noted that these images show a larger region of membrane than those in figure 2a). protein alignment that reinforces the curvature fields between proteins. even though nac - null induces a similar low - density curvature field and has the same depth and same order parameter asymmetry (supplemental figure 4), at high density it does not display the same curvature - field reinforcement as -syn100 (figure 3d, middle). quantification of these surfaces shows a 50% decrease in curvature capacity for high - density nac - null (figure 3c), matching the experimentally observed decrease in tubulation (figure 3b) remarkably well. figure 3c shows that in the case of the native -syn100 sequence, the high - density per - protein curvature field recovers that of the low - density (single - protein) case. this recovery is absent in the case of nac - null, suggesting a loss of time - averaged helical alignment compared with the native sequence. we quantified this effect by time - averaged distance matrices for amino acids in nearest - neighbor protein protein pairs (supplemental figure 5). by observing the temporal stability within these distance matrices (or lack thereof), we asked the following question : does the stability of specific protein protein alignment motifs (which may correlate with binding energy) correlate with stabilization of long - range curvature fields ? despite limited sampling, the data are consistent with the notion that the reinforcement of the curvature fields in -syn100 correlates with stabilization of protein protein alignments. as hypothesized, in the case of -syn100 the proteins sample a relatively tight window of possible alignment states (25.2% unique states sampled). most interestingly, the nac - null results show a marked increase in the transitions between states (50.4%). the most recent theory for ah - induced curvature induction suggests a small range of depths over which positive curvature will be induced for a given hydrophobic thickness. as proteins partition more deeply into the bilayer, the effect is lost and can even be reversed to produce negative curvature. the data presented thus far do not directly address this because relative to dc the simulated proteins all partitioned to the same depth. in a purely computational experiment, we manipulated this partitioning by artificially varying the charge of -syn100 (via computational point mutations) within the same lipid mixture (i.e., the same dc). indeed, the results confirmed a high sensitivity of the curvature to subtle changes in depth. for example, we showed the ability to turn a 100% pc bilayer with -syn100 (deep partitioning, small dc, low curvature) into a pg - like bilayer (shallow partitioning, small dc, high curvature). while -syn100 does not bind pure pc bilayers in the fluid phase, this computational exercise is valuable in the context of understanding the driving forces for curvature induction. context, interpretation of the lost tubulation in the pg : pc mixture, where the protein depth relative to dc is invariant, is complicated by the concomitant decreases in curvature and affinity. in order to exaggerate the relationship among depth, hydrophobicity, and curvature, we simulated a 400:1 system in a 1:3 pg : pc mixture. in this mixture, the protein again partitions to the same position relative to dc as in popg. surprisingly, when we calculated the distance matrix for interacting proteins in pg : pc mixtures, we found similar (or even reduced) mobility as in the wild type (supplemental figure 5d). similar to the 400:1 popg system, this reduced mobility is accompanied by a reinforcement of local curvature fields (figure 3c, d), although the stabilized curvature field is less than half as intense. we can explain the reduced curvature intensity as a result of a 0.14 nm reduction in dc in going from the 400:1 popg system to the 1:3 pg : pc system. however, in view of the dramatically reduced binding affinity that we measured experimentally, along with the observation of increased mobility of nac - null, the reduced mobility of the protein was perplexing. however, in the context of a two - stage binding process and the fact that our experiments were performed at equal bound protein density, interpretation of the data becomes possible. the loss in binding affinity likely reflects a loss in electrostatic attraction between the unfolded, soluble protein and the lipid headgroups. however, once adsorbed, the folded and bound protein is stabilized by a solvating lipid shell, primarily through lys pg contacts. in 100% pg, the lipids are free to bind and unbind the protein without great penalty, as the lys groups can be immediately stabilized by another pg. on the other hand, in the pg : pc mixture, lipid exchange is likely slowed by the penalty of replacing a lys pg contact with a lys pc contact. thus, lipid diffusion (binding / unbinding) could be expected to be slowed in the pg : pc mixture. on average this may slow protein diffusion because the time - averaged lipid protein complex will be more stable. our simulations are not sampled well enough to test this long - time - scale phenomenon with statistical certainty. figure 4 summarizes much of the relevant data presented thus far. as stated above, there is a systematic increase in hydrophobic thickness of native -syn100-containing bilayers as the pg density increases (figure 4, black dashed line). as the hydrophobic thickness decreases from 2.0 to 1.9 nm, there is reduced lipid order asymmetry that correlates with a reduction in curvature and is consistent with loss of tubulation capacity. when we incorporate the data for the nac - null systems, in particular the result at high density, this correlation fails. the nac - null protein partitions less shallow than -syn100, inducing an even greater hydrophobic thickness and lipid order asymmetry (curvature) (figure 4, black) ; experimentally, however, nac - null has a reduced tubulation capacity. for these reasons, we conclude that lipid order asymmetry (lost in the native sequence at 1:3 pg : pc) is necessary but not sufficient (present in nac - null) for tubulation. colors demarcate low - density (1600:1, black) vs high - density (400:1, blue). figure 4 also reiterates the point that at high density (400:1), pg : nac - null and 1:3 pg : pc + -syn100 induce approximately the same amount of per - protein curvature. this shows that these induced curvature fields alone do not directly correlate with tubulation, as the nac - null variant does tubulate vesicles (albeit 50% less than pure pg). the data suggest that even if a protein partitions to the appropriate depth in a membrane with sufficient hydrophobic thickness (and therefore imparts sufficient lipid order asymmetry), the tubulation capacity is reduced if stable protein lipid complex interactions are not established (as in nac - null). however, in the 400:1 1:3 pg : pc + -syn100 system, stable protein lipid complex interactions are formed, as evidenced by the stabilized curvature field (figure 3d) and the reduced protein mobility (supplemental figure 5d), yet reduced lipid order asymmetry reduces the stress on the membrane below the tubulation threshold. therefore, we conclude that stability of the protein in the bound state (lost in nac - null) is a necessary but not sufficient (present in the native sequence at 1:3 pg : pc) component of the tubulation mechanism. thus, it appears that two constraints must be met in order for -syn100 to tubulate a vesicle : (1) it must bind with sufficient energy to slow the protein / lipid dynamics and allow for nucleation of pretubule assemblies and (2) it must bind in a narrow window of depths, inducing a particular hydrophobic thickness that can promote sufficient per - protein curvature. furthermore, because the nac - null variant still maintains some tubulation activity, there must be a driving force present in the nac - null and popg -syn100 systems but not in 1:3 pg : pc, one that is not dependent on strong binding affinity and drives the formation of a tubule. we propose that this driving force is the energetic penalty of induced lipid order asymmetry across the leaflets. for -syn100 to induce tubulation of a large vesicle we speculate that there exists a reinforced / nucleating -syn100 structure with high order asymmetry and that resolution of this asymmetry may drive tubulation. no experimental information is available to suggest that such structures exist or to provide hints concerning what those structures might look like. in a modest effort to gain insight into how large - scale assemblies may promote tubulation, we ran three simulations with hand - built assemblies (consisting of 48 proteins) embedded in a popg membrane. we recognize that these tubule simulations sample only three possible nucleating structures and acknowledge that other computational techniques (including more aggressive coarse graining and mesoscopic modeling) may be better - suited for such investigations. nonetheless, this approach has allowed us to investigate the possible behaviors of acyl chains in the remodeling process. each system contained 85 296 lipids (5 200 608 total cg beads) and 48 proteins arranged in one of three unique conformations (spoke, circumferential, and carpet). the local protein density for each system (defined as the protein : lipid ratio within 1 nm of the protein) was set at 1:50, just below the experimental saturation density observed with anionic and zwitterionic / anionic lipid mixtures. figure 5 displays the initial starting configuration and the final snapshot for the spoke conformation. for all three protein conformations, membrane remodeling occurred very rapidly. during the first 40 ns, an initial invagination spanned the central ring of the membrane encompassing the inner 5 nm of the protein spokes. by 100 ns, by 300 ns there was a fully formed nascent tubule (25 nm in height) surrounded by undulating popg bilayer. it is possible that if these simulations were allowed to run for much longer times, the highly ordered protein conformation might diffuse apart. while we can not rule this out, we do note that after 300 ns the carpet conformation began to realign into radial (spoke) and circumferential orientations to orient -syn to the tubule s curvature field (supplemental figure 9d). the system includes 48 -syn100 proteins (yellow) and 85 296 popg lipids (blue). the n - terminus of each protein is indicated by. (b) snapshot at 300 ns simulation time. figure 6a shows the total lipid order parameter sz(x, y) for the protein monolayer (top) and the opposite monolayer (bottom) calculated over the first 20 ns (left) and the last 20 ns (right) of the simulation of the spoke conformation (results for the circumferential and carpet conformations are presented in supplemental figures 8 and 9, respectively). in all three systems, there is a large shift in the total lipid order toward greater disorder and eventually the formation of antialigned chains [i.e., negative sz(x, y) ]. as the tubule forms, this shift in order is accompanied by a transition toward a symmetric sz(x, y) profile across the bilayer (figure 6b, c and supplemental figures 8 and 9). by the end of the tubulation event, the two monolayers have similar sz(x, y) profiles [see supplemental figure 10 for the detailed time course of sz(x, y) for the spoke conformation ]. this change is different than the changes observed for the low - density -syn100 systems (figure 2d and supplemental figure 3) where a stable asymmetry is established. because of the high protein density, the limited numbers of lipids near the protein are forced to accommodate the void volume beneath each protein, inducing increased splay away from the local normal. (a) sz(x, y) for the protein - containing leaflet (top) and opposite leaflet (bottom) at the early stage (0 to 20 ns, left) and the late tubule stage (830 to 850 ns, right). the color map reference (black) is set at sz for pure popg. (b) time course of the mean sz for the protein (black) and opposite (red) monolayers as the tubule develops. (c) time course of the average difference sz across the membrane (sz = sz, opposite sz, protein). at the core of the protein assembly, the increased splay is significant enough to drive antialignment of the acyl chains (chain orientation orthogonal to the local bilayer normal). we quantified this monolayer coupling by determining the total number of interleaflet acyl - chain contacts in regions near the protein and regions in the bulk membrane. the number of first - shell acyl - chain - to - acyl - chain interactions (inset) exhibits an 2-fold increase in the number of contacts near the protein relative to the bulk. this is a consistent trend for all three protein conformations (see supplemental figure 11). total number of interleaflet contacts (acyl - chain - to - acyl - chain) for lipids near the protein (black) vs lipids in the bulk (red) in the spoke conformation. numerous computational studies have explored protein - driven membrane remodeling. a major focus of many of these studies bar - domain proteins are a curvature - inducing class of proteins that can contain both a rigid scaffolding and an ah domain, and similar to -syn, these proteins have been shown to both sense and generate curvature. in n - bar proteins, the ah plays an essential role in curvature generation and the stability of the membrane tube by stabilizing dimer furthermore, evidence exists that the ah, and not the scaffolding domain, is responsible for the protein s curvature - sensing abilities. ah curvature induction can be so extreme as to promote membrane scission, as is the case for the enth domain of epsin and the monomeric family of synucleins (-syn, -syn, and -syn). there is a balance between the remodeling effects induced by the rigid scaffold and those driven by the ah, and elucidating the role of each mechanism remains an active area of investigation. there are several potential complementary mechanisms for -syn - induced membrane curvature and tubulation. we have shown that at equal bound density the protein has a dramatically reduced effect on tubulation of pg : pc mixtures compared with popg bilayers. we correlated this effect with an experimentally measured decrease in binding affinity (60-fold) and a simulated increase in hydrophobic thickness, partition depth, and order parameter asymmetry. this finding raised the question of whether depth and order asymmetry alone can explain tubulation differences. to at least in part address this, we designed an -syn variant lacking the nac domain (nac - null), which we predicted would have reduced affinity but partition to approximately the same depth as wild - type -syn in popg vesicles having consistent hydrophobic thickness. removal of the nac did in fact reduce binding to popg vesicles (by 1 order of magnitude). this more mild reduction in affinity correlates with a more mild reduction in tubulation, despite the fact that the nac - null mutant partitions to a slightly less shallow depth than the wild - type protein (and actually slightly increases the induced curvature field) and has the same impact on the order parameter asymmetry. this finding suggests that depth and order asymmetry alone do not explain the reduction in tubulation, though it certainly does not rule out their potential contribution in the case of the pg : pc mixture (supplemental figure 5d). rather, our simulations suggest that the nac domain may be essential in stabilizing protein lipid complexes and, in so doing, promoting organization on the bilayer surface. indeed, a very recent study supports our findings on the importance of the hydrophobic core of the nac domain in -syn - induced membrane remodeling. using supported lipid bilayers, that study shows a loss of induced membrane defects and reduced membrane - bound protein cluster size with an -syn variant lacking the hydrophobic sixth heptad. in a recent study, lipowsky discussed how the adhesion energy of an adsorbing particle can induce spontaneous membrane curvature. using n - bar as an example, the theory predicted that the remodeling capacity of the n - bar scaffolding domain is directly coupled to the adhesion energy it gains upon interacting with the membrane. if the adsorbing n - bar protein imparts sufficient adhesion energy with the membrane (i.e., greater that the bending energy required to deform the bilayer), the membrane will buckle and adopt the intrinsic curvature of the protein. recent work from the voth group has characterized the role of binding energy for exactly this n - bar / membrane system. using their recently developed hybrid cg model, the authors varied the cg n - bar binding affinity for the lipid headgroup. in doing so, they were able both to inhibit protein aggregation and macroscopic membrane remodeling (low adhesion energy) and to induce disruptions and tears within the membrane (high adhesion energy). because -syn lacks a scaffolding domain, the notion of adhesion energy must be taken in a slightly different context. instead of the adhesion energy coupling the membrane to a rigid scaffold domain, weaker binding would increase lipid exchange within the lipid solvent shell around the protein. lipid complex, potentially accelerating the protein dynamics on the surface and reducing the likelihood of nucleating stable protein assemblies. this is not to say that partition depth and hydrophobic thickness are not significant. it was surprising to us that given similar relative partition depths, such a small change in the hydrophobic thickness in the pg : pc mixture (0.16 nm thinner than pg) might correspond to a such a large experimental observable. however, the recent theory of ah curvature induction developed by may s group predicts a partition depth and hydrophobic thickness dependence on the spontaneous curvature and bending rigidity of a membrane. as the hydrophobic thickness increases, the range and magnitude of the predicted spontaneous curvature is expanded (e.g., an increase of 0.2 nm in 2dc on the order of the difference we observed between pure pg and the 1:1 mixture corresponds to a doubling of the curvature intensity for the same partition depth). all of this leads to the following question : are individual curvature fields the necessary piece, or is it organization of the fields that dominates ? perhaps it is a combination of the two. our simulation and experimental results suggest that lipid order asymmetry (either through protein partition depth or membrane hydrophobicity) and binding affinity are both necessary but not sufficient components of the ah tubulation mechanism. more generally speaking, increased spontaneous curvature is the result of an area mismatch between monolayers, and it has been shown experimentally through the use of transiently asymmetric lipid vesicles (protein - free) that an increase in area mismatch by as little as 1% is enough to initiate the macroscopic remodeling of lipid vesicles. in those experiments, the transient asymmetry relaxed with time as a result of lipid flip - flop. the rate of lipid flip - flop is typically very low (corresponding to a time scale of minutes to hours) in pure lipid vesicles. however, ahs have been shown to greatly enhance lipid flip - flop rates. in the case of -syn tubulation experiments, where a high concentration of -syn is added to solution, the relationship among binding kinetics, protein reorganization (e.g., into organized pretubule structures), the development of local curvature stresses and the resulting curvature fields, lipid flip - flop, and tubulation remains unknown. in one scenario, where binding is presumed to be much faster than tubulation, every lipid in the outer leaflet of the vesicle would be occupied in forming the solvation shell of a neighboring protein. in that case our simulations were designed to test a second scenario in which the nucleation of tubules occurs rapidly and locally. indeed, high local densities can induce curvature and recruit more proteins. in this case, accelerated local lipid flip - flop seems likely in order to relieve the local curvature strain. in order to test this, we eliminated the area mismatch between the leaflets : the small systems had protein on both leaflets, while in the large systems (protein in only one leaflet) we eliminated the excess area by removing lipids. this choice was made in order to probe whether the proteins themselves can still cause remodeling and tubulation in the case where there is time for lipid flip - flop. previous work by our group has focused on the coupling of lipid order across the bilayer leaflets, where we identified acyl - chain interdigitation as central to the propagation of order across monolayers in phase - separated bilayers. in the case of the -syn tubulation simulations presented here, we have also observed interleaflet coupling, though the character of the interdigitation is quite different. as a tubule develops, lipids in both monolayers adopt an antialigned conformation, increasing favorable chain chain interactions between the monolayers (see figure 6 and supplemental figure 11). the drive toward order parameter symmetry observed in the tubulation simulations, which is coupled to these additional contacts, may provide an important additional piece of the biophysical driving force for tubulation. in this study we have discussed -syn100-induced membrane remodeling / tubulation in the context of in vitro studies showing that -syn tubulates synthetic vesicles. in vivo, -syn has been shown to interact with both the inner and outer membranes of the mitochondria. overexpressed -syn, which is associated with parkinson s disease, induces fragmentation of mitochondria and impairs mitochondria complex 1 activity. mitochondria have 15% anionic lipids, the majority of which is cardiolipin (an anionic lipid with four acyl chains and a headgroup charge of 2). this association of -syn with mitochondria coupled with its affinity for cardiolipin lead us to predict that many of the mechanisms discussed here are at play in this pathological condition. the different structural characteristics of cardiolipin may result in a unique remodeling capacity relative to more typical anionic phospholipid mixtures (e.g., popg : popc). | we have investigated the membrane remodeling capacity of the n - terminal membrane - binding domain of -synuclein (-syn100). using fluorescence correlation spectroscopy and vesicle clearance assays, we show that -syn100 fully tubulates popg vesicles, the first demonstration that the amphipathic helix on its own is capable of this effect. we also show that at equal density of membrane - bound protein, -syn has dramatically reduced affinity for, and does not tubulate, vesicles composed of a 1:1 popg : popc mixture. coarse - grained molecular dynamics simulations suggested that the difference between the pure popg and mixture results may be attributed to differences in the protein s partition depth, the membrane s hydrophobic thickness, and disruption of acyl chain order. to explore the importance of these attributes compared with the role of the reduced binding energy, we created an -syn100 variant in which we removed the hydrophobic core of the non - amyloid component (nac) domain and tested its impact on pure popg vesicles. we observed a substantial reduction in binding affinity and tubulation, and simulations of the nac - null protein suggested that the reduced binding energy increases the protein mobility on the bilayer surface, likely impacting the protein s ability to assemble into organized pretubule structures. we also used simulations to explore a potential role for interleaflet coupling as an additional driving force for tubulation. we conclude that symmetry across the leaflets in the tubulated state maximizes the interaction energy of the two leaflets and relieves the strain induced by the hydrophobic void beneath the amphipathic helix. |
fibrillary glomerulonephritis (fgn), a rare glomerular disease was first described in 1977. this rare entity is characterized by the presence of elongated, nonbranching randomly arranged microfibrils (1030 nm) in the mesangium and/or glomerular capillary wall. ultrastructurally, the microfibrils seen in fgn are indistinguishable from amyloid fibrils however differ from amyloid by its larger size and lack of reactivity to reagents that detect amyloid including congo red. different histological patterns of glomerular injury have been described in the kidney biopsy series of fgn including mesangioproliferative, membranoproliferative, membranous, diffuse sclerosing, and rarely endocapillary proliferative and crescentic. clinically, fgn presents commonly with hypertension, proteinuria, hematuria, and renal insufficiency. a recent single - institution study of 66 cases with fgn showed a mean serum creatinine of 2.1 mg / dl at the time of kidney biopsy ; however, one - third of cases had normal range serum creatinine levels (1.2 mg / dl). rapidly progressive glomerulonephritis (rpgn) is a rare clinical syndrome that is associated with extensive crescent formation surrounding 50% of glomeruli on kidney biopsy. we present an interesting adult case of fgn who presented with renal failure and massive proteinuria secondary to crescentic glomerulonephritis. we also review the few cases of fgn presenting as rapidly progressive or advanced renal failure and crescentic glomerulonephritis that have been reported in the literature. a 66-year - old caucasian female presented to the emergency room (er) with 4-week history of intermittent abdominal pain, nausea, vomiting, and poor oral intake. nephrology consultation was called for an evaluation of increased creatinine (17.54 mg / dl) found on laboratories performed in the er. serum creatinine performed approximately 4 months prior to the er visit was within normal range (1.24 mg / dl). her medication included amlodipine, benazepril, hydrochlorothiazide, and pantoprazole. on physical examination, urinalysis was significant for 3 + proteinuria and 2550 red blood cells per high power field. initial laboratory evaluation also revealed elevated blood urea nitrogen of 119 mg / dl and low hemoglobin level of 8.7 g / dl. serological testing for hepatitis b surface antigen, hepatitis c antibody, antinuclear antibody, antineutrophilic cytoplasmic antibodies, anti - glomerular basement membrane antibody, cryoglobulins, and human immunodeficiency virus antibody were all negative. of the remaining four glomeruli that did not have crescents, one was globally sclerosed and one showed segmental mild mesangial matrix expansion. eighty percent of the tubules were intact and there was a mild degree of tubular atrophy. however, there was mild to moderate interstitial infiltrates comprising of lymphocytes and occasional eosinophils and neutrophils. immunofluorescence microscopy showed 1 + fine granular deposits of igg along the capillary loops and 1 + deposits of c3 predominately in the mesangium. there was no glomerular staining for iga, igm, c1q, kappa, or lambda light chains. structured electron - dense material was seen predominately in the mesangium with some capillary loops having subendothelial deposition [figure 1 ]. structures are randomly oriented fibrils (27,000) in view of the light microscopy findings of crescentic glomerulonephritis, the patient was initiated on intravenous pulse corticosteroid (methylprednisolone 1 g) daily for 3 days followed by daily oral prednisone (1 mg / kg) therapy. the patient was also initiated on daily oral cyclophosphamide (1.5 mg / kg) therapy awaiting results of electron microscopy. the patient continued to remain dialysis - dependent despite receiving approximately 7 weeks of combined corticosteroid and cyclophosphamide therapy. fgn is a rare glomerular disease seen in 0.51% of native kidney biopsy series. clinically, the mean age of presentation of fgn is 5560 years. the male to female ratio described in larger case series of fgn varies from 1:1.21.8 and the ratio of white americans to african americans ranges from 8.39.5:0.2 - 1. clinical features at the time of kidney biopsy commonly include hypertension, proteinuria, hematuria, and varying degree of renal insufficiency. nephrotic syndrome can be seen in one - third of the cases. although thought to be an idiopathic condition, a recent single - institution study of 66 cases with fgn found an association with malignancy in 15 (23%) patients. the presence of crescents on light microscopic examination of kidney biopsy specimens of fgn have varied from none in some large kidney biopsy series to up to 20% in other such series. however, crescentic glomerulonephritis defined by the presence of crescent affecting 50% of glomeruli has rarely been reported. nasr. reported three cases of crescentic and necrotizing glomerulonephritis in their kidney biopsy study of 66 cases of fgn. however, the clinical presentation, management, and outcomes of these three fgn cases with crescentic glomerulonephritis were not presented in this study. few case reports of c - fgn cases presenting clinically with rapidly progressive or advanced renal failure have been described in the literature. table 1 summarizes the clinical presentation, treatment, and renal outcome of these cases. clinical features, treatment and renal outcome in reported cases of crescentic fibrillary glomerulonephritis with renal failure in the literature fgn is associated with poor prognosis and 50% progress to end - stage kidney disease (eskd) within 2 years of diagnosis. optimal approach and therapy for fgn are not known. while the use of both renin - angiotensin system (ras) blockers alone or in combination with immunosuppressive agents has been reported in the literature, renal outcome to such therapeutic strategies a retrospective study reported long - term renal outcomes in 27 cases with fgn who received either ras blockers alone or in combination with immunosuppressive agents, mainly rituximab and cyclophosphamide. as compared to the group that received immunosuppressive therapy, more (10 of 14) patients from the group that only received ras blockers progressed to eskd after a median 46-month follow - up period. of note, none of these patients had crescentic gn. eight out of the 12 cases had mpgn pattern of glomerular injury and none had crescentic gn. the authors concluded that patients who did not progress with rituximab therapy had better renal function and shorter time from diagnosis to treatment than those who progressed during the study period. based on our literature review, optimal therapy of patients presenting with fgn and crescentic gn is not known and it seems that this group of patients may have a much poorer renal outcomes with rapid progression to eskd than other patterns of glomerular injury described in patients with fgn. our patient continued to remain dialysis - dependent despite receiving high - dose corticosteroid and cyclophosphamide for nearly 7 weeks. | fibrillary glomerulonephritis (fgn) is a rare primary glomerular disease that commonly presents clinically with hypertension, proteinuria, microscopic hematuria, and varying degree of renal insufficiency. histologically, fgn can present with different patterns of glomerular injury, more commonly mesangioproliferative, membranoproliferative, and membranous nephropathy. while crescent formation has been described in some kidney biopsy series of fgn, crescentic glomerulonephritis pattern of glomerular injury has been rarely described. optimal therapy and outcomes in fgn presenting with crescentic gn is not currently known. we report an adult patient who presented with massive proteinuria and severe renal failure. the kidney biopsy revealed crescentic fgn (c - fgn). the patient remained dialysis dependent despite immunosuppressive therapy. we also briefly review fgn, and the few reported cases of c - fgn that presented as rapidly progressive or advanced renal failure in the literature. |
coagulase - negative staphylococci (cons), especially staphylococcus epidermidis, are considered as normal epithelial flora of every human in different parts of the body such as nares, head and axilla, with an essential role in maintaining the normal flora of healthy skin (1). s. epidermidis is also recognized as an important opportunistic pathogen in nosocomial infections, particularly in indwelling medical device users and contaminant agent of blood cultures. nosocomial infection with s. epidermidis should be more complicated when bacteria are resistant to beta - lactam antibiotics such as methicillin. these bacteria are common causes of nosocomial infections worldwide in immunocompromised patients or patients who carry indwelling devices (2). reduced affinity for beta - lactam antibiotics in methicillin - resistant staphylococci mediates by a penicillin - binding protein (pbp2a) encoded by meca gene located in staphylococcal chromosome cassette mec (sccmec). furthermore, it is believed that s. epidermidis acts as a reservoir for meca and transfers it to s. aureus by horizontal gene transfer. recent data indicate that not only meca, but also other mobile genetic elements can be transferred from s. epidermidis to s. aureus (3). the aim of this study was to determine the prevalence of s. epidermidis as well as antibiotic susceptibility pattern and meca prevalence in s. epidermidis isolated from intensive care unit (icu) patients. a cross - sectional study was conducted from september 2010 to september 2011. a collection of 184 coagulase - negative staphylococci isolated from blood (n = 135), urine (n = 17), tracheal (n = 19), wound (n = 6), cerebrospinal fluid (csf) (n = 2), pleural fluid (n = 2), synovial fluid (n = 2), and peritoneal fluid (ascitic fluid) (n = 1), from three different teaching hospitals of tehran, iran, was included in this study the sample was enriched in tryptic soy broth, and grown on mannitol salt agar, then catalase, tube coagulase and urease tests, and carbohydrate fermentation were performed. s. epidermidis is catalase positive, urease positive, unable to ferment d - mannitol and d - trehalose, and able to ferment d - mannose and d - maltose (4, 5). antibiotic susceptibility testing was performed for s. epidermidis isolates using disk diffusion method on mueller - hinton agar plates (merck, germany) and commercial antibiotic disks including : vancomycin (30 g), linezolid (30 g), rifampin (5 g), ciprofloxacin (5 g), trimethoprim - sulfamethoxazole (1.25/23.75 g), tetracycline (30 g), and erythromycin (15 g), (mast, uk), according to the clinical and laboratory standards institute (clsi) guidelines (6). dna of each s. epidermidis isolate was extracted from 1ml of overnight bacterial culture. extraction was performed by adding te [10 mm tris, 1 mm edta (ph = 8) ] buffer and lysostaphin, 95c for 5 minutes, then 0c for 5 minutes, and centrifuged in 10000 rpm for 5 minutes. dna was measured using a biophotometer (eppendorf, germany) to determine the concentration and purity. (7) with the following specific primers : forward : 5-gtgaagatataccaagtgatt-3 reverse : 5-atgcgctatagattgaaaggat-3. amplification was performed in a mj mini. u.s.a ; initial denaturation step at 94c for 5 minutes, followed by 30 cycles of 45 seconds at 94c,45 seconds at 55c, and 90 seconds at 72c, ending with a final extension step at 72c for 10 minutes, followed by a hold at 4c. the amplicon size was 147 bp (figure 1). in the pcr reaction, s. epidermidis atcc 12228 and s. aureus atcc 33591 1, marker, 100 bp ; 2, s. aureus atcc 33591 (147 bp) ; 3 - 5, positive clinical strains. confidence interval test was used to assess the statistical significance with confidence level of 95% (= 0.05). a cross - sectional study was conducted from september 2010 to september 2011. a collection of 184 coagulase - negative staphylococci isolated from blood (n = 135), urine (n = 17), tracheal (n = 19), wound (n = 6), cerebrospinal fluid (csf) (n = 2), pleural fluid (n = 2), synovial fluid (n = 2), and peritoneal fluid (ascitic fluid) (n = 1), from three different teaching hospitals of tehran, iran, was included in this study the sample was enriched in tryptic soy broth, and grown on mannitol salt agar, then catalase, tube coagulase and urease tests, and carbohydrate fermentation were performed. s. epidermidis is catalase positive, urease positive, unable to ferment d - mannitol and d - trehalose, and able to ferment d - mannose and d - maltose (4, 5). antibiotic susceptibility testing was performed for s. epidermidis isolates using disk diffusion method on mueller - hinton agar plates (merck, germany) and commercial antibiotic disks including : vancomycin (30 g), linezolid (30 g), rifampin (5 g), ciprofloxacin (5 g), trimethoprim - sulfamethoxazole (1.25/23.75 g), tetracycline (30 g), and erythromycin (15 g), (mast, uk), according to the clinical and laboratory standards institute (clsi) guidelines (6). extraction was performed by adding te [10 mm tris, 1 mm edta (ph = 8) ] buffer and lysostaphin, 95c for 5 minutes, then 0c for 5 minutes, and centrifuged in 10000 rpm for 5 minutes. dna was measured using a biophotometer (eppendorf, germany) to determine the concentration and purity. pcr was performed as described by zhang. (7) with the following specific primers : forward : 5-gtgaagatataccaagtgatt-3 reverse : 5-atgcgctatagattgaaaggat-3. amplification was performed in a mj mini. u.s.a ; initial denaturation step at 94c for 5 minutes, followed by 30 cycles of 45 seconds at 94c,45 seconds at 55c, and 90 seconds at 72c, ending with a final extension step at 72c for 10 minutes, followed by a hold at 4c. the amplicon size was 147 bp (figure 1). in the pcr reaction, s. epidermidis atcc 12228 and s. aureus atcc 33591 were used as negative and positive controls, respectively (7). 1, marker, 100 bp ; 2, s. aureus atcc 33591 (147 bp) ; 3 - 5, positive clinical strains. confidence interval test was used to assess the statistical significance with confidence level of 95% (= 0.05). in this study, frequency of s. epidermidis was 34.8% (95% ci : 46.5 - 23.1). of the 64 s. epidermidis isolates, 52 (81.3%) were from blood, 6 (9.4%) tracheal, 4 (6.2%) urine, and 2 (3.1%) wound samples. meca was detected in 92.2% (95%ci : 98.8 - 85.6) of s. epidermidis isolates. resistance to rifampin and ciprofloxacin were 9.4% (95%ci : 16.5 - 2.3) and 23.4% (95%ci : 33.8 - 13), respectively. resistance to trimethoprim - sulfamethoxazole was observed in 51.6% (95% ci : 63.8 - 39.4) of s. epidermidis isolates, while 46.9% (95% ci : 59.1 - 34.7) were susceptible to trimethoprim - sulfamethoxazole. susceptibility to tetracycline was observed in 42.1% (95% ci : 54.2 - 30) of s. epidermidis isolates, while resistance was observed in 57.8% (95%ci : 69.9 - 45.7) and 59.4% (95%ci : 71.4 - 47.4) to tetracycline and erythromycin, respectively abbreviations : cip, ciprofloxacin ; e, erythromycin ; meca, presence of meca ; rp, rifampin ; sxt, trimethoprim - sulfamethoxazole ; te, tetracycline, three of the 64 isolates (4.7%) were resistance to six antibiotics, 4 (6.3%) to five antibiotics, 20 (31.3%) to four antibiotics, 9 (14%) to three antibiotics, 13 (20.3%) to two antibiotics, 14 (21.9%) to one antibiotic, and 1 (1.7%) was susceptible to all the antibiotics. the most common resistance pattern was trimethoprim - sulfamethoxazole, tetracycline, erythromycin, and methicillin resistance, found in 15 isolates (23.4%), followed by resistance to methicillin only, the second - most common resistance pattern, observed in13 (20.3%) of the isolates. in the recent years, s. epidermidis has been isolated as an infective agent in nosocomial infections. some reasons for such infections increasing are increased number of immune - deficient patients, use of indwelling medical devices, and use of antibiotics and disinfectants (8). methicillin resistance was observed in 75 - 90% of isolated s. epidermidis from nosocomial infections, which was higher than the resistance rate for s. aureus isolates (40 - 60%). noticing the presence of s. epidermidis as a human commensal flora, it is assumed to be carrier and reservoir for different genes such as antimicrobial resistance gene (2). frequency of s. epidermidis isolates in this study was significantly lower than reports of okee. (9), eftekhar and raei (10), bouchami. (11), barbier. (12), ruppe. (13), and mombach pinheiro machado ab. (14). in the present study, all the s. epidermidis isolates were susceptible to vancomycin, which was in accordance with the result of mendes. (15), eftekhar and raei (10), abd el hafez. (19). in our study, all the s. epidermidis isolates were susceptible to linezolid, which was in accordance with the results of hellmark. (18), but significantly higher than delgado. (19) report. in the present study, resistance to rifampin (17), and bouchami. (11) reports, and it also was significantly higher than abd el hafez. (17), bouchami. (11), abd el hafez., resistant to trimethoprim - sulfamethoxazole was significantly lower than report of delgado. (17), but was significantly higher than the report of haque. (16) report. in this study, susceptibility to tetracycline was significantly lower than the report of mendes. (19) ; also, resistance to tetracycline was significantly higher than the report of bouchami. (11). in this study, 92.2% of s. epidermidis rohde. (21) reported that 87.5% of s. epidermidis isolates harbored meca. our data did not show significant difference with these studies, but was significantly higher than lduma. (9) results, which reported only 10% meca distribution, and mendes. (15) study, which reported 73.2% methicillin resistant. in our study, 56.3% of s. epidermidis isolates were resistant to three antibiotics or more. considering the presence of s. epidermidis in icu patients, multidrug - resistant bacteria can cause infection and would be more complicated in treatment. in conclusion, presence rate of meca and susceptibility to linezolid and vancomycin did not show significant differences with other investigations, while susceptibility to trimethoprim - sulfamethoxazole was significantly higher, and susceptibility to tetracycline was significantly lower than other investigations. presence of meca - positive s. epidermidis isolates in icu patients, especially in ones with compromised immune systems, may cause infection and would be more complicated in the case of antibiotic resistance. | background : coagulase - negative staphylococci (cons), especially staphylococcus epidermidis, are considered as normal flora of human epithelia and also important opportunistic pathogens for nosocomial infections. s. epidermidis can also act as a reservoir for meca, responsible for high - level resistance to methicillin and transferring it to s. aureus.objectives:the aim of this study was to determine the prevalence of s. epidermidis as well as antibiotic susceptibility pattern and meca prevalence in s. epidermidis isolated from intensive care unit (icu) patients.materials and methods : a cross - sectional study was conducted from september 2010 to september 2011 and 184 coagulase - negative staphylococci were collected from different clinical samples in three hospitals. s. epidermidis was identified by conventional bacteriological tests. antibiotic susceptibility testing was performed using disk diffusion method. frequency of meca was detected by specific pcr.results:frequency of s. epidermidis was 34.8%, the most susceptibility was seen to linezolid and vancomycin, and the least susceptibility was seen to tetracycline.majority of the s. epidermidis isolates carried meca (92.2%). the most common resistant pattern was trimethoprim - sulfamethoxazole, tetracycline, erythromycin, and methicillin resistance, found in 23.4% of the isolates, followed by resistance to methicillin as the second - most common resistant pattern, observed in 20.3% of the isolates.conclusions:frequency of s. epidermidis was significantly lower, compared to other studies. presence rate of meca and susceptibility to linezolid and vancomycin did not show significant differences with other investigations, while resistant to trimethoprim - sulfamethoxazole was significantly lower compared to other investigations, and resistance to tetracycline was significantly higher in comparison to other investigations. presence of methicillin - resistant s. epidermidis in icu patients, especially in individuals with compromised immune systems, may cause infection and would be more complicated in the case of antibiotic resistance. |
pemphigus vulgaris (pv) is a chronic autoimmune mucocutaneous disease that usually manifests first in the oral cavity and may later spread to the skin or other mucous membranes. apart from ulcers, vesicles, bullae and pustular lesions, it can present solely as mucosal erosions. herein we report a case of intraoral pv presenting as intense erythema and erosions involving the gingiva. a 52-year - old female presented to the department of periodontics, al - badar dental college and hospital, gulbarga with complaints of bright red gingivae and discomfort in her normal oral function. the site most severely affected was her upper front buccal gingivae. the considerable pain and discomfort that the patient felt hindered her from carrying out effective oral hygiene measures and this in turn exacerbated the gingival symptoms. she had essential hypertension, diabetes mellitus and hyperlipidemia and her daily medications included insulin, antihypertensive drugs and statins. on intraoral examination, there were multiple large irregular erosions and areas of intense erythema involving particularly the gingivae of both upper and lower arch, buccally and palatally / lingually [figures 1 and 2 ]. orthopentamogram showed a combination of horizontal and vertical bone loss. the nikolky 's sign (loss of epithelium occasioned by rubbing apparently unaffected skin) that is a feature of pv was positive. the patient had no cutaneous involvement and other mucosal sites such as conjunctiva, nasal passages and oesophagus were free of lesions. intensely erythematous gingivae with multiple erosions erosive lesions on the gingiva palatally based on the history, multiple erosions and the apparent fragility of the gingivae experienced during examination, a vesiculobullous disorder was suspected. is usually seen in the elderly, the former was thought to be more likely. the other common conditions that can present with similar manifestations are oral lichen planus, drug hypersensitivity or idiopathic. histopathological examination revealed suprabasal blister formation associated with extensive acantholysis of keratinocytes, suggestive of pv [figure 3 ]. photomicrograph showing suprabasilar split and acantholytic cells since the patient only had isolated gingival involvement, she was started on systemic corticosteroids (prednisolone 0.5 mg / kg / day). this initial dose was stepped up to 1mg / kg / day, which gave marked improvement in two weeks. the patient was instructed to apply twice daily topical steroid oral paste (triamcinolone acetonide 0.1%) along with local anesthetic gel. sometime after commencement of corticosteroid therapy, scaling and root planing was performed. over the past 1 year currently, the patient is on this daily low - dose systemic corticosteroid therapy (prednisolone), topical steroid oral paste (triamcinolone acetonide 0.1%) and supplementary medications. till date, no other sites are involved. pemphigus is a group of potentially life - threatening autoimmune mucocutaneous diseases characterized by epithelial blistering affecting cuteneous and/or mucosal surfaces, the term being derived from the greek word pemphix (bubble or blister). although vulgaris means common in latin, the worldwide incidence of pv is low and has been reported to be 0.10.5 per 100,000 persons per year. pv is the most common variant of pemphigus, comprising of 80% of the disease entity. pv frequently involves the mouth and has a fairly strong genetic background ; ethnic groups such as ashkenazi jews and people of mediterranean and indian origin are particularly susceptible and there is a link to hla class ii alleles. clinically, pv appears to occur in males and females in an equal ratio, and is most frequently reported in patients between the fourth and sixth decades of life. mortality from pv before the development of effective therapies was as high as 90%, mainly due to dehydration and secondary systemic infection. oral lesions are common and early manifestations of pv, seen typically in adults (rarely in childhood). one recent multicenter study in several countries showed that bulgarian patients less frequently had oral mucous membrane lesions (66%) compared with italian (83%) and israeli (92%) patients. initially vesiculobullous, the oral lesions readily rupture and as the older ones rupture and ulcerate, new bullae develop. gingival lesions are less common and usually comprise severe desquamative or erosive gingivitis, characterized by red erosions or deep ulcerative craters. in the present case, gingiva was the only site involved ; the gingiva was intensely erythematous and erosive involving both attached and marginal gingiva. however, the patient 's oral hygiene was very poor and this in turn contributed further to gingival inflammation, leading to generalized periodontitis. the classic signs of oral or gingival pv are multiple erosions or desquamation and a positive nikolsky sign which both of which were present in our case. histologically, there is an intraepithelial blister associated with acantholytic cells, features which were evident in this patient. systemic corticosteroids are the treatment of choice in patients with pv ; topical steroid therapy alone is insufficient for sustained control of the disease because of the systemic autoimmune nature of pv. in the present case, systemic and topical corticosteroid treatment and adjuvant therapy of antifungal mouthwash, use of soft brush and vitamin supplementation were instituted. pv is a chronic autoimmune mucocutaneous disease that often primarily involves the oral cavity. as it is a life - threatening disease condition, it is important for the clinician to be able to recognize oral manifestations of pv at an early stage and treat or refer appropriately. dental professionals can thus play an important and significant role in the early diagnosis and management of pv. | pemphigus vulgaris (pv) is an autoimmune blistering disease affecting the mucous membrane and skin. typically, oral lesions appear before skin lesions, and in a majority of the cases only oral lesions are present. the dentist may then be the first to recognize and diagnose this disease. it is unusual for pv to present over the gingiva as a primary site of involvement. diagnosis is based on clinical presentation and confirmed by histopathological study. early diagnosis and management can prevent the uneven life- threatening effects of this potentially chronic mucocutaneous disorder. the case serves to enhance our awareness of the gingiva as a site at which systemic disease can manifest itself. |
. basically, there are two different outcomes of drowning, death on the spot of drowning, and survival from the initial apnoea. however, with the lower respiratory tract challenged by water, the survivor may suffer acute lung injury (ali), which is characterized by developing pulmonary inflammation and edema. it was reported that inflammation factor secretion, pulmonary edema, and inflammatory spreading to entire lung or even both lungs were closely related to the alteration of communication between cells. gap junction channels (gjcs), connecting the cytoplasm between adjacent cells, are cell membrane channels, which provide a pathway for rapid exchange of ions, metabolites, and small intracellular signal molecules, such as ca, cyclic amp, and so on. the critical contribution of gjcs to disease etiology has been intensively researched in recent years, and connexin 43 (cx43), as the main mode of connection between alveolar epithelial cells, participates in a variety of acute / chronic lung disease occurrence and development. evidence proven that cx43 may regulate ca signal path way, and this would play a pivotal role in acute lung injury. 3,5,4-tri - o - acetylresveratrol (figure 1), with three hydroxyls replaced by acetyls, is an analog of resveratrol. several studies demonstrated that it exerted anti--irradiation effects by inhibiting the expression of reactive oxygen species (ros) and acted as inhibitors of paf - induced washed rabbit platelet aggregation. besides, our previous results showed that 3,5,4-tri - o - acetylresveratrol might trigger the accumulation and concentration of resveratrol in lungs. based on the previous evidence, we put forward and proved in the present research the hypothesis that cx43 participated in the inflammation induced by seawater instillation via affecting intracellular communication. while 3,5,4-tri - o - acetylresveratrol could protect lungs by enhancing the expression of cx43, suppressing inflammatory reaction and reconstructing intercellular communication male sprague - dawley rats, weighing 180220 g each, were obtained from the animal center (fourth military medical university, xi'an, china). the rats were housed in air - filtered, temperature - controlled units with 12-hour light - dark cycles and had free access to food and water. all experiments were approved by the animal care and use committee of the fourth military medical university and were in accordance with the declaration of the national institutes of health guide for care and use of laboratory animals (publication no. seawater (osmolality 1300 mmol / l, ph 8.2, sw 1.05, nacl 6.518 g / l, mgso4 3.305 g / l, mgcl2 2.447 g / l, cacl2 1.141 g / l, kcl 0.725 g / l, nahco3 0.202 g / l, nabr 0.083 g / l) was prepared according to the major composition of the east china sea provided by chinese ocean bureau. 3,5,4-tri - o - acetylresveratrol was obtained from the pharmacy department of medicinal chemistry with hplc purity > 99%. resveratrol was purchased from xi'an grass plant technology corporation (xi'an, china), purity > 98%. lucifer yellow ch dilithium salt was purchased from sigma chemical company (st. louis, mo, usa). enzyme - linked immunosorbent assay (elisa) kits of tnf- and il-1 were purchased from r&d corporation (r&d systems inc., elisa kit of il-10 was purchased from senxiong science and technology industrial corporation (shanghai, china). anti - connexins 43 and anti--actin monoclonal antibodies were obtained from anbo biotechnology company (changzhou, china). real time sd rats were randomly assigned into 5 groups (n = 8).control group : rats without any intervention.seawater drowning group : the rats were anesthetized with pentobarbital sodium (100 mg / kg of body wt, administered i.p.). a heparin - filled blunt - ended polyethylene catheter was inserted into the left carotid artery to monitor the mean arterial pressure and obtain blood samples. after exposure of the trachea, a 20 min stable baseline period was followed, then a syringe (1 ml) was inserted into the trachea and seawater (4 ml / kg) was instilled at a steady speed within 4 min into both lungs. all rats were sacrificed at 4 h after seawater instillation.3,5,4-tri - o - acetylresveratrol (50 mg / kg) + seawater drowning group : 3,5,4-tri - o - acetylresveratrol was administered daily orally for 7 days before modeling.3,5,4-tri - o - acetylresveratrol (150 mg / kg) + seawater drowning group : 3,5,4-tri - o - acetylresveratrol was administered daily orally for 7 days before modeling.3,5,4-tri - o - acetylresveratrol (450 mg / kg) + seawater drowning group : 3,5,4-tri - o - acetylresveratrol was administered daily orally for 7 days before modeling. seawater drowning group : the rats were anesthetized with pentobarbital sodium (100 mg / kg of body wt, administered i.p.). a heparin - filled blunt - ended polyethylene catheter was inserted into the left carotid artery to monitor the mean arterial pressure and obtain blood samples. after exposure of the trachea, a 20 min stable baseline period was followed, then a syringe (1 ml) was inserted into the trachea and seawater (4 ml / kg) was instilled at a steady speed within 4 min into both lungs. 3,5,4-tri - o - acetylresveratrol (50 mg / kg) + seawater drowning group : 3,5,4-tri - o - acetylresveratrol was administered daily orally for 7 days before modeling. 3,5,4-tri - o - acetylresveratrol (150 mg / kg) + seawater drowning group : 3,5,4-tri - o - acetylresveratrol was administered daily orally for 7 days before modeling. 3,5,4-tri - o - acetylresveratrol (450 mg / kg) + seawater drowning group : 3,5,4-tri - o - acetylresveratrol was administered daily orally for 7 days before modeling. the doses of 3,5,4-tri - o - acetylresveratrol (50, 150, and 450 mg / kg) used here were based on previous dose - response and time - course studies carried out in our laboratory. the lungs were moved out rapidly from thoraxes and processed in the manners described below. at the end of the experiments, lung tissues of the same lobe from every rat were fixed with 10% formalin for 24 h, and then embedded in paraffin. after deparaffinization and dehydration, the lungs were cut into 5 m - thick sections with a microtome and stained with haematoxylin and eosin. lung wet - to - dry ratio (w / d) was used to quantify the magnitude of pulmonary edema. the lung tissues, obtained 4 h after modeling, were weighed immediately, and then dried to constant weight at 70c for 72 h and weighed again. the wet - to - dry ratio was calculated through dividing the wet weight by the dry weight. levels of tnf-, il-1, and il-10 in the lung tissues were determined by using commercially available elisa kits. briefly, lung tissues were homogenized in cool phosphate - buffered saline (lung tissue to pbs 1 : 5). the lungs were perfused with ph 7.4 pbs to remove the blood cells from the pulmonary circulation, and then, the tissue samples from each group were collected and the total proteins were extracted. the protein samples were boiled, separated on a 12% sds - polyacrylamide gel, electrotransferred to nitrocellulose membranes, blocked with 5% nonfat dry milk in tris - buffered saline with tween 20, and incubated overnight at 4c with monoclonal antibodies against cx43 (1 : 200) and -actin (1 : 5000). after repeated washing, the secondary antibody (anti - rabbit igg peroxidase conjugated, 1 : 10000) was incubated, and bands were visualized by using the enhanced chemiluminescence (ecl) system (amersham pharmacia biotech, arlington heights, il, usa). the results were expressed as the ratio to -actin level in the same protein samples. the total rna of the lung samples was extracted with trizol reagent (takara). cx43 and -actin were examined by real time pcr following the manufacturer 's instructions (takara perfect real time). genes and primers are listed as follows : cx43, (forward) 5-ggaaatcgaacggctgggcgt-3, (reverse) 5-tcgcgtgaagggaagaagcgat-3 ; -actin (forward) 5-gcactgtgttggcatagaggtc-3, (reverse) 5-acggtcaggtcatcactatcgg-3. amplification and detection were carried out by using bio - rad my iq detection system (edinburgh biological science and technology development co., ltd, shanghai, china). the human lung epithelial cell line, a549 (obtained from atcc, rockville, md, usa), was maintained in 1640 medium supplemented with 10% fetal calf serum. the human umbilical vein endothelial cell (huvec) line was maintained in ham 's f12medium supplemented with 10% fetal calf serum. both of the two cell lines were treated with 100 u / ml of penicillin and 100 g / ml of streptomycin at 37c in a humidified atmosphere containing 5% co2 and 95% air. after incubated in the presence or absence of resveratrol (200 mol / l), seawater (0.25 ml per 1 ml total volume) was added to a549 and huvec cells and the cells were stimulated for the 4 h. a dye transfer assay was used to assess gap junction communication. a549 cells were rinsed with 2 ml pbs after medium was removed from the plates. 2 milliliters of 0.075% lucifer yellow ch(ly), a fluorescent membrane - impermeable cell marker dye dissolved in pbs, were added to the cells, and two scrape lines (parallel, equidistant scrapes per well) were made by gently passing a diamond - tipped pen (tip diameter, 0.25 mm) across the cultures. the plates were placed for 5 minutes at 37c in a humidified 5% co2 incubator. the dye solution was then discarded, and the dishes were rinsed twice with pbs to remove background fluorescence. after that, cells were examined with an inverted confocal microscope (fv1000 ix81, olympus) at emission / excitation wavelengths of 528/425 nm. the images were quantified by counting the number of donor and recipient cells and calculating a cell coupling index with the ratio of recipient to donor cells. the value of dye transfer, defined as the number of secondary recipient cells visualized by lucifer yellow ch, was recorded for only one side of the scrape (figure 8). statistically significant differences between experimental conditions were performed by one - way analysis of variance (anova) followed by dunnett 's test. the results showed that 4 hours after seawater inhalation induced pulmonary edema, alveolar damage, and infiltration of inflammatory cells in the lung tissues and alveoli (figure 2(b)), but pretreatment with different doses of 3,5,4-tri - o - acetylresveratrol could significantly improve the lung injury (figures 2(c) and 2(d)). to observe the lung edema, we observed the lung wet / dry weight ratios (figure 3). the wet / dry ratios significantly increased in seawater drowning group compared with the control (n = 8, p < 0.05). we also examine the effects of 3,5,4-tri - o - acetylresveratrol on the levels of tnf-, il-1, and il-10 in the lung tissues. as shown in figure 4, four hours after seawater instillation, tnf- and il-1 contents significantly increased. pretreatment with 3,5,4-tri - o - acetylresveratrol markedly inhibited the expression of these inflammatory mediators. cx43 mrna expression was examined using real time pcr on the lung samples 4 h after seawater exposure (figure 5). the results showed that seawater aspiration increased the cx43 mrna level (p < 0.05), while pretreatment with different doses of 3,5,4-tri - o - acetylresveratrol obviously upregulated the level of cx43 mrna (p < 0.05). we then performed western blot to study the changes of cx43 in protein levels (figure 6). when challenged with seawater, the reactive pattern of cx43 protein was opposite to that of its mrna. namely, the protein levels of cx43 in lungs decreased 4 h after seawater exposure (p < 0.05). similar cell numbers were initially loaded with dye in all treatment groups (figures 7(a) and 7(b)). however, compared with ratios of the control group, there were fewer labeled neighboring cells of seawater groups, and the treatment with resveratrol significantly increased the number of labeled neighboring cells. in the present study, we demonstrated that (i) intratracheal instillation of seawater (4 ml / kg) induced obvious histological changes, pulmonary edema, and inflammation in a dosage dependence manner ; (ii) seawater stimulation could obviously suppress the cellular communication between cultured cells ; (iii) pretreatment with 3,5,4-tri - o - acetylresveratrol could effectively alleviate the seawater - induced lung injuries ; (iv) resveratrol, intermediate metabolite of 3,5,4-tri - o - acetylresveratrol, could rebuild or strengthen the seawater impaired communication between cells. although several promising pharmacological therapies have been studied for patients with ali and ards, none of these pharmacological treatments obviously reduced mortality. resveratrol (3,5,4-tri - o - acetylresveratrol) is a polyphenolic compound which is a phytoalexin synthesized by a wide variety of plant species. it has lots of pharmacological properties, such as antioxidation, anti - inflammatory, cardioprotection, cell cycle inhibition, and neuroprotection. for example, it is not stable with short half - life and it has a low bioavailability. while resveratrol 's analog, 3,5,4-tri - o - acetylresveratrol may overcome some of those disadvantages to some extent. evidence showed that this analog was effective in inhibiting the expression of reactive oxygen species (ros) and paf - induced washed rabbit platelet aggregation. ali / ards can be divided into two categories based on origin : direct (or pulmonary) ali and indirect (extra - pulmonary) ali. seawater drowning - induced acute lung injury (swd - ali) belongs to direct ali. clinical studies indicated that white blood cell and neutrophils apparently increased in most acute lung injury (ali)/acute respiratory distress syndrome (ards) patients ' plasma with bilateral diffuse or localized alveolar infiltrates on chest x - ray. our previous results also showed that infiltration of inflammatory cells and permeability of alveolar wall to evans blue apparently increased in swd - ali rat model, which to some extent explained the possible mechanism of seawater drowning - induced acute lung injury. inflammation response is a series of complex pathological process, including release of cytokines, growth factors and chemokine, and migration of neutrophils, monocytes and lymphocytes to tissue spaces. evidence showed that communication between flanking cells stimulated by cx43 provided the foundation for the onset and development of inflammation in lung tissues. we found, in the present study, that seawater exposure resulted in upregulation of cx43 in gene level, deregulation of cx43 protein, and secretion of inflammatory factors, such as tnf- and il-1, while pretreatment with 3,5,4-tri - o - acetylresveratrol reduced the contents of inflammation factor. besides, il-10, as a famous inhibitory inflammation factor, was increased by 3,5,4-tri - o - acetylresveratrol. more importantly, 3,5,4-tri - o - acetylresveratrol markedly upregulated the expression of cx43 in gene and protein levels, and its intermediate metabolite contributed to the reconstruction of cellular communication in a549 and huvec. pulmonary edema is another critical issue of acute lung injury, which results from breathing membrane barrier (bbb) dysfunction, and capillary permeability increase [18, 19 ]. accumulation of liquid rich in protein in alveolar space may increase the mortality of ali / ards. it was demonstrated, in a gunshot lung injury rabbit model, that cellular communication played an important role in capillary permeability increase and leakage of liquid into alveolar space. in addition, it was found that inhibition of cx43 mrna and protein expression led to the increase of single cell permeability. we found, in the present study, that seawater inhalation led to lung edema and seawater stimulation also suppressed cellular communication of huvec. however, 3,5,4-tri - o - acetylresveratrol could alleviate the lung edema in rats suffering seawater instillation, and its metabolic intermediates, resveratrol, recovered the cellular communication restrained by seawater exposure. connexin 43 (cx43), which plays a key role in regulating of inflammation and microvascular permeability, increased in gene level while was inhibited in protein level upon seawater stimulation. this may mean that seawater stimulation would enhance the transcription but inhibit the translation of cx43. we observed the facilitating effect of 3,5,4-tri - o - acetylresveratrol on cx43 in swd - ali and two kinds of cells stimulated by seawater. our results suggested that 3,5,4-tri - o - acetylresveratrol alleviated seawater instillation induced inflammation and pulmonary edema probably via activation of cx43. | the aim of the present study was to examine the effects of 3,5,4-tri - o - acetylresveratrol on connexin 43 (cx43) in acute lung injury (ali) in rats induced by tracheal instillation of artificial seawater. different doses (50, 150, and 450 mg / kg) of 3,5,4-tri - o - acetylresveratrol were administered orally for 7 days before modeling. four hours after seawater inhalation, histological changes, contents of tnf-, il-1 and il-10, and the expression of cx43 in lungs were detected. besides, the gap junction communication in a549 cells and human umbilical vein endothelial cells (huvecs) challenged by seawater was also evaluated. histological changes, increased contents of inflammatory factors, upregulation in gene level, and deregulation in protein level of cx43 in lungs stimulated by seawater were observed. on the other hand, pretreatment with 3,5,4-tri - o - acetylresveratrol significantly inhibited infiltration of inflammation, development of pulmonary edema, and contents of inflammatory mediators in lungs. above all, 3,5,4-tri - o - acetylresveratrol upregulated the expression of cx43 in both gene and protein levels, and its intermediate metabolite, resveratrol, also enhanced the gap junction communication in the two cell lines. the results of the present study suggested that administration of 3,5,4-tri - o - acetylresveratrol may be beneficial for treatment of inflammatorycellsin lung. |
the success of contemporary and modern endodontics relies upon adequate knowledge of root canal anatomical variations and use of advanced diagnostic and treatment modalities. recent studies and reviews have shown success rates up to 95% in teeth with irreversible pulpitis and 85% in teeth with necrotic root canals. mandibular molars are the first permanent teeth to erupt in the oral cavity at 67 years followed by completion of calcification at 89 years of age. the completion of canal differentiation commences at 36 years after closure of the apical foramen. many variations exist with regards to its root and root canal anatomy thus necessitating critical evaluation of each individual case for variations. this case report describes the successful non - surgical endodontic management of a three - rooted mandibular first molar with two canals in the mesial root, one canal each in both distobuccal and distolingual roots using helical computed tomography (ct) imaging. a 21-year - old male reported to the outpatient department of conservative dentistry and endodontics with a chief complaint of pain in the lower left back region since 2 months. clinical examination revealed the decayed distal surface in the left mandibular first molar (tooth # 36) [figure 1 ] with no fistulae or edema. there was tenderness to palpation and vertical percussion, but the tooth mobility was within normal physiological limits. the pre - operative radiograph of tooth # 36 taken from 20 degrees mesial angulation showed the presence of three roots with slight widening of the periapical periodontal ligament space in relation to the mesial root apex and periapical radiolucency measuring about 1 mm with respect to the periapex of the distobuccal root [figure 2 ]. from the clinical and radiographic findings, a diagnosis of irreversible pulpitis with acute apical periodontiitis with tooth # 36 was made. to confirm the presence of extra root and to get detailed information of the anatomical variation in tooth # 36, three - dimensional reconstructed [figures 3 and 4 ] and axial images [figure 5 ] dentascan was the case of radix entomolaris with vertucci type i root canal in both distobuccal and distolingual root and type ii in mesial root. the distal surface of the tooth was restored with composite resin (z100 ; 3 m dental products, st paul, mn, usa) after caries excavation to enable better isolation. tooth # 36 was anesthetized by using 1.8 ml (30 mg) of 2% lidocaine containing 1:200,000 epinephrine (xylocaine ; astrazeneca pharma ind ltd., bangalore, india). a rubber dam was placed and a modified endodontic access opening was established in tooth # 36. the pulp chamber floor was shown to have four canals connected by the developmental root fusion line (drfl). coronal enlargement was done with a nickel titanium (niti) protaper sx rotary file (dentsply maillefer, ballaigues, switzerland) to improve the straight - line access. working length was determined with the help of an apex locator (root zx ; morita, tokyo, japan) and later confirmed by using a radiograph [figure 6 ]. cleaning and shaping was performed under rubber dam isolation by using protaper niti rotary instruments (dentsply maillefer) with a standardized technique. irrigation was performed using normal saline, 5.25% sodium hypochlorite solution and 17% ethylenediaminetetraacetic acid. after completion of cleaning and shaping, the root canals were dried with absorbent points (dentsply maillefer). calcium hydroxide (calcicur ; voco, cuxhaven, germany) was placed as an intracanal medicament with a lentulo spiral (dentsply maillefer) for 1 week and the access cavity was sealed with cavit (3 m espe dental products, st paul, mn, usa). the patient was asymptomatic on the next visit ; therefore, tooth # 36 was obturated using protaper gutta percha and ah 26 root canal sealer [figure 7 ]. the tooth was permanently restored using composite resin (z100 ; 3 m dental products). pre - operative intraoral view pre - operative intraoral periapical radiograph d reconstructed image axial section at apical level working length intraoral periapical radiograph post - operative intraoral periapical radiograph mandibular molars being the cornerstones of dental occlusion are reported to have the incidence of a third root in 13% of the cases, and this was strongly correlated with the ethnicity of the studied population. three canals are present in 61.3%, four canals in 35.7% and five canals in approximately 1%. root canal configuration of the mesial root revealed two canals in 94.4% and three canals in 2.3%. the most common canal system configuration was vertucci type iv (52.3%), followed by type ii (35%). root canal configuration of the distal root revealed type i configuration in 62.7%, followed by types ii (14.5%) and iv (12.4%). the presence of isthmus communications averaged 54.8% on the mesial and 20.2% on the distal root. presence of extra roots in the mandibular molars was first reported in the literature by carabelli. the presence of extra root on the buccal surface is termed as radix paramolaris and on the distal surface is termed as radix entomolaris. carleson and alexandersen described that root canal morphology in extra root was found to be vertucci 's type i mostly. ethnicity is a predisposing factor for anatomical variations such as number of roots, but there is no direct relationship between ethnicity and configuration of the root canal system. the incidence of three - rooted mandibular first molars in the indian population is 5.97%, in europeans is 3.44.2%, in africans 3% and in eurasians less than 5%. the prevalence in the taiwanese population is 33.33%, with a bilateral incidence of a symmetrical distribution of 53.65%. there was a significantly greater incidence of three - rooted teeth on the left side of the mandible than on the right, but gender did not show a significant relationship with this variant prevalence. the mongoloid population exhibited significantly more mandibular first molars with three roots, with a 3:1 ratio when compared with caucasians and african americans, with the frequency ranging from 5% to 30%. thus, this trait is considered as eumorphic variation in people with the mongoloid traits. thorough knowledge of root canal anatomical variations predispose to the success of endodontic treatment performed in a non - invasive manner. use of advanced diagnostic and treatment aids help in managing various challenges faced by endodontists in day - to - day practices. | this case report describes the successful non - surgical endodontic management of carious exposed three - rooted mandibular molar with four root canals detected on the pre - operative radiograph taken with 20 degrees mesial angulation and confirmed with a 64-slice helical computed tomography scan - assisted 3-d - reconstructed images. access cavity shape was modified to locate the extra canal with respect to the distolingual root in the left mandibular first molar. copious irrigation was accomplished with 5.25% sodium hypochlorite and 17% edta. biomechanical preparation was done using protapers. calcium hydroxide dressing was done for 1 week. the tooth was obturated using gutta percha and ah 26 root canal sealer, and it was permanently restored with composite. clinical examination on follow - up visits revealed no sensitivity to percussion and palpation in the left mandibular first molar. thorough knowledge of root canal variations and use of advanced diagnostic modalities lead to successful non - surgical management of the complex cases. |
spontaneous pseudoaneurysm of the femoral artery is extremely a rare entity.(1) it can be secondary to the rupture of a true aneurysm, but in most events the cause is unclear.(2) most patients with non - atherosclerotic pseudoaneurysms of sfa are asymptomatic initially and later present with a pulsatile, expanding mass along the anatomical course of sfa in the thigh and have a history of some trauma or surgical procedure.(3) due to their complications, large pseudoaneurysms require some kind of intervention. if not treated, they are at risk for rupture, infection, distal embolization or arteriovenous fistula formation. (1) we report a young patient with spontaneous femoral artery pseudoaneurysm without any history of trauma or vascular intervention successfully treated surgically. a 29-year - old healthy man presented with right thigh swelling since 2 months which is painful throbbing in nature and aggravated by walking. there was no history of local trauma and he had never suffered peripheral vascular disease. on presentation, he was hemodynamically stable and there was a tender swelling at the medial aspect of right mid thigh which was pulsatile. ct angiogram was done which showed a pseudoaneurysm of the lower third of femoral artery. ct angiogram showed a pseudoaneurysm of the lower third of femoral artery a surgical excision of the pseudo - aneurysm was done under general anesthesia. after evacuation of hematoma, there was a large defect in the wall of the artery. therefore, a greater saphenous vein interposition graft was used to maintain the vascular continuity. formation of a femoral artery pseudoaneurysm is not infrequent after femoral arterial access, and it occurs in up to 0.7% of the diagnostic procedures and in up to 8% of the interventional procedures. (4) recently, there has been a significant decrease in both major and minor vascular complications after diagnostic and therapeutic procedures with an incidence of pseudoaneurysm less than 0.3 %. (5) spontaneous pseudoaneurysm of the superficial femoral artery in the young age group is a rare occurrence. some of the false aneurysm may close spontaneously but rupture is a major concern followed by thrombosis, distal embolization and compression of adjacent structures. (3) atherosclerosis has been proposed by several authors as an etiologic factor in spontaneous perforation of the femoral arteries or its branches. (6 - 8) in these case reports, the patients were elderly with atherosclerotic changes in their peripheral arteries and weakening of the arterial walls could have led to spontaneous rupture. patients who have inherited connective tissue disorders such as ehlers - danlos have also been reported to present with spontaneous rupture of their arteries because of the fragility of their arterial walls. (9). our patient, however, was young and there was no evidence of atherosclerotic changes in his arteries on the angiogram or during surgery. furthermore, he did not have any clinical features to suggest an inherited connective tissue disorder. there was also no history for any trauma and for which we labeled him to have a spontaneous femoral artery pseudoaneurysm. therapeutic options for femoral pseudoaneurysm include open surgical repair, ultrasound - guided compression, ultrasound - guided thrombin injection, coil embolization, and endovascular repair using stent - grafts. (10) particularly in young patients, an appropriate approach is surgical exploration with hematoma evacuation and arterial repair by means of arterial suture, patch angioplasty, or graft interposition. (2) femoral artery pseudoaneurysm has been a common complication after the wide use of femoral artery access for diagnostic and therapeutic procedures. however, spontaneous rupture of the artery without trauma remains a rare entity. as there are many evolving types of management, the surgical intervention may has superiority when there is a suspicious of underlying vascular pathology. | spontaneous femoral artery pseudoaneurysm is a rare disease and reported cases are very few. most of them are related to underlying pathology either atherosclerotic disease or connective tissue disease. we present a 29-year - old healthy man with two months history of a painful pulsating mass at the level of the lower right thigh without any previous history of trauma, surgery or puncture of the femoral artery. an angiogram revealed a right superficial femoral artery pseudo - aneurysm. it was treated surgically by resection of the aneurysm, reconstruction with inter - positional saphenous vein graft. we reported this case because of its rare incidence in the young patient with no underlying pathology. |
obesity is one of the most significant and rapidly increasing health problem today, giving rise to an explosion in health care costs. the amount of energy we consume and how much we move around are the key factors in body weight regulation, but why a positive energy balance emerges in the majority of individuals in societies with access to abundant food calories is not completely understood. our lifestyle has changed dramatically since the beginning of industrialization, and some of these changes probably affect energy balance in ways that favour weight gain. some experts consider the dogma that voluntary changes in food intake and physical activity are the principal causes of the obesity epidemic to be circumstantial. moralizing the problem and socially stigmatizing the obese has done nothing to stem the epidemic. results from both human and animal studies indicate the existence of an intricate homeostatic system for maintaining body weight within a fairly narrow range and the presence of a powerful hedonic system that can interfere with this relatively finely tuned regulation. if central mechanisms in this homeostatic system, which involves endocrine feedback from the periphery to the brain, are influenced by changes in the environment, then even small environmental changes could potentially cause changes in body weight. we propose a novel hypothesis involving a mechanism in which a small change in blood ph caused by increased concentrations of co2 in our environment may contribute to the development of obesity. coinciding with the sharp increase in the prevalence of human obesity over the last 50 years, weight gain has also been observed in animals. a recent study found that 24 populations of 8 different species, including laboratory animals that had been fed the same diets for decades, all displayed significant weight gain. this suggests that a shared environmental factor, favouring weight gain, may be involved in the regulation of energy balance. the atmospheric concentration of co2 has remained between 180280 p.p.m. over the past two million years. however, with the increasing industrialization of modern societies, co2 concentration has risen to 400 p.p.m., with an additional increase to 550 p.p.m. thus, the partial pressure of co2 in the outside air has increased by nearly 40% over the last century. henry 's law states that the solubility of a gas is directly proportional to the partial pressure of that gas above the solvent, so we can assume that humans now absorb around 40% more co2 in body tissue. this increased acidic load is only partially counterbalanced by increased excretion of renal acid (h). it is therefore reasonable to assume that blood and cerospinal fluid are now slightly more acidic than 100 years ago. additionally, in industrialized cultures, we spend 8090% of our time indoors, where co2 concentrations are often higher. it is well established that acidity of the blood increases (decreased ph) with increasing co2 concentration in the inhaled air. in a number of studies of a duration of several weeks, a lower ph was maintained throughout, indicating that full renal compensation did not occur. acidosis is also compensated by increased ventilation, but apparently, this does not happen before co2 concentration in inspired air is about 10 000 p.p.m. in parts of the brain (hypothalamus), there are specialized nerve cells (orexin neurons), which are involved in the regulation of appetite, energy metabolism, wakefulness, feeding behaviour and libido. these orexin neurons are extremely sensitive to changes in ph, a decrease of only 0.1 ph units leading to a doubling in their activity. given the observed increase in atmospheric co2 concentration from 280 to 400 p.p.m., and the decrease in the ph of blood observed in animals and humans exposed to co2 concentrations ranging from 7000 to 15 000 p.p.m., we estimate that the firing rate of the orexigen neurons has increased by approximately 1% in the course of the last century. the basis for the calculation : if the ph decrease with 0.1 ph units, the firing rate will change nearly 100%. during the past century additionally, we assume that there is a linear relationship between the ph in the blood and the co2 concentration in the inspired air. two studies was identified where blood ph was measured in relation to exposure to low concentration of co2. a human study with exposure to 7000 p.p.m. co2 and a blood ph decrease of 0.05 units and a guinea pigs study with exposure to 15 000 p.p.m. (7000400 p.p.m.). according to (williams.,) will a drop in ph of 0.05 ph units means a 50% increase in the firing rate. a rough estimate of the proposed increase in the firing rate would be : 50%/6600 p.p.m.=0.008%/p.p.m we hypothesize that a lower blood ph, caused primarily by rising co2 concentrations in air, indoors as well as outdoors, leads to elevated activity in parts of the hypothalamus, resulting in increases in appetite and energy intake. these ph - sensitive neurons also regulate wakefulness, and increased activity in the neurons will lead to increased wakefulness, which has also been associated with weight gain. interestingly, numerous studies have shown an association between short sleep duration and increased body mass index. the national sleep foundation (www.sleepfoundation.org) finds that sleep duration has been decreasing steadily over the past century. activation of the orexin system will lead to less sleep, and lack of sleep has been reported to decrease levels of the satiety hormone leptin, increase levels of the hunger hormone ghrelin, and alter glucose homeostasis. additionally, in patients with obstructive sleep apnoea, characterized by both a hypercapnic and a hypoxic state, energy expenditure (ee) has been found to decrease with increasing severity of the affliction. leptin concentrations in these patients are only half that found in healthy control subjects matched for age, sex, body mass index, waist / hip ratio and fat mass. leptin is involved in the regulation of both energy intake and of ee, and narcoleptic patients, despite eating less than normal people, they still become obese. lower orexin levels decrease appetite, so the weight gain may be a result of decreased ee caused by lower circulating plasma leptin levels. this effect of leptin may be the same as that linking shorter duration of sleep to obesity. orexin neurons are inhibited by glucose and leptin, and leads to decreased appetite, wakefulness, sympathetic stimulation, libido and need for pleasure / reward. in addition, the orexin neurons stimulate centres in the brain forming adrenocorticotrophic hormone, which in turn leads to increased production of cortisol by the kidneys. increased cortisol levels are known to increase appetite and favour abdominal energy storage in pathological doses (cushing disease). interestingly, short sleep duration and chronic stress cause increased levels of cortisol and have resulted weight gain in some individuals. if elevated environmental levels of co2 reduce blood ph, and this then leads to an increased firing rate in the orexin neurons in the hypothalamus, then several key processes in the brain could be affected, leading to greater appetite and increased energy storage. the relationship between increased exposure to co2 and the processes described above has not been investigated previously. an obvious way to test the hypothesis is to conduct long - term animal studies, in which the effects of exposure to elevated levels of co2, as the sole intervention factor, on the amount of food eaten, weight gain, growth rate, and if there are measurable effects, whether they might be explained by a decrease in blood ph. by use of metabolic cages with different co2 concentrations, differences in ee (respiratory calorimetry) and ideally, the animals should be followed over several generations, because the acidic stress is likely to influence the production of cortisol, which is known to induce foetal programming, resulting in increased risk of obesity and related diseases in the offspring. long - term studies in humans prohibited by ethical considerations, but short - term studies to test the effects of moderately elevated levels of co2 on appetite regulation could be performed in respiration chambers. we conducted a small, short - term, crossover design, pilot study, with 6 young healthy males (body mass index 2025 kg m and 2235 years) randomly exposed to ambient air or 8000 p.p.m. the study was reviewed by the science ethics committee for the capital region of denmark, journal number : h-4 - 2011-fsp, and the committee judged that because of the very limited intervention that the project was not notifiable. before the study, we have obtained informed consent from all the participants. subjects ' ad libitum energy consumption increased numerically by 6.1% when exposed to co2 (95% ci : 5.7 to 17.9%, s.d. : 14.8%). though not statistically significant, probably due to insufficient statistical power, the results was in the expected direction and suggests that the hypothesis is worthy of exploration in larger cohorts, both animal and human, using suitable designs to minimize individual variability in the measured outcomes. the cost of health care for an increasingly overweight and obese population has increased dramatically, and efforts to halt the obesity epidemic have failed. we propose that exposure to increasing amounts of co2 in the atmosphere changes regulatory neuronal mechanisms in the hypothalamus, resulting in decreased ee and sleep, and increased appetite and obesity. if substantiated, this could prove to be an additional mechanism in the multifactorial causes of obesity, which will necessitate major political, economic and environmental interventions to combat the obesity epidemic. | human obesity has evolved into a global epidemic. interestingly, a similar trend has been observed in many animal species, although diet composition, food availability and physical activity have essentially remained unchanged. this suggests a common factor potentially an environmental factor affecting all species. coinciding with the increase in obesity, atmospheric co2 concentration has increased more than 40%. furthermore, in modern societies, we spend more time indoors, where co2 often reaches even higher concentrations. increased co2 concentration in inhaled air decreases the ph of blood, which in turn spills over to cerebrospinal fluids. nerve cells in the hypothalamus that regulate appetite and wakefulness have been shown to be extremely sensitive to ph, doubling their activity if ph decreases by 0.1 units. we hypothesize that an increased acidic load from atmospheric co2 may potentially lead to increased appetite and energy intake, and decreased energy expenditure, and thereby contribute to the current obesity epidemic. |
in the aged one, the auditory loss, is one of the three more prevalent chronic conditions, being behind only of the arthritis and the hypertension1 2. according to3, approximately 90% of the people with superior age the 80 years present auditory loss. when it occurs in function of the aging process it is known as presbycusis and it generates one of the crippling riots of the communication, hindering the aged ones to play in the society, because it not only provokes the sensorial privation to hear, as the difficulty of understanding of speaks of that they surround it making it difficult the full communication1 4 5 6. besides, it causes a series of social problems, amongst them : the removal of the social and familiar activities, low self - worth, isolation, solitude, depression and irritability1 7 8. the aging process does not only represent the loss of the auditory threshold, it generates structural and functional modifications compromising all the components of the postural, sensorial control (visual, somatic sensorial and vestibular contest), effector (force, amplitude of movement, biomechanical alignment, flexibility) and the central processing9 10 the physiological reduction of the vision, the hearing, the corporal stability, the articulate alterations and of the muscular power can facilitate to the risks of accidents and fall for the slowing of the defensive reactions11. the falls are dealt with as factor great epidemiologist relevance, social and economic in the whole world, therefore, it is the type most common of accident between the aged ones12. the complications lead the causes of deaths in people above of 65 years and promote physical, psychological, and social deficiency, being able to take the individual the dependence, reduction of the daily activities and the confidence, alteration of the life style13 generating negative consequences in relation to the quality of life14. as the life expectancy is increasing, it is important to adopt measured in which they minimize the damages caused for it a time that the surgical and medicinal indications are rare, taking advantage the use of adaptations of device of individual sonorous amplification (aasi). the use allows the rescue of the perception of the sounds of speaks, beyond the ambient sounds, promoting the improvement of the communication ability15. the increase of the social visibility of the segment of people older than 60 years, in brazil, was one of the factors that had instigated the mobilization of governmental bodies and not governmental for the attendance of the new demands appeared in the scope of the health, the assistance and the social security16. in 2004 the health department, instituted the national politics of attention to the auditory health and through n would carry sas / ms. 587 had distribution of the state net for action in the basic attention, the average and high complexity (brazil, health department, 2004). by means of deliberation cib - sus - mg n 156 in 21/03/2005, the association of parents and friends of exceptional (apae) of patos de minas was credential and qualified to the rendering of services of the related program having guaranteed to the individual the diagnosis, adaptation and supply of the device of individual sonorous amplification - aasi17. in this context, the objective of this study was to evaluate balance, fear of fall and aged quality of life of with bilateral sensorineural auditory loss before and after the adaptation to the device of individual sonorous amplification (aasi). this study was previously approved by the committee of ethics and research with human beings of the unitri (register n 647923) and followed resolution 196/96 of the national advice of health. all the procedures had been explained as well as described at great length in the assent term, that was signed by the volunteers, and from now on, it was initiated collects it of data. the sample was constituted by 56 individuals (32 of the masculine sex) between 60 and 84 years, carriers of bilateral sensorineural auditory loss, chosen teams from handbooks of the service of attention to hearing care (sasa). the handbooks had been constituted by evaluation of the otoloraryngologist, audiological evaluation, speech therapy evaluation, interview with social assistant and psychologist. the aged inserted carriers of bilateral sensorineural loss auditory in the system of attention to hearing care (sasa) had been enclosed of patos de minas / mg brazil, with age between 60 and 84 years ; e excluded the users of prosthesis or orthesis of inferior members ; carriers of neurological and behavioral riots ; individuals previously submitted to the auditory rehab ; not signature of the term of free and clarified assent ; not understanding of the commands for the application of the tests. in the evaluation the identification data had been collected, physicians, anthropometrics (weight, height and index of corporal mass - imc). the volunteers had answered to the questionnaires of quality of life (questionnaire sf-36)18 and of fear to fall (falls efficacy scale - international /fes - i)19 and had carried through the balance test (berg balance scale / bbs)20. after 4 months, the volunteers who have adapted to the aasi, had returned for reevaluation, they had remade the balance test, and had answered beyond the questionnaires cited to the satisfaction with amplification in daily life (sadl)21. all the questionnaires applied in this research previously had been validated and possess adequate psychometrics properties. the prevalence of the variable : sex, degree of auditory loss, presence of humming, presence of vertigo, the sample had been dichotomized and analyzed for the test of the independence qui - square. for comparative analysis of you prop up them of the domains of the sf-36 was used the test of mann whitney. the values of fes - i, berg and bbs between the adapted, not - adapted groups and reevaluated had been compared by means of the analysis of the variance (anova two way) and the test of post - hoc de tukey. to correlate the follow up of the domains of the sf-36, scale bbs, to the fes - i, the presence of vertigo, the presence of humming and to the age of the volunteers the correlation of spearman was used. a level of significance of 5% (p < 0,05) in all the prevalence of the variable : sex, degree of auditory loss, presence of humming, presence of vertigo, the sample had been dichotomized and analyzed for the test of the independence qui - square. for comparative analysis of you prop up them of the domains of the sf-36 was used the test of mann whitney. the values of fes - i, berg and bbs between the adapted, not - adapted groups and reevaluated had been compared by means of the analysis of the variance (anova two way) and the test of post - hoc de tukey. to correlate the follow up of the domains of the sf-36, scale bbs, to the fes - i, the presence of vertigo, the presence of humming and to the age of the volunteers the correlation of spearman was used. a level of significance of 5% (p < 0,05) in all it is noticed in table 1, a homogeneous distribution between the gender and how much to the degree of auditory loss and one high prevalence of humming and vertigo between the participants. table 2 presents the anthropometrics characteristics, age and props up of scales fes - i and bbs of the volunteers. one verifies overweight in the sample, good level of balance and little fear to fall. we observe that volunteers present good shore up in domains of sf-36, what indicates that does not have reduction in the quality of life in aged with auditory loss, however is distinguished reduction of the item vitality (table 3). cf, functional capacity ; af, physical aspect ; egs, general state of health ; vit, vitality ; as, social aspects ; ae, emotional aspects ; sm, mental health. after 4 months of the rank of the auditory device the volunteers who adapted itself to the auditory device had been reevaluated. in our study, 50% of the volunteers adapted to the auditory device. in table 4, it is noted a homogeneous distribution of the anthropometrics variable and age between the groups. how much to the adaptation to the aasi, it is noticed that the masculine sex had greater difficulty in adapting to the auditory device and that the degree of auditory loss, complaints of humming and vertigo, are variable that had not influenced in the adaptation of the aasi (table 5). as pointed in table 6, in relation the humming complaint, only 4 of the 20 volunteers had not related benefits with the use of auditory prosthesis in this variable. how much to questionnaire sadl, in the referring item self - confidence, 100% of the volunteers had told to improvement of this variable after adaptation, being that 90% had answered much improvement of the self - confidence. it is observed that the aged ones that if they did not adapt they present greater functional capacity that the aged ones of the suitable group. however, in the reevaluation of the suitable group (frog), a significant improvement when comparing with the evaluation was verified. furthermore, it had significant improvement of the egs of the suitable group when comparing the values of the evaluation and reevaluation. cf, functional capacity ; af, physical aspect ; egs, general state of health ; vit, vitality ; as, social aspects ; ae, emotional aspects ; sm, mental health. a, suitable group ; in, the group not adapted ; frog, reevaluation of the adapted ones. p < 0,05 comparing the x in + p < 0,05 comparing the x frog in the analysis intergroup, did not have difference between props up them of the bbs and of the fes - i, however, in the analysis intra - group, the suitable group presented minor fear to fall (table 8). a, suitable group ; in, the group not adapted ; frog reevaluation of the adapted ones. p 0,05 comparing the x in + p < 0,05 comparing x r table 9 presents the gotten correlations of the groups evaluated between the domains of the sf-36 and you prop up them of scales fes - i, bbs, presence of vertigo, presence of humming and the age. it is verified that in the suitable group it had positive correlation between the bbs and the domains cf, pain, egs and a negative correlation between the fes - i and the domains cf and pain. cf, functional capacity ; af, physical aspects ; egs, general state of health ; vit, vitality ; as, social aspects ; ae, emotional aspects ; sm, mental health ;, suitable group ; in, the group not adapted, frog, reevaluation of the adapted ones. p 0,005 ; + p < 0,0 after the adaptation of the aasi, notices positive correlation between the bbs and domains cf and egs, as well as, between age ae and. a negative correlation between fear of fall is noticed and domains cf, egs and vit. in the present study it had for purpose to evaluate the contribution of auditory device aasi in the quality of life, balance and fear to fall in aged with bilateral sensorineural auditory loss. to observe greater prevalence of auditory loss in men (57%) corroborating with other studies7 8. the complaint of humming (69.6%) and vertigo (57%) associate to the auditory loss was highly prevalent in the sample a time that inherent degenerative processes to the age are responsible for the occurrence of vertigo and humming in the geriatric population10. the score of the questionnaire that evaluates balance was 51 points, what it represents a good functional balance, probably for the fact of that our sample was constituted by aged not institutionalized with auditory loss and good functional capacity, a time that had been excluded individuals that made use of device of aid to the march and that they presented neurological illnesses. how much to the fear of fall one was observed score average of 25,5 in the volunteers of this research. similar results had been found by22 that when evaluating physically active the aged fes - i of aged of the community and found one medium score of 24,9 and 27,5 respectively. in such a way we can infer that, the auditory loss did not influence in the fear of fall. probably, this is related to the good balance that the aged ones had presented what it diminishes the fear of fall. in general, the averages of the scores of the domains of the sf-36 had been above of 50% demonstrating that the volunteers have good quality of life. second23 in the sf-36 one props up high in the domain physical aspect and functional capacity can indicate that the aged ones possess little limitation to the work and in activities of daily life, presents good health and functional ability. we know that the concept of quality of life (qv) is very ample, subjective, dependent of the socio - cultural level, the personal ambitions of the individuals and not only of the structure attack24. this can be observed in the present study, therefore, nor always a structure attack, as the auditory loss intervenes directly with the quality of life of the volunteers, being the qv a product of some factors. all the volunteers of the sample had putted prosthesis. after 4 months of use of aasi, 50% of the volunteers adapted to the auditory device. in the suitable volunteers it had reevaluation of the variable : this stated period was established in virtue of studies to demonstrate that 90 days are enough for a good adaptation and acclimatization of the aasi25. how much to the factors related to the adaptation to the aasi, our findings are in accordance with study previously carried through that demonstrated that presence of humming and vertigo do not influence in the adaptation to the aasi26. however, valley to point out that, previous studies demonstrate that factors as story of satisfaction with the device, the number of hours / day, participation of the family5, expectations, necessities of communication, degree of loss, tolerance for intense sounds, expectations, motivation4 and auditory whitewashing2, is important criteria for a successful adaptation. one of the main complaints of the aged ones with presbycusis is the presence of the humming that frequent is a factor of great negative repercussion in the life of the individual, compromising the quality of life, making it difficult sleep, the concentration in the daily and professional activities, as well as the social life27. the humming is an extremely frequent clutter in patients with auditory loss, reaching about 40 million people in u.s.a. the practical clinic has demonstrated that carrying individuals of auditory loss associated the humming benefit themselves with the use of auditory prosthesis, therefore these, beyond improving the understanding of the conversation, alleviates the humming,29, had demonstrated that the use of prosthesis auditory improved the humming in 78,2% of the individuals and our sample 79% of the suitable volunteers if they had benefited with reduction of the humming. in accordance with6 the auditory use of prosthesis benefits to the daily activities and the functionality in carrying individuals of sensorineural auditory loss. this corroborates with our findings, therefore we find significant improvement of the functional capacity (cf) and of the general state of health (egs). probably due to adaptation to the aasi, improvement of the hearing, the ability of communication occurred and security feeling, which had also influenced the increase of egs and cf, and consequently improves of the quality of life7. having as endorsement the findings of30 we believe that the increase of the egs resulted consequently in the increase in the daily activities of life and of the cf what it reflected in reduction of the fear of fall of the volunteers. another factor associated with this reduction was possibly the increase of the confidence and security told by the volunteers from the positive results of questionnaire sadl. aged with auditory sensorineural loss they had presented good quality of life and balance and had related little concern in falling. however after adaptation to the aasi, had improvement of the quality of life (egs and cf), reduction of the fear of fall and the humming and increase of the auto - confidence after adaptation to the aasi. valley to point out despite 50% of the volunteers if only adapted and that the masculine sex had greater difficulty in adapting to the auditory device and that the variable age, degree of loss, presence of humming and vertigo, they had not been factors that had intervened with the adaptation to the aasi. from this study we can infer that so important the aasi is for the aged one, how much the direct benefit of it in the physiotherapist intervention is. the well adapted patient will have greater communication capacity, increase of the self - confident, greater attention and understanding of the information that are important factors in a preventive and rehabilitative interaction. valley to point out that the development of new studies that they aim at to elucidate the factors associates to the adhesion to the aasi would be of great interest therefore would prevent unnecessary expenses of the public health. | summary introduction : the aging process provokes structural modifications and functional to it greets, compromising the postural control and central processing. studies have boarded the necessity to identify to the harmful factors of risk to aged the auditory health and security in stricken aged by auditory deficits and with alterations of balance. objective : to evaluate the effect of auditory prosthesis in the quality of life, the balance and the fear of fall in aged with bilateral auditory loss. method : carried through clinical and experimental study with 56 aged ones with sensorineural auditory loss, submitted to the use of auditory prosthesis of individual sonorous amplification (aasi). the aged ones had answered to the questionnaires of quality of life short form health survey (sf-36), falls efficacy international scale- (fes - i) and the test of berg balance scale (bbs). after 4 months, the aged ones that they adapted to the use of the aasi had been reevaluated. results : it had 50% of adaptation of the aged ones to the aasi. it was observed that the masculine sex had greater difficulty in adapting to the auditory device and that the variable age, degree of loss, presence of humming and vertigo had not intervened with the adaptation to auditory prosthesis. it had improvement of the quality of life in the dominance of the state general health (egs) and functional capacity (cf) and of the humming, as well as the increase of the auto - confidence after adaptation of auditory prosthesis. conclusion : the use of auditory prosthesis provided the improvement of the domains of the quality of life, what it reflected consequently in one better auto - confidence and in the long run in the reduction of the fear of fall in aged with sensorineural auditory loss. |
the transportation sector of the united states accounts for over 70% of the nation 's total petroleum consumption, and 57% of the petroleum is imported. in addition, use of petroleum - based fuels contributes to accumulation of greenhouse gases (ghg) in the atmosphere. due to concerns of energy security and ghg emissions, it becomes crucial to develop domestic sustainable alternatives to petroleum - based transportation fuels. biofuels produced from cellulosic biomass (herbaceous, woody, and generally inedible portions of plant matter) are a sustainable alternative to petroleum - based fuels. the united states has the resource to produce over 1 billion dry tons of biomass with more than 80% of cellulosic biomass including about 320 million dry tons of woody biomass annually [5, 6 ]. this amount of biomass is sufficient to produce 90 billion gallons of liquid fuels that can replace about 30% of the nation 's current annual consumption of petroleum - based transportation fuels. in contrast to grain - based biofuels, cellulosic biofuels do not compete for the limited agricultural land with food or feed production. figure 1 shows the major processes of converting woody biomass to ethanol (the most common form of biofuels). size reduction of woody biomass is necessary because large - size woody biomass can not be converted to biofuels efficiently with the current conversion technologies [810 ]. machines available for wood chipping include disk, drum, and v - drum chippers [1214 ]. straight knives are mounted on a flywheel that revolves at a speed ranging from 400 to 1000 revolutions per minute (rpm). wood chips produced by wood chipping usually have sizes ranging from 5 to 50 mm. the second step is biomass milling to further reduce the wood chips into small particles. this step is usually conducted on knife mills or hammer mills [1719 ]. wood particles produced by biomass milling usually have sizes ranging from 0.1 to 10 mm. energy consumption of this step ranged from 0.15 to 0.85 wh / g [15, 20, 21 ]. sieves are installed on knife mills and hammer mills to control the size of wood particles. during biomass milling, wood particles that are smaller than the sieve size (the size of the openings on a sieve) will pass through the sieve ; those larger than the sieve size will be recirculated and milled further. in this study, sieves and sieve size are reserved to describe the sieves installed on knife mills or hammer mills. there are reported studies about the effects of sieve size on energy consumption in woody biomass milling using knife mills or hammer mills. a consistent observation was that energy consumption increased dramatically as sieve size became smaller [2224 ]. however, these reports did not present sugar yield (proportional to ethanol yield) results using the wood particles produced by biomass milling. it was reported that woody biomass with smaller particle size had higher sugar yield [2528 ]. however, particle size in these reported studies was defined differently from the sieve size in this paper. in these studies, wood particles produced by knife mills or hammer mills using a certain sieve size the term particle size was actually the particle size range determined by the sizes of the openings on the screens. in this paper, the size of the openings on the screen is called screen size. moreover, previously reported studies did not present energy consumption data for the biomass milling process used to produce the wood particles from which the sugar yield measurements were performed. the lack of comprehensive studies about the effects of sieve size on energy consumption in size reduction (biomass milling) and sugar yield in hydrolysis makes it difficult to decide which sieve size should be selected in order to minimize the energy consumption in size reduction and maximize the sugar yield in hydrolysis. the purpose of this study is to fill this gap in the literature by studying the effects of sieve size on energy consumption in size reduction and sugar yield in hydrolysis simultaneously. since the purchased wood chips had a wide distribution in size, the wood chips were separated into three groups using two screens with screen size of 5 and 12.5 mm, respectively. are those that passed through the 12.5 mm screen but not the 5 mm screen. examples of large, medium, and small wood chips are shown in figure 3. the moisture content of the wood chips (as purchased) was 1.2%, measured by following the asae standard s358.2. to adjust the moisture content of wood chips to a desired level, distilled water was added (by spraying evenly) to the wood chips. to achieve wood chips of 10% and 18% moisture content, 96 and 233 ml after moisture content adjustment, the wood chips were placed in the sealed ziploc bags and stored in a refrigerator at 4c for at least 72 hours before knife milling. three knives (95 mm long and 35 mm wide) were mounted on the rotor inside the milling chamber. a sieve (145 mm long and 98 mm wide) was mounted at the bottom of the milling chamber. sieves with three sieve sizes (4, 2, and 1 mm, resp.), as shown in figure 6, were used in this study. sieve sizes of 1 and 4 mm were selected because they were the minimum and maximum sieve size, respectively, that could be practically investigated in this study. as described in section 2.1, the wood chips prior to milling had a range of 5 to 12.5 mm. if any available sieve size larger than 4 mm was used, some of the wood chips would fall through the sieve without being cut. furthermore, based on previous experience, if any available sieve size smaller than 1 mm (the next one was 0.5 mm) was used, some of the sieve openings would be blocked by milled particles, causing significant increase in milling time and energy consumption. at the beginning of each test, the knife mill was run for 10 seconds before loading any wood chips to avoid the current spike (this would happen if the knife mill started with wood chips already in the milling chamber). this amount of wood chips was enough to keep the milling chamber approximately full (in volume). during knife milling, more wood chips were loaded into the milling chamber using a scoop as shown in figure 7. the amount of wood chips loaded by the scoop at each time was 5 1 g. these additional wood chips were loaded at a rate that would keep the milling chamber approximately full (in volume) but without causing over loading. in each test, the total amount of wood chips loaded into the milling chamber was 200 g. the milling time was different under different conditions. when a smaller sieve size was used, it took a longer time to mill the same amount of wood chips. after each test, wood particles in the receiving container were collected, weighed, and kept in the sealed ziploc bags. the amount of wood particles collected by the receiving container in each test was less than 200 g, because some wood chips (or particles) did not pass through the sieve yet when the knife mill was turned off. before starting the next test, the milling chamber was opened and any remaining wood chips were cleaned using a brush. to allow the motor to cool down, there was a waiting period (at least five minutes) between two successive tests. in this study, energy consumption is the electricity consumed by the electric motor of the knife mill. as shown in figure 4, electric current to the motor was measured using a fluke 189 multimeter and a fluke 200 ac current clamp (fluke corp., everett, wa, usa). data acquisition began after the first 50 g of wood chips was loaded into the milling chamber and stopped when additional 150 g of wood chips was all loaded into the chamber. the voltage (vln) was 208 v. the energy consumed during each test (that lasted for t seconds) (et) was calculated using the following : (1)et=3iavevlnt3600(wh). dividing et by the weight (w) of the wood particles collected from the receiving container after the test gives energy consumption (e) per unit weight, as expressed in (2) : (2)e = etw(whg). sugar yield in hydrolysis is the amount of glucose produced from hydrolyzing cellulose using enzymes. it was expressed as the concentration of glucose (mg / ml) in the measurement sample. figure 8 shows the four steps in sugar yield measurement. in this study, 10 g of biomass and 200 ml of 2% sulfuric acid were loaded in the 600 ml vessel of a parr pressure reactor (model 4760a, parr instrument co., moline, il, usa). after pretreatment, biomass was washed with hot distilled water using a centrifugal (model pr-7000 m, international equipment co., needham, ma, usa). the purpose of biomass washing was to remove the acid residues and inhibitors (substances that would bind to enzymes and decrease their activity to depolymerize cellulose to glucose) formed during pretreatment. each biomass sample was washed three times, and each time lasted for 15 minutes. rochester, ny, usa) enzyme complex was used for hydrolysis of wood particles into sugars in solution with sodium acetate buffer (50 mm, ph 4.8) and 0.02% (w / v) sodium azide to prevent the microbial growth during hydrolysis. enzymatic hydrolysis was carried out in 125 ml flasks with 50 ml of slurry in the water bath shaker (model c76, new brunswick scientific, edison, nj, usa) with agitation speed of 110 rpm at 50c for 72 hours. the dry mass content of the hydrolysis slurries was 5% (w / v) and the enzyme loading was 1 ml / g of dry biomass. sugar analysis was done using an hplc (shimadzu corp., kyoto, japan) equipped with an rpm - monosaccharide column (300 7.8 mm ; phenomenex, torrance, ca, usa) and a refractive index detector (rid-10a, shimadzu, kyoto, japan). the mobile phase was 0.6 ml / min of double - distilled water, and oven temperature was 80c. particle size distribution was determined using a screen shaker (model ro - tap 8 rx-29, w.s. tyler industrial group, mentor, oh, usa) as illustrated in figure 9. a stack of screens from the bottom to the top were arranged from the smallest to the largest in screen size. the screen sizes used were 0.1, 0.2, 0.4, 0.6, 1.2, 2.4, 5.6, and 6.3 mm. a pan (no openings) the screen shaker provided circular motion to the stack of screens at the rate of 278 rpm. simultaneously, the tapping hammer hit the top of the stack at the frequency of 150 times per minute. the percentage of the wood particles in each of the nine particle size ranges (6.3 mm) was translated to particle size distribution. for instance, when knife milling of wood chips with moisture content of 1.2%, energy consumption was as high as 1.38 wh / g for 1 mm sieve size and only 0.16 wh / g for 4 mm sieve size. the same trend was observed for the other two levels of moisture content. in the literature, there are no reports about the effects of sieve size on energy consumption in knife milling of poplar wood chips. phanphanich and mani used a knife mill (of the same model as the one used in this study) to reduce the size of pine wood chips (including chips, branches, barks, leaves, and small particles). the hammer mill was manufactured by sears roebuck and co. (hoffman estates, il, usa). the size of the willow wood chips (three dimensions) was 1350, 13 - 76, and 525 mm. energy consumption in hammer milling using the 1, 2, and 4 mm sieves was 1.55, 0.66, and 0.39 wh / g, respectively. moisture content of poplar wood chips also affected energy consumption in knife milling. as shown in figure 11, energy consumption in knife milling increased when moisture content increased from 1.2% to 10% and decreased slightly when moisture content increased from 10% to 18%. the literature does not have any reports about the effects of moisture content on energy consumption in knife milling of wood chips using the knife mill of the same model as the one used in this study. however, there are reports on these effects in knife milling of herbaceous biomass (such as miscanthus, switchgrass, and wheat straw). miao. investigated energy consumption in knife milling of miscanthus and switchgrass using the same model of knife mill. it was found that when moisture content increased from 710% to 15%, energy consumption in knife milling increased significantly. the same trend was also found in size reduction of wheat straw, barley straw, corn stover, and switchgrass using a hammer mill. according to mani., an increase in moisture content of cellulosic biomass would increase the shear strength of the biomass ; therefore, more energy was consumed in milling of cellulosic biomass. materials used for sugar yield evaluation were the particles produced by knife milling of wood chips with the moisture content of 1.2%. for each sieve size, there were two independent samples processed for sugar yield evaluation. the results showed that wood particles processed using the 4 mm sieve had the highest sugar yield while sugar yields of wood particles processed using the 1 and 2 mm sieves were approximately the same. wheat straw particles milled using the 2 mm sieve had higher sugar yield than those milled using the 1 mm sieve. in their work, wheat straw, corn stover, and big bluestem were milled using a hammer mill (model 18 - 7 - 300, schuttle - buffalo hammermill, buffalo, ny, usa) using 3.2 and 6.5 mm sieves. for these three types of cellulosic materials, biomass particles milled using the 6.5 mm sieve yielded more sugar than those milled using the 3.2 mm sieve (figure 14). both these reported studies involved a pelleting process (agglomerating biomass particles produced by milling into pellets) before sugar yield. figures 15 and 16 show the effects of woody biomass particle size on sugar yield reported in the literature. in dasari and berson 's study, red oak saw dust was screened into four particle size ranges. as shown in figure 15, particles in the size range of 0.030.08 mm yielded 80% more sugar than those in the size range of 0.590.85 mm. in zhu. 's study, spruce wood chips were hammer milled in three successive steps using sieve sizes of 12.7, 4.8, and 0.8 mm, respectively. as shown in figure 16, particles in the size range of smaller than 0.32 mm yielded 1.6 times more sugar than those in the size range of larger than 1.27 mm. figure 17 shows the wood particles produced by knife milling using the three different sieve sizes (4, 2, and 1 mm resp.). the particles produced using the same sieve did not have a uniform size. their size distribution is shown in figure 18. in himmel. 's study, poplar wood chips were processed by a knife mill (mitts & merrill frmag group, harvard, il, usa) using 1/16, 1/8, and 3/32 inch (1.59, 3.18, and 2.38 mm) sieves. the results from this study and the studies conducted by zhang. and theerarattananoon. however, results reported by dasari and berson and zhu. show that wood particles in the smaller size range had higher sugar yield. at this point in time however, some differences in test conditions were noticed. in the studies reported by dasari and berson and zhu., wood particles were from relatively narrow size ranges. in this work, further investigations will be carried out to study the effects of particle size distribution on woody biomass sugar yield. in this study, effects of sieve size on energy consumption in knife milling of poplar wood chips and sugar yield in hydrolysis were studied. poplar wood particles processed by knife milling using the 4 mm sieve had higher sugar yield than those processed by knife milling using the 1 and 2 mm sieves. knife milling of wood chips using the 4 mm sieve consumed less energy in size reduction than using the 1 and 2 mm sieves. the wood particles knife milled using the 4 mm sieve had higher sugar yield in hydrolysis than those milled using the 1 and 2 mm sieves. this finding is very important when deciding what sieve size is to be used in knife milling of wood chips to minimize energy consumption in size reduction and maximize sugar productivity in hydrolysis. in future study, the authors will also use 0.25, 0.5, and 8 mm sieves to further investigate the effects of sieve size on energy consumption in size reduction and sugar yield in hydrolysis. a hammer mill will be utilized to see if similar results can be obtained on different types of milling machines. more types of cellulosic materials will be tested to see if conclusions obtained in this study can be extended to different types of cellulosic biomass. | size reduction is the first step for manufacturing biofuels from woody biomass. it is usually performed using milling machines and the particle size is controlled by the size of the sieve installed on a milling machine. there are reported studies about the effects of sieve size on energy consumption in milling of woody biomass. these studies show that energy consumption increased dramatically as sieve size became smaller. however, in these studies, the sugar yield (proportional to biofuel yield) in hydrolysis of the milled woody biomass was not measured. the lack of comprehensive studies about the effects of sieve size on energy consumption in biomass milling and sugar yield in hydrolysis process makes it difficult to decide which sieve size should be selected in order to minimize the energy consumption in size reduction and maximize the sugar yield in hydrolysis. the purpose of this paper is to fill this gap in the literature. in this paper, knife milling of poplar wood was conducted using sieves of three sizes (1, 2, and 4 mm). results show that, as sieve size increased, energy consumption in knife milling decreased and sugar yield in hydrolysis increased in the tested range of particle sizes. |
osteopontin (opn) is a matricellular protein originally isolated from the bone, expressed by various cell types including macrophages, dendritic cells, and activated t cells. opn mediates several biological functions such as bone remodeling, macrophage response, cell migration, and adhesion, and it is involved in the pathogenesis of several diseases including atherosclerosis, cancer, chronic inflammatory diseases, and several autoimmune diseases [13 ]. opn costimulates t cell activation and supports differentiation of proinflammatory t helper 1 (th1) and th17 cells [4, 5 ]. opn is a member of the sibling (small integrin binding ligand n - linked glycoprotein) protein family. it has two calcium binding sites, two putative heparin binding domains, and multiple adhesion motifs which allow interaction with several receptors and cell types. opn biological functions are influenced by posttranslational modifications, such as phosphorylation, glycosylation, and protein cleavage mediated by thrombin and metalloproteinases [79 ]. opn has rgd (arginine - glycine - aspartate) integrin binding domain, through which it mediates interactions with v1, v3, v5, v6, 81, and 51 integrins [1013 ]. moreover, 91, 41, and 47 integrins bind to a cryptic svvyglr (slayglr in mice) sequence which is exposed upon thrombin cleavage occurring at the arg168-ser169 site near to the rgd motif [1416 ]. this cleavage generates two fragments, the n- and c - terminal, displaying functional differences from the full length protein. the n - terminal fragment (opn - n, approx. 35 kda) binds to integrins through the rgd or the cryptic binding site, promotes ifn- secretion in t cells, and stimulates cell migration by binding to 91 and 41 [18, 19 ]. the c - terminal fragment (opn - c, approx. 25 kda) inhibits il-10 secretion and stimulates cell - cell adhesion by interacting with cd44 isoforms containing the v6 and v7 domains [20, 21 ]. opn is highly expressed in several tumors, where it can be cleaved by thrombin and acts as a proangiogenic factor [2225 ]. moreover, the opn : cd44 interaction promotes metastasis dissemination in a variety of malignancies. the individual role of opn - n and opn - c has been investigated mainly in cancer cells because of the expression of both opn and activated thrombin in the microenvironment of several tumors [22, 27, 28 ]. on the contrary full length opn (opn - fl) is expressed at similar levels in the synovial fluid of patients with rheumatoid arthritis (ra) and in those with osteoarthritis, but opn - n levels in ra synovial fluid samples are around 30-fold higher than in those from osteoarthritis and correlate with the disease status. a considerable body of evidence suggests that opn plays a detrimental role in multiple sclerosis (ms) and its animal model, experimental autoimmune encephalomyelitis (eae) [3032 ]. in ms lesions, high opn levels are present in the perivascular cuff that surrounds inflamed blood vessels, contains inflammatory lymphocytes, and is delimited by the endothelium and the basement membrane. at this site, opn plays a role in lymphocyte recruitment into the ms lesion, which involves 41 integrin that is the target of the anti - ms drug natalizumab, a humanized monoclonal antibody that has had benefic effects for relapses prevention in rr - ms. opn binds to 41 integrin only upon cleavage by thrombin which unmasks two 41 integrin binding sites located into the n - terminal fragment. showed that the administration of recombinant opn - fl exacerbates eae, but the relative role of opn - n and opn - c is not known. however, thrombin - mediated cleavage may play a role, since thrombin activity increases with the progression of neuroinflammation, and it is detectable in the demyelinating lesions where also opn is present at high levels [35, 36 ]. moreover, in vivo administration of hirudin, a thrombin inhibitor, decreases clinical severity, demyelination, and secretion of th1- and th17-type cytokines in eae [37, 38 ]. opn modulates several cell activities in vitro, but the role of opn cleavage and the relative role of opn - c and opn - n are only partly known. moreover, several available data are difficult to compare because they have been obtained with heterogeneous approaches, such as antibody - mediated blockage of the opn receptors, use of opns produced in bacteria and eukaryotic cells, which influence the posttranslational modifications involved in opn function, or use of a mixture of opn fragments obtained by in vitro treatment with thrombin. the aim of our research was to recapitulate, in vitro, the opn effects on human cells with a particular focus on processes involved in ms relapse, that is, t cell and monocyte activation, t cell apoptosis, lymphocytes, and endothelial cell migration and adhesion, by using recombinant forms of opn - fl, opn - n, and opn - c produced in an eukaryotic system in order to ensure their posttranslation modifications. moreover, we investigated the activity of these opns and a point - mutated form of opn resistant to thrombin cleavage (opn - fl) on the eae course in vivo in mice, since the functional in vivo role of the opn cleavage and opn fragments is far from being elucidated. the coding sequences lacking the signal sequence, of the opn full length (opn - fl) and the two thrombin - cleaved fragments (opn - n and opn - c), were amplified by pcr with specific oligonucleotides and cloned into pmb - sv5 vector downstream from the immunoglobulin leader sequence. the thrombin - uncleavable opn constructs (opn - fl) was generated by mutating the mouse opn sequence from aggtca coding for amino acids r153 and s154 to the agcttt coding for s153 and f154. this substitution has been described as yielding a thrombin cleavage resistant opn. in order to introduce the mutation, we performed site - directed mutagenesis using a mutagenic oligonucleotide specific for each sequence in combination with the reverse oligonucleotide mapping on the c - terminal end of the molecule. after amplification and restriction digestion, each mutated fragment was ligated with the wild - type upstream fragment obtained by restriction digestion to reconstitute the full length sequence. all these opn constructs were subsequently amplified with specific oligonucleotides and cloned as six histidine- (6xhis-) tagged molecules into pucoe vector. for this cloning, we used a common forward oligonucleotide annealing on the kozak sequence of the pmb - sv5 vector and a specific reverse oligonucleotide carrying 6xhis sequence. recombinant opn molecules were produced in chinese hamster ovary suspension cells (chos ; invitrogen, burlington, on, canada, usa). cells were cultured in chos - sfmii medium (invitrogen) and transfected with the pucoe - opn6xhis plasmids using freestyle max reagent (invitrogen) according to the manufacturer 's instructions. to maintain stable transgene expression, transfected cells were cultured under selective pressure using 200 g / ml of hygromycin b (invitrogen). the presence of each recombinant opn construct in the cell supernatant was verified by western blotting using either an antibody directed against the his tag (tetra - his antibody, qiagen, valencia, ca, usa) or an anti - opn antibody directed against an epitope located in the n- or c - terminal half of the molecule : spp1 polyclonal antibody (invitrogen) and polyclonal anti - osteopontin antibody (millipore, billerica, ma, usa), respectively. these antibodies were detected using the appropriate horseradish peroxidase - conjugated secondary antibody (sigma - aldrich, st. the reactive proteins were visualized by the ecl (perkin elmer, waltham, ma, usa). all recombinant proteins were correctly recognized, and they displayed the expected size (i.e., 60 kda for opn - fl and opn - fl, 35 kda for opn - n, and 25 kda for opn - c). cells were grown at high density using celline devices (bd biosciences, san diego, ca, usa) and collected twice a week. after centrifugation at 400 g for 10 minutes, cell supernatants were collected and each recombinant protein was purified on his trap excel ni - sepharose resin (ge healthcare, uppsala, sweden), dialyzed overnight against pbs, and analyzed by western blotting and coomassie gel staining (sigma - aldrich). peripheral blood mononuclear cells (pbmcs) were separated from human blood samples obtained from healthy donors, who signed their written informed consent, by density gradient centrifugation using the ficoll - hypaque reagent (lympholyte - h, cedarlane laboratories, burlington, on, canada). the use of pbmcs was approved by the ethics committee of the azienda ospedaliera universitaria maggiore della carit of novara (prot. cd4 t cells and monocytes were negatively purified from pbmcs using the easysephuman cd4 negative selection kit and easysep human cd14 negative selection kit, respectively (stem cells technologies, vancouver, bc, usa). cell purity was checked by immunophenotypic analyses and was higher than 95%. peripheral blood lymphocytes (pbl) cultures were performed in rpmi 1640 supplemented with 10% fetal bovine serum (fbs), and 100 u / ml penicillin, 100 g / ml streptomycin (invitrogen). for interferon- (ifn-), interleukin- (il-) 17a, and il-10 secretion and intracellular staining, 0.1 10 cd4 t cells were activated with anti - cd3 (1 g / ml, clone : okt3) and anti - cd28 (2 g / ml, ancell, bayport, mn, usa) in the presence or absence of opn - fl 1 g / ml, opn - n, or opn - c 0.5 g / ml for 5 days. for tissue inhibitor metalloproteinase-1 (timp-1) and il-6 secretion, 0.1 10 monocytes were cultured in the presence or absence of opn - fl 1 g / ml, opn - n, or opn - c 0.5 g / ml for 2 days. the effects of opn - fl were compared to that of a commercial opn - fl purchased from r&d system, and we obtained the same results. human umbilical vein endothelial cells (huvecs) were isolated from human umbilical veins via trypsin treatment (1%) and cultured in m199 medium (sigma - aldrich) with the addition of 20% fcs (invitrogen) and 100 u / ml penicillin, 100 g / ml streptomycin, 5 ui / ml heparin, 12 g / ml bovine brain extract, and 200 mm glutamine (hyclone laboratories, south logan, usa). the purity of the ec preparation was evaluated using morphologic criteria and positive immunofluorescence for factor viii. the use of huvecs was approved by the institutional review board of the presidio ospedaliero martini of turin (prot. 263 - 07/nf) that waived the need for consent ; the data were analyzed anonymously and conducted in accordance with the declaration of helsinki. concentrations of il-17a, ifn-, il-10, il-6, and timp-1 were measured in culture supernatants by elisa according to the instructions of the manufacturers (r&d system, minneapolis, mn, usa ; ebioscience san diego, ca, usa and biolegend, san diego, ca, usa). absorbance was detected with a microplate reader (bio - rad, hercules, ca, usa), and the i - smart program was used to calculate the standard curve. cd4 t cells (1 10) were cultured for 5 days in round - bottomed 96-well plates in the presence of anti - cd3 (1 g / ml) plus anti - cd28 (1 g / ml) mab and in the presence of recombinant opn - fl 1 g / ml, opn - n, or opn - c 0.5 g / ml. after 5 days of culture, the cells were restimulated with phorbol 12-myristate 13-acetate (pma, 50 ng / ml ; sigma - aldrich) plus ionomycin (500 ng / ml ; sigma - aldrich) for 5 hours in the presence of bfa (10 g / ml ; sigma - aldrich). then, cells were permeabilized, stained with a pe - conjugated anti - il-10 mab (miltenyi biotec gmbh, bergisch gladbach, germany) and apc - conjugated anti - il-17a mab (ebioscience, san diego, ca, usa), and analyzed by flow cytometry. activation induced cell death (aicd) was evaluated on t cell lines obtained by activating pbmc with phytohemagglutinin (pha, sigma - aldrich ; 1 g / ml) and cultured in rpmi 1640 medium + 10% fbs + il-2 (2 u / ml ; sigma - aldrich) for 6 days. in the aicd assay, cells (5 10/well) were cultured in wells coated with anti - cd3 mab (okt3, 1 g / ml) with rpmi + 5% fbs + 1 u / ml il-2 in the presence or absence of opn - fl (1 g / ml), opn - n, or opn - c (0.5 g / ml). assays were performed in triplicate and results were expressed as relative cell survival % calculated as follows : (total live cell count in the assay well / total live cell count in the respective control well) 100. in the boyden chamber (bd biosciences) migration assay, resting pbl or huvecs (5 10 or 2 10, resp.) were plated onto the apical side of 50 g / ml matrigel - coated filters (8.2 mm diameter and 0.3 m or 0.5 m pore size ; neuro probe, inc. ; biomap snc, milan, italy) in rpmi or m200 serum - free medium, with or without opn - fl (10 g / ml), opn - n (5 g / ml), or opn - c (5 g / ml). mediums containing 1 ng / ml rantes (r&d system) or 10 ng / ml vascular endothelial growth factor (vegf-, r&d system) were placed in the basolateral chamber as a positive chemoattractant stimuli for pbl and huvecs, respectively. after 20 h, the cells on the apical side were wiped off with q - tips. the cells on the bottom of the filter were stained with crystal violet, and all were counted (fourfold filter) with an inverted microscope (magnification 40x). data are shown as percentages of the treated cells migration versus the control migration measured for untreated cells. control migration is (mean sem) 263 45 cells for huvecs (n = 5) and 155 25 for lymphocytes (n = 5). huvecs were grown to confluence in 24-well plates in complete m200 medium (promocell gmbh, heidelberg, germany) and then treated or not with opn - fl (10 g / ml), opn - n (5 g / ml), or opn - c (5 g / ml) for 30 min, washed with fresh medium twice, and incubated for 1 h with resting pbl (5 10 cell / well). the 1 h incubation time was chosen to allow full sedimentation of the adhering cells, but similar results were obtained with a shorter incubation time (30 min). after incubation in the adhesion assay, nonadherent cells were removed by washing three times with m200. adherent cells were counted by the image - pro plus software for microimaging (media cybernetics, bethesda, md, version 5.0). data are shown as percentages of the treated cells adhesion versus the control adhesion measured for untreated cells. this control adhesion was (mean sem) 35 4 cells per microscope field (n = 5). in the tube formation assay, huvecs were cultured in m200 serum - free medium and seeded onto 48-well plates (2.5 10/well) previously coated with 150 l of growth factor - reduced matrigel (bd biosciences) in the presence of opn - fl (10 g / ml), opn - n (5 g / ml), opn - c (5 g / ml), or control medium with vegf- (10 ng / ml, r&d system). the morphology of the capillary - like structures formed by the huvecs was analyzed after 6 h of culture using an inverted microscope (leica microsystem ; magnification 10x) and was photographed with a digital camera (leica microsystem). tube formation was analyzed and the number of tubes (with branching at both ends) was counted with an imaging system (image - pro plus software for microimaging, media cybernetics, version 5.0, bethesda, md, usa). tube formation was evaluated by counting the total number of tubes in three wells (n = 5) as previously described. specific pathogen - free female c57bl/6 mice were purchased from harlan (harlan laboratories, indianapolis, in, usa). the experimental protocol and animal handling were approved by cesapo, the ethical committee of the university of piemonte orientale (permit number : 10/2013). to induce eae, eight - week - old mice (n = 48) were immunized with 200 g of mog3555 peptide (espikem, firenze, italy) emulsified in complete freund adjuvant (sigma - aldrich) containing 4 mg / ml heat - killed mycobacterium tuberculosis (difco laboratories, detroit, mi, usa). on the day of mog3555 immunization and 48 h later, the mice were injected intraperitoneally (i.p.) with pertussis toxin (sigma - aldrich, 500 g in 0.1 ml of pbs). twenty days after the remission, mice were divided into different experimental groups receiving daily injection of 5 g of different opn variants (opn - fl, opn - n, opn - c, or a mixture of opn - c + opn - n). moreover, since opn - fl is cleaved by thrombin in vivo, we also injected opn - fl, lacking the thrombin cleavage site. animal health was evaluated daily, throughout the duration of the experiment. since the applied eae protocol and the treatments with recombinant opns do not cause a long and intense suffering, we never administered an analgesic therapy. to prevent malnutrition in palsy mice, starting from eae score 3.5 on, we put food and water directly into the cage, where they could easily reach. euthanasia was performed by cervical dislocation after a light inhalational anesthesia with isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro - ethane) (isoflurane vet fl vt, merial italia spa, pd, italy) using a precision out - of - circuit vaporizer (isotec 4 series 1789, 2biological instruments snc, va, italy) in a rodent induction chamber (2biological instruments snc). the statistical analyses were performed with graphpad prism 3.0 software (san diego, ca, usa) using the wilcoxon 's signed rank test. the friedman anova test for repeated measures followed by dunn 's multiple comparison was used to analyze the daily clinical eae score. both the human and murine leaderless opn sequences, lacking the signal sequence, were cloned into pucoe vector (opn - fl). in order to assess the role of thrombin cleavage on opn activity, we also cloned the following mouse and human opn variants : opn - n including aa 17168 (human) or 17153 (mouse) of opn ; opn - c including aa 169314 (human) or 154294 of opn ; opn - fl carrying a mutated thrombin cleavage site (from r153-s154 to s153-f154) (figure 1(a)). the cdna coding for all these variants was cloned as fusion proteins with the 6xhis tag and stably transfected into cho cells. the presence of the recombinant proteins was verified in the culture supernatants by coomassie staining and by western blotting using antibodies designed against different epitopes of opn or the his tag (figure 1(b)). all recombinant proteins displayed the expected sizes, that is, 60 kda for opn - fl and opn - fl, 35 kda for opn - n, and 25 kda for opn - c, without presence of degradation products and/or contamination by other proteins. as expected, we evaluated the effect of the human opn - fl, opn - n, and opn - c on secretion of ifn-, il-17, and il-10 by t cells, since opn is known to stimulate secretion of ifn- and il-17 and to inhibit secretion of il-10 [43, 44 ]. cd4 t cells from healthy donors were activated by triggering cd3 and cd28 and cultured in the presence and absence of opn - fl, opn - n, and opn - c for 5 days. then, secretion of il-17a, ifn-, and il-10 was evaluated by elisa in the culture supernatants. the results showed that all opn preparations were similarly active in increasing secretion of ifn- (figure 2(a)), whereas secretion of il-17a was significantly increased by opn - fl and opn - n but not opn - c (figure 2(b)), and secretion of il-10 was significantly decreased by opn - fl and opn - c but not opn - n (figure 2(c)). il-10 and il-17 expression was evaluated also by intracellular staining of cells restimulated for 5 h with pma and ionomycin after the 5-day cultures. cytofluorimetric analysis showed that the proportion of il-17a single positive cells was significantly increased by opn - fl and opn - n but not opn - c, whereas the proportions of il-10 single positive cells and il-17a / il-10 dual positive cells were significantly decreased by all opn preparations (figure 2(d)). we evaluated the effect of the human opn - fl, opn - n, and opn - c on secretion of il-6 and timp-1 in monocytes, since opn is known to stimulate secretion of both molecules which play a key role in inflammation and several pathological conditions [45, 46 ]. monocytes from healthy donors were cultured in the presence and absence of opn - fl, opn - n, and opn - c for 2 days. then, secretion of il-6 and timp-1 was evaluated by elisa in the culture supernatants. the results showed that secretion of il-6 was induced mildly by opn - fl and strikingly by opn - n, whereas opn - c had no significant effect (figure 2(e)). by contrast, secretion of timp-1 was similarly induced by opn - fl, opn - n, and opn - c (figure 2(f)). we evaluated the effect of the human opn - fl, opn - n, and opn - c in aicd of t cells, since opn is known to inhibit t cell aicd, which is a key mechanism of peripheral tolerance. pha - activated t cells obtained from healthy donors were treated with anti - cd3 mab to induce aicd in the presence and absence of opn - fl, opn - n, and opn - c, and cell survival was evaluated after 16 h. the results showed that aicd was inhibited to the same extent by opn - fl, opn - n, and opn - c (figure 3). we evaluated the effect of the human opn - fl, opn - n, and opn - c on lymphocyte migration and adhesion to vascular endothelial cells, since opn is known to induce both cell adhesiveness and migration. in the migration experiments, pbl were seeded in the upper side of a boyden chamber in serum - free medium ; opn - fl, opn - n, and opn - c and rantes, used as positive controls for chemoattraction of lymphocytes, were loaded in the lower side of the boyden chamber. the results showed that migration of lymphocytes was induced by opn - fl and, to a greater extent, opn - n, but not opn - c (figure 4(a)). in the adhesion experiments, huvecs were treated with opn - fl, opn - n, and opn - c for 30 min, washed, and then used in the adhesion assay with pbl. the results showed that adhesion was induced by both opn - fl and, to a greater extent, opn - c but not opn - n (figure 4(b)). we evaluated the effect of the human opn - fl, opn - n, and opn - c in angiogenesis in vitro by assessing migration and tube formation on huvecs, since opn is known to induce angiogenesis. the results overlapped those obtained with pbl, since migration was induced by opn - fl and, to a greater extent, opn - n, but not opn - c (figure 5(a)). in the tubulogenesis assay, huvecs were seeded onto growth factor - reduced matrigel in the presence or absence of opn - fl, opn - n, and opn - c, and the morphology of capillary - like structures formed by huvecs was analyzed after 6 h. the results showed that opn - n and opn - c induced high levels of tube formation, whereas opn - fl had a mild, although significant, effect (figure 5(b)). the first part of this work has been performed on human cells which are the therapeutic targets in human diseases. to assess whether the opn forms exert different effects also in vivo, we moved to mouse eae, a model of ms. eae is a t cell - mediated autoimmune disease characterized by perivascular cd4 t cell and mononuclear cell infiltration, causing demyelination areas in the central nervous system, leading to progressive hind - limb paralysis. several works showed that, in the mouse, opn displays similar effects than in human cells in terms of cytokine secretion, cell migration [50, 51 ], and angiogenesis. moreover, we performed pilot experiments using t cells and opn variants from mice, which confirmed the pattern of opn - fl, opn - c, and opn - n effects detected in the human model in terms of secretion of ifn-, il-17, and il-10, migration, and adhesion, which are key factors in eae pathogenesis (data not shown). eae can be induced by immunization with myelin proteins, such as proteolipid protein (plp) or myelin basic protein (mbp) or myelin oligodendrocyte glycoprotein (mog) in complete freund 's adjuvant (cfa). in c57bl/6 mice, the disease can be induced by immunization with the mog immunodominant epitope corresponding to amino acids from 35 to 55 (mog3555). in this model, mice develop progressive paralysis (relapse) followed by a stable remission. therefore, we induced eae in c57bl/6 mice and waited for the remission and, 20 days later, we started daily injections with the opn variants and monitored relapse development. we compared the effect of the mouse opn - fl, opn - n, opn - c, or opn - c + opn - n. since opn - fl in vivo is cleaved by thrombin, we also injected opn - fl, lacking the thrombin cleavage site. the results showed that opn - fl, opn - c, and opn - c + opn - n induced a similar strong relapse of the disease (p < 0.001) (figure 6), whereas opn - n induced a mild relapse (p < 0.05) and opn - fl had no significant effect. in this study, we produced recombinant proteins corresponding to the human opn - fl, opn - n, and opn - c. these proteins were produced in an eukaryotic system in order to ensure the posttranslational modifications influencing opn functions, and they were used to investigate their individual activity on key players in the immune response, such as lymphocytes, monocytes, and endothelial cells. we also used the same approach to produce the mouse opn - fl, opn - n, and opn - c, together with a thrombin - resistant form of opn - fl in which the thrombin cleavage site was mutated (opn - fl) and assessed their individual effect in vivo on mouse during eae relapse. in t cells, opn cleavage seems not to influence the effect on ifn- secretion, which marks activation of th1 cells since secretion of this cytokine is similarly costimulated by opn - fl, opn - n, and opn - c. this suggests that production of ifn- is similarly costimulated by the triggering of either integrins or cd44, and no incremental effect is ascribable to the integrin cryptic site exposed in opn - n. a similar reasoning may be applied to inhibition of aicd, which plays a key role in switching off the immune response and was similarly inhibited by opn - fl, opn - n, and opn - c. this was not surprising, since both the rgd - dependent triggering of integrins and the triggering of cd44 v6 - 7 have been shown to protect cells from apoptosis [53, 54 ]. by contrast, secretion of il-17 and il-10, respectively, upmodulated and downmodulated by opn - fl, differentially involves the two opn fragments, since il-17 upmodulation is mainly ascribable to opn - n, whereas il-10 downmodulation is mainly ascribable to opn - c. these results are in line with reports showing that the opn effects on il-17 and il-10 are selectively inhibited by anti - integrin and anti - cd44 antibodies, respectively. the il-17a secretion data were confirmed by intracytoplasmic expression of il-17a. by contrast, the different effects of the opn preparations on il-10 secretion were not confirmed by intracytoplasmic staining of il-10 since all opns similarly decreased the proportion of il-10 cells. since cell staining was performed at day 5, whereas supernatant analysis evaluated the overall secretion during the whole culture time, it is possible that the supernatant differences were ascribable to initial phases of the culture. another point is that all opn preparations decreased the proportions of il-10/il-17a double positive cells, which suggest that they can work in pushing these cells toward a frankly proinflammatory th17 effector function [55, 56 ]. a similar coordinated effect of opn fragments can be envisaged on lymphocyte adhesion to endothelial cells and migration, which are two key steps of lymphocyte extravasation and homing into tissues. our results, indeed, showed that both adhesion and migration are supported by opn - fl, but adhesion is ascribable to opn - c, whereas migration is ascribable to opn - n, and each fragment displays its effect at higher levels than opn - fl. the experiment on huvecs showed that also the opn effect on migration of endothelial cells was ascribable to opn - n and not to opn - c. by contrast, both opn - n and opn - c displayed a strikingly higher effect than opn - fl in inducing tubulogenesis, which is an in vitro assay of neoangiogenesis. these results are in line with the reports from senger and colleagues showing that vegf induces opn and v3 expression in endothelial cells and stimulates cleavage of opn by thrombin and that the resulting opn fragments are strongly chemotactic for endothelial cells and promote angiogenesis. however, these authors used a mixture of the two opn fragments obtained by in vitro thrombin - mediated cleavage of opn - fl, and they could not distinguish the individual role of opn - n and opn - c. moreover, other studies have shown that, in vascular endothelial cells, opn enhances vegf- expression, which, in turn, mediates a positive feedback on opn expression ; the blocking of this feedback signal by anti - vegf- antibodies partially inhibits the opn - induced huvecs motility, proliferation, and tube formation. in monocytes, the main finding was a marked putative effect of thrombin cleavage on il-6 secretion, which was induced moderately by opn - fl and strikingly by opn - n, whereas opn - c displayed no effect. this differential effect was detected on il-6 but not on timp-1 whose secretion was similarly induced by all opn preparations, and this suggests that opn - n fine - tunes monocyte activation [32, 49, 59, 60 ]. altogether our in vitro experiments show not only that the opn effects on il-17 and il-6 secretion and cell migration are mainly ascribable to opn - n, whereas those on il-10 secretion and cell adhesion are mainly ascribable to opn - c, but also that these effects are exerted at higher levels by the appropriate opn fragment than by opn - fl, which suggests that thrombin - mediated cleavage plays a key role in opn activity. a striking gain of function was also detected in the tubulogenesis assay in which both opn - n and opn - c were much more active than opn - fl. in the case of opn - n, this gain of function may be ascribed to exposure of the cryptic integrin binding site that allows binding to integrins unbound by opn - fl. by contrast, it is difficult to explain it for opn - c, whose only known binding site is that for cd44, which is present also in opn - fl. besides the possibility that opn - c exposes unknown cryptic binding site(s) for unknown ligand(s), the gain of function of opn - c might be ascribed to removal of the n - terminal portion of opn - fl, which may exert a partial steric or functional interference on cd44 triggering. also the in vivo experiments showed that thrombin - mediated cleavage of opn plays a key role in opn function, since opn - fl was much more effective in inducing eae relapses than opn - fl, which is resistant to thrombin - mediated cleavage. therefore, the effect of opn - fl must be ascribed to the fragments produced by thrombin cleavage in vivo. use of the recombinant opn - c and opn - n showed that the effect was ascribable to opn - c, whereas opn - n was active only weakly. the critical role of opn - c was surprising, since the presence of the cryptic binding sites for 41 would instead draw the attention to opn - n because 41 is involved in the homing of t cells into the central nervous system, and it is the target of the anti - ms drug natalizumab. substantial evidence indicates that opn - fl plays a detrimental role in ms and eae [32, 46, 61 ]. opn - fl has been found to be the most abundantly expressed cytokine in the lesions of ms patients and eae mice. moreover, opn deficient mice and mice injected with anti - opn antibodies develop a mild eae, whereas administration of opn - fl triggers relapses in several eae models. recently, proteomic analysis of ms lesions has unraveled a potential link between the coagulation cascade and ms pathology, which is supported by eae data showing that administration of the thrombin inhibitor hirudin decreases clinical severity, demyelination, and th1 and th17 cytokines secretion [36, 38 ]. intriguingly, thrombin activity is increased in the demyelinating lesions where opn is expressed at high levels. our data confirm that thrombin - mediated cleavage of opn plays a key role in ms relapse by exerting a dual effect. on the one hand, they may play a key role in the homing of autoreactive lymphocytes in the central nervous system lesions, since opn - n increases production of il-17 involved in breaking the blood brain barrier and stimulates lymphocyte migration, whereas opn - c increases lymphocyte adhesion to vascular endothelial cells. on the other hand, they may support local inflammation, since opn - n induces secretion of il-6 in monocytes, opn - c inhibits production of il-10, and both increase secretion of ifn-. in conclusion, this study shows that the opn fragments generated by thrombin exert distinct effects on cells involved in the immune and inflammatory response, and it suggests that drugs targeting each fragment may be used to fine - tune the wide effects of this cytokine. | osteopontin is a proinflammatory cytokine and plays a pathogenetic role in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (eae), by recruiting autoreactive t cells into the central nervous system. osteopontin functions are modulated by thrombin cleavage generating n- and c - terminal fragment, whose individual roles are only partly known. published data are difficult to compare since they have been obtained with heterogeneous approaches. interestingly, thrombin cleavage of osteopontin unmasks a cryptic domain of interaction with 41 integrin that is the main adhesion molecule involved in lymphocyte transmigration to the brain and is the target for natalizumab, the most potent drug preventing relapses. we produced recombinant osteopontin and its n- and c - terminal fragments in an eukaryotic system in order to allow their posttranslational modifications. we investigated, in vitro, their effect on human cells and in vivo in eae. we found that the osteopontin cleavage plays a key role in the function of this cytokine and that the two fragments exert distinct effects both in vitro and in vivo. these findings suggest that drugs targeting each fragment may be used to fine - tune the pathological effects of osteopontin in several diseases. |
chalcones (1,3-diphenyl-2-propen-1-ones) are known to possess a range of important biological activities, such as antibacterial, antifungal, antileishmanial, antimalarial, antifilarial anti - inflammatory [68 ], antiprotozoal (antileishmanial and antipanosomal), antimicrobial [1013 ], larvicidal, anticonvulsant, anti - hiv, antitumor, and anticancer activities, and they could be readily transformed into varieties of other compounds, many of which are biologically active heterocycles [19, 20 ]. owing to such biological activities of chalcones, the chemical literature shows the synthesis of a wide range of chalcones and their analogues [4, 813, 2123 ]. again, since macrocyclic compounds are well - known for their ability to show the important property of molecular recognition, macrocyclic systems containing the chalcone moiety are expected to generate compounds having interesting biological and material properties [24, 25 ]. our research on such compounds has been initiated through the synthesis of a number of macrocyclic bis- and monochalcones reported recently. in continuation of that study we have synthesized a benzo - fused 26-membered macrocyclic bischalcone, namely, (19e,43e)-2.11.27.36-tetroxaheptacyclo [44.4.0.0.0.0.0.0 ] pentaconta-1(46),4(9),5,7,12(17),13,15,19,21,23,25,29,31,33,37,39,41,43,47,49-icosaene-18,45-dione (3), and a benzo - fused 13-membered macrocyclic monochalcone, namely, (19e)-2.11-dioxatetracyclo[19.4.0.0.0 ] pentacosa-1(25),4(9),5,7,12(17),13,15,19,21,23-decaen-18-one (5), by use of readily available starting materials. alkylation products of salicylaldehyde and o - hydroxyacetophenone by the use of 1,2-(bis - bromobenzyl)benzene as alkylating agent were first prepared. the resulting dialdehyde (1) and diketone (2), both new compounds, were then subjected to claisen - schmidt reaction under high dilution condition when the macrocyclic bischalcone 3 was obtained in moderate yield (scheme 1). in order to achieve the synthesis of 5, 2,2-dihydroxychalcone (4) the reaction between 4 and 1,2-(bis - bromobenzyl)benzene was then done by following the typical procedure for alkylation of phenols (k2co3/acetone, reflux, 12 h) (scheme 2). this reaction gave 5 in very good yield and no trace of any macrocyclic bischalcone 7 (a possible product through bimolecular cyclization of the intermediate 6) (scheme 3). the new compounds 3 and 5 were characterized from their analytical and spectral data which are presented in the experimental section as well as in the supplementary file in the supplementary material available online at http://dx.doi.org/10.1155/2014/485014. ir spectra were recorded on a perkin elmer ft - ir spectrophotometer (spectrum bx ii) in kbr pellets. h and c nmr spectra were recorded in cdcl3 on a bruker av-300 (300 mhz) spectrometer. microanalytical data were recorded on a perkin - elmer 2400 series ii c, h, n analyzer. esims(+) mass spectrum of 3 was measured with a waters micromass q - tof micro instrument. column chromatography was performed with silica gel (100200 mesh) and tlc with silica gel g made of srl pvt. h and c nmr and mass spectra of different compounds can be found in the supplementary material. this compound was prepared from o - xylene by benzylic bromination with nbs [nbs (2.2 equiv.), (phcoo)2 (trace), refluxed in ccl4, 12 h, yield : 83%), m.p. s, 4h, ch2-br), 7.297.31 (m, 2h, ar - h), 7.337.38 (m, 2h, ar - h). a mixture of salicylaldehyde (2 mmol) and 1,2-bis(bromomethyl)benzene (1 mmol) was refluxed in methanolic koh (5%, 25 ml) for 7 h. removal of methanol by distillation and addition of water followed by extraction with ethyl acetate gave crude alkylation product 1, which was purified by rapid column chromatography followed by crystallization from chcl3-petroleum ether (yield : 66%). the physical and spectral data of 1 were as follows : colourless needles, m.p. 118120c ; h nmr (300 mhz, cdcl3) : 5.31 (br. s, 4h, 2 och2), 7.05 (dt, 4h, j = 7.2 and 1.5 hz), 7.427.45 (m, 2h, arh), 7.507.57 (m, 4h), 7.82 (dd, 2h, j = 7.8 and 1.8 hz), and 10.45 (br. a mixture of o - hydroxyacetophenone (2 mmol), 1,2-bis(bromomethyl)benzene (1 mmol) and anhydrous k2co3 (3 g.) was refluxed in dry acetone for 12 h. usual work - up followed by purification of the resulting crude material by column chromatography over silica gel afforded pure 2 (yield : 78%). the physical and spectral data of 2 were as follows : colourless needles (chcl3-petroleum ether), m.p. 74c ; h nmr (300 mhz, cdcl3) : 2.53 (s, 6h, 2 coch3), 5.27 (br. s, 4h, och2), 7.03 (dt, 4h, j = 8.4 and 1.8 hz), 7.407.46 (m, 4h), 7.54 (dt, 2h, j = 8.4 and 1.8 hz), and 7.71 (dd, 2h, j = 7.8 and 1.8 hz). a mixture of the dialdehyde 1 (1 mmol) and the diketone 2 (1 mmol) was dissolved in a koh solution (10%, 75 ml) in meoh - h2o (3 : 1) and the mixture was stirred at room temperature. the solid thus obtained was almost pure and it was further purified by column chromatography over silica gel followed by crystallization from chcl3-petroleum ether (yield : 49%). the analytical and spectral data of the macrocyclic product 3 were as follows : light yellow cubes ; m.p. 205207c ; ir (kbr, cm) : 1598 (c = o), 1567, 1485, 1446, 1384 cm ; h nmr (300 mhz, cdcl3) : 4.85 (s, 4h, o ch2), 5.06 (s, 4h, ch2o), 6.77 (d, 4h, j = 7.2 hz), 6.80 (t, 2h, j = 7.5 hz), 6.97 (t, 4h, j = 7.8 hz), 7.187.26 (m, 10h), 7.27 (d, 2h, j = 16.2 hz, 2 h-), 7.38 (dt, 2h, j = 7.5 and 1.5 hz), 7.47 (dd, 2h, j = 8.4 and 1.5 hz), 7.70 (d, 2h, j = 16.2 hz, 2 h-) ; c nmr (75 mhz, cdcl3) : 68.23, 68.48, 112.47, 112.75, 121.04, 121.21, 123.98, 127.65, 128.10, 128.11, 128.48, 129.40, 129.60, 130.10, 130.21, 131.47, 132.23, 133.81, 134.42, 140.17, 156.42, 157.10, 194.64 (c = o) ; ms (tof ms es) : m / z 707.18 (m+na), 685.19 (m+h). elemental analysis : calcd. for c46h36o6:c, 80.68 ; h, 5.30. found : c, 80.56 ; h 5.42%. this chalcone was prepared by condensation of o - hydroxyacetophenone and salicylaldehyde with aqueous alkali, m.p. d, 1h, j = 8.1 hz), 6.947.07 (m, 3h), 7.32 (dt, 1h, j = 7.8 and 1.8 hz), 7.52 (dt, 1h, j = 7.5 and 1.5 hz), 7.63 (dd, 1h, j = 7.8 and 1.9 hz), 7.87 (d, 1h, j = 15.6 hz, h-), 7.96 (dd, 1h, j = 8.1 and 1.5 hz), 8.21 (d, 1h, j = 15.6 hz, h-), 12.92 (s, 1h, 2-oh). to a mixture of 4 (1 mmol) and 1,2-bis(bromomethyl)benzene (1 mmol) in dry acetone (25 ml), anhydrous k2co3 (3 g) was added and the mixture was refluxed with stirring for 12 h. usual work - up of the reaction mixture followed by chromatography of the crude product over silica gel using petroleum ether - ethyl acetate (90 : 10, v / v) gave pure 5 as light yellow crystals (yield : 57%), m.p. 160162c, ir (kbr) cm : 3028, 2903, 1585, 1562, 1470, 1453, 1436, 1297, 1249, 1152, 1058, 960, 745. h nmr (300 mhz, cdcl3) : 5.28 (s, 4h, 2 arch2o), 7.017.10 (m, 2h), 7.11 (d, 1h, j = 16.5 hz, h-), 7.237.38 (m, 7h), 7.457.51 (m, 2h), 7.58 (dd, 1h, j = 7.5 and 1.8 hz, proton ortho to c = o), 7.66 (d, 1h, j = 16.5 hz, h-), c nmr (75 mhz, cdcl3) : 68.65, 71.70, 113.51, 119.01, 121.67, 123.46, 128.43, 128.81, 129.19, 129.77, 129.79, 129.87, 130.68, 130.88, 131.89, 132.62, 135.18, 135.29, 141.56, 156.71, 156.97, 195.23. ms (tof ms es) : m / z (m+na) 365.00. elemental analysis : calcd. for c23h18o3 : c, 80.68 ; h, 5.30. found : c, 80.45 ; h, 5.44%. thus, we report very simple syntheses of two new benzo - fused macrocycles incorporating chalcone moiety by the use of very common starting materials. the compounds may find important applications. | simple syntheses of the benzo - fused 26-membered macrocyclic bischalcone (19e,43e)-2.11.27.36-tetroxaheptacyclo[44.4.0.04,9.012,17.021,26.029,34.037,42]pentaconta-1(46),4(9),5,7,12(17),13,15,19,21,23,25,29,31,33,37,39,41,43,47,49-icosaene-18,45-dione (3) and the benzo - fused 13-membered macrocyclic chalcone (19e)-2.11-dioxatetracyclo[19.4.0.04,9.012,17]pentacosa-1(25),4(9),5,7,12(17),13,15,19,21,23-decaen-18-one (5) using very common starting materials and reagents are described. the compounds are new and they have been characterized from their analytical and spectral data. |
the measuring health and disability in europe (mhadie) was funded as part of the 6th framework programme by the european commission to be developed between 2005 and 2008. the main aim of the project was to demonstrate the feasibility and utility of the world health organisation s international classification of functioning, disability and health (icf) as a cross - cutting, universal framework and international standard to influence and support new european policy guidelines on health and disability by means of statistical, clinical and experimental research. icf s universal approach constitutes a paradigm shift in our understanding of disability, one that underscores the need to integrate individual functioning with the complete physical and social environment in order to capture the full - lived experience of disability that links health and social policy to promote social integration and increase participation, thereby enhancing opportunities for persons with disabilities. as part of this co - ordination action, institutions and researchers from 11 countries distributed across all the european union, have been able to demonstrate the application of the icf model in the collection of health and disability data using this common framework the project has established a network of european partners that are involved at regional, national and international level with the implementation of icf, icf children version and icf related instruments in clinical samples of selected conditions. specifically, this co - ordination action has been able to : 1) employ the icf model of functioning and disability to analyse existing general population health surveys and education statistics data ; 2) demonstrate that the icf model is adequate for describing and measuring patterns of disability in clinical samples of selected conditions, cross - sectionally and over time ; and 3) demonstrate the feasibility and usefulness of the icf for collecting and managing data in educational sectors. among the results, mhadie partners have produced specific policy recommendations and guidelines designed for use in health disability policy planning and development across the european union. | introductionthe measuring health and disability in europe (mhadie) was funded as part of the 6th framework programme by the european commission to be developed between 2005 and 2008. the main aim of the project was to demonstrate the feasibility and utility of the world health organisation s international classification of functioning, disability and health (icf) as a cross - cutting, universal framework and international standard to influence and support new european policy guidelines on health and disability by means of statistical, clinical and experimental research. icf s universal approach constitutes a paradigm shift in our understanding of disability, one that underscores the need to integrate individual functioning with the complete physical and social environment in order to capture the full - lived experience of disability that links health and social policy to promote social integration and increase participation, thereby enhancing opportunities for persons with disabilities.descriptionas part of this co - ordination action, institutions and researchers from 11 countries distributed across all the european union, have been able to demonstrate the application of the icf model in the collection of health and disability data using this common framework the project has established a network of european partners that are involved at regional, national and international level with the implementation of icf, icf children version and icf related instruments in clinical samples of selected conditions.resultsspecifically, this co - ordination action has been able to:1) employ the icf model of functioning and disability to analyse existing general population health surveys and education statistics data;2) demonstrate that the icf model is adequate for describing and measuring patterns of disability in clinical samples of selected conditions, cross - sectionally and over time ; and3) demonstrate the feasibility and usefulness of the icf for collecting and managing data in educational sectors.recommendationsamong the results, mhadie partners have produced specific policy recommendations and guidelines designed for use in health disability policy planning and development across the european union. |
the onset of seizures is usually before the age of 12 months in developmentally normal infants. the first seizure type is usually clonic, generalized or unilateral and triggered by fever. the diagnosis of ds is based on the clinical phenotype ; however, mutations in the scn1a gene can be found in 7585% of patients with ds 2, 3. the estimated incidence of ds ranges from 1 in 15 700 to 1 in 40 000 live births 2, 4. the current treatment strategy for patients with ds involves the use of multiple antiepileptic drugs (aeds), including combinations of valproate, topiramate, clobazam, stiripentol and others 5, 6. in a recent survey of european patients with ds, about 40% of patients were treated with three aeds and approximately 40% were treated with four aeds 6. despite these multidrug regimens, 45% continue to experience 4 tonic clonic seizures / month 6. to reduce frequent and disabling seizures, fenfluramine (3trifluoromethylnethylamphetamine), originally approved as an appetite suppressant 7, has also been reported to exhibit activity in refractory epilepsy 8. due to the retrospective character of previous studies 9, 10, we wanted to confirm our data in a welldesigned prospective trial in a new group of patients with ds. here we describe the effectiveness, tolerability and safety of fenfluramine in a new cohort of patients with ds who began treatment after 2010. patients of between 6 months and 50 years of age with a ds diagnosis based on the core phenotype 1 with or without scn1a mutation and whose epilepsy was uncontrolled despite adequate therapy with standard aeds were eligible. the major exclusion criteria included cardiovascular pathology, hypertension treated with medication, glaucoma, or hypersensitivity to fenfluramine or any other amphetaminelike drug. all patients who enrolled between january 2011 and december 2015 and who had at least 3 months of observation after starting fenfluramine were included in this analysis. this prospective openlabel study began with a 3month baseline period during which patients continued treatment with their current aeds. a daily diary was used to record the date, time, type and duration of seizures. a 24h electroencephalographic study was conducted during the baseline period as was an echocardiographic examination to evaluate cardiac valvular structure and function. after the 3month baseline period, fenfluramine was added at a dose of 0.251.0 mg / kg / day. the daily dose could be adjusted during the study based on efficacy or tolerability issues, with a maximum of 20 mg / day. concomitant aeds were kept stable during the first 3 months, thereafter adjustments could be made if necessary (figure s1). echocardiographic examinations were conducted every 3 months during the first year of treatment, every 6 months during the second year of treatment and annually thereafter. beginning in 2014 all echocardiograms were evaluated by a pediatric (f.m.) and an adult (b.p.p.) all data from inclusion until the last study visit (before april 2016) were analyzed. fenfluramine was obtained from zogenix international limited, emeryville, ca, usa and its purity was assessed and confirmed by the belgian pharmaceutical association. fenfluramine was compounded into capsules by the pharmacy of antwerp university hospital at doses of 2.5, 5, 10 and 20 mg / capsule. most patients in this study swallowed the capsules but, for a few, the contents were sprinkled prior to consumption. the key objective of this study was to assess the overall change in frequency of all major motor seizures during the fenfluramine treatment duration compared with the 3month baseline period. in addition, the change in major motor seizure frequency was assessed at specific time points (3, 6, 9 and 12 months postinitiation of fenfluramine treatment). clonic, tonic, clonic, atonic and myoclonic seizures lasting > 30 s. seizure frequency (seizures / month) during the baseline period was calculated as the total number of seizures divided by the number of days in the baseline period multiplied by 30. during treatment with fenfluramine, seizure frequency taking into account the small sample size and the nongaussian distribution, a wilcoxon signedrank test for paired data was used to analyze the treatment effect. statistical analysis was performed with spss software (ibm corp spss statistics for windows, version 23.0, armonk, ny, usa). global impression of change, quality of life (qol) and sleep quality were assessed at each study visit since june 2015. the qol and sleep quality of parents and patients were assessed using a visual analog scale (0, extremely bad to 10, very good). global impression of change was evaluated with a fivepoint scale (2, very much improved ; 1, much improved ; 0, no change ; 1, much worse ; and 2, very much worse). following the withdrawal of fenfluramine in 1997, its use as an aed for the treatment of intractable epilepsy was allowed in belgium following the issuance of a royal decree (kb 2002/22215). the study protocol was reviewed and approved by the ethics committee at antwerp university hospital prior to the initiation of this study (registration no. the parents or guardians of each patient were fully informed about the study and provided written informed consent prior to participation in the study. patients of between 6 months and 50 years of age with a ds diagnosis based on the core phenotype 1 with or without scn1a mutation and whose epilepsy was uncontrolled despite adequate therapy with standard aeds were eligible. the major exclusion criteria included cardiovascular pathology, hypertension treated with medication, glaucoma, or hypersensitivity to fenfluramine or any other amphetaminelike drug. all patients who enrolled between january 2011 and december 2015 and who had at least 3 months of observation after starting fenfluramine were included in this analysis. this prospective openlabel study began with a 3month baseline period during which patients continued treatment with their current aeds. a daily diary was used to record the date, time, type and duration of seizures. a 24h electroencephalographic study was conducted during the baseline period as was an echocardiographic examination to evaluate cardiac valvular structure and function. after the 3month baseline period, fenfluramine was added at a dose of 0.251.0 mg / kg / day. the daily dose could be adjusted during the study based on efficacy or tolerability issues, with a maximum of 20 mg / day. concomitant aeds were kept stable during the first 3 months, thereafter adjustments could be made if necessary (figure s1). echocardiographic examinations were conducted every 3 months during the first year of treatment, every 6 months during the second year of treatment and annually thereafter. beginning in 2014 all echocardiograms were evaluated by a pediatric (f.m.) and an adult (b.p.p.) all data from inclusion until the last study visit (before april 2016) were analyzed. fenfluramine was obtained from zogenix international limited, emeryville, ca, usa and its purity was assessed and confirmed by the belgian pharmaceutical association. fenfluramine was compounded into capsules by the pharmacy of antwerp university hospital at doses of 2.5, 5, 10 and 20 mg / capsule. most patients in this study swallowed the capsules but, for a few, the contents were sprinkled prior to consumption. the key objective of this study was to assess the overall change in frequency of all major motor seizures during the fenfluramine treatment duration compared with the 3month baseline period. in addition, the change in major motor seizure frequency was assessed at specific time points (3, 6, 9 and 12 months postinitiation of fenfluramine treatment). clonic, tonic, clonic, atonic and myoclonic seizures lasting > 30 s. seizure frequency (seizures / month) during the baseline period was calculated as the total number of seizures divided by the number of days in the baseline period multiplied by 30. during treatment with fenfluramine, seizure frequency taking into account the small sample size and the nongaussian distribution, a wilcoxon signedrank test for paired data was used to analyze the treatment effect. statistical analysis was performed with spss software (ibm corp spss statistics for windows, version 23.0, armonk, ny, usa). adverse events were monitored at each visit during the study. global impression of change, quality of life (qol) and sleep quality were assessed at each study visit since june 2015. the qol and sleep quality of parents and patients were assessed using a visual analog scale (0, extremely bad to 10, very good). global impression of change was evaluated with a fivepoint scale (2, very much improved ; 1, much improved ; 0, no change ; 1, much worse ; and 2, very much worse). following the withdrawal of fenfluramine in 1997, its use as an aed for the treatment of intractable epilepsy was allowed in belgium following the issuance of a royal decree (kb 2002/22215). the study protocol was reviewed and approved by the ethics committee at antwerp university hospital prior to the initiation of this study (registration no. were fully informed about the study and provided written informed consent prior to participation in the study. nine patients with ds between the ages of 1.2 and 29.8 years enrolled in the study between january 2011 and december 2015, and had been treated with fenfluramine for a median duration of 1.5 (range, 0.35.06) years at the time of the last assessment. the initial dose of fenfluramine in each patient was 5 or 10 mg / day, divided and administered as two separate doses, with a mean weightadjusted daily dose of 0.24 (range, 0.160.50) mg / kg / day. fenfluramine doses were increased in six patients in order to optimize effectiveness during their treatment and, at the most recent study visit, the mean dose was 0.35 (range, 0.160.69) mg / kg / day. individual patient demographic and clinical information clb, clobazam ; ffa, fenfluramine ; lev, levetiracetam ; stp, stiripentol ; tpm, topiramate ; vns, vagal nerve stimulation ; vpa, valproic acid clonic, tonic, clonic, atonic and myoclonic seizures lasting > 30 s. monthly seizure frequency was calculated as the total number of seizures during the treatment period divided by the total number of treatment days multiplied by 30 days / month. percent reduction refers to the entire treatment period compared with the seizure frequency per month in the baseline period. tonic clonic seizures were the only major motor seizures observed in these patients both before and during treatment with fenfluramine. during the 3month baseline period, the frequency of major motor seizures ranged from 0.4 to 39.7/month (median, 15.0/month). only tonic clonic seizures were observed in patients 16, whereas additional types of major motor seizures were observed in patients 79. all patients experienced a decrease in the frequency of major motor seizures after treatment with fenfluramine was initiated (table 2). compared with the 3month baseline period, a reduction in the median frequency of major motor seizures was observed from baseline (15.0/month) to 3 months (2.0/month), 6 months (1.1/month), 9 months (1.1/month) and 12 months (1.6/month) (fig. 1). a wilcoxon signedrank test showed a significant difference between the 3month baseline period compared with the first 3 months of treatment with fenfluramine (z = 2.67, p = 0.008). over the entire treatment period the median percentage reduction in major motor seizures was 75% (range, 28100%). the effect of addon fenfluramine on the frequency of major motor seizures in patients with dravet syndrome. significant differences in wilcoxon nonparametric test (p 60 mg / day), often in combination with phentermine, in the treatment of adult obesity translates to its use in low doses in children with ds. it was removed from the market in 1997 following a report of 113 cases of valvulopathy described by the united states centers for disease control and prevention. in those cases, only 2% involved fenfluramine monotherapy and 79% occurred with both fenfluramine and phentermine 16. although no echocardiographic evidence of changes in cardiac valve structure or function during fenfluramine treatment was observed in the present study, the relatively short period of exposure in this new cohort of patients may be insufficient for valvulopathy to become evident. in the original cohort of patients with ds treated with fenfluramine, eight of 10 patients had normal echocardiograms at their most recent study visit (average treatment duration of 16.6 years), whereas two patients had slight, but stable, cardiac valve thickening without any clinical manifestations 11. it is important to note that cardiac valvulopathy has been reported to be dose related. showed that, in adult obese patients, severe valvulopathy was more common with 60 mg / day compared with patients treated with < 40 mg / day 17. in the present study, lower doses of fenfluramine were employed ; the maximum dose in these patients was 20 mg / day and the median dose was 10 mg / day. major limitations of the current study include the small sample size, use of a study diary, openlabel design, absence of systematic electroencephalographic monitoring and lack of a control group. sleep quality, qol and global impression were not assessed at baseline for the majority of the patients. it is, however, encouraging that the findings in this group of patients are consistent with those reported in the original cohort 10. the ongoing phase 3 randomized, placebocontrolled studies on the use of fenfluramine in ds will be required to confirm these initial observations and aid in further defining the benefit risk profile in patients with ds. these results on the use of lowdose fenfluramine in this new cohort of patients with ds further support the observations reported in the original cohort. patients with ds experienced sustained periods of clinically meaningful improved seizure control during fenfluramine treatment with a favorable tolerability and no echocardiographic or clinical evidence of cardiac valvulopathy or pulmonary hypertension. a.s.s. is a phd student at the university of antwerp and received an educational grant from zogenix. the seizure frequency is indicated by a red line (, left yaxis). | background and purposedravet syndrome (ds) is a severe, drugresistant epilepsy. fenfluramine has been reported to have a longterm clinically meaningful anticonvulsive effect in patients with ds.methodsthis prospective, openlabel study assessed the safety and effectiveness of lowdose fenfluramine in a new cohort of patients with ds. following a 3month baseline period, fenfluramine was added to each patient 's current antiepileptic drug regimen at a dose of 0.251.0 mg / kg / day (max. 20 mg / day). the incidence of major motor seizures (tonic, clonic, tonic clonic, atonic and myoclonic seizures lasting > 30 s) in both the baseline and treatment periods was assessed via a seizure diary. periodic echocardiographic examinations during the treatment period were used to assess cardiovascular safety.resultsnine patients (aged 1.229.8 years) enrolled in the study and were treated with fenfluramine for a median duration of 1.5 (range, 0.35.1) years. median frequency of major motor seizures was 15.0/month in the baseline period. all patients demonstrated a reduction in seizure frequency during the treatment period with a median reduction of 75% (range, 28100%). seven patients (78%) experienced a 50% reduction in major motor seizure frequency. the most common adverse events were somnolence (n = 5) and anorexia (n = 4). no evidence of cardiac valvulopathy or pulmonary hypertension was observed.conclusionsthe effectiveness and safety of lowdose fenfluramine as an addon therapy for ds in this new prospective cohort supports previous findings. |
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