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eyes were obtained from 53 dogs including 5 beagles (23 years old), 1 chihuahua (10 years old) and 47 mixed breeds (1 to 13 years old) of each gender that were euthanized by carbon dioxide overdose (47 mixed breeds) or deep anesthesia using pentobarbital sodium (100 mg / kg, i.p.) (the other 6 dogs) for an unrelated experiment to the eye by other group in our faculty. both eyes of all animals were removed within 2 hr of death. for a macroscopic study (all cases), the unilateral eyes were cut along the equatorial plane, and the tapetal fundus was photographed by digital camera (ex - z400, casio, tokyo, japan). for a histological study (20 cases including 5 beagles, 1 chihuahua and 18 mixed breeds), following macroscopic observations, the unilateral eyes were injected with 3% glutaraldehyde in 10% formalin into the vitreous cavity. after fixation, the posterior cup of each eye was dehydrated, embedded in nitrocellulose (collodion, wako, osaka, japan), cut at 3040 m and then stained with hematoxylin - eosin or thionine. in 5 beagles, 40-m sections of tapetum with the retina and choroid were cut at 0.5 to 2 mm intervals and reconstructed after measurement of the tapetum thickness and observations of the rpel. these procedures were carried out in accordance with the guidelines of the ethical committee of tottori university, japan. macroscopic observations : the normal tapetum was triangular with rounded angles and smooth contour. its base had usually contact with the optic nerve disc (od), but separated from the od in rare cases. thus, the nasal angle of the tapetum was more acute than the temporal angle. the color tone of the tapetum was not even ; its center was brightest (fig. 1fig. 1.the normal tapetum is almost triangular with rounded angles and smooth contour usually contacting with the optic nerve disc (arrow). n, nasal ; t, temporal. bar=5 mm.). based on histological observation, the color tone of the tapetum reflected the thickness of the tapetal tissue. the normal tapetum is almost triangular with rounded angles and smooth contour usually contacting with the optic nerve disc (arrow). the atypical tapetum was smaller and more variable in shape, contour and color than the normal one (fig. the tapetal area decreases from the nasal and ventral parts (left and ventral sides in each tapetum, respectively) of the tapetum (a d). the chihuahua dog (d) lacks the tapetum, and the dorsal part of its fundus is reddish - brown, which correspond approximately to the normal tapetal area both in location and size.). the atypical tapetum disappeared from the periphery, especially from the nasal and ventral parts. in mild cases, the atypical tapetum was irregular in contour and faded in the nasal part (fig. in severe cases (the chihuahua), the fundus was devoid of the tapetum and had a reddish - brown area that was similar in both location and extent to the normal tapetum (fig. the tapetal area decreases from the nasal and ventral parts (left and ventral sides in each tapetum, respectively) of the tapetum (a d). the chihuahua dog (d) lacks the tapetum, and the dorsal part of its fundus is reddish - brown, which correspond approximately to the normal tapetal area both in location and size. bilateral differences in appearance of the tapetum were few through the normal and atypical tapeta. regarding the relationship of the atypical tapetum to age, the occurrence ratio was 31% in the atypical tapetum and 69% in the normal tapetum in dogs under age 5 (35 cases) and was 72% in the atypical tapetum and 28% in the normal tapetum in dogs over age 6 (18 cases). histological observations : in the normal tapetum, the rpel was almost unpigmented on the tapetum (fig. 3fig. the retinal pigment epithelial layer (arrow) on the tapetum is unpigmented. bar=100 m.). the narrow peripheral zone of the tapetum was covered by the lightly pigmented rpel. thus, the distribution of tapetal tissue closely corresponded to the area of the macroscopic tapetum. the tapetum was thick in its central part, and its thickest part was located dorso - temporally to the od (fig. the thickest part of the tapetum (central black area) is located dorso - temporally to the optic nerve disc (white circle). dots show the periphery of the tapetum covered by the lightly pigmented retinal pigment epithelial layer. t, temporal.). the thickest part of the typical tapetum varied in thickness, from 20 to 70 m (average : 53 m, standard deviation : 17 m) and consisted of maximum 20 layers of tapetal cell. the thickest part of the typical tapetum (t). the retinal pigment epithelial layer (arrow) on the tapetum is unpigmented. bar=100 m. reconstructed topographic distribution of the tapetum thicknesses. the thickest part of the tapetum (central black area) is located dorso - temporally to the optic nerve disc (white circle). dots show the periphery of the tapetum covered by the lightly pigmented retinal pigment epithelial layer. t, temporal. in the atypical cases, in which the tapetum was markedly small and very irregular in contour, the tapetum was very thin. in these cases, regardless of the shape of the atypical tapetum, the unpigmented rpel showed a similar extent to that in the normal tapetum. pigmentation of the choroid and pigmented rpel was normal in all cases (fig. 5fig. 2c. a, the thickest part of the tapetum (t) is covered by unpigmented retinal pigment cells (arrow). b, unpigmented retinal pigment epithelial cells (arrow), which normally cover the tapetal tissue, are found in the broad area without tapetal tissue. bar=100 m.). while the chihuahua dog macroscopically lacked the tapetum, unpigmented rpel showed histologically a similar distribution to that in the normal tapetum (fig. a, the unpigmented retinal pigment epithelial layers (arrow) are found in the reddish - brown area without the tapetum. b, although the macroscopic tapetum is not observed, fragmented tapetal tissues (t) are rarely found in the reddish - brown area. 2c. a, the thickest part of the tapetum (t) is covered by unpigmented retinal pigment cells (arrow). b, unpigmented retinal pigment epithelial cells (arrow), which normally cover the tapetal tissue, are found in the broad area without tapetal tissue. bar=100 m. histological photographs of fig. a, the unpigmented retinal pigment epithelial layers (arrow) are found in the reddish - brown area without the tapetum. b, although the macroscopic tapetum is not observed, fragmented tapetal tissues (t) are rarely found in the reddish - brown area. a wide variation in extent and color of the tapetum has been reported in normal dogs [9, 11, 16 ]. the tapetum is poorly developed in the toy breeds including the chihuahua and in dogs with a merle coat color [11, 16 ]. pigmentation of the choroid correlates closely with the development of the tapetum [10, 11 ]. interestingly, the albino ferret and the siamese cat lack choroidal pigmentation, but have the tapetum [22, 23 ]. according to thibos., the choroid in the siamese cat contains melanocytes, but is defective in pigment production. thus, the siamese is completely free from pigment in the choroid but has the tapetum, because tapetal cells derive from neural crest cells as well as choroidal melanocytes. the blue - eyed white cat has no tapetum and no choroidal pigmentation, because neural crest cells do not migrate into the choroid. all cases observed in this study were normal in pigmentation of the choroid. in this study, unpigmented rpel was equally distributed in the dog retina regardless of the extent and shape of the tapetal area. in the chihuahua, the tapetum was not found, but the unpigmented rpel was similar in distribution to that of the normal tapetum. the developmental relation of the unpigmented rpel to the tapetum has not been studied in dog. according to a study of the bovine eye, the adult pattern of pigmented rpel in nontapetal zones and unpigmented rpel in the presumptive tapetal zone is well established in the early stage of pregnancy. in other word, the extent of the unpigmented rpel is decided before the development of the tapetum. thus, compartmentalization of unpigmented and pigmented rpel would not be directly related to the development or regression of the tapetum. in this study, histologically, tapetal layers decreased in number throughout the atypical tapetum. therefore, thin tapetal parts, namely the periphery of the tapetum, disappeared first, and the contour of the tapetum became irregular with shrinkage of the tapetum. according to wyman and donovan, the degree of beadiness of the tapetum increases with aging. beadiness refers to the mosaic pattern observed in ophthalmoscopic examination at the periphery of the tapetum. thus, it is suggested that the tapetum in the dog tends to decrease in size macroscopically with aging as the number of tapetal cell layers decreases due to tapetal cell degeneration. it is speculated that the atypical tapetum results from tapetal cell degeneration in addition to congenital hypoplasia of the tapetum. for example, smaller - sized breeds have significantly smaller tapetal area. in this study, the normal tapetum had a wide variety of thickness (53 17 m). several studies have shown selective tapetal cell degeneration following administration of drugs, including a beta - adrenergic blocking agent sch 19927, a macroride antibiotic (rosaramicin), an azaride antibiotics (cp 62,993), an aromatase inhibitor (cgs 14796c) and an antipsychotic agent (1192u90). tapetal cells in the dog are packed with long slender rods, which are rich in zinc [12, 24, 25 ]. zinc is essential for preservation of the structure of normal tapetal rods, because the congenitally abnormal tapetum which lacks zinc in its tapetal rods is not visible in ophthalmoscopy and shows an absence of the regular arrays of tapetal rods. drugs that chelate zinc cause a reversible tapetal decoloration in dogs [8, 19 ]. even in human in developed countries, zinc deficiency is common in the elderly, and zinc supplementation delays the progression of age - related macular degeneration and reduces the risk of loss of vision. if zinc deficiency is not uncommon in elderly dogs, tapetal cells may gradually degenerate for zinc deficiency with aging. cysteine, to which zinc readily binds, is necessary for the biosynthesis of glutathione, which is quantitatively the most important antioxidant and radical scavenger. the age - related decrease in the body s cysteine and /or glutathione level plays a causative role in various ageing related degenerative processes. as the age - related decrease in zinc and cysteine may synergistically increase tapetal cell degeneration, there may be a trend toward an increase of tapetal cell degeneration with aging. | abstractwe aimed to document macroscopic variations in the cellular tapetum in the dog, to provide a histologic description of the macroscopic results and to evaluate the correlation between the macroscopic appearance and aging. fifty three dogs including 5 beagles, 1 chihuahua and 47 mixed breeds of each gender were used. for a macroscopic study, the fresh tapetal fundi were photographed using digital camera. for a histological study, the glutaraldehyde - formalin fixed eyes were embedded in nitrocellulose and stained with hematoxylin - eosin or thionine. the normal tapetum was triangular with the rounded angles and the smooth contour. the atypical tapetum was smaller and more variable in shape, contour and color than the normal one. in severe cases, the fundus was devoid of the tapetum. the atypical tapetum tended to increase in frequency with aging. retinal pigment epithelial cells on the normal tapetum were unpigmented. in the eye with the atypical tapetum, regardless of tapetal size and shape, unpigmented retinal pigment epithelial cells showed a similar distribution to that on the normal tapetum, even in a dog without a tapetum. although there is a congenitally hypoplastic tapetum, the atypical tapetum tends to increase in incidence and severity with aging. |
the comprehensive aortic root and valve repair (carvar) procedure is, basically, one of the valve sparing surgical techniques for aortic valve or aortic root disease. three basic surgical procedures of this technique are annular reduction, reduction of sinotubular junction (stj) and leaflet correction.1)2) for reduction of the annulus or stj, a commercially available sizing device, strip or ring and template (sciencity co, seoul, korea) are used.3) carvar procedure and devices, named and designed by dr. mg song, are under evaluation for list up process as new health technology by the health insurance review and assessment service (hira) from 2007. during this process, han.4) reported 5 clustered cases of coronary ostial stenosis after carvar procedure. they also reported several cases of re - operation from valvular dysfunction or endocarditis and even cases of mortality after operation to the korea food and drug administration. under the debates on the safety and effectiveness of the cavar procedure, the ministry of health and welfare, korea offered a prospective randomized study. for the design and production of patient information for prospective study, it was required to establish a data of safety from previously performed operations.5) vitualy the entire carvar procedure was performed by a single operator (dr. myeong gun song) at 2 university hospitals : from march 2007 to july 2007 at asan medical center (seoul, korea) and from october 2007 to november 2009 at konkuk university hospital (seoul, korea). three hundred ninety seven patients (26 cases at asan medical center, 371 cases at konkuk university hospital) received carvar operation during this period and were enrolled for this study. data from medical records were gathered by a trained investigator from national evidence - based health care collaborating agency (neca). to obtain more complete information, a follow up was performed via 2 tracks including medical records of the out patient department and claim data of medical service in hira. four major threatening clinical events including serious surgical bleeding requiring transfusion over 20 units of packed red blood cell, endocarditis, re - operation from any cause and death were focused for evaluation of safety. clinical events at out patients ' department were followed up till march 2010. to compare with conventional valve replacement surgery, 337 patients without aortic root disease { aortic valve disease (avd) only group } were analyzed separately. total incidence during the follow up period (%) and 1-year cumulative incidence (% /year) were calculated by the kaplan - meier method. this study was approved by the institutional review board of neca (necairb 10 - 001) and organized data review board with 6 professional experts from the korean society of thoracic and cardiovascular surgery and the korean society of cardiology for peer data review. importantly, there were 9 cases of age under 19 and 8 cases of older than 80 years - old. there were 20 fatal cases and 25 re - operated cases and 19 cases of endocarditis. even in the avd only group, the 1 year cumulative death rate was 4.41%/year and risk of re - operation was 5.81%/year. for the treatment of avd, aortic valve replacement is regarded as a standard treatment. but, aortic valve sparing surgery can be applied to aortic regurgitation due to dilatation of the aortic root or ascending aorta without significant leaflet deformity. theoretically, carvar is one of the aortic valve sparing surgical techniques. hahm.1) described that the advantages of this technique are a wide range of indication, preservation of aortic root function, easy surgical technique and low recurrence of valvular dysfunction. but, from the results of this study, the carvar technique failed to show any advantage in mortality and morbidity. in a recently reported 28 years experience (from 1982) of the bentall procedure, the operative mortality was 5.5%, and risk of late reoperation and endocarditis were 3.2% and 1.4% respectively.6) moreover, lim.6) reported that, in patients with aortic root dilatation with advanced aortic regurgitation, operative mortality was 2.4% in the bentall procedure group and 0% in the valve - sparing operation group.7) compared with those data, in patients with carvar surgery, mortality seems quite higher than recently reported results and the risk of endocarditis and re - operation are much higher than in previous reports. in the least, it was restricted in gathering enough clinical data from medical records, hence we could not evaluate data comprehensively. nontheless, the primary aim of this study is to provide basic safety data for further prospective randomized study. therefore, it can be suggested that the carvar procedure should undergoe more in - depth investigation in consideration for the safety of patients. | comprehensive aortic root and valve repair (carvar) is a recently introduced surgical technique for aortic valve disease. the national evidence - based health care collaborating agency was offered by the ministry of health and welfare, korea to perform a restrospective outcome analysis for this surgical procedure. the aims of this study were to evaluate the safety of patients who underwent carvar surgery and to provide a rationale for further prospective randomized study. during the period of march 2007 to november 2009, 397 patients received this procedure and enrolled in this study. clinical events including major bleeding, endocarditis, re - operation and death were followed - up till march 2010 by medical records. during the follow - up periods, 1-year cumulative incidence of major bleeding, re - operation, endocarditis and death were 3.55, 5.65, 5.05 and 5.33%/year respectively. this study showed that the carvar technique is not beneficial, and is indeed even more harmful than conventional valve replacement surgery. |
an 11-year - old spayed female domestic shorthair cat with a history of hyperthyroidism treated with carbimazole for 7 months was presented for a check - up after a few episodes of vomiting. the cat had been receiving prednisolone at 0.5 mg / kg po q12h for recent pancreatitis and concurrent inflammation of liver and small intestines confirmed by biopsies. clinical examination revealed pale mucous membranes with a capillary refill time of 120 s) and fibrinogen serum concentration (3.5 morphological changes of thrombocytes in the absence of thrombocytopenia were also noted. in - saline agglutination test was positive. feline leukaemia virus and feline immunodeficiency virus tests were negative. on the basis of these findings, immune - mediated anaemia secondary to chronic carbimazole administration prednisolone was increased to 2 mg / kg po q24h and carbimazole tablets were stopped. despite close monitoring and intensive care, this report suggests that severe haemotoxicity may occur as a sequel of chronic carbimazole administration in cats. routine bloodwork and accurate follow - up of cats under treatment with thyrotoxic therapy may be advisable, in order to detect haematological changes before lethal complications occur. hyperthyroidism is one of the most common endocrine disease in cats, especially in middle - aged to older cats. owing to concurrent geriatric problems, which are likely to increase the risk of anaesthetic - related complications, medical treatment is often preferred over surgical thyroidectomy. serious haematological side effects are a well - known complication for patients on thyrotoxic treatment, although only two cases of methimazole - induced haemotoxicity have been reported in cats. we report a life - threatening haematological complication occurring in a cat after chronic carbimazole administration. an 11-year - old spayed female domestic shorthair cat weighing 5.9 kg, with a history of hyperthyroidism treated with carbimazole for the past 7 months, was presented for a check - up after a few episodes of vomiting. total thyroxine, haematology and biochemistry were monitored routinely every 3 months. since the beginning of the therapy, the treatment started on carbimazole 15 mg po q24h and the dosage was fixed accordingly with monitoring to reach a dose of 10 mg po q24h where the cat was stable. the cat was also receiving prednisolone at 0.5 mg / kg po q12h for recent pancreatitis and concurrent inflammation of liver and small intestines, confirmed with biopsies. at presentation, the cat was alert and responsive. its body condition score was 2/5 and the cat had gained 200 ; g in body weight during the previous month. heart rate was 164 beats per minute, with no identifiable heart murmur and the lung sounds were clear. blood was collected for a comprehensive panel and manual packed cell volume (pcv). g / l ; reference interval [ri ] 6080), mild hypoalbuminaemia (22 ; g / l ; ri 2546) and decreased creatinine serum concentration (69 mol / l ; ri 88177). serum total thyroxine was within normal limits for the species (45 nmol / l ; ri 1950 ; nmol / l) but borderline for a cat on treatment with carbimazole. reticulocyte count revealed a regenerative anaemia (0.17 ; 10/ml) and on the blood smear red blood cells showed polychromasia and anisocytosis. few lymphocytes appeared reactive with deeply basophilic cytoplasm and occasionally with abundant pale basophilic cytoplasm. feline leukaemia virus (felv) and feline immunodeficiency virus (fiv) tests were negative (idexx snap test). a coagulative panel showed that prothrombin time (42 s ; ri 7.011.0 s), partial thromboplastin time (> 120 s ; ri 13.020.0 s) and fibrinogen serum concentration (3.5 abdominal ultrasound and t - fast were also normal and no pericardial, pleural or peritoneal effusion were found. the liver appeared heterogeneous with no overt masses ; the spleen was homogenous and small. the right kidney was 4.3 cm in length, the left one measured 3.7 cm and the architecture of both appeared normal. based on these results, we could rule out the most common causes of immune - mediated anaemia and conclude that the haematological disorder was secondary to chronic carbimazole administration. doxycycline was started at 5 ; mg / kg po q12h with the purpose of treating an undetected mycoplasma haemofelis infection. omeprazole was also started at 1 mg / kg po q24h to prevent gastrointestinal ulceration due to the prednisolone immunosuppressive dose. the possibility of a blood transfusion was mentioned to the owner, who declined the procedure owing to the potential for complications. blood pressure was measured every 2 h, while pcv was monitored every 8 h. body temperature was maintained within normal ranges for the species by active warming. despite close monitoring and intensive care, the cat died the same evening of admission to the hospital. differential diagnoses for immune - mediated anaemia in cats include infectious, neoplastic and chronic diseases, as well as drugs and toxins. in our case, the diagnostic investigations were suggestive of immune - mediated anaemia secondary to chronic carbimazole administration. infectious diseases were excluded on the basis of the negative results for fiv and felv testing of serum. furthermore, the cat was an indoor cat and had no possibility of contact with other cats. there was no evidence of neoplastic disease, as corroborated by the ultrasonographic and radiographic findings, which failed to identify the presence of masses, organ enlargement or cavitary effusions. a haematological disorder resulting from chronic disease can not be completely ruled out. a chronic pancreatitis, with concurrent inflammation of liver and small intestine, the coagulation abnormalities detected in this cat, namely the increased prothrombin time, partial thromboplastin time and fibrinogen serum concentration, together with the immunomediated origin of the anaemia, as confirmed by the positive in - saline agglutination test, seem to indicate the carbimazole toxicity as the most likely cause. similar cases of severe anaemia caused by methimazole were treated by discontinuing the treatment, with no re - challenge with antithyroid drugs. alongside this, it has been recommended to administer prednisolone (12 mg / kg po q1224h) and doxycycline (5 mg / kg po q12h). in order to prevent gastric ulceration due to a high dose of prednisolone, proton pump inhibitors (omeprazole 0.51.0 mg / kg po q24h) must be used. h2-receptor antagonists (ranitidine 1 mg / kg po q12h) can also be used to decrease pepsin and gastric acid secretions and so have a secondary efficacy. clinical side effects typically associated with chronic carbimazole therapy include anorexia, vomiting and lethargy, but these signs are usually mild and transient, and similar to those reported with methimazole. more serious side effects are reported for methimazole, such as hepatopathy, bleeding diathesis (prolonged proteins induced by vitamin k absence or antagonists [pivka ] clotting time) and marked thrombocytopenia. anaemia, including aplastic anaemia, was reported by chapman, after 3 years of treatment, and also by weiss in 2006. it is also of note that there may be synergistic side effects with the combination of other drugs, even if the severity is unknown. when used concurrently, there may be a chance that carbimazole competes with this other drug for protein binding and so causes adverse side effects. carbimazole is a well - studied thyrotoxic drug which is considered to have fewer life - threatening side effects than methimazole. although carbimazole may be better tolerated in cats than methimazole, the potential for serious adverse reactions still exists. in order to detect them, mooney recommended carrying out a complete blood count every 2 weeks, at least for the first 3 months of therapy, when these reactions are most likely to occur. owing to the rapid course of events, as well as to the severity of the condition at presentation, the cat died on the same night of admission to the hospital. a closer follow - up of the cat during carbimazole treatment could have allowed for an earlier detection of the haematological changes, in order to undertake preventive measures before lethal complications occurred. life - threatening side effects, occasionally reported with methimazole in cats, may occur also with carbimazole. routine bloodwork and accurate follow - up of cats under treatment with thyrotoxic therapy may be advisable, in order to detect haematological changes before lethal complications occur. | case summaryan 11-year - old spayed female domestic shorthair cat with a history of hyperthyroidism treated with carbimazole for 7 months was presented for a check - up after a few episodes of vomiting. the cat had been receiving prednisolone at 0.5 mg / kg po q12h for recent pancreatitis and concurrent inflammation of liver and small intestines confirmed by biopsies. clinical examination revealed pale mucous membranes with a capillary refill time of 120 s) and fibrinogen serum concentration (3.5 g / l). morphological changes of thrombocytes in the absence of thrombocytopenia were also noted. in - saline agglutination test was positive. abdominal radiographic and ultrasonographic examinations excluded the presence of organ abnormalities and peritoneal effusion. blood biochemistry was unremarkable. feline leukaemia virus and feline immunodeficiency virus tests were negative. on the basis of these findings, immune - mediated anaemia secondary to chronic carbimazole administration was suspected. prednisolone was increased to 2 mg / kg po q24h and carbimazole tablets were stopped. despite close monitoring and intensive care, the cat died the same evening of admission to the hospital.relevance and novel informationthis report suggests that severe haemotoxicity may occur as a sequel of chronic carbimazole administration in cats. routine bloodwork and accurate follow - up of cats under treatment with thyrotoxic therapy may be advisable, in order to detect haematological changes before lethal complications occur. |
during ahi, hiv-1 replicates in cd4 t cells and causes profound immune activation and cd4 t - cell depletion [15, 25, 26 ]. after acute infection, cd4 t cells become the main targets of virus infection during the transition from naive to effector and memory t cells [27, 28 ]. in addition, cytokines and high levels of viremia are responsible for elevated levels of immune activation in ahi, resulting in infection of both naive and memory cd4 t cells. thus, hiv-1 integrates into the genome of a diverse population of activated proliferating cd4 t cells. after immune control of virus replication immune activation declines and proviral cd4 t cells return to a resting phase while harboring the provirus that can persist in this silent stage for the remaining of the life span of these cells. the wide variety of cd4 t cells among the latent reservoir, and their differential susceptibility to activation stimuli, represent significant hurdles to their elimination by immune responses or by combination antiretroviral therapy (cart). one mechanism that has been proposed to eradicate the latent reservoir relies on the administration of latency - reversing agents [31, 32 ] that can reactivate provirus for viral antigen expression, followed by elimination of the infected cell either through direct cytolytic effects of the virus life cycle or with endogenous immune responses. however, this strategy, originally called shock and kill [31, 32 ], has proved difficult to implement because of (1) low levels and impaired function [3436 ] of hiv-1specific cd8 t cells ; (2) insufficient activation of hla class i restricted antigen - specific cd8 effector t cells [30, 33, 37 ] ; (3) inadequate expression of hiv-1 antigens by reactivated cd4 t cells ; (4) localization of reactivated cd4 t cells in protected sites, such as lymph node b cell follicles [39, 40 ] ; and (5) viral escape mutants that limit virus - infected t - cell clearance by cd8 cytotoxic t cells [41, 42 ]. to overcome the natural limitations of the immune system in targeting the chronic phase of hiv-1 infection and the latent reservoir, new strategies have been proposed that harness the antiviral activities of nab or non - nab monoclonal antibodies (mabs) ; mabs can be selected based on their ability to target conserved regions of hiv-1 env on virus - infected cd4 t cells (ie, non - nabs) or on their breadth of neutralization of hiv-1 isolates (ie, broadly neutralizing antibodies [bnabs ]) [8, 43 ]. non - nabs capable of mediating adcc by recruiting fc receptor (fcr) iiia bearing cells such as, nk cells, are of interest as immune therapies to reduce the size of the latent reservoir because these antibodies can target env on the surface of primary virus infected cells. the use of mab - based molecules that can recognize and promote infected cell killing combined with cart is a promising new strategy for treating hiv-1 infection [4547 ]. moreover, mabs and derivative molecules could be used in combination with latency - reversing agents to augment endogenous immune effector functions for the elimination of latently infected cells on reactivation of the provirus. the bnabs bind to 1 of 6 sites on the virion env and prevent virion infection (figure 1). these sites of neutralization vulnerability include the gp41 membrane external proximal region, the gp120-gp41 interface, the cd4-binding site (cd4bs) [5053 ], variable regions 1 and 2 (v1/v2) glycan, the fusion domain, and variable region 3 (v3) glycan [56, 57 ]. although the development of nab in hiv-1infected patients is rarely associated with control of disease progression, passive administration of bnabs in nonhuman primates individually or in combination can prevent simian - human immunodeficiency virus (shiv) infection [5967 ]. the protective effect of bnabs also requires fc - mediated effector functions [6873 ]. thus far, no vaccine to date has been successful at eliciting bnabs, and this failure seems to be linked to certain antibody characteristics, such as long and hydrophobic heavy chain complementarity determining region 3 loops, high degrees of somatic hypermutation, and/or autoreactivity all traits of antibodies controlled by immune tolerance mechanisms [7476 ]. bnabs do arise in about 50% of hiv-1infected individuals, but only after 24 years of infection. it has been demonstrated that virus diversity in infected individuals arises because of pressure exerted by the immune system and the ability of hiv-1 to mutate, leading in turn to the ability of the immune response to develop more bnab responses [10, 13, 78 ]. this cycle of events may explain the delay in the appearance of hiv-1 bnab responses. sites of vulnerability on the human immunodeficiency virus (hiv) type 1 envelope glycoprotein (env) spike. the structure of a hiv-1 prefusion trimer is displayed and the 6 major sites of vulnerability targeted by broadly neutralizing antibodies discussed in this review are indicated : the variable regions 1 and 2 (v1/v2) loop (green), the base of the variable region 3 (v3) loop (maroon), the cd4-binding site (lavender), the interface between gp120 and gp41 proteins (magenta), the fusion peptide region (orange), and the membrane proximal external region (mper). because of the limited structural information, the mper near the base of the env trimer is represented by a red cylinder. in the humanized mouse model (see the review by nixon in this special issue for the discussion of animal models for hiv-1 latency), passively administered postexposure prophylaxis with a combination of cd4bs bnab 3bnc117, v3 glycan bnab 101074, and v1/v2 glycan bnab pg16 decreased viremia in about 50% of the mice and substantially delayed virus rebound compared to cart treated animals. moreover, the investigators observed a decline in the level of cellular - associated dna in the aviremic mice treated with the 3-mab cocktail. in addition, it was demonstrated that critical mutations that nullified the interactions of their fc domains with fcrs on effector cells attenuated the therapeutic potential of the mabs. the majority (60%) of mice treated with the fcr antibody cocktails showed early rebound viremia after treatment, whereas early rebound viremia was observed in only 5% of the mice treated with the cocktail of antibodies with unmutated fc regions. therefore, bnabs can significantly affect both plasma viremia and the pool of latently infected cells through recognition of hiv-1 env on the host cell membrane and engagement of fcr - bearing cells, as shown elsewhere. several preclinical studies conducted in nonhuman primates support the observation in the humanized mouse model that bnabs can not only successfully reduce the level of plasma viremia during chronic infection [8183 ] but might also reduce levels of proviral dna. the passive administration of the v3 glycan bnab, pgt121, and the cd4bs bnabs 3bnc117 and b12 in combination reduced shiv proviral dna levels in the gut - associated lymphoid sites, lymph nodes, and peripheral blood. moreover, the vrc07/pgt121 combination administered during acute shiv infection in rhesus macaques not only induced a lower level of peak viremia and reduced the pool of latently infected cells but could also control virus replication and prevent the establishment of latency. whether the observed reduction of provirus dna in tissue compartments can be sustained needs to be further investigated (see also the review by henrick on the measurement of the latent reservoir in this special issue). recent human trials have evaluated the safety and efficacy of passively infused bnabs as hiv therapeutics. longitudinal observation of the individuals enrolled in this study revealed that some of the participants were capable of developing broader nab responses than those present before the infusion of the 3bnc117 mab, suggesting bnab selection of new virus variants that drove endogenous nab breadth. therefore, one positive aspect of bnab - based immunotherapy resides in the possibility that it may positively alter the natural humoral responses. the vrc01 cd4bs bnab was also tested in hiv-1infected individuals, and the administration of a single dose of mab induced a plasma viremia reduction of almost 2 log10. however, in both studies, resistant virus isolates were either observed before treatment, hampering the therapeutic effect of the mab, or appeared after infusion of the bnabs during analytical treatment interruption. the appearance of virus escape mutants should be regarded as a cautionary aspect of the administration of individual bnabs, and advocates for the search of more potent antibodies or evaluations of bnab combinations that target multiple epitopes to minimize the potential for virus escape. the alvac / aidsvax b / e rv144 vaccine trial conducted in thailand showed an estimated 31.2% vaccine efficacy. an immune correlates analysis indicated that non - nabs targeting the v2 region that were capable of mediating adcc were correlated with decreased transmission risk. adcc antibody responses in hiv-1 infection may also control virus replication [9395 ] and delay disease in adults [96, 97 ] and children. although non - nabs have failed to protect from infection in nonhuman primate challenge studies, they limit simian immunodeficiency virus or shiv [64, 73 ] replication and limit the number of transmitted / founder viruses. during the course of study of rv144 immune correlates, it was found that the adcc - mediating antibodies, while nonneutralizing, bound to the surface of transmitted / founder virus infected cd4 t cells. thus, these types of antibodies were well suited for use in developing therapeutic antibodies that can target virus - infected cd4 t cells in the setting of chronic hiv-1 infection. the therapeutic usage of mab and mab - derived molecules to eliminate latently infected cells on spontaneous or pharmacologically induced reactivation of replication competent provirus would rely on the ability of these molecules to recognize hiv-1 envelope as soon as it is expressed on the surface of infected cells. among the non - nabs, it has been reported that those directed against the cd4-inducible constant regions 1 and 2 of the gp120 could be among the first to bind to the surface of infected cells [100, 101 ]. these observations have prompted interest in developing antibody - derived molecules that will be further discussed. of note, the epitope recognized by these mabs is also one of the most conserved sequences of the hiv-1 envelope, providing a unique target for broad recognition of circulating hiv-1 isolates [45, 102 ]. to treat cancer and other human diseases, new immune therapeutics have been developed by combining 2 or more antigen - binding variable fragments of immunoglobulins into a single molecule termed bispecific (bsab) or trispecific antibodies. these antibodies can either bind to antigens presented on the surface of an individual cell, or on 2 distinct cells. in general, the bsabs can be divided in 2 major categories based on the presence or absence of the fc region. its presence can be beneficial during the production and purification of these molecules, but it also provides better solubility, stability, effector functions related to the ability to recruit fcr - bearing cytotoxic effector cells and complement, and half - life by binding to the fcr neonatal. in contrast, bsabs lacking the fc region rely exclusively on their ability to bind specific antigens for their biological effects. novel bsabs based on bnabs targeting neutralization - sensitive regions of the hiv-1 envelope have been successfully developed and tested in vitro for their neutralization potency (figure 2a) ; bsabs were found to retain the potency and the breadth of the parental individual mabs and were also able to neutralized hiv-1 isolates that were resistant to the individual bnabs. the same studies demonstrated that the pharmacokinetic properties of the bsabs were equal to those of the parental antibodies. these data were encouraging and indicated that further evaluation should be conducted in animal models to validate their activity and determine bispecific bnab safety and efficacy. alternative strategies for bsabs have been explored and led to the design of imabm36 and 10e8v2.0/imab (figure 2a). one immunoglobulin arm in these bsabs is specific for human cd4 (imab), which blocks the gp120-binding site on the cd4 molecule, and the second arm is specific for hiv-1 gp120 (m36 or 10e8v2.0). thus, these novel bsabs use a dual mode of action to prevent virus entry by interacting both with the virus itself and with the host cell surface receptor required for infection. the latter bsab (10e8v2.0/imab) has also been tested in the humanized mouse model and was demonstrated to both reduce virus load and provide protection from virus challenge. these findings support further evaluation of the therapeutic efficacy of bsabs in clinical trials. anti human immunodeficiency virus (hiv) type 1 envelope (env) antibody - derived molecules. a, bispecific antibodies combine 2 antigen (ag)binding site variable fragments (fvs) into a single immunoglobulin. the 2 fvs (fv1 and fv2) can recognize 2 different antigenic regions of the hiv-1 env [104 ], or the hiv-1 env and cellular receptors involved in virus entry [105, 106 ] or in cytotoxic functions [107 ]. b, bispecific t - cell engagers (bites), which are generated by using a single polypeptide linker to join 2 single - chain fvs (scfvs) with different antigen specificities. the first - generation hiv bite was designed to bind the hiv-1 envelope cd4-binding site and recruit cytotoxic t cells by engaging cd3. the variable domains of the 2 scfvs are not only linked by a polypeptide linker to create a diabody-type structure but also stabilized into a disulfide - linked heterodimer with different antigen specificity. one arm can bind the hiv-1 envelope, and the other can bind cytotoxic effector cells. dart molecules can also be designed to include the antibody fc region to improve the half - life of the molecules. a further technological development of the bsab concept was to engineer molecules that consisted of 2 single - chain variable fragments (scfvs) from different antibodies joined by a single polypeptide linker. these new molecules were designed with the specific goals of improving the size, valency, flexibility, half - life, and biodistribution. the first - generation molecule included 1 scfv that bound to cd3 t cells (cd4 or cd8) via the cd3 receptor, and the other scfv was specific for the b6.2 molecule expressed on tumor cells ; these molecules were first defined as bispecific t - cell engagers (bites). of note, they can redirect t - cell killing in an antigen - specific manner that is independent of major histocompatibility class i recognition of the antigen - bearing cells and the presence of costimulatory molecules [109, 110 ]. subsequent work was performed to improve the stability, potency, and manufacturability of the bites, which led to the generation of dual - affinity retargeting (dart) molecules. in dart molecules, the variable domains of the 2 specificities are incorporated into a disulfide - linked heterodimer in which short linkers between the variable light chain and variable heavy chain segments promote a diabody-type association, with the disulfide bond stabilizing the structure [112114 ]. both bites and dart strategies have been explored to develop novel classes of therapeutics that can be used to treat chronic hiv-1 infection with the specific goal of eliminating latently infected cells on the reactivation of the provirus. pegu developed a bite molecule capable of recruiting cd3 cytotoxic t cells and redirecting their killing by virtue of the anti hiv-1 arm based on the vrc07 mab targeting the hiv-1 cd4bs envelope region (figure 2b). the molecule was named vrc01-cd3 and, as previously observed for the other bites, it was shown to induce activation of cd8 or cd4 t cells only in presence of target cells expressing the hiv-1 envelope. one peculiar characteristic of this molecule was its ability to reduce the frequency of proviral dna positive cd4 t cells in 5 of 8 subjects during a 2-day in vitro tissue culture system, suggesting that this molecule could be effective when used as a component of hiv cure treatment strategies. as further development of the dart molecules to treat hiv-1 infection, sung explored the ability of novel hiv - specific dart molecules to eliminate hiv - infected cells (figure 2c). the dart molecules used in this study were composed of a cd3-specific arm for recruitment of cytotoxic effector t cells and an hiv - specific arm based on the cd4-inducible constant regions 1 and 2 and gp41 cluster 1 non - nabs a32 and 7b2, respectively, for recognition of hiv-1 envelope on the surface of infected cells. these molecules were demonstrated to be able to redirect the killing of hiv-1infected cells and reduce the amount of virus recovered from virus outgrowth assays performed with cell cultures from antiretroviral therapy treated patients on incubation with the latency - reversing agent vorinostat. similar results were observed by sloan, who developed dart molecules with the hiv-1 arm based not only on the a32 and 7b2 non - nabs but also on bnabs that target the n332 glycan (pgt121), v1/v2 (pgt145), cd4bs (vrc01), and mper (10e8). most of these dart molecules had 50% effective concentrations in the picomolar range, suggesting that they are suitable for clinical applications. of note, when dart molecules specific for different hiv envelope regions were evaluated in combination, the investigators did not observe either antagonistic nor synergistic effects for their cytotoxic activity, suggesting that it may be possible to combine dart molecules to expand the breadth of activity against the diversity of hiv-1 isolates present in clinical settings. interestingly, both bites and dart molecules were demonstrated to be capable of redirecting normal resting cytotoxic t cells for killing of hiv - infected cells, bypassing the need of effector cell preactivation, which has been described elsewhere as a hurdle in the elimination of the latent reservoir by shock - and - kill strategies because endogenous hiv-1specific hla class i - restricted cd8 t cells require preactivation for effective cytolysis of reactivated latently infected cells. novel treatment strategies that will rely on the use of antibody - based immune therapies can overcome the hurdles thus far identified for the hiv-1specific immune responses to eliminate latently infected cells alone or in combination with latency - reversing agents and antiretroviral therapy. however, more work is needed to determine the clinical impact that these molecules may have in treating latent hiv-1 infection. the minimum amount of env antigen expressed on the surface of hiv-1infected cd4 t cells that will be needed for antibody recognition is not known. moreover, it will be crucial to determine whether new molecules such as bites and dart molecules may have a superior biodistribution profile compared with other bsabs, which would allow them to penetrate and recruit effector cells in the anatomic sites harboring the latently infected cells [39, 115 ]. passive administrations of antibody have relied on the parenteral administration of these molecules and their derivatives. therefore, a pressing challenge resides in the design of new molecules that have a longer half - life to require less frequent administration regimens while retaining the specificity of the parental mab. in the case of dart molecules, for instance, there has already been an initial attempt at increasing the half - life of the molecules by engineering them to express a fc region that can bind to the fcr neonatal and provide increased recirculation of the molecule without promoting other fc - related effector functions (figure 2d). moreover, recombinant adeno - associated viruses have been engineered to deliver fusion proteins that resemble antibodies and antibody - based molecules. gardner have been able to deliver a cd4-immunoglobulin fusion protein in combination with a small ccr5 mimetic sulfopeptide that provided protection from challenge with shiv - ad8 for up to 40 weeks after infusion, without adverse effects. it is important to perform human clinical trials with these agents to evaluate their safety, immunogenicity and, ultimately, efficacy. finally, considering the advances in cancer therapy obtained by using mabs that target immune checkpoints, and the fact that some of the same checkpoints have been demonstrated to be involved in the exhaustion of anti - hiv - specific cellular immune responses, such as the expression of pd-1 [3436 ], efforts should be devoted to the antibody - mediated rescue of exhausted effector t cells by interfering with the interaction of programmed cell death protein 1 (pd-1) and/or cytotoxic t - lymphocytes antigen 4 (ctla-4) with their ligands [118120 ]. from the work of understanding the immune correlates of the rv144 vaccine trial and the immunobiology of bnab development have come new non - nab and bnab reagents that either as whole molecules or as engineered bispecific or trispecific antibody molecules the field is presently in the early stages of evaluation of these new reagents for treatment of hiv-1 infection, for targeting the latently infected pool of cd4 t cells, and to address their safety, immunoregulatory functions, and immunogenicity. additional ongoing work is aimed at improving antibody - based reagent half - life and potency to increase the anti the hope is that new antibody formulations, in combination with cart and latency - reactivating agents, can promote nk or cd8 t - cell killing of infected cd4 t cells, thus adding a new class of drugs for the treatment, and eventual cure, of hiv-1 infection. | abstracthuman immunodeficiency virus (hiv) type 1 uses the cd4 molecule as its principal receptor to infect t cells. hiv-1 integrates its viral genome into the host cell, leading to persistent infection wherein hiv-1 can remain transcriptionally silent in latently infected cd4 + t cells. on reactivation of replication - competent provirus, hiv-1 envelope glycoproteins (env) are expressed and accumulate on the cell surface, allowing infected cells to be detected and targeted by endogenous immune responses or immune interventions. hiv-1 env - specific antibodies have the potential to bind hiv-1 cell surface env and promote elimination of infected cd4 + t cells by recruiting cytotoxic effector cells, such as natural killer cells, monocytes, and polymorphonuclear cells. harnessing humoral and innate cellular responses has become one focus of research to develop innovative strategies to recruit and redirect cytotoxic effector cells to eliminate the hiv-1 latently infected cd4 + t - cell reservoir. |
chronic kidney disease (ckd) is a major global health burden due to its high prevalence and associated risk of end - stage renal disease (esrd), cardiovascular disease (cvd), and premature death (13). the global burden of disease study 2013 estimated 956,200 deaths worldwide were directly attributable to ckd in 2013, which represents a 134.6% increase from 1990 (4). in addition, ckd was ranked as the 19 highest cause of years of life lost in 2013 (4). this number of deaths and years of life lost has almost certainly underestimated the disease burden of ckd, as it probably only captures deaths due to esrd. it is well documented that cvd causes most of the deaths in patients with ckd (2,3). worldwide, an estimated 1.9 million esrd patients were on renal replacement therapy in 2010 (5). medical costs for the treatment of ckd and esrd are enormous and represent an immense financial burden to families and society as a whole. for example, overall us medicare expenditures for ckd and renal replacement therapy in 2010 were 41.0 and 32.9 billion us dollars, respectively, accounting for 24% of the total medicare budget (6). diabetes and hypertension are the leading causes of ckd in all high - income countries and many low- and middle - income countries (1). the global epidemic of diabetes and hypertension could lead to a worldwide increase in prevalence and in the number of persons with ckd and its complications without effective interventions (7,8). although the prevalence of ckd has been reported in individual countries, global estimates of ckd prevalence and absolute burden are not available. accurate estimates of the worldwide prevalence of this condition are essential as a source of primary information and for rational planning of health services. quantifying the global burden of ckd will allow public - health policy - makers around the world to assign sufficient priority and resources to its prevention and treatment. we aimed to estimate the global prevalence and absolute burden of ckd in 2010 by pooling data from population - based studies worldwide. a total of 35 reports from 33 studies conducted in 32 countries, which represent 48.6% of the global population 20 years old, were included in the analysis (figure 1) (943). among the included studies, 16 were conducted in nationally representative samples and the rest were in multisite or regional samples (table 1). the crude prevalence of ckd stages 15 varied from 4.5% in south korea to 25.7% in el salvador in men, and from 4.1% in saudi arabia to 16.0% in singapore in women ; stages 35 varied from 1.3% in china to 15.4% in nepal in men and from 1.7% in singapore to 21.3% in nepal in women. the age - standardized global prevalence of ckd stages 15 among adults aged 20 years in 2010 was 10.4% in men (95% ci 9.3 to 11.9%) and 11.8% in women (11.2 to 12.6%). the age - standardized prevalence was 8.6% in men (7.3 to 9.8%) and 9.6% in women (7.7 to 11.1%) in high - income countries, and 10.6% in men (9.4 to 13.1%) and 12.5% in women (11.8 to 14.0%) in low- and middle - income countries (table 2). the age - standardized global prevalence of ckd stages 35 among adults aged 20 years in 2010 was 4.7% in men (95% ci 3.4 to 6.7%) and 5.8% in women (4.4 to 8.1%). the age - standardized prevalence was 4.3% in men (3.5 to 5.2%) and 5.7% in women (4.4 to 7.6%) in high - income countries, and 4.6% in men (3.1 to 7.7%) and 5.6% in women (3.9 to 9.2%) in low- and middle - income countries. the estimated total number of adults with any stage of ckd in 2010 was 225.7 million (205.7 to 257.4 million) men and 271.8 million (258.0 to 293.7 million) women worldwide. the estimated total number of adults with any stage of ckd was 48.3 million (42.3 to 53.3 million) men and 61.7 million (50.4 to 69.9 million) women in high - income countries, and 177.4 million (162.9 to 209.0 million) men and 210.1 million (200.8 to 231.7 million) women in low- and middle - income countries (table 2). the estimated total number of adults with ckd stages 35 in 2010 was 100.6 million (72.0 to 146.4 million) men and 135.8 million (102.7 to 193.9 million) women worldwide. the estimated total number of adults with ckd stages 35 was 26.1 million (22.0 to 31.1 million) men and 44.1 million (35.3 to 54.8 million) women in high - income countries, and 74.5 million (50.3 to 123.5 million) men and 91.7 million (66.7 to 143.1 million) women in low- and middle - income countries. the prevalence of ckd (both stages 15 and stages 35) increased with age (figure 2). subsequently, women had higher prevalences than men in middle - age groups, and this sex - difference became even greater in older age groups, especially for stages 35. the pattern of age - related increase in ckd prevalence in men and women was consistent in high - income and low- and middle - income countries (table 2). in general, age - specific prevalences of ckd were higher in low- and middle - income countries compared to high - income countries, except in those aged 70 years, whose prevalence was higher in high - income countries for both men and women. the absolute numbers of men and women with ckd were much greater in low- and middle - income countries compared to high - income countries. approximately 78.6% of men and 77.3% of women with ckd stage 15 and 74.1% of men and 67.5% of women with ckd stage 35 were living in low- and middle - income countries. our analysis indicated that over 497 million adults aged 20 years and older in the world had ckd stages 15 in 2010. of them, 236 million had moderate or severe decreases in kidney function, i.e., ckd stages 35. the majority of individuals with ckd were living in low- and middle - income countries. the age - adjusted prevalences of ckd were higher in low- and middle - income countries compared to high - income countries. our results also suggest that women had a higher prevalence of ckd and that prevalences increased with age consistently in high - income and low- and middle - income countries. however, the estimates in our analysis are limited by available data from published studies, including the use of only a single creatinine measurement to calculate estimated glomerular filtration rate (egfr). it has been suggested that creatinine - based equations might systematically underestimate the true gfr, particularly among individuals with higher gfr levels (60 ml / min per 1.73 m) (44). in addition, the majority of population - based surveys did not follow patients for 3 months to confirm ckd. eriksen and ingebretsen reported that among 6,863 individuals in the municipality of troms with an initial egfr of 3059 ml / min/1.73 m, 2,175 (31.7%) had subsequent estimates 60 ml / min/1.73 m within 3 months (45). it is uncertain whether these findings are applicable to other populations with different races / ethnicities, geographic regions, and diets. nevertheless, these data indicate that a single measure of egfr may substantially overestimate the prevalence of ckd due to false positives. the high prevalence of ckd worldwide has led to an increased prevalence of esrd and mortality from cvd (13). it was estimated that more than 1.9 million esrd patients were on renal replacement therapy in 2010 (5). esrd causes reduced life expectancy and poor quality of life in patients and presents an enormous financial burden to families and society as a whole (1,6). cvd is the leading cause of death in the world, and ischemic heart disease and stroke collectively killed 12.9 million people in 2010, which represents one in four deaths worldwide (4). both reduced gfr and albuminuria or proteinuria are associated with increased risk of esrd, cvd and premature death (2,3). therefore, the prevention, detection, and treatment of ckd should be an effective approach for reducing esrd, cvd, and total mortality in the world. our study reported a high prevalence of ckd in the elderly, especially for ckd stages 35. this high prevalence of ckd is mirrored by an increased number of elderly on kidney dialysis (46). the national kidney foundation kidney disease outcomes quality initiative (nkf kdoqi) or the kidney disease : improving global outcomes (kdigo) clinical guidelines were used to define ckd in studies included in this analysis (47,48). the appropriateness of egfr < 60 ml / min/1/73 m as the cut - point for ckd in the elderly is debatable since older age is associated with a physiological decline in gfr, and moderately reduced gfr might have less risk predictive value for esrd, cvd, and total mortality in the elderly (44,49). therefore, use of a non - age - specific threshold for gfr to define ckd is most likely to substantially overestimate ckd prevalence in older age groups. further studies are warranted to define an appropriate gfr threshold for ckd in the elderly. our study also documented that women had a higher prevalence of ckd than men, and the gender difference was more pronounced in older age groups. these findings are consistent with large, nationally - representative studies from the united states, finland, and china (12,29,33). data from the us national health and nutrition examination survey (nhanes) showed that the higher prevalence of ckd in women compared to men was consistent when egfr was calculated based on the modification of diet in renal disease (mdrd) equation, chronic kidney disease epidemiology collaboration (ckd - epi) creatinine - based equation, and ckd - epi cystatin c - based equation (6). however, the prevalence and incidence of esrd are significantly higher in men than in women (6). one possible explanation for this discrepancy is a faster progression of ckd to esrd in men compared to women (45). our findings indicated that ckd affected many more individuals in low- and middle - income countries than in high - income countries. a higher prevalence of ckd and a much larger population resulted in a considerably greater absolute burden of ckd in low- and middle - income countries. in fact, more than three quarters of patients with ckd were living in low- and middle - income countries in 2010. lifestyle changes due to rapid urbanization and globalization have led to the increased prevalence of ckd risk factors in low- and middle - income countries, while limited healthcare resources result in extremely low treatment and control rates of ckd risk factors in those countries (7,8). our study results call for global collaborative efforts to combat ckd in low- and middle - income countries. the world health organization global action plan for the prevention and control of noncommunicable diseases 20132020 mentions kidney disease as a condition associated with two of its main diseases of interest, cvd and diabetes, but does not include ckd as a main priority (50). the worldwide epidemics of diabetes and hypertension have likely played a major role in the increased global burden of ckd (1,7,8). in addition, changes in lifestyle risk factors, environmental exposures, and the aging of the population have all likely contributed to the increased burden of ckd (1). these risk factors are also associated with an increased risk of esrd, cvd, and total mortality in patients with ckd. despite having an estimated prevalence similar to diabetes in many countries, much less attention and resources have been allocated to ckd surveillance, prevention, and management compared with many other chronic diseases (51). to reduce the global burden of ckd and related morbidities and mortalities, a comprehensive approach is needed that focuses on several inter - related risks to health, including hypertension, high ldl cholesterol, tobacco use, obesity, physical inactivity, poor diet, and diabetes (1,47,48). in conclusion, our findings indicate that ckd is an important global - health challenge, especially in low- and middle - income countries. national and international efforts on the prevention, detection, and treatment of ckd are needed to reduce its morbidities and mortalities worldwide. literature search, study selection, and data extraction were conducted independently and in duplicate by at least two investigators using a standardized protocol and data - collection form. we searched medline using key words prevalence, cross - sectional study, survey, renal insufficiency, renal disease, chronic kidney disease, kidney function, glomerular filtration rate, albuminuria and proteinuria. the search was restricted to studies in humans published from january 1, 1990, to december 31, 2014. a manual search was performed using references cited in reviews and original research articles. in addition, we searched projects awarded by the international society of nephrology global outreach program from 2005, when the first clinical research program projects were funded, to 2014. eligibility criteria for inclusion were : (1) population - based original studies in which prevalence of ckd (or data to calculate it) was reported ; (2) sex- and age - specific prevalence of ckd was provided ; (3) ckd was defined according to the nkf kdoqi or kdigo guidelines (47,48) and (4) serum creatinine values were standardized. the nkf kdoqi guidelines were selected because most nationally representative studies reporting ckd prevalence use these guidelines to define ckd. if a national study was available for a country, we used its data. if not, we used data from the largest and most recent multisite or regional study. if data from multiple regional studies within a country were available (15,16,30,31), they were pooled to provide national estimates by weighting by age- and sex - specific sample size. if the most recent prevalence estimates were not published and if study datasets were available, age- and gender - specific prevalence estimates were calculated from the datasets. we calculated age- and sex - specific prevalence of ckd stages 15 and stages 35 in the us population using data from nhanes 20072012 (29) and ckd stages 35 in argentina and uruguay using data from the study on the detection and follow - up of cardiovascular disease and risk factors in the southern cone of latin america (cescas) (30). if egfr from multiple equations was reported, we preferred the ckd - epi equation over the mdrd equation. likewise, we preferred urinary albumin excretion 30 mg / day (or albumin - to - creatinine ratio 30 mg / g) over urinary protein excretion 300 mg / day or a positive dipstick test of 1 + or greater to define kidney damage if multiple markers were reported. ckd stages 15 was defined as kidney damage or egfr < 60 ml / min/1.73 m, while ckd stages 35 was defined as egfr < 60 ml / min/1.73 m (47). countries were grouped into high - income countries and low- and middle - income countries using the world bank classification system updated in july 2013 (52). high - income countries are defined as those with a gross national income (gni) of $ 12,616 or more, and low - and middle - income countries are those with a gni less than $ 12,616 (52). for countries without prevalence estimates meeting our inclusion criteria, we applied data from the country with the closest geographic proximity within the same geographical region defined using the world bank classification system (52). if more than one country with data neighbored a country with no data, the country with the most similar gni was used. not all studies provided prevalence data for the full age range under consideration (from 2029 years to 70 years). incomplete age - specific prevalence was estimated using logistic regression to model the relationship between age and prevalence of ckd for each sex in high - income countries and low- and middle - income countries, separately. median age and prevalence of ckd within each reported age group from available studies were used to create the predictive models. goodness of fit was tested by cragg and uhler 's pseudo r - square, and all models were well fit in high - income countries (r between 0.55 and 0.84) and had poorer fit in low- and middle - income countries (r between 0.27 and 0.59). the prevalence and mean age in men and women from each study with missing age - specific prevalence were used to calibrate the predictive equation before it was used to estimate the age - specific prevalence for that country. if studies did not provide age - specific data in the 10-year increments of interest (i.e., 2029, 3039, etc), we averaged the prevalence from the two closest 10-year age groups weighting by age - specific population size from the country to estimate the prevalence of ckd for the age ranges of interest. for countries that reported only the prevalence of ckd stages 35, logistic regression was used to predict the age - specific prevalence of stages 15 in men and women for high - income countries and low- and middle - income countries, separately. eight high - income countries (48 age - specific prevalence estimates) and five low - income countries (30 age - specific prevalence estimates) reported both prevalence of ckd stages 15 and stages 35 in men and women. these data were used to generate the predictive models for ckd stages 15 prevalence estimates from the age - specific prevalence of ckd stages 35. for studies that reported only the prevalence of ckd stages 15, these models all had good fit assessed by cragg and uhler 's pseudo r - square (r between 0.56 and 0.89). sex- and age - specific prevalences of ckd for each country were applied to the who sex- and age - specific population counts in 2010 to estimate the number of people with ckd in the country for each sex- and age - group (53). the total number of persons with ckd in high - income countries and low- and middle - income countries was summed separately to provide an estimate of the total number of persons with ckd for each income category by sex- and age - groups, and the number from each income category was added to obtain the worldwide count. the sex- and age - specific prevalences of ckd for 2010 in high - income countries and low- and middle - income countries were calculated by dividing the total number of people with ckd in each income category by the number of people in that income category by sex- and age - groups. worldwide prevalence was estimated by dividing the total number of persons with ckd by the total adult world population. the prevalences of ckd in high - income countries and low- and middle - income countries were standardized by age to the 2010 world population separately for each sex and overall using the direct method. confidence intervals for all prevalence estimates were calculated using bootstrap methods (54). for each prevalence estimate, we chose n studies, where n is the number of countries used to estimate that prevalence estimate, with replacement and calculated a mean prevalence. then we repeated this process 10,000 times and choose the values at 2.5% and 97.5% of the distribution as our bootstrap 95% confidence intervals. confidence intervals for all absolute burden estimates were calculated by multiplying the bootstrap 95% confidence intervals by the population for that prevalence estimate to get the 95% confidence intervals for the number of people with ckd. all analyses were done using sas (version 9.3), r (version 3.1.2), and microsoft excel software. we searched medline using key words prevalence, cross - sectional study, survey, renal insufficiency, renal disease, chronic kidney disease, kidney function, glomerular filtration rate, albuminuria and proteinuria. the search was restricted to studies in humans published from january 1, 1990, to december 31, 2014. in addition, we searched projects awarded by the international society of nephrology global outreach program from 2005, when the first clinical research program projects were funded, to 2014. eligibility criteria for inclusion were : (1) population - based original studies in which prevalence of ckd (or data to calculate it) was reported ; (2) sex- and age - specific prevalence of ckd was provided ; (3) ckd was defined according to the nkf kdoqi or kdigo guidelines (47,48) and (4) serum creatinine values were standardized. the nkf kdoqi guidelines were selected because most nationally representative studies reporting ckd prevalence use these guidelines to define ckd. if a national study was available for a country, we used its data. if not, we used data from the largest and most recent multisite or regional study. if data from multiple regional studies within a country were available (15,16,30,31), they were pooled to provide national estimates by weighting by age- and sex - specific sample size. if the most recent prevalence estimates were not published and if study datasets were available, age- and gender - specific prevalence estimates were calculated from the datasets. we calculated age- and sex - specific prevalence of ckd stages 15 and stages 35 in the us population using data from nhanes 20072012 (29) and ckd stages 35 in argentina and uruguay using data from the study on the detection and follow - up of cardiovascular disease and risk factors in the southern cone of latin america (cescas) (30). if egfr from multiple equations was reported, we preferred the ckd - epi equation over the mdrd equation. likewise, we preferred urinary albumin excretion 30 mg / day (or albumin - to - creatinine ratio 30 mg / g) over urinary protein excretion 300 mg / day or a positive dipstick test of 1 + or greater to define kidney damage if multiple markers were reported. ckd stages 15 was defined as kidney damage or egfr < 60 ml / min/1.73 m, while ckd stages 35 was defined as egfr < 60 ml / min/1.73 m (47). countries were grouped into high - income countries and low- and middle - income countries using the world bank classification system updated in july 2013 (52). high - income countries are defined as those with a gross national income (gni) of $ 12,616 or more, and low - and middle - income countries are those with a gni less than $ 12,616 (52). for countries without prevalence estimates meeting our inclusion criteria, we applied data from the country with the closest geographic proximity within the same geographical region defined using the world bank classification system (52). if more than one country with data neighbored a country with no data, the country with the most similar gni was used. not all studies provided prevalence data for the full age range under consideration (from 2029 years to 70 years). incomplete age - specific prevalence was estimated using logistic regression to model the relationship between age and prevalence of ckd for each sex in high - income countries and low- and middle - income countries, separately. median age and prevalence of ckd within each reported age group from available studies were used to create the predictive models. goodness of fit was tested by cragg and uhler 's pseudo r - square, and all models were well fit in high - income countries (r between 0.55 and 0.84) and had poorer fit in low- and middle - income countries (r between 0.27 and 0.59). the prevalence and mean age in men and women from each study with missing age - specific prevalence were used to calibrate the predictive equation before it was used to estimate the age - specific prevalence for that country. if studies did not provide age - specific data in the 10-year increments of interest (i.e., 2029, 3039, etc), we averaged the prevalence from the two closest 10-year age groups weighting by age - specific population size from the country to estimate the prevalence of ckd for the age ranges of interest. for countries that reported only the prevalence of ckd stages 35, logistic regression was used to predict the age - specific prevalence of stages 15 in men and women for high - income countries and low- and middle - income countries, separately. eight high - income countries (48 age - specific prevalence estimates) and five low - income countries (30 age - specific prevalence estimates) reported both prevalence of ckd stages 15 and stages 35 in men and women. these data were used to generate the predictive models for ckd stages 15 prevalence estimates from the age - specific prevalence of ckd stages 35. for studies that reported only the prevalence of ckd stages 15, these models all had good fit assessed by cragg and uhler 's pseudo r - square (r between 0.56 and 0.89). sex- and age - specific prevalences of ckd for each country were applied to the who sex- and age - specific population counts in 2010 to estimate the number of people with ckd in the country for each sex- and age - group (53). the total number of persons with ckd in high - income countries and low- and middle - income countries was summed separately to provide an estimate of the total number of persons with ckd for each income category by sex- and age - groups, and the number from each income category was added to obtain the worldwide count. the sex- and age - specific prevalences of ckd for 2010 in high - income countries and low- and middle - income countries were calculated by dividing the total number of people with ckd in each income category by the number of people in that income category by sex- and age - groups. worldwide prevalence was estimated by dividing the total number of persons with ckd by the total adult world population. the prevalences of ckd in high - income countries and low- and middle - income countries were standardized by age to the 2010 world population separately for each sex and overall using the direct method. confidence intervals for all prevalence estimates were calculated using bootstrap methods (54). for each prevalence estimate, we chose n studies, where n is the number of countries used to estimate that prevalence estimate, with replacement and calculated a mean prevalence. then we repeated this process 10,000 times and choose the values at 2.5% and 97.5% of the distribution as our bootstrap 95% confidence intervals. confidence intervals for all absolute burden estimates were calculated by multiplying the bootstrap 95% confidence intervals by the population for that prevalence estimate to get the 95% confidence intervals for the number of people with ckd. all analyses were done using sas (version 9.3), r (version 3.1.2), and microsoft excel software. | chronic kidney disease (ckd) is a major risk factor for end - stage renal disease, cardiovascular disease and premature death. here we estimated the global prevalence and absolute burden of ckd in 2010 by pooling data from population - based studies. we searched medline (january 1990 to december 2014), international society of nephrology global outreach program funded projects, and bibliographies of retrieved articles and selected 33 studies reporting gender- and age - specific prevalence of ckd in representative population samples. the age standardized global prevalence of ckd stages 15 in adults aged 20 and older was 10.4% in men (95% confidence interval 9.311.9%) and 11.8% in women (11.212.6%). this consisted of 8.6% men (7.39.8%) and 9.6% women (7.711.1%) in high - income countries, and 10.6% men (9.413.1%) and 12.5% women (11.814.0%) in low- and middle - income countries. the total number of adults with ckd was 225.7 million (205.7257.4 million) men and 271.8 million (258.0293.7 million) women. this consisted of 48.3 million (42.353.3 million) men and 61.7 million (50.469.9 million) women in high - income countries, and 177.4 million (159.2215.9 million) men and 210.1 million (200.8231.7 million) women in low- and middle - income countries. thus, ckd is an important global - health challenge, especially in low- and middle - income countries. national and international efforts for prevention, detection, and treatment of ckd are needed to reduce its morbidity and mortality worldwide. |
pseudomonas aeruginosa is a common gram - negative rod - shaped bacterium that leads to significant morbidity and mortality in humans. p. aeruginosa is a prototype of multidrug - resistant pathogen and is recognized for its ubiquitous distribution, advanced antibiotic resistance mechanisms, and nosocomial infections. p. aeruginosa has the ability to survive on minimal nutrition and tolerate a variety of physical insults leading to its persistence in the community and hospital settings. despite the wide distribution of p. aeruginosa in nature and the potential for community - acquired infections, serious infections with this bacterium p. aeruginosa was identified as the fifth most frequently isolated nosocomial pathogen by the surveillance data from the united states. p. aeruginosa is one of the major causes of hospital - acquired infections and is responsible for about 1020% of nosocomial infections in patients admitted in the intensive care units (icus). p. aeruginosa can be isolated from a wide variety of sources in the hospital, including respiratory equipment, antiseptics, soaps, sinks, mops, medicines, and physiotherapy tools. p. aeruginosa is divided into different phenotypes based on the drug resistance patterns of the organism. multidrug - resistant (mdr) phenotype is defined as p. aeruginosa, which is resistant to more than one antimicrobial agent in three or more antimicrobial categories. a similar resistance to more than one antimicrobial agent in <3 antimicrobial categories is defined as drug - resistant (dr) p. aeruginosa. the details of the antimicrobial agents and categories used to define the phenotypes are given in table 1. extensively dr (xdr) phenotype is defined as p. aeruginosa, which is resistant to more than one antimicrobial agent in all the antimicrobial categories, except in two or less. pan - dr (pdr) phenotype is defined as a bacterium which is resistant to all antimicrobial agents in all antimicrobial categories. antimicrobial categories and agents used to define the pseudomonas aeruginosa phenotypes along with percentage of resistant strains mdr, xdr, and pdr phenotypes elaborate inactivating enzymes, such as extended - spectrum beta - lactamases (esbl) and metallo--lactamases (mbl), that make beta - lactams and carbapenems ineffective. esbl - producing p. aeruginosa was initially detected in europe in the mid-1980s, and mbl - producing p. aeruginosa was first reported from japan in 1991. literature is sparse regarding the incidence of different phenotypes of p. aeruginosa from our country as we could retrieve only two articles in the pubmed with the relevant (p. aeruginosa, india, phenotype, incidence) keywords. hence, we conducted this study to find the incidence of mdr, xdr, and pdr phenotypes of p. aeruginosa in the icu of a tertiary care hospital. this prospective, observational study was conducted at the department of microbiology of a tertiary level hospital for over a period of 2 years from january 2014 to december 2015. we included all patients who were at least 10 years of age at the time of the admission to the icu of this hospital. as per our hospital policy, patients below 10 years of age requiring intensive are admitted to the pediatric icu. our study does not include data from the pediatric icu, thereby resulting in the exclusion of patients below 10 years of age. the patient with evidence of septicemia and known diagnosis of p. aeruginosa infection at the time of admission was excluded from the study. routine hematology and biochemistry tests were conducted as per the request of the clinical team. microbiological specimens were collected from the blood, urine, and sputum samples of the patients. other samples were collected based on the clinical suspicion from a site harboring infection, such as throat swab, wound discharge, and body fluid culture. samples obtained after an invasive procedure and from the indwelling catheters were also included in the study. a total of 100 environmental samples were collected as part of active surveillance of the hospital environment and staff, which were grouped as group 2. these samples were collected from the medical staff (hand, throat, and nasal swabs) and the hospital environment (air, surgical instruments, dressing material, floors, door, walls, beds, sinks, and antiseptic solutions). all blood culture and fluid samples with a minimum volume of 3 ml the bact / alert system is a continuously monitored blood culture system for detecting bacteremia and fungemia. bottles flagged as positive by the bact / alert system were subcultured and interpreted according to the standard laboratory protocols. samples other than the blood and body fluids of group 1 and all group 2 were cultured on macconkey agar and blood agar. these plates were incubated overnight at 37c and all the bacteria grown in these culture media were processed for identification and antibiotic susceptibility testing using automated bacterial system vitek 2 system (biomrieux). vitek 2 system uses a new fluorescence - based technology used for the identification and susceptibility testing of bacteria and is compliant with the clinical and laboratory standard institute (clsi) guidelines 2014. p. aeruginosa was identified by its colony characteristics, pigment production, grape - like odor, oxidase positivity, motility, gram - negative character of the bacilli, ability to decarboxylate arginine and to grow at 42c. antibiotic sensitivity patterns of these isolates were studied by minimal inhibitory concentration using the vitek 2 system as per the clsi 2014 guidelines. strains which had the same types of resistance patterns (antibiotype) were considered to be from the same clone. this prospective, observational study was conducted at the department of microbiology of a tertiary level hospital for over a period of 2 years from january 2014 to december 2015. we included all patients who were at least 10 years of age at the time of the admission to the icu of this hospital. as per our hospital policy, patients below 10 years of age requiring intensive are admitted to the pediatric icu. our study does not include data from the pediatric icu, thereby resulting in the exclusion of patients below 10 years of age. the patient with evidence of septicemia and known diagnosis of p. aeruginosa infection at the time of admission was excluded from the study. routine hematology and biochemistry tests were conducted as per the request of the clinical team. microbiological specimens were collected from the blood, urine, and sputum samples of the patients. other samples were collected based on the clinical suspicion from a site harboring infection, such as throat swab, wound discharge, and body fluid culture. samples obtained after an invasive procedure and from the indwelling catheters were also included in the study. a total of 100 environmental samples were collected as part of active surveillance of the hospital environment and staff, which were grouped as group 2. these samples were collected from the medical staff (hand, throat, and nasal swabs) and the hospital environment (air, surgical instruments, dressing material, floors, door, walls, beds, sinks, and antiseptic solutions). all blood culture and fluid samples with a minimum volume of 3 ml the bact / alert system is a continuously monitored blood culture system for detecting bacteremia and fungemia. bottles flagged as positive by the bact / alert system were subcultured and interpreted according to the standard laboratory protocols. samples other than the blood and body fluids of group 1 and all group 2 were cultured on macconkey agar and blood agar. these plates were incubated overnight at 37c and all the bacteria grown in these culture media were processed for identification and antibiotic susceptibility testing using automated bacterial system vitek 2 system (biomrieux). vitek 2 system uses a new fluorescence - based technology used for the identification and susceptibility testing of bacteria and is compliant with the clinical and laboratory standard institute (clsi) guidelines 2014. p. aeruginosa was identified by its colony characteristics, pigment production, grape - like odor, oxidase positivity, motility, gram - negative character of the bacilli, ability to decarboxylate arginine and to grow at 42c. antibiotic sensitivity patterns of these isolates were studied by minimal inhibitory concentration using the vitek 2 system as per the clsi 2014 guidelines. strains which had the same types of resistance patterns (antibiotype) were considered to be from the same clone. the mean age of the patients who were included in the study was 36.2 years. a total of 597 pathogenic bacteria were isolated from the 1915 clinical specimens, giving a culture positivity rate of 31.2% for the clinical specimen. the details of all the bacterial species isolated from the study samples are shown in figure 1. other bacteria such as coagulase - negative staphylococci, staphylococcus aureus, escherichia coli, and proteus spp. accounted for the rest of the bacterial cultures. hospital staff and group 2 samples did not grow any pathogenic bacterium, including p. aeruginosa. pattern of microbes isolated in the study sample the incidence rate of p. aeruginosa was highest (78%) in the age group of patients between 20 and 50 years. urine (47 out of 88) and wound (38 out of 88) samples accounted for the majority of the positive isolates. the prevalence of p. aeruginosa phenotypes based on the resistance patterns is shown in figure 2. our data showed that 47.7% strains were dr, 50% were mdr, and 2.3% were of xdr phenotype. no strains were resistant to all antibiotics in all antimicrobial categories as all the strains were found to be sensitive to fosfomycin and colistin (100%). our study showed the prevalence of drug resistance in almost 98% of the isolates of p. aeruginosa in the icu. the only solace is the findings of the 0% presence of pdr phenotype, thereby showing the efficacy of certain antibiotics against this organism. piperacillin resistance was found to be 70% in our study, which is comparable to a report showing it to be 73%. emergence of resistance to beta - lactams in p. aeruginosa has become a serious threat, particularly against the third- and fourth - generation cephalosporins. there are a lot of molecular mechanisms involved for resistance against these antibiotics, generation of esbl enzyme, incorporation of bla genes in integrons, and inability of porin genes to enhance their expression level and/or alteration of antibiotic target sites. ceftazidime and cefepime are the prescribed antipseudomonal third- and fourth - generation cephalosporins, respectively. the resistance to ceftazidime and cefepime observed in our study was similar to that of a previous report from india. our study showed that 80% of p. aeruginosa were resistant to carbapenem antibiotics such as imipenem and meropenem. previous studies showed a similar trend from other countries and a few authors had shown 100% resistance against carbapenems. this suggests the declining efficacy of the carbapenems as an effective antibiotic in the management of nosocomial infections. fluoroquinolone compounds are one of the important antimicrobial agents that have been used for a variety of infections. new generation fluoroquinolones are beneficial against gram - negative and gram - positive bacteria, whereas older fluoroquinolones were effective against aerobic gram - negative bacteria only. our data showed 96% resistance against ciprofloxacin and 67% resistance against levofloxacin while 100% resistance against ciprofloxacin was demonstrated earlier. our data showed similar resistance pattern between amikacin and gentamicin, which was similar to that of the published literature. similar data were not available for comparison with other authors about the sensitivity to aztreonam. similar data were reported from other countries where there is a high prevalence of p. aeruginosa infection. to the best of our knowledge, this is the first study from india that defines the prevalence of mdr, xdr, and pdr p. the strengths of our study include long duration of observation and the use of highly advanced automated bacteria identification system in the identification of p. aeruginosa. the limitations of our study include data derived from a single hospital may not be applicable to other geographic locations and inability to check resistance pattern to all the antipseudomonal drugs. mdr p. aeruginosa is responsible for serious nosocomial infections among the patients admitted in the icus of the hospital. epidemiological studies and strict laws regarding antibiotic policies should be constructed to limit the unnecessary use of antibiotics so that spread of multidrug resistance can be avoided. | background : infection by pseudomonas aeruginosa is common in the intensive care unit (icu), leading to increased morbidity and mortality. the organism is classified into various phenotypes based on the drug resistance pattern, namely, drug - resistant (dr), multi - dr (mdr), extensively dr (xdr), and pan - dr (pdr). we aim to study the incidence of p. aeruginosa phenotypes in a tertiary level icu.materials and methods : we conducted this prospective, observational study for 2 years (january 2014-december 2015) and collected appropriate clinical samples (blood, urine, wound discharge, etc.,) from all the patients admitted to icu. we excluded patients with known septicemia and p. aeruginosa infection. group 1 comprised a total 1915 patient samples and group 2 comprised 100 active surveillance samples, collected from the medical staff and the hospital environment. the data were analyzed using appropriate statistical methods, and a p < 0.05 was considered statistically significant.results:we isolated 597 pathogenic bacteria out of 1915 specimens, giving a culture positivity rate of 31.2%. klebsiella (43%), acinetobacter (22%), and p. aeruginosa (15%) were the top three isolated bacteria. none of the surveillance samples grew p. aeruginosa. antibiotic resistance studies revealed that 47.7% of p. aeruginosa isolates were dr, 50% were mdr, and 2.3% were xdr phenotype. none of the strains showed pdr phenotype.conclusion:our data revealed a high prevalence of dr phenotypes of p. aeruginosa in the icu. judicious use of antibiotics and strict infection control measures are essential to reduce the prevalence of drug resistance. |
there are about 190 cases reported with higher prevalence of diagnosis in pediatric age group. with first histological confirmation in 1895 polyorchidism can clinically have varied presentations as inguinal swelling, cryptorchidism, testicular torsion, hydrocele and varicocele with majority being asymptomatic. the most common form of polyorchidism is triorchidism (or 3 testes) with left sided predominance (65%) over the right. a higher tendency for malignant transformation in non - functional testis necessitates the radiological and functional assessment of polyorchid testes. we hereby report a case of 24-year old male with left side super - numerary non - functional testis, presenting as left sided inguinal hernia. a 24-year old young male came with reducible swelling in left inguinal region since 4 years without associated history of trauma or pain. on detailed local examination, a reducible swelling in the left inguinal region with positive cough impulse and positive deep ring occlusion test was noted. the right testis was normal in size, whereas left testis was smaller in size 332 cm (about 50%) compared to the right side. intraoperative findings of indirect inguinal hernia sac was present with small undescended super - numerary testis of size 2.52.51 cm at the deep inguinal ring (figure 1) having short vas deferens and a normally located left testis with epididymis and vas deferens in the scrotal sac. due to anomalous anatomy of super - numerary inguinal testis, orchidectomy with excision of indirect sac was performed. the histopathological examination of the specimen reported thickened capsule of testis and tubules with arrest of development of the germ cells (figure 2). a prominence of sertoli and leydig cells along with hyalinised, thickened basement membrane of seminiferous tubules and dense intervening stroma was found. a follow up tumor marker assay showed negative study, which can likely be present in burned out tumor in spite of negative histology. polyorchidism presents itself more commonly in the 2 to 3 decade, with approximately 50% of cases reported between 15 to 25 years of age. the common presentations of polyorchidism manifests as maldescent (40%), hernia (30%), torsion (15%), hydrocele (9%), malignancy (6%), infertility and epididymitis. the testis takes its origin from the medial part of primitive genital ridge while the epididymis and vas deferens develop from the wolffian duct. the exact etiology of polyorchidism is unclear, however accidental division of genital ridge before 8 week of gestation could be a possible cause. there are theories proposing the probable presence of multiple testes first being duplication of longitudinal genital ridge resulting into separate testes so total volume of testis exceeds of one. the second theory explains transverse division of genital ridge at different levels resulting into different combination of testes, epididymis and vas deferens. based on site of testes they can be classified as scrotal (66%), inguinal (23%) and abdominal (9%). leung (1988) classified polyorchidism based on embryological development with possible anatomical distinct epididymis or vas deferens. singer (1992) and colleagues proposed classification based on anatomical and functional potential of supernumerary testis to epididymis and finally by vas deferens. an effort to restore the functionality of undescended testes can be attempted if surgically corrected before age of 4 - 5 years. a significant risk of malignancy is reported in undescended non - functional testis before puberty being 2.2% while after puberty it rises substantially to 5.4%. recent publications also suggest higher incidence of testicular malignancies (4 to 6%) in cryptorchid testis compared to normal testis. there is higher risk of testicular malignancy in polyorchidism cases of about 7% especially in non - scrotal testis. of these the management of supernumerary testes has been a topic of much debate and different factors like age of patient, location of testes, functional status and lastly compliance and accessibility for follow up have to be taken into consideration. broadly in pediatric age group or patients with reproductive desire and uncomplicated polyorchidism should have conservative approach like orchidopexy, however a strict vigilant follow up is mandatory. while adults without reproductive desire, complicated polyorchidism and with inaccessibility for follow up should not be considered for conservative management. in our view supernumerary testis with complications like cryptorchidism, torsion and intraoperative non - functional testis i.e., without a draining vas deferens or epididymis, excision can be performed due to higher risk of malignancy in polyorchidism. while conservative approach is recommended for uncomplicated polyorchidism in functional testis with a dedicated follow up. current review of literature recommends conservative management for uncomplicated functional testes and regular follow up in polyorchidism. a surgical approach is advisable in complicated, non - compliant and non - reproductive patients in view of higher risk of malignancy in these patients. | triorchidism is the commonest variety of polyorchidism, an entity with more than two testis is an extremely rare congenital anomaly of the testis. although excision of the abnormal testis is a safer alternative proposed, recent literature suggests more conservative approach in normal testes with watchful regular follow up to screen for malignancy. this case presented as a left inguinal swelling diagnosed as indirect left inguinal hernia. the left side testis was of smaller size (about half) with normal sperm count, morphology and motility. intraoperatively indirect inguinal hernia was noted with supernumerary testis at deep ring in addition to normal left testis in left scrotal sac. the ectopic testis were small (2.52.51 cm) lacking epididymis and with short vas deferens. an evident normal semen analysis and varied anatomy, the decision for orchidectomy of ectopic testis was taken. the histopathological finding was consistent with arrest in germ cell development. |
the incidence of natural pop ranges from 1:60 000 to 1:1 500 deliveries, accounting for 3.3% of all ectopic pregnancies. since the report of the first successful human in vitro fertilization embryo transfer (ivf - et) cycle resulting in ectopic pregnancy, the association of ectopic pregnancy and ivf cycles has been well established. despite the increased incidence of ectopic pregnancy associated with assisted reproductive technologies, the overall prevalence of ovarian pregnancy has been estimated at 0.3%, representing 6% of all ectopic pregnancies. with new technologies being introduced for ivf - et in recent years, pop has been reported after some of these procedures. thus, 3 reports highlight pop occurrence after intracytoplasmic sperm injection (icsi) and blasto - cyst transfer. donor embryo transfer is a relatively new procedure and, to our knowledge, has not been reported in association with ovarian pregnancy. we report a case of pop that occurred after early donor embryo transfer in a woman with no predisposing factors for ectopic implantation. this case is unique because the ovarian implantation occurred after ovarian suppression rather than with hyperstimulation, retrieval, and formation of corpora lutea cysts as reported in prior published case reports. a 39-year - old nulligravid woman presented to our fertility center for evaluation of primary infertility. on initial examination, a transverse vaginal septum obscuring the cervix and a 1.5-cm subserosal fibroid were identified. the husband was diagnosed with azoospermia and had twice undergone testicular biopsy with insufficient spermatogenesis, preventing sperm retrieval. they elected and arranged for a donor embryo transfer at a fertility center in their home country. the patient then had an ovarian suppression with a long protocol using buserelin acetate and estradiol valerate. the initial value was only 5 iu / l on posttransfer day 14 with an increase to 11, 27, 47 and 653 iu / l on days 16, 18, 20 and 27, respectively (figure 1). a repeat ultrasound was performed 8 days later after the patient experienced vaginal spotting with rising hcg. the hcg level reached 4811 iu / l. at that time, an ultrasonogram was significant for a 1.5 cm x 1.5 cm x 1.4 cm solid echogenic structure with reinforced vascular marking within to the left ovary suggesting primary ovarian pregnancy (figure 2). the left ovary was enlarged by a 3.0 cm x 2.0 cm heterogeneous mass consisting of several hemorrhagic bluish cysts, with a smooth external surface (figure 3). intraoperative frozen section and final histology confirmed the presence of the conception products associated with ovarian tissue. a small amount of serous fluid was in the posterior cul - de - sac. follow - up quantitative hcg levels declined appropriately and reached an undetectable level (< 2 transvaginal ultrasound on day 40 post embryo transfer : echogenic structure with reinforced vascular marking within the left ovary. although a rare phenomenon, primary ovarian pregnancy is an important complication associated with ivf - et treatment. theoretical risk factors for ovarian implantation include reduction in tubal contractility as a result of high progesterone levels from multiple corpus lutea, ovarian hypervascularity after hyperstimulation and egg retrieval, excessive endometrial cavity distension with media during embryo transfer, a high number of the transferred embryos, and transfer of blastocyst. in contrast, the donor embryo ivf cycle requires ovarian suppression, eliminating most hypothesized risk factors. however, in our case, a high number of transferred embryos is a possible explanation of ovarian implantation. interestingly, the most frequently reported site of ovarian pregnancy is the left ovary, leaving the question of exact mechanism of retrograde embryo migration open. because the occurrence of ovarian pregnancy is difficult to predict, its early recognition is imperative in patients undergoing ivf treatment. in fact, up to 50% of primary ovarian pregnancies might be diagnosed at an asymptomatic state mainly based on a physician 's suspicion and experience, preventing serious complications such as massive hemoperitoneum. although not specific for ovarian pregnancy, abnormally low- and slow - rising hcg levels aid in early recognition of abnormal implantation.. the initial sonographic picture of ovarian pregnancy might be obscured by multiple corpora lutea cysts after hyperstimulation and egg retrieval in a standard ivf - et cycle. the decisive ultrasonographic characteristics are the visualization of gestational sac structures and the presence of fetal heart beat within the ovary. however, even the subtle sonographic findings, such as a round echogenic mass with reinforced vascular marking within the ovary and free peritoneal fluid, are consistent with ovarian pregnancy in case of ovarian suppression for donor embryo transfer (figure 2). laparoscopic management with resection of ovarian gestation and preservation of remaining ovarian tissue is the preferred treatment for ovarian pregnancy. to avoid misdiagnosis, surgical frozen section at the time of surgery additionally, successful medical management with methotrexate as an alternative to surgery and treatment for a persistent trophoblast after laparoscopic resection of ovarian gestation has been reported. however, the absence of a histopathological diagnosis needed to meet the widely accepted spiegelberg 's criteria of primary ovarian pregnancy precludes using methotrexate as the first line of treatment. it is important for clinicians and patients to recognize the possibility of developing a primary ovarian pregnancy after ovarian suppression and donor embryo transfer in an ivf program. our case demonstrates that close follow - up of abnormally rising hcg, clinical suspicion, and sono - graphic appearance of an adnexal mass can lead to the early diagnosis and successful laparoscopic ovary preserving treatment. | primary ovarian pregnancy is a rare type of ectopic pregnancy, particularly following in vitro fertilization. although there have been a few reported cases of primary ovarian pregnancy following in vitro fertilization embryo transfer, we believe this is the first report involving donor embryo transfer. a high index of clinical suspicion, abnormal human chorionic gonadotropin levels, and early ultrasound evaluation may aid timely diagnosis and appropriate management. this report provides a reminder to practitioners to advise patients undergoing embryo transfer of the primary ovarian pregnancy risk. information is provided herein regarding the diagnosis and management of primary ovarian pregnancy in women treated with in vitro fertilization. we review the criteria for early diagnosis and treatment options. |
post - translational modifications are essential for the rapid and reversible modification of the physiochemical properties of a protein, resulting in the changes of enzyme activity, oligomerization state, protein protein interaction, subcellular localization or half - life (1). protein phosphorylation is the most abundant and biologically the most important post - translational modification on the tyrosine, serine and threonine residues, and is catalyzed reversibly by specific protein kinases and phosphatases. bacteria and some plants rely on histidine autophosphorylation of the sensory kinases and aspartate phosphorylation of the response regulators (thus two - component systems) (2). protein phosphorylation is perhaps the best studied due to the close association between dys - regulated phosphorylation and human pathologies (3). therefore, it is extremely important to determine the degree and the site of the in vivo protein phosphorylation. however, due to the very low stoichiometry, limited dynamic range, high complexity and quantitative difficulties of protein phosphorylations, highly selective enrichment procedures and sensitive mass spectrometry (ms) are required to decipher the phosphoproteome (4). selective phosphopeptide enrichment has been accomplished in several ways by using anti - phosphotyrosine antibodies, immobilized metal affinity chromatography (imac), chemical modifications or strong cation exchange chromatography (5). the seamless combination of imac and nano - liquid chromatography enables reproducible separation and identification of phosphopeptides in a low - femtomole range 6, 7, and thus it is the most frequently used method in the study of cellular phosphorylation. for some time, protein phosphorylation was considered to exist exclusively in eukaryotes until the first observation of protein phosphorylation occurring in escherichia coli 8, 9. indeed, protein phosphorylation is fundamental to the regulation of all kinds of physiological processes for the bacteria, especially for several key steps in the infection process, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare, scrambling the host signaling cascades and impairing its defense mechanisms 10, 11. the global phosphoproteome has been established in a number of bacteria (table 1), including corynebacterium glutamicum (12), campylobacter jejuni (13), bacillus subtilis 14, 15, 16, e. coli (17), latococcus lactis (18), streptococcus pneumoniae (19), klebsiella pneumoniae (20), mycoplasma pneumoniae (21), pseudomonas species (22), mycobacterium tuberculosis (23), streptomyces coelicolor (24) and helicobacter pylori 13, 25. phosphorylation in the bacteria was biased toward threonine compared with serine, while serine phosphorylation may account for 80%-90% of total phosphorylation sites in the eukaryotes (26). l. lactis, c. jejuni, and s. coelicolor contain more phosphorylation sites of threonine than serine (table 1). the most abundant subset of phosphorylated proteins is the enzymes involved in the central carbon / protein / nucleotide metabolism, and some other phosphorylated housekeeping proteins are helicases, chaperones, ribosomal proteins and amino acyl trna - synthetases (27). one quantitative phosphoproteomic analysis on the model bacterium b. subtilis has been performed with stable isotope labeling by amino acids in cell culture (silac) (28). the striking distinction between the known metazoan and bacterial phosphoproteomes are the extent : 30%-50% of proteins are phosphorylated in humans 29, 30, whereas it is at least one order of magnitude lower in bacteria (27). the only notable exception is m. tuberculosis, whose yield was significantly high : 516 phosphorylation sites in 301 phosphoproteins, accounting for > 7% of m. tuberculosis proteins (23). differential roles were proposed for the protein phosphorylations in eukaryotes and prokaryotes : protein phosphorylation in the eukaryotes is extensively used for transduction of signals inter- and intra - cellularly, whereas the function may be less central in the prokaryotes (16). bacterial phosphorylation sites can be conferred by two major search algorithms netphosbac (31) and diphos (32), which were summarized in a recent review paper (27). here we will focus on an overview of the recent advances in the field of bacterial phosphoproteome, highlighting recent methods in phosphoproteomics, connectivity of the phosphorylation networks, as well as the correlation between pathological potentials and the known bacterial phosphoproteomes. the major challenges in phosphoproteomics are the generally very low stoichiometry and high complexity of phosphorylation, limited dynamic range of detection methods available, and quantitative difficulties (4). as for the complexity, the phosphosite database (www.phosphosite.org) lists over 99,000 non - redundant phosphorylation sites up to august 2011, and this number is expanding continually. the conservation of the phosphorylation sites in the bacterial phosphoproteome is extremely low, even in the case of proteins phosphorylated in most bacterial species, such as the enzymes of the central carbon metabolism (27), which suggests the diverse kinase specificities to adapt to different environmental niches. the dynamic range of the phosphoproteome is quite large (~10), from hundreds of millions to a few copies per cell. therefore, it is mission impossible to detect all the phosphopeptides upon proteolytic digestion of the whole cell lysates or tissue samples. selective enrichment of the phosphoproteins / peptides is required and has been accomplished in several ways : anti - phosphotyrosine antibodies (33), imac 34, 35, chemical modifications 36, 37 and strong cation exchange chromatography (scx) (38). figure 1 summarizes several of the most significant work over the past decade 5, 39. for a global view of serine, threonine and tyrosine phosphorylations, imac may be the best choice (6), with peptide recovery up to 90% as determined by p or p - radioactivity measurements (40). phosphopeptides are retained on nitriloacetic acid (nta) or iminodiacetic acid (ida)-linked hard metal ions, such as fe, ga, al (39) and zr (41), through their negatively charged phosphate group. non - specific binding of peptides containing glu and asp could be minimized either by carefully controlling experimental conditions or through methyl - esterification of carboxylic acids by hcl - saturated dry methanol (42). recently, titanium dioxide (tio2) chromatography has emerged as the most common method for the enrichment of global phosphoproteins / peptides (43). advances in the high accuracy ms allow for the identification of thousands of phosphorylation sites in a single experiment (44). both imac and tio2 columns can be coupled seamlessly with ms for reproducible separation, detection and identification of phosphopeptides in a low - femtomole range 7, 45, 46, 47. upon the identification of the phosphoproteome, bioinformatics analysis through gene ontology (go) annotation (48) or the bacterial localization prediction tool psortb (49) is often applied to get the relevant information of cellular function, localization and protein as a gram - negative, spiral - shaped bacterium that colonizes in the gastric mucosa of humans (51), h. pylori has been frequently associated with atrophic gastritis, peptic ulcer disease, functional dyspepsia and gastric carcinomas (52). cytotoxin - associated antigen a (caga) is a major pathogenicity protein in h. pylori and plays key roles in inducing gastric inflammation, ulcer and carcinogenesis. caga protein is translocated from the bacterium, undergoes tyrosine phosphorylation at the glu - pro - ile - tyr - ala (epiya) motif in the host cells and induces a cellular hummingbird phenotype of transformation (53). in addition, non - phosphorylated caga interacts with host proteins, such as epithelial tight junction - scaffolding protein zonulin (zo-1), cell adhesion protein e - cadherin, hepatocyte growth factor receptor c - met, cadherin - associated protein -catenin, adaptor protein grb-2 and the kinase par1, leading to a loss of cell polarity and inducing pro - inflammatory and mitogenic responses (54). besides the knowledge about caga phosphorylation in the host cells, there is no information available about the in vivo phosphorylation state of h. pylori intracellular proteins. bioinformatics indicated that the genome of h. pylori strain 26695 contains at least one protein kinase (hp0432) and one ppm - family protein phosphatase (hp0431) (55). up to now, three independent studies intended to discover the intracellular phosphorylation in h. pylori : (1) eight proteins phosphorylated at serine residues through sds - page and autoradiography (56) ; (2) 57 proteins identified through fe - imac enrichment, 2d gel electrophoresis and maldi - tof ms analysis, albeit without further phosphorylation site mapping (13) ; and (3) 82 phosphopeptides from 67 proteins with 79 class i (with localization probability higher than 0.75) phosphorylation sites : 33 (42.8%) on serine, 31 (38.7%) on threonine and 15 (18.5%) on tyrosine (25). tyrosine phosphorylation has generally been regarded as an exclusively eukaryotic phenomenon and plays roles in multicellularity, and metazoans have a higher proportion of tyrosine phosphorylation than unicellular eukaryotes (57). this is based on the virtual absence of tyrosine phosphorylation in unicellular eukaryotes, such as yeasts. most bacterial phosphorylation sites are on serine (70%) and threonine (20%), while tyrosine phosphorylation sites account for less than 10%. a noteworthy feature of h. pylori phosphoproteome is the significantly high overall abundance (~18.5%) of tyrosine phosphorylation. there are three bacterial phosphoproteomes with comparable tyrosine phosphorylation percentage : s. coelicolor, 13.6% (24) ; p. aeruginosa, 14.5% (22) ; and k. pneumoniae, 25.8% (20) ; while most other bacterial model organisms with known phosphoproteomes have less than 10% phosphorylation sites on tyrosine (table 1). p. aeruginosa phosphoproteome has a much higher tyrosine phosphorylation level (14.5%) compared with the non - pathogenic p. putida species (7.5%) (22). in fact, various findings have supported the contribution of tyrosine phosphorylation to the bacterial pathogenicity, such as pedestal formation (tir of enteropathogenic e. coli and citrobacter) (58), cell elongation, scattering and inflammation (caga of helicobacter) 53, 59, capsular polysaccharide biosynthesis (20), cell invasion (tarp of chlamydia) (60), pro - inflammatory responses and cell proliferation (bepd - f of bartonella) (61). the association has been established between protein tyrosine phosphorylation and the control of surface polysaccharide production or transport (62), as well as between protein serine / threonine phosphorylation and cell envelope biosynthesis (63). surface polysaccharides are believed to be involved in the early steps of the infection process and are considered potent virulent factors (64). some proteins from various species are homologous to a family of enzymes involved in exopolysaccharide synthesis grouped together as by - kinases (bacterial tyrosine kinases) and show similar auto - phosphorylation activities 65, 66. by - kinases, albeit only a few copies per bacterial genome, are widespread in bacteria (66) and are usually encoded by genes in the large operons involved in biosynthesis and export of sugar polymers (67). for instance, the tyrosine kinase wzc of e. coli is essential for the synthesis of the exopolysaccharide colonic acid and the assembly of group 1 capsular polysaccharide (62). it is essential for the pathogenicity of s. pneumonia to produce capsular polysaccharide (cps). the cps biosynthesis proteins cpsb, cpsc and cpsd regulate cps production via tyrosine phosphorylation of cpsd, a homologue of wzc. the mutations in some key elements of cpsc produced wild - type levels of cps, but were unable to cause bacteremia in mice upon intranasal challenge (69). it seems that the relatively high abundance of tyrosine phosphorylation in the phosphoproteomes of several pathogenic bacteria reflects in some way the pathogenicity of the bacteria, such as in the direct pathogen in eukaryotic cells, multiple phosphorylated proteins are common and serve as molecular switches for signal fine - tuning. for example, five phosphorylation sites are present in the eukaryotic initiation factor (eif) 2b catalyzed by four different protein kinases (70) : two conserved c - terminus serine sites are phosphorylated by casein kinase 2 and required for the in vivo interaction of eif2b with eif2 and in vitro eif2b activity ; the third site (ser539) is required for the recruition of the glycogen synthase kinase 3 (gsk3) to the fourth site (ser535) ; the fifth site is phosphorylated by casein kinase 1 and lies out of the catalytic domain of eif2b. one distinctive feature of h. pylori phosphoproteome is the high occurrence of multiple phosphorylation sites : 35 out of the identified 84 phosphopeptides contain at least two phosphorylation sites. one example is the peptide sakandaseita llntiayetistlsk from the enzyme of alanine racemase, which has six phosphorylation sites. this phenomenon has been noted for proteins accumulated when the bacteria were under stress or with overloaded proteolytic systems (71), and was also reported in the characterization of the phosphoproteome of the gram - positive pathogenic bacterium s. pneumoniae (19) as well as the non - pathogenic l. lactis (18). these observations indicated that one protein may be phosphorylated on multiple sites to fulfill differential roles or to function together to achieve delicate micro - regulations of the pathogenicity mechanisms, such as adhesion to the host, stimulation and regulation of pathogenic functions and impairing the host defense mechanisms (10). multiple phosphorylation sites may have similar biological implications, at least for the species of l. lactis (18) and h. pylori : both l. lactis and h. pylori have smaller genome sizes, simpler transcriptional machinery and fewer (two and three, respectively) sigma factors compared with other model bacteria such as e. coli, suggesting that more regulations are needed through delicate post - translational modifications. protein protein interaction map with the involvement of phosphoproteins is important to understand the regulatory mechanisms of post - translational modifications in the bacteria. as shown in figure 2, the phosphoprotein interaction map of h. pylori consisted of a large network covering 163 proteins, among which there are 28 identified phosphoproteins (25). the possible partners for vaca in the a category include an outer membrane porin protein hopl, a hypothetical protein hp0699, a predicted abc transporter hp1464 and a predicted atpase / dna transfer protein virb4_5. vaca is secreted from h. pylori through the syringe - like virb / vird4-like type iv secretion apparatus. it is noteworthy that virb4_5 is also identified to be phosphorylated, implicating the involvement of phosphorylation in the regulation of vaca secretion through the bacterial membrane. the two partner proteins groel and flgb were determined to be phosphorylated and were predicted to interact directly with each other (25). the in vivo interaction between groel and flgb was previously observed in b. subtilis (72), albeit without any assignment of the in vivo function for such interaction. h. pylori produces flagella to fulfill its requirement for the bacterial colonization in the human gastric mucosa. as a member in the hsp60 family, h. pylori groel has been shown to increase the risk of gastric carcinoma (73). previous studies demonstrated that the phosphorylation and dephosphorylation of groel regulated its binding and dissociation from unfolded proteins (74), possibly through the switch between the oligomeric states mediated by phosphorylation (75). further experimental investigations are needed to reveal the contribution of the groel and flgb partners to the pathology of the bacterium with regards to how the phosphorylation of these two proteins regulates the bacterial colonization. phosphoprotein interaction network through bioinformatics or experimental determinations provides some hints for the connectivity of the phosphorylation networks and the correlation between pathological potentials and the known bacterial phosphoproteomes. it is essential to determine the degree and the site of the in vivo protein phosphorylation in order to understand the underlying mechanisms between dys - regulation of phosphorylation and human pathologies. highly selective enrichment procedures and sensitive ms are required to decipher the phosphoproteome due to the very low stoichiometry, limited dynamic range, high complexity and quantitative difficulties of protein phosphorylations. recent technical developments made it possible to uncover some whole - cell bacterial phosphoproteomes, which revealed the correlation between bacterial pathological potentials and phosphorylations : the high abundance of tyrosine phosphorylations in a few bacterial phosphoproteomes indicates their roles in the pathogenicity, especially in the case of pathogen - host interactions ; the high abundance of multi - phosphorylation sites in bacterial phosphoprotein is a compensation of the relatively small phosphorylation size and an indicator of the delicate regulation of protein functions. with further development in the ms - based techniques, more information will be obtained about bacterial phosphoproteomes and the correlation between bacterial phosphorylations and potential pathogenicity. this will help us to develop protein phosphorylation - targeted prodrugs in the control of bacterial infections. | increasing evidence shows that protein phosphorylation on serine, threonine and tyrosine residues is a major regulatory post - translational modification in the bacteria. this review focuses on the implications of bacterial phosphoproteome in bacterial pathogenicity and highlights recent development of methods in phosphoproteomics and the connectivity of the phosphorylation networks. recent technical developments in the high accuracy mass spectrometry have dramatically transformed proteomics and made it possible the characterization of a few exhaustive site - specific bacterial phosphoproteomes. the high abundance of tyrosine phosphorylations in a few bacterial phosphoproteomes suggests their roles in the pathogenicity, especially in the case of pathogen host interactions ; the high abundance of multi - phosphorylation sites in bacterial phosphoprotein is a compensation of the relatively small phosphorylation size and an indicator of the delicate regulation of protein functions. |
a 6yearold male was brought to our emergency department having sustained an accidental penetrating injury to zone 1 of the neck with a sickle. on examination, there was a foreign body sickle (fig. 1) with its wooden handle as an entry point at the right submandibular region with no active bleeding and without evidence of vascular or neurological injuries. the sickle had serrated part on its concave surface and blunt part on its curvature. 2. what is the superior limit of foreign body piercing the neck in this patient ? penetrating neck injury can be life threatening because of the increased risk of injury to the vital structures such as blood vessels, airway, cervical spines, and nerves which are present in such a small confined area 1. the foreign body may be providing tamponade effect on a major blood vessel so it should not be blindly pulled out until radiological evaluation 2. our patient initially has undergone plain xray of the neck as a means of costeffective investigation. but from xray alone, it was not possible to find the accurate information regarding the relation of foreign body with the adjacent vital structures. computed tomography scan can be helpful to know the extent of the injury sustained by the patient with penetration of neck with sharp objects such as sickle, knife, broken glasses, metallic and wooden foreign bodies. in this case, the computed tomography showed the metallic foreign body penetrating the soft tissue of neck on right side reaching the opposite side orbit. intraoperatively, the foreign body was found to be penetrated to floor of mouth, involving dorsum of tongue and soft palate, then entered into left nasal cavity, piercing the middle turbinate, and finally entered into left orbital cavity lying posterior, that is, very close to the orbital apex (fig. 2, 3, 4). coronal view of ct scan showing the entry point and the extent of foreign body. axial view of ct scan showing metallic foreign body in left orbit. correlating the radiological and clinical finding, although xray plain film is a costeffective investigation method in the case of metallic foreign body penetrating the neck, one should not rely alone on it, as it can not reveal status of neurovascular structures lying adjacent to foreign body in an anatomically important area such as head and neck region. ct scan is a definitive imaging technique which yields important information and makes interpretation easy. so, it must be taken before planning the removal of foreign body in operation theater. | key clinical messagect scan is the most important investigation in patients with penetrating neck injury in which it can show the extent of internal injury which may be overlooked. without ct scan being performed, one should not try to remove foreign body by just pulling blindly, as it can injure vital structures. |
mammals are thought to develop in a bacteria - free environment within the mother 's womb. they are born through a birth canal densely populated by lactic acid bacteria (harrison., 1953 ; dominguez - bello., 2010 ; ravel., 2011) and, during a substantial period of their early development, feed exclusively on maternal milk. these conserved traits may be important for the nutrition and protection of the newborn (sela and mills, 2010). in this work, we characterized the mouse intestinal microbiota from the early stages of development until weaning. six friend leukemia virus b (fvb) female mice, 45 weeks old, were bred with males, and the intestinal contents of litters and mothers were sampled until weaning (supplementary figure s1). this work was approved by the university of puerto rico iacuc (604 - 2008). owing to the low fecal yield in newborn mice, colon contents were collected at these stages. we confirmed that colon contents were good proxies for feces (supplementary figures s2s5), as reported (peterson. dna was extracted from 81 samples using mobio powersoil kits (mobio laboratories, carlsbad, ca, usa) as recommended by the manufacturer, including bead beating. sample dna was pcr - amplified from the variable v2 region of the 16s rrna gene, then sequenced using 454 pyrosequencing (life sciences genome sequencer flx instrument, roche, branford, ct, usa) as described (andersson. sequences were processed using quantitative insights into microbial ecology (qiime) 1.4 (caporaso., 2010). rarefaction analysis was performed based on the number of operational taxonomic units (otus) and the amount of phylogenetic branch length observed in each sample (hamady., 2010). beta diversity was estimated using the unifrac metric (lozupone and knight, 2005), and hierarchical clustering was performed using the unweighted pair group method with arithmetic mean (upgma). analyses of variance (anova) were made using the statistical program r, version 2.14.0 (2011 - 10 - 31), using the r library. we obtained 121 893 v2 region 16s rrna gene sequences from 81 samples, with a mean of 1505274 sequences per sample. the global estimated coverage was 98.8%, the coverage values for the offspring at each age were as follows : day 1=96.2% day 3=98.5% day 9=99.3% day 21=97.3% adults (older than 21 days)=96.9%. adult mothers harbored 6121.7 fecal otus, with dominance of phyla firmicutes (52.3%), bacteriodetes (42.1%) and unclassified otus (4.8%) (supplementary figure s6 ; supplementary table s1). bacterial communities of the adults did not vary significantly with age (supplementary figure s7), gender (supplementary figure s8) or physiological stage associated to parturition (anova, p=0.124 ; supplementary figure s9). at day 1 after delivery, the vaginas of the six mothers harbored 3811.5 otus, with a dominance of proteobacteria (80%) and firmicutes (17%) (supplementary figure 1 ; supplementary table s2). colonic bacterial communities in newborn mice at early ages were closer to maternal communities in vaginas than to those in feces (anova, p=0.000), but by age 21 days, they clustered closer to maternal feces (anova, p<0.001), as shown by principal coordinates analysis (supplementary figure s10) and upgma clustering (supplementary figure s11). the colonic bacterial diversity decreased considerably at age 3 and 9 days (figure 1, supplementary figures s12s15, supplementary table s3), from nearly complete dominance of streptococcus to lactobacillus (figure 2, supplementary figure s16, supplementary table s2). by the time of weaning (21 days), the fecal diversity had increased to similar levels to those in the mothers (figure 1, supplementary figures s13s15, supplementary table s2), and with nearly complete disappearance of the streptococcus species (figure 2). procrustes analysis shows that these results are robust to the sequence coverage obtained here (supplementary materials), and the decrease in diversity during the mid - strict lactation period is supported by other diversity metrics, including phylogenetic diversity (supplementary figure s13), chao1 (supplementary figure s14) and shannon entropy (supplementary figure s15). consistent with prior studies in humans (matsumiya., 2002 ; dominguez - bello., 2010), site - specific selective factors exert pressure during development, and divergence of communities occurs in each body location. previous work based on cultivable bacteria in mice (schadler, 1973) has shown an initial colonization by lactobacilli, followed by coliforms, and finally by obligate anaerobes. in the present study, there was an initial bacterial bloom of streptococcus immediately after birth, which decreased after day 3 to be replaced by lactobacillus species that are facultative anaerobes that ferment milk lactose and casein, and produce lactic acid (kunji., 1996 ; jiang and savaiano, 1997 ; angelakis., 2012). lactate production acidifies (ph<5.5) the intestinal contents and inhibits the growth of anaerobes (soergel, 1994 ; jiang and savaiano, 1997), including lachnospiracea, clostridiales and bacteroidales, whose abundance was increased by the time of weaning. the introduction of solids to the milk diet increases the diversity of substrates for intestinal bacteria, and the strictly anaerobic colonizers become established with new pathways of fermentation, leading to the production of short - chain fatty acids, hydrogen, methane and co2 (ruppin., 1980). the importance of select groups of bacteria including sfb, clostridium, bacteroides, bifidobacterium and lactobacillus, in their role on the host mucosal immune system, has been recently reviewed (reading and kasper, 2011). contrary to the proposed developmental choreography with steady age - associated increase in microbiota alpha diversity (koenig., 2011), the results presented here provide evidence of a biphasic progression toward the adult colonic microbiota, with an early reduction of diversity during suckling with dominance by lactate producers, and a second phase with increased diversity by anaerobes, coinciding with the introduction of solid food. | the birth canal provides mammals with a primary maternal inoculum, which develops into distinctive body site - specific microbial communities post - natally. we characterized the distal gut microbiota from birth to weaning in mice. one - day - old mice had colonic microbiota that resembled maternal vaginal communities, but at days 3 and 9 of age there was a substantial loss of intestinal bacterial diversity and dominance of lactobacillus. by weaning (21 days), diverse intestinal bacteria had established, including strict anaerobes. our results are consistent with vertical transmission of maternal microbiota and demonstrate a nonlinear ecological succession involving an early drop in bacterial diversity and shift in dominance from streptococcus to lactobacillus, followed by an increase in diversity of anaerobes, after the introduction of solid food. mammalian newborns are born highly susceptible to colonization, and lactation may control microbiome assembly during early development. |
commercial archwires of stainless steel (ss), nickel - titanium (niti), titanium molybdenum alloy (tma), and low - friction tma (l - tma) were used (0.017 0.025 ormco, glendora, california, usa). scanning electron microscope (hitachi, su6600, japan) was used to observe the surface morphology of the archwires. elemental composition of the archwires was assessed using energy dispersive spectroscope (eds) (horiba - emax). atomic force microscope (afm) (xe-100 park, korea) was used to evaluate the three - dimensional surface ra of the archwires. a potentiostat (gill ac, acm instruments, england) was used to perform the linear polarization test, a fast and nearly nondestructive electrochemical technique. a saturated calomel electrode and platinum were used as the reference electrode and counter electrode, respectively. modified fusayama artificial saliva (nacl [400 mg / l ], kcl [400/mg / l ], cacl2 h2o [795 mg / l ], na2h2 po4 h2o [690mg / l ], kscn [300 mg / l ], na2 s.9h2o [5 mg / l ], and urea [1000 mg / l ]) with a ph of 6.5 at 37c was used as the corrosion test electrolyte. to evaluate the effect of fluoride concentration on these archwires, 0.5% of naf (simulating the fluoride concentration contained in commercial fluoridated toothpastes) eight groups were taken, and the sample size was 10 for each of these groups. the experimental groups are mentioned below : group 1 : ss in artificial salivagroup 2 : niti in artificial salivagroup 3 : tma in artificial salivagroup 4 : l - tma in artificial salivagroup 5 : ss in artificial saliva with 0.5% nafgroup 6 : niti in artificial saliva with 0.5% nafgroup 7 : tma in artificial saliva with 0.5% nafgroup 8 : l - tma in artificial saliva with 0.5% naf. group 1 : ss in artificial saliva group 2 : niti in artificial saliva group 3 : tma in artificial saliva group 4 : l - tma in artificial saliva group 5 : ss in artificial saliva with 0.5% naf group 6 : niti in artificial saliva with 0.5% naf group 7 : tma in artificial saliva with 0.5% naf group 8 : l - tma in artificial saliva with 0.5% naf. the linear polarization tests were carried out after dipping the archwire samples into the test electrolyte for 2 h. the linear polarization values were measured from 10 mv to + 10 mv with a scan rate of 0.1 mv / s. from the present test, the polarization resistance (rp [cm ]) was obtained which is inversely proportional to the corrosion rate and directly proportional to corrosion resistance. eight groups were taken, and the sample size was 10 for each of these groups. the experimental groups are mentioned below : group 1 : ss in artificial salivagroup 2 : niti in artificial salivagroup 3 : tma in artificial salivagroup 4 : l - tma in artificial salivagroup 5 : ss in artificial saliva with 0.5% nafgroup 6 : niti in artificial saliva with 0.5% nafgroup 7 : tma in artificial saliva with 0.5% nafgroup 8 : l - tma in artificial saliva with 0.5% naf. group 1 : ss in artificial saliva group 2 : niti in artificial saliva group 3 : tma in artificial saliva group 4 : l - tma in artificial saliva group 5 : ss in artificial saliva with 0.5% naf group 6 : niti in artificial saliva with 0.5% naf group 7 : tma in artificial saliva with 0.5% naf group 8 : l - tma in artificial saliva with 0.5% naf. the linear polarization tests were carried out after dipping the archwire samples into the test electrolyte for 2 h. the linear polarization values were measured from 10 mv to + 10 mv with a scan rate of 0.1 mv / s. from the present test, the polarization resistance (rp [cm ]) was obtained which is inversely proportional to the corrosion rate and directly proportional to corrosion resistance. figure 2 and table 1 show the afm observations and the corresponding surface (ra, nm). statistical analysis using one - way analysis of variance (anova) showed a significant influence on the ra value (p tma > niti > ss. table 3 reveals that rp value of all archwires decreased drastically in artificial saliva with 0.5% naf. the collected data were subjected to statistical analysis using one - way anova and was found that there was a statistically significant influence on the rp value (p < 0.001). scanning electron microscopic images. niti : nickel titanium, tma : titanium molybdenum, l - tma : low - friction titanium molybdenum alloy atomic force microscopic images. niti : nickel titanium, tma : titanium molybdenum, l - tma : low - friction titanium molybdenum alloy surface roughness (ra, nm) for different wires polarization resistances (cm) for various groups in artificial saliva without sodium fluoride polarization resistances (cm) for various groups in artificial saliva with sodium fluoride the ss and titanium alloys used in orthodontic appliances rely on the formation of a passive surface oxide film to resist corrosion. the protective layer is not infallible ; it is susceptible to both mechanical and chemical disruption. potentiodynamic polarization experiments and scanning electron microscopic observations of archwires composed of ss, niti, and tma exposed to electrochemical corrosion in artificial saliva have shown evidence of pitting corrosion formed on the wire surface. as for ss archwires, it is well known that chromium element in the ss alloy can form a thin and adherent cr2o3-based protective film which provides the corrosion resistance of a substrate alloy. a minimum chromium content of around 11% is required to form a protective passive film on the ss wire. in case of niti archwires, the tio2-based (also small traces of nio) passive film can provide a good measure of niti alloy biocompatibility. tma wires form a passive film of tio2 and traces of mo3, zro2, sno, and l - tma form the same protective layer as tma wire together with the formation of traces of no due to ion bombardment of nitrogen. edie. in their study stated that the corrosion potential of ss and niti is not different. however, the present study [table 3 ] suggests that more surface pitting and corrosion occurred in ss than in niti, tma, and l - tma. these results were almost similar to the results reported by suarez. and kim and johnson. they concluded that the titanium wires appear to be the most inert wire of those tested and are unlikely to release metal ions when used intraorally. however, in this study, l - tma was found to have a slightly higher corrosion resistance than tma wires. the improvement in corrosion resistance of l - tma was believed due to the presence of no in the outermost surface of the alloys. it was found that tio2-based passive film formed on titanium metal has better corrosion resistance in acidic artificial saliva than the cr2o3-based passive film on ss. this result was also consistent with a previous study which has also concluded that tio2 passive film is more corrosion resistant than cr2o3. the better corrosion resistance of tma wires than niti might be due to the increased titanium content in them (78% in tma and 45% in niti). this is clearly seen in the present study in which the composition of the archwire is determined by energy dispersive spectroscope (eds). a previous study had suggested that surface ra of orthodontic archwires ought to be taken as an important indicator of the trend toward archwire corrosion resistance. in our study [table 1 ], tma showed the highest surface ra value and ss the least surface ra. from the potentiostatic tests, it was confirmed that tma and l - tma exhibited the highest corrosion resistance. contrary to this, earlier studies were reported but our study was consistent with the study reported by danto. from this, it can be concluded that chemical composition of the wire is the primary factor influencing the corrosion resistance and surface ra is only secondary. from [table 3 ], it is very clear that the corrosion resistance of all wires had reduced drastically in the presence of 0.5% naf (although l - tma showed the highest rp value). the cr2o3 passive film of ss reacts with naf and the following reaction takes place. cr2o3 + 2naf crf2 + na2o + cro2 in case of niti and tma wires, tio2 reacts with naf to form titanium - fluoride complex compound. tio2 + naf na2 tif6 however, tma and l - tma archwires showed a better corrosion resistance than niti. this may be due to the presence of some other oxides in the surfaces passive film (moo3, zro, and sno). this was followed by niti, l - tma, and ss in decreasing order. from the potentiostatic study, it was found that tma and l - tma had the highest corrosion resistance. this indicates that the chemical composition of the wire is the primary influential factor to have high corrosion resistance and surface ra is only secondary. in the present study, the rp values were measured after exposing the archwires to artificial saliva and artificial saliva - containing sodium fluoride (0.5%) for 2 h. this simulated the in vivo corrosion resistance of the archwires in the oral cavity. it also simulated the in vivo corrosion resistance of archwires when residual fluorinated toothpastes interacted with the archwires 2 h after brushing. it was seen that the in vivo corrosion resistance of the archwires in the oral environment might decrease if the highly fluorinated toothpastes were used and/or the exposure time of archwires to residual fluorinated toothpastes was prolonged. therefore, complete removal of residual high - fluorinated toothpastes from the crevice between archwire and bracket during tooth brushing is required. furthermore, the repair of the protectiveness of the surface oxide film on the archwires might occur after full removal of residual fluorinated toothpastes by mechanical tooth brushing. however, the effect of a lesser concentration of fluoride and variation in the exposure time to this particular concentration of fluoride on the archwires is still to be investigated. | objective:(1) to evaluate the corrosion resistance of four different orthodontic archwires and to determine the effect of 0.5% naf (simulating high fluoride - containing toothpaste of about 2250 ppm) on corrosion resistance of these archwires. (2) to assess whether surface roughness (ra) is the primary factor influencing the corrosion resistance of these archwires.materials and methods : four different archwires (stainless steel [ss ], nickel - titanium [niti ], titanium molybdenum alloy [tma ], and ion - implanted tma) were considered for this study. surface characteristics were analyzed using scanning electron microscopy, atomic force microscopy (afm), and energy dispersive spectroscopy. linear polarization test, a fast electrochemical technique, was used to evaluate the corrosion resistance, in terms of polarization resistance of four different archwires in artificial saliva with naf concentrations of 0% and 0.5%. statistical analysis was performed by one - way analysis of variance.results:the potentiostatic study reveals that the corrosion resistance of low - friction tma (l - tma) > tma > niti > ss. afm analysis showed the surface ra of tma > niti > l - tma > ss. this indicates that the chemical composition of the wire is the primary influential factor to have high corrosion resistance and surface ra is only secondary. the corrosion resistance of all wires had reduced significantly in 0.5% acidic fluoride - containing artificial saliva due to formation of fluoride complex compound.conclusion:the presence of 0.5% naf in artificial saliva was detrimental to the corrosion resistance of the orthodontic archwires. therefore, complete removal of residual high - fluorinated toothpastes from the crevice between archwire and bracket during tooth brushing is mandatory. |
atherosclerosis, one of the main complications of diabetes, constitutes the leading cause of morbidity and mortality in today 's world. macrophages, as major inflammatory contributors, are key modulators of atherosclerotic plaque formation and progression : m1 macrophages, which are related to inflammatory effects, stimulate inflammation and promote plaque progression, while m2 macrophages, which link with anti - inflammatory roles, contribute to inflammation resolution and atheroma regression [2, 3 ]. previous studies have demonstrated that excessive inflammatory m1 monocytes / macrophages emerge in peripheral blood of both prediabetic and diabetic patients [4, 5 ]. furthermore, our previous study also found that m1 monocytes / macrophages, the inflammatory subset, increased in circulation and atherosclerotic plaque in stz - induced diabetic mice and were related to enhanced inflammation and accelerated atherosclerosis. thus, the increased inflammatory macrophages in diabetes contribute to persistent low grade inflammation and accelerated atherosclerosis. various factors are responsible for macrophage inflammatory activation in diabetes. besides hyperglycemia, the advanced glycation end products (ages), which are generated irreversibly in high glucose condition, are another group of critical pathogenic factors in diabetes. because inflammatory macrophages dominate the progression of atherosclerosis, blockage or attenuation of ages - induced macrophage inflammatory response might alleviate diabetic atherosclerosis. in addition to its antihyperglycemic effects, metformin, with over half - decade use as first - line therapy for type 2 diabetes, slows down diabetic atherosclerosis development through mechanisms that are still not fully understood. recently, metformin 's anti - inflammatory properties were demonstrated in lipopolysaccharide induced macrophages [11, 12 ], and previous studies had shown that metformin could downregulate rage expression and suppress nfb signaling in various cells [14, 15 ]. as our previous work proved that rage / nfb signaling was involved in ages - induced macrophage inflammatory activation, it is reasonable to hypothesize that metformin might inhibit ages - induced inflammatory response in macrophages. therefore, the present study tested the hypothesis and could provide novel elucidation for the antiatherosclerotic effect of metformin. male c57bl/6 mice (8 weeks old) were purchased from slac laboratory animal co., ltd. the mice were housed under specific pathogen - free conditions with controlled temperature (2225c) and 12 h light / dark cycles ; they were given standard chow and water ad libitum. all animal experiments were approved by the ethics committee of xinhua hospital affiliated to shanghai jiao tong university school of medicine (approval number xhec - f-2016 - 012). mice were anesthetized with pentobarbital (50 mg / kg, i.p.) to minimize suffering and killed by cervical dislocation without recovery from anesthesia. tibias and femurs were removed and bone ends were cut off, and bone marrow was flushed with pbs supplemented with 1% fetal bovine serum (fbs, gibco, australia, cat # 10099 - 141). a single cell suspension was prepared by filtering the cells through a 40 m strainer (bd falcon, cat # 352340). the cell suspension was centrifuged at 1500 rpm for 5 min, and the cell pellet was resuspended in culture medium. bone marrow cells were cultured for 7 days at 37c with 5% co2 in dulbecco 's modified eagle 's medium (dmem), high glucose (hyclone, beijing, china, cat # sh30022.01b) supplemented with 10% fbs, 2 mm l - glutamine (sigma - aldrich, missouri, usa, cat # 59202c), 100 u / ml penicillin and 100 g / ml streptomycin (beyotime, jiangsu, china, cat # c0222), and 20 ng / ml gm - csf (peprotech, new jersey, usa, cat # 315 - 03). macrophages (> 95%) were identified by flow cytometry with anti - cd11b apc and anti - f4/80 fitc (ebioscience, california, usa, cat # 17 - 0112 and cat # 11 - 4801) staining. bmdms were seeded at a density of 0.5 10/ml and cultured overnight before stimulation. based on experimental protocol, the bmdms were treated with different concentrations of metformin (0.25, 1.0, and 2.0 m) or ages (200 mg / l) for different time periods. for pathway studies, the bmdms were pretreated with anti - rage antibody or ammonium pyrrolidinedithiocarbamate (pdtc) or compound c for 60 min. the anti - rage neutralizing antibody (r&d systems, minnesota, usa, cat # af1179) was reconstituted at 0.2 mg / ml in sterile pbs and further diluted with culture medium to the final concentration of 20 g / ml. metformin hydrochloride (abcam, cambridge, uk, cat # ab120847) was dissolved in water at a concentration of 50 mm and further diluted with culture medium to the final concentrations (0.25, 1.0, and 2.0 m). the nfb inhibitor pdtc (abcam, cambridge, uk, cat # ab141406) was dissolved in dmso at a concentration of 100 mm and further diluted with culture medium to the final concentration of 50 m (contains 0.5% dmso, v / v). the ampk inhibitor compound c (abcam, cambridge, uk, cat # ab120843) was firstly dissolved in dmso at a concentration of 10 mm and further diluted with culture medium to the final concentration of 5 m (contains 0.5% dmso, v / v). to rule out the influence of dmso, proper dosages of dmso were added to culture medium to meet the groups with the highest concentration of dmso. age - bsa was purchased from anyan - bio technology (shanghai, china, cat # ay-4710p), and the corresponding amount of bsa was used as control. total rna was extracted from bmdms using trizol reagent (takara, liaoning, china, cat # 9109), and 1 g of total rna was reverse transcribed to cdna using the primescript rt master mix kit (takara, liaoning, china, cat # rr036a). real - time pcr array analysis was performed by using the sybr premix ex taq kit (takara, liaoning, china, cat # rr420a) in a total volume of 20 l, with 2 l of cdna primers (0.2 mm each), 10 l of sybr green, and 0.4 l of rox dye ii. the standard pcr conditions consisted of 95c for 30 sec, followed by 40 cycles of 95c for 5 sec and 60c for 34 sec, with a final dissociation stage ; the samples were run on an abi 7500 detector (applied biosystems, california, usa). the amounts of target genes were determined and normalized to the amount of gapdh cdna (sangon biotech, shanghai, china, cat # b661304). the sequences of the primers (synthesized by sangon biotech, shanghai, china) for the target genes were as follows : fwd 5-ctcacaagcagagcacaagc-3 and rev 5-tccagcccatactttaggaaga-3 for il-1 ; fwd 5-tctgcaagagacttccatcca-3 and rev 5-agtctcctctccggacttgt-3 for il-6 ; fwd 5-ggtgcctatgtctcagcctc-3 and rev 5-ccacttggtggtttgtgagtg-3 for tnf- ; fwd 5-tgcactaccaaagccacaag-3 and rev 5-tgatcctcatgccagtcagt-3 for il-10. equal amounts of protein (4060 g) were resolved on sds - page and transferred to polyvinyl difluoride (pvdf) membranes. the blots were blocked with 5% milk in tbst for 1 h at room temperature and then incubated with primary rabbit anti - mouse antibodies overnight at 4c. the antibodies against p - ampk (1 : 1000, cat # ab133448) and rage (1 : 1000, cat # ab3611) were purchased from abcam (cambridge, uk), and the antibodies against nfb-65 (1 : 1000, cat # 8242s) and p - nfb-65 (1 : 1000, cat # 3033s) were purchased from cell signaling technology (massachusetts, usa). the blots were subsequently incubated with the secondary goat anti - rabbit antibodies conjugated with horseradish peroxidase (1 : 1000) for one hour and enhanced using a chemiluminescence system (chemidoc xrs+, bio - rad laboratories, usa). the intensity of each band was normalized to the loading control tubulin (beyotime, jiangsu, china, cat # at819). after different stimulations, single cell suspensions of bmdms were prepared in flow buffer and incubated with antibodies. anti - mouse cd86 pe (ebioscience, california, usa, cat # 12 - 0862) was used to identify m1 macrophages while anti - mouse cd206 fitc (biolegend, california, usa, cat # 141704) was used to identify m2 macrophages. results were acquired with a bd canto ii flow cytometer (bd biosciences, usa) and analyzed by the flowjo software (tree star, usa). after different stimulations, cells were washed thrice with cold pbs and fixed in 4% (w / v) paraformaldehyde for 20 min and then washed with pbs again. next, cells were incubated in buffered normal goat serum to prevent nonspecific binding of antibodies for 1 h at room temperature. they then were incubated overnight with antibody against nfb-65 (1 : 200, cat # 8242s) purchased from cell signaling technology (massachusetts, usa), followed by incubation with cy3 goat anti - rabbit igg (1 : 500 ; beyotime, cat # a0516) for 1 h at 37c. dapi was used to stain the cell nuclei (blue) at a concentration of 1.43 m (sigma, st. one - way analysis of variance (anova) was used to assess the effects of one factor among multiple groups, and post hoc testing was done by tukey test. two - way anova was used to assess the effects of two factors among multiple groups, and post hoc testing was done by bonferroni test. chicago, usa) for windows. a two - tailed value of p < 0.05 was considered statistically significant. our previous study elucidated that ages promoted bmdms to express proinflammatory cytokines through rage / nfb signaling. to confirm this finding, here we tested cytokine expression profile of bmdms after ages stimulation with or without anti - rage neutralizing antibody or pdtc pretreatment. bmdms were divided into 4 groups : control, ages, ages + anti - rage, and ages + pdtc group. cells in the last two groups were pretreated with anti - rage antibody (20 g / ml) or pdtc (50 m) for 60 min, respectively, and then, together with ages group, the three groups were cultured with ages (200 mg / l) for 24 h. the control group was treated with bsa (200 mrna levels of proinflammatory cytokines (il-1, il-6, and tnf-) and anti - inflammatory cytokine (il-10) were measured by real - time pcr. figure 1 shows that ages markedly increased mrna expression of proinflammatory cytokines (il-1, il-6, and tnf-) (figures 1(a), 1(b), and 1(c)), while only slightly upregulating that of il-10 (figure 1(d)), indicating that ages predominantly induced inflammatory response in murine macrophages. in addition, pretreatment with anti - rage antibody or pdtc ameliorated the proinflammatory effects of ages (figures 1(a), 1(b), and 1(c)). therefore, rage / nfb signaling is involved in ages - induced inflammatory response in macrophages. because metformin has anti - inflammatory potential [11, 12 ], next we tested whether metformin inhibited ages - induced inflammatory response in macrophages. bmdms were divided into 5 groups : control, ages, ages + met 0.25 (metformin 0.25 m), ages + met 1.0 (metformin 1.0 m), and ages + met 2.0 (metformin 2.0 m). cells in the last 3 groups were pretreated with different concentrations of metformin (0.25, 1.0, and 2.0 m) for 60 min, respectively, and then, together with ages group, the 4 groups were stimulated with ages (200 mg / l) for 24 h. the control group was treated with bsa (200 mrna levels of proinflammatory cytokines (il-1, il-6, and tnf-) and anti - inflammatory cytokine (il-10) were measured again by real - time pcr. figure 2 shows that metformin dose dependently inhibited ages ' enhancement on mrna expression of proinflammatory cytokines (figures 2(a), 2(b), and 2(c)), while promoting that of il-10 (figure 2(d)), indicating that metformin inhibited ages - induced inflammatory response in bmdms. as the results showed that 2.0 m metformin had the strongest effects on downregulating genes expressions of proinflammatory cytokines but upregulating mrna expression of anti - inflammatory cytokine, we administrated 2.0 m metformin for the following experiments. next, we tested whether inhibition of rage / nfb pathway was responsible for metformin 's suppressive effects on ages - induced inflammation. because metformin is an agonist of ampk and activation of ampk can abate inflammation in various cells [14, 16, 17 ], we also evaluated activity of ampk. firstly, bmdms were divided into 4 groups : control, ages, met, and ages + met group. in ages group, cells were cultured with ages at 200 mg / l for 24 h ; in met group, cells were cultured with metformin at 2.0 m for 24 h ; in ages + met group, cells were pretreated with metformin at 2.0 m for 60 min and then cultured with ages at 200 mg / l for 24 h ; in control group, cells were cultured with bsa at 200 mg / l for 24 h. western blot analysis was performed to measure protein levels of rage and phosphorylated ampk (p - ampk). figure 3(a) shows that ages significantly upregulated expression of rage and reduced levels of p - ampk, while metformin had the opposite effect. moreover, pretreatment with metformin significantly attenuated effects of ages on rage upregulation and ampk inactivation. then, we evaluated the effect of ages on activation of nfb pathway in macrophages with or without metformin (2.0 m) pretreatment for 60 min before ages (200 mg / l) stimulation for different time intervals (0, 30, 60, and 180 min). in western blot analysis (figure 3(b)), the p - p65/p65 ratio in the ages group markedly increased after ages stimulation, peaking at 60 min and decreasing thereafter, indicating that nfb was activated after ages stimulation. furthermore, the p - p65/p65 ratio in the ages + met group was much lower at each time point than in the ages group, suggesting that ages - induced nfb activation was partly inhibited by metformin pretreatment. taken together, these findings suggested that metformin inhibited rage / nfb signaling. because metformin is an ampk activator, we next performed experiments to confirm whether metformin 's suppression of nfb signaling is ampk dependent. bmdms were divided into 4 groups : control, ages, ages + met, and ages + met + cc group. because ages - induced nfb activation peaked at 60 min after ages stimulation, the duration of ages stimulation was set to 60 minutes. in ages group, cells were cultured with ages at 200 mg / l for 60 min ; in ages + met group, cells were pretreated with metformin for 60 min and then cultured with ages at 200 mg / l for 60 min ; in ages + met + c - c group, cells were pretreated with compound c, an ampk inhibitor, at 5 m for 60 min, and then they were treated with metformin at 2.0 m for 60 min followed by ages at 200 mg / l for 60 min ; in control group, cells were cultured with bsa at 200 mg / l for 60 min. figure 4 shows that p65 nuclear translocation in ages group was significantly higher relative to control group indicating nfb activation ; metformin pretreatment significantly inhibited p65 nuclear translocation ; and compound c pretreatment abolished metformin 's effects. subsequently, we tested whether metformin 's anti - inflammatory effect on ages - induced macrophages was dependent upon ampk activation. bmdms were divided into 4 groups : control, ages, ages + met, and ages + met + c - c group. in ages group, cells were cultured with ages at 200 mg / l for 24 h ; in ages+met group, cells were pretreated with metformin for 60 min and then cultured with ages at 200 mg / l for 24 ; in ages + met + c - c group, cells were pretreated with compound c at 5 m for 60 min, and then they were treated with metformin at 2.0 m for 60 min followed by ages at 200 mg / l for 24 h ; in control group, cells were cultured with bsa at 200 mg / mrna levels of proinflammatory cytokines (il-1, il-6, and tnf-) and anti - inflammatory cytokine (il-10) were tested again by real - time pcr. figure 5 demonstrated that pretreatment with compound c attenuated metformin 's inhibition on proinflammatory cytokines mrna expression and promotion of il-10 mrna expression in macrophages. thus, these data indicated that the anti - inflammatory function of metformin was at least partly dependent on ampk activation. m1 or m2 macrophages are, respectively, considered as pro- and anti - inflammatory macrophages. because cd86 is one of the important surface markers for m1 (proinflammatory) macrophages and cd206 for m2 (anti - inflammatory) macrophages, we next studied metformin 's impact on ages - induced surface markers (cd86 and cd206) expression on macrophages by flow cytometry analysis. bmdms were divided into 6 groups : control, ages, ages + anti - rage, ages + pdtc, ages + met, and ages + met + c - c. in ages group, cells were cultured with ages at 200 mg / l for 24 h ; in ages + anti - rage group, cells were pretreated with anti - rage neutralizing antibodies for 60 min followed by ages at 200 mg / l for 24 h ; in ages + pdtc group, cells were pretreated with pdtc for 60 min, followed by ages at 200 mg / l for 24 h ; in ages + met group, cells were pretreated with metformin at 2.0 m for 60 min and then cultured with ages at 200 mg / l for 24 h ; in ages + met + c - c group, cells were pretreated with compound c at 5 m for 60 min, and then they were treated with metformin at 2.0 m for 60 min followed by ages at 200 mg / l for 24 h ; in control group, cells were cultured with bsa at 200 mg / l for the same amount of time. m1 surface marker cd86 and m2 surface marker cd206 were detected by flow cytometry analysis. as expected, ages significantly increased cd86 but did not affect cd206 expression ; and pretreatment with anti - rage antibody or pdtc significantly abated ages ' effect. pretreatment with metformin partly reversed ages ' effects on cd86 expression and markedly increased cd206 expression ; however, compound c abated metformin 's potency (figure 6). the present study provides novel insight into a hypoglycemic agent : metformin inhibits the inflammatory response induced by advanced glycation end products in murine macrophages partly through ampk activation and suppression of rage / nfkappab signaling. macrophage is a vital player in atherosclerosis, with its inflammatory response determining progression of atherosclerotic lesions. ages are recognized as strong inflammatory mediators in the diabetic microenvironment, inducing an inflammatory response in macrophages [20, 21 ]. therefore, ages - induced macrophage activation and inflammation augmentation are critical mechanisms contributing to diabetic accelerated atherosclerosis, rendering it important to inhibit inflammatory response in macrophages in order to slow down or even block atherosclerotic progression. recently, metformin was shown to have anti - inflammatory effects on lipopolysaccharide (lps) or pma induced macrophages [11, 22, 23 ]. however, it is still unknown whether metformin was able to suppress ages - induced inflammatory response in macrophages. the present study showed that pretreatment with metformin not only reduced mrna expression of proinflammatory cytokines (il-1, il-6, and tnf-) but also upregulated mrna expression of anti - inflammatory cytokine (il-10) in macrophages. previous study has demonstrated that rage / nfb axis is important in driving inflammation in ages - induced macrophages. in the present study, we found that blockade of rage or nfb signaling could significantly attenuate ages - induced genes expression of inflammatory cytokines, which confirmed the critical role of rage / nfb signaling in ages - induced inflammatory response. in addition, although evidence is mounting that metformin downregulates rage expression or inhibits nfb activity in several cell types [1315 ], until the present study, there had been no reports on metformin 's inhibition on ages - induced rage / nfb signaling in macrophages. therefore, for the first time, we demonstrated that metformin inhibits ages - induced inflammatory response in macrophages via suppressing rage / nfb activation. of note, in the present study, we found blockade of rage or nfb signaling or administration of different concentrations of metformin could not completely abolish the proinflammatory effects of ages. such findings hint that pathways other than rage / nfb might also be responsible for ages - induced inflammatory response in macrophages. as is well known, in addition to rage, ages can bind with several other receptors such as age - receptor complex, scavenger receptor, and toll - like receptor 4 (tlr-4) to transmit messages [24, 25 ]. furthermore, besides nfb pathway, mapk and stat signaling have been proven to be implicated in driving inflammatory response in macrophages [26, 27 ]. therefore, rage / nfb signaling is just one of the many pathways mediating the inflammatory response induced by ages ; other potential pathways remain to be studied. metformin is an ampk activator, and ampk signaling suppresses nfb activation thereby attenuating inflammatory responses [28, 29 ]. in the present study, pretreatment with compound c, an ampk inhibitor, abated metformin 's suppression on nfb as well as proinflammatory cytokines expression, indicating that metformin 's inhibition of ages - induced inflammatory response in macrophages was ampk dependent. of note, in the present study, ages slightly increased mrna expression of il-10, while pretreatment with metformin significantly strengthened ages ' induction of il-10 mrna expression in a dose dependent manner. in light of the powerful anti - inflammatory function of il-10, the promotion effect of metformin upon il-10 expression furthermore, blockade of nfb signaling did not change il-10 expression while ampk blocking treatment significantly ameliorated metformin 's promotion on il-10 expression, indicating that ages - induced il-10 expression was nfb independent while metformin 's enhancement on expression of il-10 was at least partly ampk dependent. therefore, metformin not only suppressed expression of proinflammatory cytokines through ampk signaling induced nfb inhibition but also enhanced expression of anti - inflammatory cytokine (il-10) also via ampk activation. a previous study found that metformin primed macrophages into different phenotypes based on the microenvironment. in the present study, metformin inhibited ages - induced m1 surface marker cd86 expression while increasing m2 surface marker cd206 expression. based on the metformin 's effects on cytokines expression, results of the present study suggest that metformin probably inhibits ages - induced macrophage m1 polarization and might enhance macrophage m2 polarization. because macrophage polarization determines function, that is, pro- (m1) or anti - inflammatory (m2) [18, 19 ], the results further support metformin 's inhibition of ages - induced inflammatory response in macrophages. ages - induced proinflammatory status of macrophages might be a critical mechanism responsible for diabetic accelerated atherosclerosis. therefore, the present study demonstrates that metformin inhibits ages - induced inflammatory response in murine macrophages through ampk activation and suppression of rage / nfb signaling, thereby providing a novel molecular mechanism responsible for metformin 's benefits on diabetic atherosclerosis. | advanced glycation end products (ages) are major inflammatory mediators in diabetes, affecting atherosclerosis progression via macrophages. metformin slows diabetic atherosclerosis progression through mechanisms that remain to be fully elucidated. the present study of murine bone marrow derived macrophages showed that (1) ages enhanced proinflammatory cytokines (interleukin-1 (il-1), il-6, and tumor necrosis factor- (tnf-)) mrna expression, rage expression, and nfb activation ; (2) metformin pretreatment inhibited ages effects and ages - induced cluster designation 86 (cd86) (m1 marker) expression, while promoting cd206 (m2 marker) surface expression and anti - inflammatory cytokine (il-10) mrna expression ; and (3) the ampk inhibitor, compound c, attenuated metformin effects. in conclusion, metformin inhibits ages - induced inflammatory response in murine macrophages partly through ampk activation and rage / nfb pathway suppression. |
in recent years, consumers demands regarding food production and food safety have changed dramatically. vegetables represent an important part in the human diet providing a significant amount of essential vitamins, minerals and dietary fibers. subsequently, minimally processed food like fresh - cut fruits and vegetables has become an important part of the diet due to its high nutritional value, freshness and practical use. while most food processing technologies involve the stabilization of products and methods for prolonging their shelf life, minimal processing actually reduces the shelf life of food, which represents a potential microbiological risk for consumers, especially in case of improper hygienic conditions during distribution and processing. western society is facing a trend of green consumerism that sets a demand for food products preserved without synthetic additives, salt, as well as environmentally friendly food production technologies. in order to follow this trend, one of the possible solutions in assuring food safety is the use of essential oils and their constituents as antimicrobial additives. essential oils and their constituents like carvacrol and eugenol show antimicrobial activity against different toxicogenic and pathogenic microorganisms. a number of studies have shown that they possess antibacterial, antiviral, antifungal [9 - 14 ], antiparasitic, insecticidal and anticarcinogenic activity. constituents of essential oils like carvacrol and eugenol show antifungal activity against a wide range of fungi [18 - 20 ]. they could be used as a safer alternative to chemical fungicides in the food industry. carvacrol, (2-methyl-5-[1-methylethyl]-phenol), is a monoterpenoid phenol, a hydrophobic compound, soluble in ethanol, diethyl ether, carbon tetrachloride and acetone with a melting point at 1c and boiling point at 237.7c. it has been identified as the active constituent of essential oil of origanum vulgare l. (oregano) and essential oil of thymus vulgaris l. (thyme) that exhibits high antimicrobial and antioxidant activities. carvacrol has been shown to increase membrane fluidity and cause leakage of protons and potassium ions, resulting in a collapse of membrane potential and inhibition of adenosine triphosphate (atp) synthesis. aside from the inhibition of the growth of vegetative bacterial cells, carvacrol is able to inhibit the production of diarrheal toxin by bacillus cereus in broth and in real systems. mode of action of toxin limitation includes two theories : if toxin excretion is an active process, there may be insufficient atp or proton - motive force to export it from the cell. alternatively, the lower specific growth rate may mean that the cells use all the available energy to sustain viability, leaving little over for toxin production. eugenol (2-methoxy-4-[2-propenyl]phenol) is an allyl chain substituted guaiacol, a major component of syzygium aromaticum (clove) essential oil (approximately 85%), but also extracted from essential oils of myristica fragrans houtt. (nutmeg), cinnamomum cassia blume (cinnamon), ocimum basilicum l. (basil) and laurus nobilis l. (bay leaf). it is a clear to pale yellow oily liquid with a melting point at 7.5c and boiling point at 254c. sub - lethal concentrations of eugenol have been found to inhibit the production of amylase and proteases by b. cereus. cell wall deterioration and a high - degree of cell lysis were also noted. the hydroxyl group on eugenol is thought to bind to proteins, preventing enzyme action in enterobacter aerogenes. aspergillus carbonarius optimally grows at a temperature of 30c, but it can grow even at 10c. water activity growth range is between 0.96 and 0.98, while the ph growth range is from 2 to 10. it is usually found in grapes, grape juice, wine, raisins, and sometimes on raw coffee beans. a. carbonarius is the main producer of ochratoxin a, a nephrotoxin which afflicts all tested animal species, while its impact on human health is not easily determined. the connection between ochratoxin a and balcan endemic nephropathy has been the subject of numerous studies but is, as yet, not completely established. food products that could potentially be contaminated with ochratoxin a are jams and grape vinegar. penicillium roqueforti is a psychrothrophic mold that can grow well in cold temperatures and cause spoilage of refrigerated food products. however, the optimal growth temperature of p. roqueforti lies between 20c and 30c for water activity of 0.89 - 0.92. it can grow in a wide range of ph values (3 - 10). furthermore, p. roqueforti is not susceptible to inhibiting activity of weak acids which are usually used as preservatives in the food industry. although known as a starter culture in roquefort cheese production, p. roqueforti also produces certain toxins such as p. roqueforti toxin, roquefortin c, and mycophenolic acid. the aim of this study was to determine the antifungal effect of essential oil constituents carvacrol and eugenol against the foodborne pathogenic molds a. carbonarius a1102 and p. roqueforti ptfkk29 in in vitro and in situ conditions on slices of watermelon citrullus lanatus l. sorento at different incubation temperatures. fungal cultures of a. carbonarius a1102 and p. roqueforti ptfkk29 species were obtained from the collection of fungi of the faculty of food technology osijek. strains were maintained on slants of potato dextrose agar (pda) (biolife, italy) at 4c. before experiments, afterwards, the spores in the suspension were counted (brker - trk counting chamber) and their number was adjusted to 1 10 spores ml. in vitro antifungal activity of eugenol and carvacrol was evaluated by macrobroth method on fungal species of a. carbonarius a1102 and p. roqueforti ptfkk29. eugenol (concentrations applied were : 250, 500, and 750 ppm) and carvacrol (concentrations applied were : 100, 150 and 200 ppm) (sigma, germany) were dissolved in solution of 10% tween 80 (biolife, italy), 96% ethanol (kemika, croatia) and distilled water, sterilized by filtration and used immediately. yeast extract sucrose broth (yesb) was used as growth medium for measurement of the inhibitory effect of eugenol and carvacrol. medium was sterilized at 121c for 15 min followed by cooling to 50c in a water bath. after cooling, different concentrations of eugenol and carvacrol were added to sterile broth, homogenized and 5 ml of growth medium was dispensed in test tubes. fungal spores were inoculated (10 cfu / ml) in yesb and incubated at 25 1c, and the minimal inhibitory concentration (mic g / ml) was recorded after 72 h of incubation. suitable controls : broth control (without fungal spores), growth controls (with fungal spores), solvent (tween 80 and 96% ethanol) and eugenol or carvacrol controls were set under identical conditions. the last tube with no apparent growth of the organism represented the mic of the compound. to test minimal fungicidal concentration (mfc), from last tube after 72 h of incubation at 25c if no growth was observed, mfc was detected. antifungal activity of eugenol and carvacrol was tested in in situ conditions on watermelon c. lanatus l. sorento. watermelon was washed in tap water and dried with paper towels followed by surface sterilization with 70% ethanol. fruit was cut (with sterile knife) on 5 mm thick slices, and additionally slices of = 20 mm, were cut with sterile tube cap. slices were transferred in petri plates (5 slices / dish) and left in laminar hood with ultraviolet lamps turned on for 20 min. each watermelon slice was inoculated with 5 l of fungal spore suspension (1 10/ml) followed by 5 l of tested component solution. fungal colony radii were measured, in 2 perpendicular directions, until 6 day of the incubation period. results were analyzed by microsoft office excel 2003 (microsoft corporation, redmond, usa) and graphpad prism version 5.0 for windows (two - way anova with multiple comparison and bonferroni post - hoc test) (graphpad software, san diego, usa). fungal cultures of a. carbonarius a1102 and p. roqueforti ptfkk29 species were obtained from the collection of fungi of the faculty of food technology osijek. strains were maintained on slants of potato dextrose agar (pda) (biolife, italy) at 4c. before experiments, afterwards, the spores in the suspension were counted (brker - trk counting chamber) and their number was adjusted to 1 10 spores ml. in vitro antifungal activity of eugenol and carvacrol was evaluated by macrobroth method on fungal species of a. carbonarius a1102 and p. roqueforti ptfkk29. eugenol (concentrations applied were : 250, 500, and 750 ppm) and carvacrol (concentrations applied were : 100, 150 and 200 ppm) (sigma, germany) were dissolved in solution of 10% tween 80 (biolife, italy), 96% ethanol (kemika, croatia) and distilled water, sterilized by filtration and used immediately. yeast extract sucrose broth (yesb) was used as growth medium for measurement of the inhibitory effect of eugenol and carvacrol. medium was sterilized at 121c for 15 min followed by cooling to 50c in a water bath. after cooling, different concentrations of eugenol and carvacrol were added to sterile broth, homogenized and 5 ml of growth medium was dispensed in test tubes. fungal spores were inoculated (10 cfu / ml) in yesb and incubated at 25 1c, and the minimal inhibitory concentration (mic g / ml) was recorded after 72 h of incubation. suitable controls : broth control (without fungal spores), growth controls (with fungal spores), solvent (tween 80 and 96% ethanol) and eugenol or carvacrol controls were set under identical conditions. the last tube with no apparent growth of the organism represented the mic of the compound. to test minimal fungicidal concentration (mfc), from last tube after 72 h of incubation at 25c if no growth was observed, mfc was detected. antifungal activity of eugenol and carvacrol was tested in in situ conditions on watermelon c. lanatus l. sorento. watermelon was washed in tap water and dried with paper towels followed by surface sterilization with 70% ethanol. fruit was cut (with sterile knife) on 5 mm thick slices, and additionally slices of = 20 mm, were cut with sterile tube cap. slices were transferred in petri plates (5 slices / dish) and left in laminar hood with ultraviolet lamps turned on for 20 min. each watermelon slice was inoculated with 5 l of fungal spore suspension (1 10/ml) followed by 5 l of tested component solution. fungal colony radii were measured, in 2 perpendicular directions, until 6 day of the incubation period. results were analyzed by microsoft office excel 2003 (microsoft corporation, redmond, usa) and graphpad prism version 5.0 for windows (two - way anova with multiple comparison and bonferroni post - hoc test) (graphpad software, san diego, usa). the antifungal activity of eugenol and carvacrol against fungal species a. carbonarius a1102 and p. roqueforti ptfkk29 expressed as mic and mfc concentrations were presented in table 1. antifungal activity of tested compounds ranged from 1000 (mic) to > 2000 ppm (mfc) of eugenol or 250 (mic) to 2000 ppm (mfc) of carvacrol. antifungal activity of eugenol and carvacrol on a. carbonarius and p. roqueforti the activity of eugenol and carvacrol on watermelon slices (in situ conditions) at 25c and 15c was presented in figures 1 - 4. letters (a, b, and ab) : significant difference between tested treatments (p 0.05) aspergillus carbonarius colony growth inhibition by carvacrol on watermelon slices at 25 and 15c. letters (a, b, and ab) : significant difference between tested treatments (p 0.05) penicillium roqueforti colony growth inhibition by eugenol on watermelon slices at 25 and 15c. significant difference compared to control (p 0.05). letters (a, b, and ab) : significant difference between tested treatments (p 0.05) penicillium roqueforti colony growth inhibition by carvacrol on watermelon slices at 25 and 15c.. letters (a, b, and ab) : significant difference between tested treatments (p 0.05) essential oils or their components like eugenol or carvacrol used for antifungal testing are becoming more interesting in modern food technology, although their effect is well - known. nowadays, consumers concerned about their health are more interested in food produced with minimal processing or preservatives added. this represents a problem in food technology since food safety during the production process or storage is not easily attained. one of the most affected products is minimally processed food - fruit or vegetable salads. during processing minimally processed food, washing (with or without disinfectants), cutting and packaging are allowed. these factors, together with cutting that cause cellular juice leakage, facilitate microbial growth. fungi selected for this experimental study are important in the food industry for several reasons : a. carbonarius is widespread on fruits, produces a huge amount of contaminant conidia while p. roqueforti, although not common as a fruit detrimental fungus, is capable for growth at lower temperatures. watermelon, as well as other vegetables (melons, cantaloupes) and fruits (strawberries, apples, etc.) have become very popular ready - to - eat minimally processed salads for consumers worldwide. however, the absence of more intensive preservation methods increases their susceptibility to fungal (or bacterial) growth and besides spoilage, minimally processed salads are widely known as serious safety issue. the most sensitive fungal species tested was p. roqueforti ptfkk29 [table 1 ], since eugenol acted fungicidally at 2000 ppm on its growth. carvacrol, at a concentration of 1000 ppm, was even more effective in fungicidal activity on tested fungi. since fungicidal effect of carvacrol is well - known, compound, as well as oregano or thyme essential oils, can be used as efficient preservatives in food processing technology. similarly, a. carbonarius a1102 was more susceptible to carvacrol (mic value of 500 ppm compared to 2000 ppm of eugenol) although this species, compared to p. roqueforti ptfkk29 is more resistant to tested chemicals. interpretation of results obtained from antimicrobial assays is demanding, due to different methodology or different strains applied. in a similar experiment authors observed mic activity of carvacrol and eugenol on penicillium expansum at a concentration of 262 and 500 ppm, respectively, while in our experiment (unpublished data) the same species was inhibited by 500 and 2000 ppm of antifungal compounds tested. selection of different strains of the same species in antifungal assays is suggestible, since quite different results can be obtained. the results of mic activity of eugenol and carvacrol were used in in situ testing of antifungal efficacy of compounds against same species on watermelon slices incubated at 25c and 15c. the main idea of performing this assay on selected temperature regimes came from improper cold storage conditions, often observed in markets. concentrations of compounds applied were,, and of mic of eugenol and carvacrol. at a temperature of 25c, a. carbonarius a1102 grew rapidly and at 4 day of incubation, all tested concentrations of eugenol, as well as control samples, reached 22 mm (diameter of watermelon slices). this species showed rapid growth on tested fruit slices, especially at higher storage temperature applied. statistically significant difference, compared to control, was observed only at the highest concentration (750 ppm) applied at 2 incubation day while among concentration of eugenol, 250 and 750 ppm were significantly different [figure 1 ]. lower growth rate at 15c was observed where lag phase of fungal growth occurred even in the control sample until 2 day, while 500 and 750 ppm prolonged this phase until 6 day (possibly even further, although this day was the final day of incubation). carvacrol affected a. carbonarius growth in a similar way [figure 2 ] where, at 25c almost all samples reached the end of fruit slices. however, significant difference was observed between 200 ppm and 150 ppm, compared to control sample (2 incubation day). at 15c fungal growth was slower, reaching < 6 mm at the end of the incubation period (control sample). although almost no difference was observed at the start of incubation time, incubation at 4 and 6 day indicated a significant difference in activity of carvacrol, where 200 and 150 ppm were different compared to results obtained by 100 ppm, as well as a control sample. both eugenol and carvacrol acted similarly on a. carbonarius during in situ experiments on watermelon slices. compared to a. carbonarius, p. roqueforti showed slower growth rate at both selected temperature intervals [figures 3 and 4 ]. interestingly, this species grow faster at a lower temperature (15c) which shows its psychrotrophic nature. lag phase of fungal growth lasted until 4 incubation day for the control sample, while treatments prolonged this phase further to 6 day. since the growth at 25c was slow, differences between treatments and control are visible at 4 and 6 incubation day, although only difference between control and treatments was noticed. further incubation would show differentiation between control and concentration of eugenol applied in this experimental setup. higher growth rate with strong differentiation between concentrations applied was observed at 15c incubation temperature where all treatments applied were significantly different compared to control sample. although, considerably lower concentrations of carvacrol on slices were applied, similar results of fungal growth inhibition occurred at both temperatures applied [figure 4 ]. lag phase of p. roqueforti growth was shorter for 2 days compared to eugenol [figure 3 ]. although the growth rate of p. roqueforti treated with 100 ppm of carvacrol was faster, compared to control, as suspected since, if fungi are treated with chemicals applied in concentrations below their inhibitory concentration, their growth rate (and even mycotoxin production) can be even more pronounced. differences are also possible due to fruit surface structure. watermelon surface has pronounced hollows that make colony diameter assessment more complicated. this problem can be successfully resolved by an increasing number of fruit slices tested and with experiment repetitions. potential disadvantage in the application of essential oils or their components in food systems is a modification of characteristic sensory profile of the food. in this experimental study, this specific issue was not detected, since small volume of tested compounds was applied (5 l). both selected essential oil components, eugenol and carvacrol inhibited tested fungi (a. carbonarius a1102 and p. roqueforti ptfkk29) in yesb, as well as in in situ conditions (watermelon slices). lag phase of the fungal colony growth on watermelon was prolonged in response to higher concentrations of components applied, for a longer period of time. lower temperature (15c) retarded the growth of a. carbonarius a1102, while this effect was not observed during the growth of p. roqueforti ptfkk29. inhibitory effect of both tested compounds against fungal growth of selected fungal species on watermelon slices is concentration dependent. | background : essential oil components eugenol and carvacrol (ranging between 100 and 200 ppm for carvacrol and between 250 and 750 ppm for eugenol) were tested for antifungal activity against foodborne pathogenic fungal species aspergillus carbonarius a1102 and penicillium roqueforti ptfkk29 in in vitro and in situ conditions.materials and methods : in vitro antifungal activity of eugenol and carvacrol was evaluated by macrobroth method, while watermelon citrullus lanatus l. sorento slices were used for antifungal assays in situ.results:selected components, eugenol and carvacrol showed significant inhibitory effect against tested fungi (a. carbonarius a1102 and p. roqueforti ptfkk29) in yeast extract sucrose broth, as well as in in situ conditions. the minimal inhibitory concentration (mic) of eugenol against a. carbonarius a1102 determined by macrobroth method was 2000 ppm, while against p. roqueforti ptfkk29 determined mic was 1000 ppm. carvacrol inhibited growth of a. carbonarius a1102 at minimal concentration of 500 ppm, while against p. roqueforti ptfkk29, mic was 250 ppm. the assays in real food system watermelon slices for eugenol and carvacrol show that the inhibitory effect against both selected fungal species was concentration dependent. furthermore, our results showed that antifungal effect of carvacrol as well as eugenol applied on watermelon slices in all concentrations was a result of effective synergy between an active antifungal compound and lower incubation temperature (15c) in inhibition of a. carbonarius a1102.conclusion:the present study suggests that the use of eugenol and carvacrol is promising natural alternative to the use of food chemical preservatives, in order to improve safety and quality of fresh - cut and ready - to - eat fruits. |
lung cancer presenting in about 85% by non - small cell lung cancer (nsclc) is the leading cause of death among human malignancies. the ability to establish consistent human tumour xenografts in small animals is a crucial part of preclinical nsclc therapy investigations, and whole body irradiation (wbi) serves as the classic method for experimentally inducing immunosuppression in rodents improving tumor take rates for human lung cancer models in nude mice [3, 4 ] and nude rats [59 ]. nevertheless, human tumour xenografts in nude mice are an accepted model for in vivo biological and preclinical studies of nsclc [24 ]. they reach 200400 g and are more suitable to some surgical manipulations, particularly on small structures such as blood vessels or to molecular imaging applications especially by means of clinical scanners. at the same time the size of rats causes an inhomogeneous distribution of radiation dose due to the absorption of x - rays within the animal 's body reducing the effectiveness of wbi for immunosuppression. decrease of the dose together with elevated immune competence of nude rats may effects in substantially diminished tumour take rates compared to those in nude mice or even spontaneous regression of the tumours. in this paper a new design of wbi improving radiation mediated immunosuppression of rats is described and its application for the generation of human nsclc xenografts in nude rat model is discussed. the principles of human tumour xenograft engraftment in the nude rat model illustrated here for nsclc may be useful for research applications involving other types of human tumours. male athymic 5 weeks old nude rats (harlan laboratories gmbh, borchen - alfen, germany) were kept five rats per cage with water and food ad libitum at least one week before experiments. animal housing and experiments were approved by the local animal care committee according to the institutional guidelines and the german animal welfare regulations. intraperitoneal anesthesia was performed with ketamin 500 (120 mg / kg, curamed, germany)/xylazin (16 mg / kg, rompun, germany) mixture. human nsclc cells (a549, dsmz, braunschweig, germany) were transplanted by subcutaneous injection of 5 10 cells in 0.2 ml of phosphate buffer saline in the right lower limb of animals in two days after wbi. radiation exposure was carried out by means of the x - ray system (yxlon int. x - ray gmbh, germany) : 200 kv, 20 ma, 0.5 mm cu. dosimetry was performed with the clinical dosimeter unidos equipped with the semifles ionization chamber (ptw - freiburg, germany) using a set of water equivalent plate (1 5 5 cm) phantoms (ptw rw3 slab phantom 29672, siemens, germany). dose depth profile within the water equivalent phantom was computed using geant 4 monte carlo toolkit simulating the passage of x - ray photons through matter. the experimental results are expressed as the mean standard deviation of several independent experiments. the dose depth profile within the water equivalent phantom computed using the geant 4 monte carlo toolkit simulating the passage of x - ray photons through matter was compared with the measured dose depth values using a set of water equivalent plate (1 5 5 cm) phantoms (figure 1). a good agreement in the dose depth value was revealed between the simulation and measurements for wbi of rat using common 6 6 20 cm cage (figure 1, inset a). the dose reduces exponentially from its maximum value (set to 100%) at the top to ~30% at the bottom of phantom (figure 1(a)). therefore, during common wbi, for example, with 5 gy using common 6 6 20 cm cage parts of the rat 's body placed at the top of the cage could get more than 8 gy, while the parts located closer to the bottom of the cage including femur and tibia of the leg, where subcutaneous tumour transplantation was planned, could receive less then 3 gy. this inhomogeneity of the dose distribution in the 6 cm thick phantom could be substantially reduced by irradiation from opposing directions (figure 1(b)). however, such design of wbi could not be applied for the rats positioned in the standard cage due to the movement of the animals during irradiation time. even if they had the same position in the chamber at the beginning of the treatment, they could move or rotate (figure 2). it was not possible to follow position of each rat in time and to calculate the dose received by point of interest, for example, at right leg where s.c. anesthetized rats lying in physiological position on the right side (figure 1, inset b) were irradiated with half of the dose (2 gy), rotated onto the left side (figure 1, inset c), and irradiated again with another half of the dose (2 gy). in this case thickness of rats was less then 4 cm, additionally improving the homogeneity of the dose distribution (figure 1(c)). human nsclc tumour xenografts in nude rats were established by subcutaneous injection of 5 10 a549 cells after conventional wbi with dose 5 gy and after inducing the new wbi with dose 2 + 2 gy from opposite sides. in both cases s.c. injection of nsclc a549 cells after wbi induced tumour growth in nude rats (figure 3). however, after conventional wbi with a dose of 5 gy tumour nodules grew relatively slow (figure 3(a)) and reached a volume of approximately 60 mm in 40 days. at the same time, after double wbi with 2 + 2 gy from opposite sides, tumour nodules grew significantly faster (figure 3(b)) and reached a volume of approximately 1000 mm in 40 days (figure 3, inset a). the presented results show that the conventional x - ray system could provide acceptable dose uniformity across rats of typical thicknesses by irradiation of animals (2 + 2 gy) from opposite sides using a new cage which was created for irradiation of 2 rats at the same time. an advantage of the designed wbi for immunosuppression of rats and improvement of human nsclc tumour xenograft growth was illustrated by comparison of two of our attempts to establish the human nsclc tumour xenografts in nude rats after conventional wbi with dose 5 gy and after inducing the new wbi with dose 2 + 2 gy from opposite sides. in both cases subcutaneous however, after conventional wbi with a dose of 5 gy tumour nodules grew relatively slow (figure 3(a)). we had shown earlier that they achieved about 140 mm in volume in 70 days and then spontaneously regressed during the following 50 days. at the same time, after double wbi with 2 + 2 gy from opposite sides, tumour nodules grew significantly faster, reached a volume of 1000 mm in 40 days (figure 3(a)). resulted tumours were large enough to be successfully investigated by magnetic resonance imaging, positron emission tomography, and computed tomography using clinical scanners. subsequent investigations of the same human nsclc tumour xenografts in nude rat model have shown that manipulation of tumour xenograft environment could further enhance tumour growth and change tumour vascularisation by administration of exogenous growth factors, cotransplantation with endothelial cells or modified by additional tumour bed irradiation [8, 9 ]. nevertheless, the reported method of double wbi from two opposite sides can be applied using commercially available x - ray tubes ; increased homogeneity of the radiation dose distribution through the cross section of the animal 's body, allowing for producing human nsclc xenografts by subcutaneous injection of a549 cells in the leg of the nude rat, yields reliable tumour growth patterns. established human tumour xenografts were suitable for analysis in clinical scanners, offered good image quality, and presented an alternative to dedicated small animal scanners for numerous applications in cancer research. presenting method of radiation mediated immunosuppression of rats may be useful for many preclinical research applications involving other types of human tumours. | human tumour xenografts in a nude rat model have consistently been used as an essential part of preclinical studies for anticancer drugs activity in human. commonly, these animals receive whole body irradiation to assure immunosuppression. but whole body dose delivery might be inhomogeneous and the resulting incomplete bone marrow depletion may modify tumour behaviour. to improve irradiation - mediated immunosuppression of human non - small cell lung cancer (nsclc) xenografts in a nude rat model irradiation (2 + 2 gy) from opposite sides of animals has been performed using a conventional x - ray tube. the described modification of whole body irradiation improves growth properties of human nsclc xenografts in a nude rat model. the design of the whole body irradiation mediated immunosuppression described here for nsclc xenografts may be useful for research applications involving other types of human tumours. |
ultrasound imaging is one of the most inexpensive, safe, and commonly used diagnostic tools for imaging soft tissues and vasculature. the use of microbubble contrast agents enables visualization of microvasculature which can not be seen directly with doppler ultrasound. microbubbles composed of gaseous cores covered with stabilizing agents can drastically enhance the ultrasound signal because of their large compressibility, which leads to enhanced scattering of ultrasound. the echogenicity of microbubbles coupled with their physical interactions with acoustic energy can also be used for triggered release of active agents or for conversion of acoustic energy to thermal energy to enable therapeutic applications. for example, recent studies have shown that the insonation of microbubbles with low - intensity ultrasound can lead to a localized temperature increase, which in turn disrupts tumor vasculature (also known as antivascular ultrasound therapy), enabling a minimally invasive procedure to disrupt cancerous tissues. these properties of microbubbles make them ideal candidates for theranostics ; that is, the same microbubble agents can be used for diagnostics and therapeutic applications. currently available commercial agents consist of polydisperse microbubbles with size distributed over a broad range of diameters. studies have shown that the effectiveness of these agents can be significantly enhanced by making the size distribution narrow for molecular imaging and therapeutic applications. although some methods to fractionate microbubbles to enhance the uniformity of size have been reported, these techniques inevitably lead to loss of significant fraction of bubbles. another important factor that significantly affects microbubble properties for ultrasound - related applications is the surfactant that is used to stabilize microbubbles. an approach to control the molecular structure and properties of these surfactants would be highly beneficial because their structure affects the surface functionality and the echogenicity of microbubbles. the generation of monodisperse microbubbles that are stabilized with surfactants that can be precisely designed and controlled would lead to microbubbles that have ideal functionality for ultrasound imaging and novel therapeutic approaches such as targeted drug delivery and antivascular ultrasound therapy (avust). in this study, we present a method to create stable protein - shelled microbubbles using a microfluidic flow focusing device that uses an air - actuated membrane valve, which enables the production of highly monodisperse sub-10 m microbubbles. although other studies have shown that monodispserse bubbles can be generated based on microfluidic techniques, the size range of microbubbles that can be generated from such devices is somewhat limited. a method based on the dissolution of highly soluble gas such as co2 in a long microfluidic channel has shown to generate monodisperse bubbles of varying sizes. a method that enables the formation of bubbles over a wide range of size without using soluble gas and long channels would provide a complementary method that can further expand the use of microfluidic techniques to generate monodisperse microbubbles. the air - actuated membrane valve enables precise control over the size of microbubbles while producing highly monodisperse microbubbles. to stabilize the microbubbles generated by the microfluidic technique unlike common proteins that have been used to stabilize microbubbles, oleosin potentially provides versatility in imparting additional functionality via recombinant protein technology. we demonstrate an example of such modularity by expressing and incorporating fluorescent oleosin into the microbubble shell. we demonstrate that careful tuning of the composition of the stabilizing agents is critical in the formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. microfluidic flow focusing devices with expanding nozzle design (figure 1a) are fabricated using single layer soft lithography in poly(dimethylsiloxane) (pdms). negative photoresist su-8 2010 (microchem, newton, ma), thinned to a 3:1 ratio with su-8 developer, is spin - coated onto a clean silicon wafer to a thickness of 5 m and patterned to uv light through a transparency photomask (cad / art service, bandon, or) using a karl suss ma4 mask aligner (suss microtec inc., sunnyvale, ca). to incorporate an air - actuated valve, we use single - layer membrane valves, which exist in the same plane as the microfluidic channel, allowing us to fabricate the entire microfluidic device in a single layer mold. sylgard 184 poly(dimethylsiloxane) (dow corning, midland, mi) is mixed with cross - linker (ratio 12:1), degassed thoroughly and poured onto the photoresist pattern, and cured for 1 h at 65 c to make the membrane highly compliant. the pdms replica are peeled off the wafer and bonded to a pdms membrane fabricated by spin - coating pdms on a glass slide after oxygen plasma activation of both surfaces. having a microchannel fully enclosed in pdms allows for more efficient use of the valve membrane. (a) schematic illustration of a pdms microfluidic device used to generate monodisperse microbubbles of different sizes. (b) cross - sectional geometry of the nozzle (see figure s1 for junction dimensions). (c) schematic of a microbubble stabilized with a mixture of oleosin and (peo)n-(ppo)m-(peo)n triblock copolymer. multiple rounds of pcr are used to create the oleosin gene 42 - 30g-63 and egfp-30g-63. the genes are inserted into the expression vector pbamuk, a pet series derivative constructed by the duyne laboratory (som, penn). pbamuk adds a 6-histidine tag to the c - terminus of the protein for imac purification. protein is expressed in the e. coli strain bl21 de3 (stratagene) controlled by the lac promoter. cultures are grown at 37 c in luria bertani (lb) with kanamycin (50 g ml) until od600 0.70.9. protein expression is induced with isopropyl -d-1-thiogalactopyranoside (iptg) to a final concentration of 1.0 mm. cells are harvested by centrifugation, and cell pellets are frozen at 20 c prior to purification. b - per protein extraction agent (fisher scientific) is used for protein purification. 42 - 30g-63 is expressed in inclusion bodies whereas egfp-42 - 30g-63 is expressed in the soluble fraction of the cell. 42 - 30g-63 is purified according to the b - per protocol for inclusion bodies, and egfp-42 - 30g-63 is purified according to the protocol for soluble proteins. the concentration of purified protein is measured with a nano - drop 1000 (thermo scientific). all analysis is completed in pbs unless otherwise noted. to establish the purity of the proteins, sds / page gels are run on nupage novex 412% bis - tris mini gels (invitrogen) in mes buffer. the gel is destained overnight in water and imaged with a kodak gel logic 100 imaging system. sample spots are created with 0.5 l protein in 1 pbs and 0.5 l of saturated sinapinic acid solution (50/50 acetonitrile / water + 0.1% tfe). spectra are collected on an ultraflextreme maldi - tof (bruker, billerica, ma) (see figure s5 for egfp spectra). to measure the protein secondary structure, far - uv cd spectra are collected at 25 c on an aviv 410 spectrometer (aviv biomedical inc.) using a 1 mm quartz cell. naf is used to replace nacl due to the strong absorbance of the cl ion. the liquid phase containing the shell material consists of oleosin or a solution containing oleosin proteins and (peo)78-(ppo)30-(peo)78 or (peo)100-(ppo)65-(peo)100 diluted in phosphate - buffered saline (pbs) (ph 7.2, sigma - aldrich, st. the liquid phase consisting of oleosin and (peo)n-(ppo)m-(peo)n triblock copolymers at the optimal concentration dispersed in pbs is supplied to the device using a syringe pump (harvard apparatus phd ultra) at flow rates between 500 and 1000 l h. to connect the channels to syringes, polyethylene tubing with an i.d. of 0.38 mm and an o.d. of 1.09 mm (bb31695-pe/2, scientific commodities inc, lake havasu city, az) is used. the gas phase consists of 99.999% pure nitrogen gas (n2, gts welco, richmond, va) or octafluorocyclobutane (c4f8) (synquest laboratories, alachua, fl) supplied to the device using a pressure regulator (type 700, controlair inc., polyethylene tubing with an i.d. of 0.86 mm and an o.d. of 1.32 mm (bb31695-pe/5, scientific commodities inc, lake havasu city, az) the membrane valve is actuated using a dual - valve pressure controller (pcd-100psig - d - pcv10, alicat scientific, tucson, az) at pressure between 0 and 40 psi. microbubbles are produced by first applying a small pressure to the gas inlet (24 psi) immediately followed by injecting the liquid phase at the desired flow rate (5001000 l h). the gas pressure is then increased slowly until steady state of bubble generation is reached. images of microbubbles production are captured using an inverted microscope (nikon diaphot 300) connected to a high speed phantom v7 camera. for microbubbles that remain stable during generation and collection, water interface in 35 mm petri dishes, acquiring images under an upright microscope (carl zeiss axio plan ii) connected to a qimaging retiga 2000r camera. microbubbles diameter variation over time is measured and images are analyzed using imagej (v 1.47v, nih). a nicolet 8700 ft - ir spectrophotometer (thermo scientific) is used to obtain the ft - ir spectra of microbubbles and their constituent solutions on znse windows (phoenix infrared, lowell, ma). samples are prepared by placing a small aliquot of solution on top of the window and are fully dried before measurements are performed. the spectra are taken between 5000 and 600 cm, at 1.93 cm wavenumber resolution. microbubbles for ultrasonic imaging are collected and imaged directly in 16 mm membrane dialysis bag, which is prefilled with buffer solution and sealed at one end. after a desired amount of bubbles is collected, the tube is sealed at the other end, carefully avoiding formations of air pockets. the collected microbubbles are imaged using a clinical ultrasound scanner hdi 5000 (phillips / atl, bothell, wa) which is equipped with a broadband high - frequency ultrasound transducer at 715 mhz. grayscale b - mode images are acquired with a mechanical index (mi) of 0.37 and 0.47 with focus between 0.51.5 and 12 cm, respectively. for a variety of applications that involve microbubbles and ultrasound, the size distribution of microbubble agents drastically influences the efficacy of the image contrast enhancement and therapeutic methods. to enable formation of microbubbles with high monodispersity and, at the same time, tunable size, we use an expanding nozzle flow - focusing microfluidic device with a single - layer membrane valve at the orifice as schematically illustrated in figure 1. a previous study has shown that the size of liquid emulsion droplets produced by a flow - focusing microfluidic device can be controlled by changing the size of the orifice via the actuation of the valve. likewise, this design gives us the flexibility to tune the size of gas microbubbles in the same chip without changing the continuous phase or gas flow rates, by only changing the size of the orifice through the application of pressure to the valve. furthermore, the use of the single - layer membrane valve overcomes the low resolution that is typically achieved by using polymeric photomasks (smallest feature 10 m). for the initial testing of the microfluidic device to control the size of microbubbles, we use nitrogen gas and a common surfactant, sodium dodecyl sulfate (sds, sigma - aldrich, st. louis, mo), at a concentration of 20 mg ml in the aqueous phase to stabilize microbubbles. we are able to produce monodisperse microbubbles with radius ranging from approximately 2 to 10 m for several hours without changes in the bubble size. an advantage of this microfluidic device is that the size of microbubbles that can be generated from a single microfluidic device can be controlled over a wide range, unlike most flow - focusing microfluidic devices that have limited range of size control. by increasing the pressure that is applied to the single - layer valve, we can control the size of the nozzle and, in turn, the size of microbubbles as shown in figure 2. we observe that the diameter of the microbubbles, db, decreases linearly with the width of the nozzle, wn. interestingly, the microbubble generation frequency (f = the number of microbubbles generated per second) is inversely proportional to the volume of microbubbles as shown in the inset of figure 2b (f db). such a trend indicates that the gas flow rate, calculated to be qg 62 l h (= 8.4 l h), remains more or less constant under varying nozzle size. the constant gas flow rate under varying nozzle width may be attributed to the change in the cross - sectional shape of the channel, from a horizontal slit to a square or hourglass shape. although sds enables the investigation of microfluidic device performance, microbubbles formed using sds are not stable upon collection. (a1a9) series of micrographs of the microfluidic device during the generation of microbubbles using a solution containing sds at a concentration of 20 mg ml in the aqueous phase. by changing the size of the nozzle, which is controlled by an air - actuated valve placed at the orifice the inset shows the microbubbles generation frequency (f) vs volume of microbubbles (db). the linear relationship between the two quantities indicates that the gas flow rates remains more or less constant under varying nozzle size. to produce stable microbubbles with high monodispersity, size tunability, and structural modularity the protein has a natural amphiphilic structure with n- and c - terminal hydrophilic arms and a central hydrophobic core containing a proline knot forcing the protein into a hairpin structure. oleosin has been used in various biotechnology and biomedical applications exploiting its amphiphilic properties. in its native state, eliminating a large portion of the hydrophobic domain and removing the majority of the secondary structure in the protein backbone have been shown to yield a oleosin mutant that becomes highly soluble in water and naturally self - assembles into micelles. the soluble oleosin mutant is named 42 - 30 - 63 defining the number of amino acids in each domain : the n - terminal hydrophilic arm, the central hydrophobic core, and the c - terminal hydrophilic arm, respectively. this molecule is produced by truncating the wild - type molecule without changes in the sequence of amino acids. the 42 - 30 - 63 oleosin mutant is further modified by inserting five glycines into the hydrophobic core (see supporting information for protein sequences) creating a mutant we refer to as 42 - 30g-63. the protein is expressed in the escherichia coli strain bl21 (de3) with isopropyl -d-1-thiogalactopyranoside (iptg) induction. protein is purified using immobilized metal affinity chromatography through a 6-histidine tag on the c - terminus of the protein, leading to highly purified products (figure 3). protein molecular weight is confirmed with sds - polyacrylamide gel electrophoresis (sds - page) and matrix - assisted laser desorption / ionization - time - of - flight (maldi - tof) mass spectroscopy (figure 3). the addition of the five glycines to the 42 - 30 - 63 mutant increases the protein expression, stability, and solubility while abolishing secondary structure, as shown by circular dichroism (figure 3). in contrast, the cd spectra of wild - type (wt) oleosin shows -sheet character as previously reported (see supporting information for detailed analysis). (a) sds - page gel showing > 95% purity for 42 - 30g-63. (b) madli - tof spectra confirming the molecular weight for 42 - 30g-63 (expected : 15 027 ; measured : 15 025). (c) far - ultraviolet circular dichroism (uv cd) spectra of 42 - 30g-63 and wild - type oleosin. when we produce microbubbles using oleosin, at concentrations between 1 and 2 mg ml, we can only stabilize bubbles with radius above 10 m. during the generation of microbubbles with radii smaller than 10 m, bubbles are observed to undergo coalescence within and outside of the microfluidic device (figure s2). in addition, the relatively high surface tension between the liquid and the gas phases makes the generation of such microbubbles challenging, often resulting instability of microbubbles in the microfluidic device. interestingly, a number of microbubble systems that are currently being investigated (e.g., phospholipid - stabilized microbubbles) often have extra components such as poly(ethylene glycol)-based surfactants such as amphiphilic triblock copolymers to enhance the microbubble stability and generation process. thus, we add widely used poly(ethylene glycol)-b - poly(propylene glycol)-b - poly(ethylene glycol) triblock copolymers ((peo)n-(ppo)m-(peo)n where n and m denote the number of ethylene oxide and propylene oxide repeat units, respectively ; these polymers are also known as pluronic and polxamer) to the oleosin solution to test whether the production of microbubbles can be facilitated. we test two different types of (peo)n-(ppo)m-(peo)n triblock copolymers : (peo)100-(ppo)65-(peo)100 and (peo)78-(ppo)30-(peo)78. when we use a mixture containing 12 mg ml oleosin and 520 mg ml (peo)100-(ppo)65-(peo)100 (average molecular weight 12 600), we are able to consistently generate monodisperse microbubbles at the nozzle ; however, these microbubbles undergo significant coalescence upon collection. in contrast, when we add (peo)78-(ppo)30-(peo)78 (average molecular weight 8400) to oleosin solutions, we are able to generate microbubbles at the nozzle and very limited coalescence is observed upon collection. we find that the optimal concentration for stable microbubble formation requires an aqueous phase containing 1 mg ml of oleosin and 10 mg ml of (peo)78-(ppo)30-(peo)78. (peo)78-(ppo)30-(peo)78 is known to be more effective in stabilizing gas bubbles than (peo)100-(ppo)65-(peo)100, which may explain the effectiveness of the former in facilitating the microbubble production. to further understand the possible role of (peo)78-(ppo)30-(peo)78 in facilitating the formation of microbubbles and the role of oleosin in imparting long - term stability to microbubbles, we perform ft - ir spectroscopy of four different samples : pure oleosin, pure (peo)78-(ppo)30-(peo)78, a mixture of oleosin and (peo)78-(ppo)30-(peo)78 with the same composition as the solution used for microbubble generation (1:18 mole ratio), and microbubbles. ft - ir spectra remarkably show that the composition of microbubble shell is very different from that of the solution as shown in figure 4. the concentration of oleosin present in the microbubble shell is significantly higher than that of the original solution used for microbubble generation, as evidenced by the prominent presence of peaks associated with pure oleosin in the microbubble spectrum (e.g., peaks found around 1535, 1650, and 3290 cm). although it is not straightforward to quantify the composition of the microbubble shell based on ft - ir, the comparison of the four spectra shows that oleosin seems to be the major species that is stabilizing microbubbles. these results suggest that (peo)78-(ppo)30-(peo)78 present in the solution facilitates microbubble production by lowering the surface tension and rapidly covering the microbubbles upon breakup at the nozzle. once microbubbles are generated and flow through the channel, oleosin starts to adsorb and possibly displace some of (peo)78-(ppo)30-(peo)78 that are on the microbubble surface. the spectra of pure oleosin and microbubbles are amplified by factors of 2.5 and 5, respectively, to clearly show the features. micrographs of microbubbles produced using a solution containing 1 mg ml oleosin and 10 mg ml (peo)78-(ppo)30-(peo)78. (a) a small number of large bubbles are present upon collection via plastic tubing. (b) big bubbles disappear 24 h after collection, leaving monodisperse microbubbles. in the samples that are collected through polyethylene tubing, we typically observe that there are a small number of fairly large bubbles (> 20 m in diameter). although the physical mechanism behind the appearance of these large bubbles is not known, their number fraction is extremely small, typically less than 1%. interestingly, these large bubbles disappear completely approximately 24 h after collection, leaving behind a collection of highly monodisperse microbubbles as shown in figure 5. since we do not see any major coalescence between microbubbles occurring within the pdms microfluidic device, we believe these large bubbles likely form during transfer of the microbubbles from nozzle to a container via polyethylene tubing. possibly, abrupt changes in dimensions and relative shear stress experienced by microbubbles between the pdms device and the collection tube as well as the lower speed at which the microbubbles travel in the polyethyelene tube before being released in a petri dish may lead to collision between bubbles and eventual coalescence. another possibility is that these large bubbles have slightly different surface composition since they are observed to undergo dissolution when they are stored for an extended period, whereas the monodisperse bubbles that were originally generated at the nozzle do not dissolve completely over a long period of time. interestingly, we are able to collect highly monodisperse microbubbles without any large bubbles if we collect the produced bubbles straight into a well that is position in the same plane as the microfluidic channel (figure 6). the high monodispersity of microbubbles is illustrated by their ability to pack into hexagonal array, which indicates that the coefficient of variation (cv) is less than 5% around the average bubble size, consistent with our optical microscopy - based analysis. these results show that even small perturbations can lead to disruption of microbubbles that are generated using microfluidic devices, and extra care must be taken in collecting microbubbles for clinical applications since large bubbles in blood vessels can lead to serious problems such as embolism. micrograph of monodisperse microbubbles produced using a solution containing 1 mg ml oleosin and 10 mg ml (peo)78-(ppo)30-(peo)78 and collected into a well in the pdms device without the use of plastic tubing.,, and cv in the inset represent the average (in m), standard deviation (in m), and coefficient of variation, respectively. microbubbles generated using the mixture of oleosin and (peo)78-(ppo)30-(peo)78 (molar ratio of oleosin : triblock copolymer = 1:18) are remarkably stable once they are collected. when microbubbles are collected and stored in water (microbubbles reside at the air water interface due to their buoyancy), microbubble radius decreases by about 13% during the first few days and eventually ceases to shrink further. these microbubbles remain stable at least for 4 weeks, and their size does not show any changes after 5 days as shown in figure 7, suggesting that these microbubbles will not undergo dissolution even after 4 weeks. the stability of these microbubbles does not depend on whether n2 or c4f8 is used as the gas phase. in contrast, microbubbles generated solely with (peo)78-(ppo)30-(peo)78 do not exhibit such excellent stability. these results indicate that oleosin plays a critical role in stabilizing the shell of microbubbles, which likely consists of a mixture of oleosin and (peo)78-(ppo)30-(peo)78, to prevent complete dissolution or coalescence of microbubbles upon their collection. similar examples, in which shells suppresses the dissolution of microbubbles, have been observed in microbubbles that have been stabilized with other types of proteins, nanoparticles, or synthetic polymers. micrographs showing microbubbles stability over time for microbubbles produced using a solution containing 1 mg ml oleosin and 10 mg ml (peo)78-(ppo)30-(peo)78. (b) microscope images of 24 days after collection. as discussed briefly above, one of the unique aspects of oleosin is that the molecular structure and thus the properties of the monolayer that contains this molecule can be engineered using recombinant protein technology. recombinant protein technology allows for precise molecular engineering of proteins generated from microorganisms such as bacteria and thus can be used to generate oleosin species with different functionality and properties. to demonstrate proof - of - principle that this molecule has such modularity, we express a green fluorescent protein mutant oleosin by fusing enhanced green fluorescent protein (egfp) to the n - terminus of the 42 - 30g-63 oleosin. the modified oleosin genes are constructed using standard molecular biology techniques and cloned into the expression vector pbamuk. it is evident that the microbubbles produced with the blend of the two oleosin species (pure at 1 mg ml, mutant at 0.05 mg ml) along with 10 mg ml (peo)78-(ppo)30-(peo)78 have the egfp mutant species incorporated in the bubble shell, whereas the microbubbles generated without the egfp mutant species do not show surface fluorescence (figure 8). also, fluorescence intensity is observed to be fairly uniform on the surface of the bubbles. our results clearly indicate that that oleosin with different functionalities can be generated and incorporated into the microbubble shell and that oleosin distributes uniformly on the surface of microbubbles. confocal fluorescent microscopy images of bubbles produced with (a, b) oleosin and (c, d) with a blend containing the egfp mutant. in both cases a solution containing 1 mg ml oleosin and 10 mg ml (peo)78-(ppo)30-(peo)78 is used to produce microbubbles. echogenicity measurements are carried out using microbubbles generated with a solution containing 1 mg ml oleosin and 10 mg ml (peo)78-(ppo)30-(peo)78. we collect microbubbles directly in a 3 cm long dialysis tubing with a diameter of 16 mm, which is sealed at one end and prefilled with pbs solution containing 10 mg ml (peo)78-(ppo)30-(peo)78. microubbles are transferred directly into the dialysis tube from the pdms device outlet using polyethylene tubing, which is submerged in the pbs solution. after collecting a desired amount of microbubbles, the tube is sealed on the other end to avoid introducing any air pockets and is stored in 50 ml centrifuge tubes filled with pbs solution containing 10 mg ml (peo)78-(ppo)30-(peo)78. the tube is kept on a spinning wheel rotating at 60 rpm to induce continuous motions of the microbubbles and more importantly to remove large bubbles that may have been collected. since antivascular and other microbubble - based therapies are monitored using high - frequency ultrasound, the echogenicity of the microbubbles is tested using a broadband high - frequency ultrasound transducer at 715 mhz in brightness mode (b - mode). the microbubbles are acoustically active along the entire length of the dialysis tube as shown in figure 9. in contrast, a pbs solution containing 10 mg ml (peo)78-(ppo)30-(peo)78 without any microbubbles does not show any acoustic signal, indicating that the oleosin - stabilized microbubbles are highly echogenic. microbubbles remain acoustically responsive 30 min after the initial measurement and even 1 week after the first measurement, showing nondetectable changes in the signal brightness (figure 9). these results clearly indicate that these microbubbles stabilized with oleosin are highly stable and echogenic and thus could have significant potential for theranostic applications. ultrasound sonography images of c4f8 microbubbles generated with a solution containing 1 mg ml oleosin and 10 mg ml (peo)78-(ppo)30-(peo)78. ultrasound images of microbubbles (a, b) 12 h after generation and (c, d) 30 min and (e, f) 7 days after initial imaging. ultrasound images of control samples are reported in panels g and h. the microbubbles have a radius of about 4 m. we have shown that a recombinant mutant oleosin, in combination with a triblock copolymer, (peo)78-(ppo)30-(peo)78, can be used to successfully produce stable and monodisperse microbubbles with high echogenicity. we demonstrate that the use of a pdms microfluidic device with an air - actuated valve is an effective method to control the size of microbubbles while maintaining narrow size distribution. microbubbles incorporating oleosin show high stability and can be further functionalized using recombinant protein technology, which we demonstrated by the incorporation of egfp mutant oleosin into microbubbles. we envisage that the combination of microfluidic generation and oleosin - based stabilization of microbubbles will represent a promising platform for ultrasound - related applications. in particular, by functionalizing oleosin with specific targeting ligands via recombinant protein techniques, it will be possible to enable localized microbubble - based ultrasound therapy. also, by varying the molecular structure of oleosin (e.g., controlling the structure of hydrophobic domain), microbubble shells with different rheological properties could be generated. | microbubbles are used as contrast enhancing agents in ultrasound sonography and more recently have shown great potential as theranostic agents that enable both diagnostics and therapy. conventional production methods lead to highly polydisperse microbubbles, which compromise the effectiveness of ultrasound imaging and therapy. stabilizing microbubbles with surfactant molecules that can impart functionality and properties that are desirable for specific applications would enhance the utility of microbubbles. here we generate monodisperse microbubbles with a large potential for functionalization by combining a microfluidic method and recombinant protein technology. our microfluidic device uses an air - actuated membrane valve that enables production of monodisperse microbubbles with narrow size distribution. the size of microbubbles can be precisely tuned by dynamically changing the dimension of the channel using the valve. the microbubbles are stabilized by an amphiphilic protein, oleosin, which provides versatility in controlling the functionalization of microbubbles through recombinant biotechnology. we show that it is critical to control the composition of the stabilizing agents to enable formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. our protein - shelled microbubbles based on the combination of microfluidic generation and recombinant protein technology provide a promising platform for ultrasound - related applications. |
it is found in countries and regions with inadequate sanitary infrastructure and insufficient health education. the life cycle of the parasite includes the adult stage, the egg, and the larval stage (cysticercus). when a person ingests raw or semi - cooked pork meat with cysticerci, the scolex evaginates and attaches with its double row of 22 to 32 hooks and its 4 suckers to the mucosa in the upper third section of the small intestine, which is the duodenum - jejunum, and in 3 - 4 months transforms into a fully developed adult tapeworm. t. solium tapeworms measure 1 - 5 m long, and the scolex is followed by the neck, from which the strobila is formed, resembling a ribbon formed by 700 to 1,000 immature, mature, and gravid proglottids. the immature proglottids have not yet developed sexual organs while the gravid proglottids resemble sacs full of eggs. tapeworms are hermaphroditic organisms because each mature segment contains 350 to 600 testes and 3 ovary lobes. gravid proglottids are released with feces, starting at 3 - 5 months after infection, and each proglottid contains between 50,000 and 60,000 eggs, located inside the multilobulated uterus. as proglottids are located farther away from the scolex they become bigger ; mature proglottids measure 2.1 to 2.5 mm long and 2.8 to 3.5 mm wide, while gravid segments measure 3.1 to 10 mm long and 3.8 to 8.7 mm wide. they range in size from 26 to 34 m while cysticerci and adult worms are macroscopic and have a similar scolex that measures 0.6 to 1.0 mm. eggs are conformed by the oncoshpere, or hexacanth embryo, and the embryophore that surrounds it. when swine ingest eggs, bile and enzymes disaggregate the embryophoric blocks and digest the oncospheral membrane ; in this way the oncosphere, with the aid of its hooks and enzymes, can cross the mucosa and circulate until it transforms into the larval stage. cysticerci establish primarily in the skeletal and cardiac muscles, as well as in the brain of pigs, a process that takes approximately 3 months. they remain viable for at least 1 year, when pigs are usually sent to slaughter. in humans, the former is a small, spherical or oval, white or yellow, vesicle that measures between 0.5 and 1.5 cm and has a translucent bladder wall, through which the scolex can be seen as a small solid eccentric granule. this type of cysticerci is generally separated from the host tissue by a thin collagenous capsule, within which it remains alive. the racemose cysticercus appears in the human brain either as a large, round, or lobulated bladder circumscribed by a delicate wall, or resembles a cluster of grapes, and measures up to 10 or even 20 cm and may contain 60 ml fluid. interestingly, the scolex can not be seen, and in some cases only detailed histological studies reveal its remains. cellulose type cysticerci are conformed by 2 chambers ; the inner one contains the scolex and the spiral canal and is surrounded by the outer compartment that contains the vesicular fluid, usually less than 0.5 ml. when a person ingests a living cysticercus, the first event that takes place is the widening of the pore of the bladder wall for the scolex and neck to emerge, leaving the bladder wall and vesicular fluid to disintegrate in the digestive tract. the domestic pig is the intermediate host because it harbors the cysticercus ; however, humans can acquire cysticercosis after accidentally ingesting t. solium eggs. cysticerci in humans develop mainly in the central nervous system, eyes, striated and heart muscles, and subcutaneous tissues. in the brain clinical manifestations are pleomorphic, and depend on the location, number, and stages of the lesions as well as characteristics of the immune response of the host., parasites can be palpated when superficial, or seen in x - rays as white dots or nodules, but generally they do not interfere with the muscle function, except in massive infections, when they cause muscle pseudo - hypertrophy with intense inflammation and pain., parasites are found in the subretinal space, close to the macula, vitreous fluid, or anterior chamber. inflammation may cause blindness or decreased vision secondary to proliferative vitreo - retinopathy and retinal detachment. although some books state that tapeworms can survive for about 25 years in the human intestine, recent experience indicates that t. solium remains only for around 1 year releasing along this time a few gravid proglottids in feces 2 - 3 times a week. interestingly, the prevalence of taeniosis among patients with neurocysticercosis (ncc) is higher than previously reported. in addition, a clear association between the presence of taeniosis and the severity of ncc has been reported, since most massive cerebral infections (with more than 100 cysticerci) were present in patients who harbored the adult tapeworm in the intestine. therefore, the perception that tapeworms are silent guests, causing no harm to humans, is erroneous and tapeworm carriers should be regarded as potential sources of contagion to themselves and to those living in their close environment. taenia asiatica differs from taenia saginata and t. solium in 4 aspects ; 1) the scolex has 2 rows of rudimentary hooklets, which is considered as a wart - like formation. 2) the number of the lateral branches of the uterus is different and allows identifying if the proglottids belong to t. solium (7 - 11 branches), t. saginata (14 - 32 branches), or t. asiatica (12 - 26 branches). 3) t. asiatica cysticerci in pigs are not found in muscles, they can be recovered from the liver, omentum, serosa, and lung. between 1940 and 1970, necropsy reports in mexico indicated that around 2% of adults had ncc, and between 50 and 80% of them had no symptoms. in 1972, when i started my ph.d. project, no computed tomography or magnetic resonance existed, and the only diagnostic procedure was using the cerebrospinal fluid, where antibodies by complement fixation and increase of eosinophils by differential count of blood cells, were the only methods to suggest cysticercosis in patients with neurologic symptoms such as convulsive crisis or hydrocephalus. therefore, along the next 20 years, immunoelectrophoresis, enzyme immunoassays, including elisa, and western blot were standardized with the aim of defining the real magnitude of t. solium in mexico by detecting specific anti - cysticercus antibodies in serum [7,9 - 13 ]. immunoelectrophoresis was the first technique used in communities ; 1,610 samples obtained from 9 communities, mostly rural, in the state of chiapas were analyzed. interestingly, in spite of the low sensitivity of immunoelectrophoresis (50%), its high specificity (100% because practically no echinococcosis is found in mexico) allowed to identify a clear correlation. communities with less than 4,000 inhabitants had 1 - 8% seropositivity, while populations with up to 35,000 citizens had 1% or less antibody frequency ; indicating that the parasite was more frequent in small, and thus less developed, towns. afterwards, a study performed in 20,000 samples from a national survey, we were able to identify by immunoelectrophoresis a central area in mexico in which people had between 0.6 and 1% of anti - cysticercus antibodies ; remarkably, this geographic area, called " el bajio " is the most important pig breeding area in mexico for national consumption of pork meat. a few years later in a small field practice for a group of epidemiology students, we identified the main risk factor for acquiring cysticercosis ; the tapeworm carrier, probably my main contribution to science. this was recognized because there were " clusters " in the community (125 inhabitants) where people with convulsive crisis and/or antibodies by elisa and pigs with cysticerci in the tongue lived close to a tapeworm carrier. a survey was performed in a soldier camp in mexico city, blood and fecal samples from 1,000 militaries and their families were analyzed for t. solium serum elisa antibodies, copro - antigen elisa, and coproparasitoscopic detection of eggs. demographic results were gathered in relation to positivity to any assay ; 86.7% of family members of positive soldiers ate in street food stores, as compared to 62.5% of family members of negative soldiers, while having a history of proglottid release was 12% vs 3.7% in the same family groups. probably the study that was better known around the world, but certainly was not the first one, was that with the 4 families of orthodox jews that along 2 years each had a member that had convulsive crisis, which was confirmed at the center for disease control, atlanta, usa as due to ncc. peter schantz to identify the risk factor, because being orthodox they did not want to know anything about " the pig tapeworm ". he found out that they spent the previous vacation together and took with them a maid from mexico, and obtained the data from the maid ; we fetched her in the state of puebla and identified that she had a tapeworm, her brother and son had ncc ; also 7 other asymptomatic members from the jewish families were antibody positive by western blot. thus, 1 tapeworm carrier in the household, who cooked and most probably had inadequate hygienic habits, infected 13 people. starting 1990 we performed studies with the support of the international development research center, ottawa, canada in order to evaluate different intervention measures for control of human cysticercosis ; health education related to the life cycle of t. solium and the risk factors for acquiring cysticercosis, as well as mass treatment with praziquantel against taeniosis [7,20 - 24 ]. a huge field study was established along 2 years in which all the inhabitants of 3 communities (around 3,000 in each) were asked for blood and fecal samples ; in depth sociological interviews were performed in 10% of the people in order to develop the educational intervention. praziquantel (kindly donated by merck mexico) was provided in 2 communities and health education in the other 2, in such a way that 1 community received only the cestocide drug, 1 only the education program, and 1 community both measures. praziquantel was used at 5 mg / kg weight instead of 10 mg / kg in order to avoid neurologic exacerbation if cysticerci were lodged in the brain ; but unfortunately its cestocidal effect was also reduced to 54% instead of the 95% expected. in contrast, health education was so effective that no pigs born after the intervention had cysticerci up to 40 month after this measure was implemented. this formidable effect could be due to the fact that people learned that if their pigs had no access to human feces (in latrines, garbage, or due to free roaming) they would not acquire the disease, and thus could be sold in a better price. marshall lightowlers, and was evaluated in mexico, and later on in peru and cameroon. this is, up to date, the vaccine against any parasite with the highest efficiency ; 100% of immunized pigs did not develop cysticerci after experimental infections with t. solium eggs or in spite of living in highly contaminated environments. another intervention was assessed in a jurisdiction of " el bajio " with more than 750,000 inhabitants, in which doctors in health centers were given a small flask with some t. solium proglottids, and invited to ask the patients that attended for any reason, if they or someone at home released segments as those in the flask. patients were also asked if a member of the household had convulsive crisis or if they had " measly pigs " at home. to those with a positive answer, praziquantel at 10 mg / kg was offered. announcements in walls, radios, newspapers, and short courses were provided in the entire jurisdiction. the year before and at the end of the year in which the intervention took place, annual data were obtained from the official disease notification system at the ministry of health ; 7 cases vs 41 of taeniosis were detected before and after ; also ncc increased from 16 to 68 cases and swine cysticercosis from 2 to 17, because people became aware of the disease. all tapeworm carriers were treated (4 worms were t. solium, the remainings were t. saginata), as expected only 10% of the tapeworms were t. solium. i consider that i am closing my research circle on cysticercosis, since this disease has been controlled in mexico. this reflection is based on the dramatic decrease in the frequency of human ncc and of human taeniosis that has been taking place in mexico in the last 20 years, according to the official national notification system that is collected by the ministry of health. the explanation for this reduction is based on a sequence of events that started with the publications produced along the last 40 years by mexican scientific and medical communities working on cysticercosis. the vast amount of information published most probably promoted the establishment of a national program for the control of t. solium (guidelines for the prevention and control of taeniosis and cysticercosis, nom 021 ssa2 - 1994, 2004) that are obligatory to follow in the whole nation, lead by the ministry of health. finally, in mexico, a general improvement of living conditions has taken place, which has had a positive impact in the reduction of ncc and swine cysticercosis. the last aspect of this review is to focus towards comparing research related to t. solium and t. asiatica. the main concept is to understand the difference between having the disease and being a tapeworm carrier. cruz., in their operational studies on the control of t. solium taeniasis / cysticercosis in ecuador, calculated that expulsion of a tapeworm by at least 148 carriers reduced the exposure to t. solium eggs both for man and pigs in the study areas, and considered that with a relatively low reinfection rate, this human reservoir of infection would remain diminished for several years. in this way, the real disease, ncc, can be controlled. on the other hand, the group of jeon. described that nationwide surveys conducted every 5 years in korea revealed that the positivity rate for taeniid eggs decreased from 1.9% in 1969 to 0.02% in 1997, and human taeniosis was no more detected in the 2004 survey.. also indicated that during the period of 1998 and 2002, a total of 19,894 chinese inhabitants of 3 ethnic minorities in guangxi province, china were surveyed for taenia worms. a total of 927 (4.7%) persons discharged proglottids, and, in 2002, 108 were treated, and 117 adult tapeworms were recovered. using surveys in order to retrieve tapeworms in asian countries suggests that carriers do not attend health institutions for clinical symptoms, indicating that taeniosis does not cause a disease. i personally consider that the 3 species of human tapeworms should be dealt as different concepts. t. solium has to be eliminated from carriers because it is the main risk factor for ncc, a devastating disease ; t. saginata causes minor symptoms due to the discharge of proglottids. the adult parasites of t. asiatica do not cause disease, since they are not associated with symptomatology that requires medical treatment. the 2 most important justifications of my reasoning are : 1) in the last few years, clinical and basic studies are demonstrating with increased numbers of publications and of clinical successes that helminths participate in immunomodulating the intestinal milieu in order to reduce inflammatory bowl diseases (usually referred to as ibd), such as crohn 's disease, that are becoming very prevalent in developed countries around the world [32 - 37 ]. 2) the world 's biodiversity is being threatened because of the huge human development ; there is no need to further reduce it. | three species of tapeworms infect humans in their adult stage (taenia solium, taenia saginata and taenia asiatica). the 3 are flat, opaque white or yellowish, and exceptional long segmented parasites, measuring 1 to 12 m in their adult stage. in this review, the development of the knowledge regarding the first species, mainly focused on understanding how the larval stage or cysticercus is transmitted to humans, is described. the second species is a cosmopolitan parasite that only causes taeniosis and not cysticercosis ; therefore, it will not be included. information on the third species, which is presently being produced, since this species was recognized as such only at the end of the 20th century, will be discussed at the end of this review. |
although fish in natural populations may carry high body burdens of both organic and inorganic mercury, the effects of this divalent metal on such lower vertebrates is poorly understood. in this report, inorganic mercury in the form of mercuric chloride (hgcl2) is shown to produce both high - dose inhibition and low - dose activation of leukocytes in a marine teleost fish, sciaenops ocellatus. concentrations of inorganic mercury > or = 10 microm suppressed dna synthesis and induced rapid influx of radiolabeled calcium, as well as tyrosine phosphorylation of numerous cellular proteins. lower concentrations (0.1 - 1 microm) of hgcl2 that activated cell growth also induced a slow sustained rise in intracellular calcium in cells loaded with the calcium indicator dye fura-2, but did not produce detectable tyrosine phosphorylation of leukocyte proteins. these studies support the possibility that subtoxic doses of hgcl2 may inappropriately activate teleost leukocytes, potentially altering the processes that regulate the magnitude and specificity of the fish immune response to environmental pathogens.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 6.figure 7. |
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in order to achieve the national vision of reaching and sustaining routine immunization coverage of 90% for all vaccines by the year 2020, there was need to involve both the private and public health facilities in providing immunization services. there is paucity of information on the success of private - public partnership in improving free immunization coverage in africa. however, in tuberculosis (tb) management, private - public partnership has been shown to have contributed in detecting more than 25% of tb cases in china, india, nigeria and philistines and in maintaining a high treatment among tb patients. utilization of private health facilities is common in nigeria. in some towns ; the private sector had outgrown the public sector in health care provision. a study in nigeria showed that 78% of people treated their last case of diarrhea and 65% treated their last case of acute respiratory infection outside the public health sector. this preference for private health facilities might be due to proximity of private health facilities to the people, ability to have a flexible payment schedule, constant availability of a medical doctor in private health facilities and poor services in some government hospitals. this preference for health facilities informed the need to bring in the private sector into the immunization program while maintaining the high quality of services provided. the realization of the importance of the private health facilities in meeting the immunization target while maintaining the quality of care led to the signing of a memorandum of understanding (mou) in 2009 between the abia state ministry of health (moh) and private health facilities. the mou sought to formally involve private health facilities in the provision of free immunization services in the state. the collaboration involves : i) supply of free vaccines to private health facilities ; ii) provision of cold chain equipment to ensure vaccines are potent ; iii) provision of data tools e.g. tally sheets, registers etc. ; and iv) capacity building for the private service providers. on their part, the private health facilities are to : i) provide the vaccination free to clients ; ii) record immunization data and render monthly data returns as required ; iii) collect vaccines from local government areas (lgas) cold store and account for all vaccines and materials collected ; iv) provide weekly scheduled routine immunization services, and v) allow access to moh officers or its agents to enter their facilities for purposes of supervision, surveillance, monitoring and evaluation. the private - public partnership was open to all private health facilities in abia state, but is mainly functional in four local governments areas of aba south, aba north, osisioma and umuahia north. these four local government areas are urban / semi - urban lgas and have many private health facilities operating within them. this private - public partnership (ppp) is unique because the private facilities provide the services free without being paid any money by government or development partners. our literature search did not reveal any documented evidence of this type of partnership in any other part of africa. this study seeks to assess the contribution of the private health facilities in providing immunization services in these four local government areas. this is a retrospective study of data submitted monthly by the local government immunization officers to the primary health department of abia state ministry of health between january 1, 2011 and december 31, 2011. the local government immunization officers collect vaccines from the state cold store and store them in the local government cold stores. however, some large tertiary hospitals collect their vaccines directly from the state cold store. the health facilities supply data monthly to the local government immunization officers (lios) on vaccines and consumables (syringes, injection water) used. the data from the local government immunization officers contains the names of the different health facilities, the status of the health facility (whether private or public), and the number of children that received the different vaccines in each of the health facilities. the data also contains the number of children that received different doses of each of the vaccines in each health facility. for instance, for diphtheria - pertussis - tetanus (dpt) vaccine, the number of children that received the first dose, the second dose and the third dose of dpt vaccine are all indicated in separate columns. we used the third dose of dpt (dpt3) which is usually given at 14 weeks to measure the routine immunization coverage in the local government areas because unlike some other vaccines, dpt is not given during supplementary immunization activities and is a more accurate indicator of vaccines given through routine immunization. the estimated local government population is based on the 2006 population census with an annual growth rate of 2.7%. the estimated monthly birth cohort is the estimated yearly number of births divided by 12. abia state, like other states in nigeria, operates the expanded program on immunization with five prescribed visits to receive one dose of bacille calmette guerin, four doses of oral polio virus vaccine, three doses of dpt, three doses of hepatitis b vaccine, one dose of measles and yellow fever vaccines. the result in table 1 showed that in aba north, 74% (28/38) of the health facilities that offered immunization services in 2011 were private health facilities and 26% (10/38) were public health facilities. however, 40% of the immunization services took place in private health facilities while 60% took place in public health facilities. in umuahia north, 20% (10/50) of the health facilities that offered immunization services in 2011 a percentage of 13% of the immunization services took place in private health facilities while 87% took place in public health facilities (table 1). furthermore, in aba south, 53% (26/49) of the health facilities that offered immunization services in 2011 were private health facilities and 47% (23/49) were public health facilities. however, 24% of the immunization services took place in private health facilities while 76% took place in public health facilities (table 1). in osisioma, 39% (15/38) of the health facilities that offered immunization services in 2011 however, 15% of the immunization services took place in private health facilities while 85% took place in public health facilities (table 1). in summary, in the four local governments, 45% (79/175) of the health facilities that offered immunization services in 2011 were private health facilities and 55% (96/175) were public health facilities. however, 21% of the immunization services took place in private health facilities while 79% took place in public health facilities. the ppp has been beneficial in providing immunization services to most parts of these four local government areas involved in the ppp. forty five percent of the health facilities offering immunization in these four local government areas are private facilities. this has helped to get the health facilities closer to the people and has also helped to provide improved access to immunization for families that prefer patronizing private health facilities. distance from health facilities has been shown as a barrier to accessing routine immunization by caregivers. although 45% of the health facilities offering immunization are private facilities, only 21% of the immunization services occur in these facilities. this is compared to 22% of immunization delivered by non - profit private sector facilities in urban areas in bangladesh. in ghana, it is estimated that 40% of total immunization are delivered by non - profit civil society organizations and mission hospitals through their outreaches and community based outreaches. the large proportion of immunization in public health facilities in abia state could be due to the fact that some of the public health facilities are large general hospitals, and teaching hospitals that have large clientele and serve as referral hospitals. in addition, many people might not yet be aware that some nearby private health facilities provide immunization services and so still travel far to public health facilities to immunize their children. furthermore, public health facilities carry out immunization outreach activities to neighboring communities and immunize children at their homes and communities and this increases the number of people that are immunized by the public health facilities unlike most of the private health facilities that only immunize at their facilities. in 2010, the mean dpt3 coverage was 95% in the 4 ppp lgas and 59% in the other 13 lgas (target 80%). this discrepancy in immunization coverage between ppp local governments and other local government areas might be due to the fact that health facilities offering immunization services might be more easily accessible to people in ppp local governments. contracting services to non - profit non - governmental organizations has also been showed to improve access to immunization and other primary health care services in bangladesh, cambodia, senegal etc. however, unlike the case of abia state, these non - profit organizations were paid to carry out these services. the data also showed wide variations in the proportion of facilities in each of the local government areas that offered immunization services which were privately owned. while 74% and 53% of facilities offering immunization in aba north and aba south local government areas respectively were privately owned, only 20% were privately owned in umuahia north. this could be explained by the fact that aba north and aba south are commercial cities with a lot of private facilities and few public health facilities while umuahia is the state capital with a lot of public health facilities. one limitation of this public - private partnership is that all the local government areas actively involved in this partnership are urban or semi - urban. thus the findings from this study can not be generalized to rural local government areas which usually have fewer privately owned health facilities. private health facilities have been shown to have a modest contribution to immunization in the four local governments practicing the ppp. in addition, they have also helped to make routine immunization accessible in most parts of the local governments involved in the ppp. efforts should be made to expand this ppp in immunization nationally to improve immunization services in nigeria.. however, there is need to ensure that the potency of vaccines are maintained by an effective monitoring of the cold chain system and that staff at the private health facilities are regularly trained. this is because private health facilities have been shown to lack knowledge on immunization schedule, waste and vaccine management and lack correct cold chain equipment. | the vision of nigeria s immunization program is to reach and sustain routine immunization coverage of greater than 90% for all vaccines by 2020. in order to achieve this, abia state embarked on a unique private - public partnership (ppp) between private health facilities and the abia state ministry of health. the aim of this partnership was to collaborate with private health facilities to provide free childhood immunization services in the state - the first of its kind in nigeria. this is a retrospective study of the 2011 abia state, nigeria monthly immunization data. in the 4 local governments operating the ppp, 45% (79/175) of the health facilities that offered immunization services in 2011 were private health facilities and 55% (96/175) were public health facilities. however, 21% of the immunization services took place in private health facilities while 79% took place in public health facilities.private health facilities were shown to have a modest contribution to immunization in the 4 local governments involved in the ppp. efforts should be made to expand ppp in immunization nationally to improve immunization services in nigeria. |
the use of light - activated composite resins has increased considerably because they provide better esthetics and an opportunity to restore extensive restorations. the structure of resin matrices of polymerized composite resins comprises bisphenolglycidyl dimethacrylate (bis - gma), triethylene glycol dimethacrylate (teg - dma) or urethane dimethacrylate (udma). in the polymerization of resin - based composites, shrinkage occurs as a result of the change from carbon single to double bonds. this event, called polymerization shrinkage, is the largest cause of stress on the cavity walls, separating the composite material from the cavity walls. separation of the composite material from the cavity walls reportedly causes microleakage at the margins of the restoration and secondary caries, post - operative sensitivity and dental pulp pathology. to overcome the problems associated with polymerization shrinkage, early attempts focused on the type and amount of the particles included in composite resins and on different applications to particle surfaces. later studies focused on the relationship between the polymerization shrinkage and the monomers composing the organic matrix of composite resins. for this reason, the 3m - espe company developed the silorane matrix system, which differs from methacrylate - based monomers and released the first composite filler material in which this matrix system was used : filtek silorane. siloxane increases the hydrophobic features of the composite and oxirane lowers the level of polymerization shrinkage compared with methacrylate - based composites. in addition, a polymerization reaction peculiar to this composite occurs on the monomer level. the manufacturer has announced that this composite can be used easily on posterior class i and ii restorations. polymerization of light - activated composite resins starts at the surface, where light is applied. for this reason, sufficient polymerization can not be provided toward the deepest parts of the restoration when using bulk technique. insufficient polymerization may result in sensitivities, discoloration and the formation of marginal gaps in the restoration. the degree of polymerization of resin - based restorative materials can be analyzed directly or indirectly using the different techniques. direct methods such as laser raman spectroscopy and infrared spectroscopy are complicated, expensive and time - consuming. surface hardness is an accepted indicator of the polymerization degree and has been used in many studies. it is simpler than other techniques and shows correlations with data gathered from the infrared spectroscopy method. surface hardness of composite resins can be affected by factors such as the density and application time of the curing light as well as the structure, thickness and color of the material. ideally, surface hardness of the composite resin should be equal or close to equal throughout the restoration and for the life of the restoration. many studies have analyzed the surface hardness of methacrylate - based composite resins with various techniques. the aim of this study was to evaluate the change in surface hardness of silorane- and composite resin - based restorative materials at different time intervals and compare the findings with the surface hardness of two different methacrylate - based composite resins. the composite resins used in the study to evaluate the surface hardness of each composite material, 18 cylindrically shaped samples were prepared using 5 mm diameter and 2 mm deep teflon molds. during the sample preparation, the teflon molds were positioned over an acetate strip on a glass plaque. after composite resin insertion, a second acetate strip was placed on top of the mold with slight pressure to remove excess material from the mold. the composite materials were then light - cured with a light - emitting diode (led) device (1000 mw / cm) (hilux, benliolu, turkey) for 40 s according to the manufacturer 's instructions and the acetate strips were removed. the top and bottom hardness of the samples was measured using vicker 's hardness tester (hmv - ii ; shimadzu, japan). a 100-g load was applied through the indenter with a dwell time of 15 s. measurements was performed 3 times for each sample at intervals of 0.5 mm. samples were stored at 37c and 100% humidity for 24 h and 7, 30 and 90 days. afterward, by dividing the bottom surface hardness value by that of the top, the hardness ratio of the material was calculated. statistical analysis was performed using the one - way analysis of variance, multiple comparisons were conducted using tukey 's test and binary comparisons were made by t - test at a significance level of p = 0.05. mean top and bottom surface hardness values of the composite resins are given in [tables 24 ] and the change in surface hardness values over time is shown in figure 1. time - dependent change in the surface hardness of filtek silorane samples time - dependent change in the surface hardness of filtek supreme samples time - dependent change in the surface hardness of majesty posterior samples the change in surface hardness values over time when the surface hardness values of the three composites were compared, only the silorane - based restorative material (filtek silorane) showed statistically significant differences (p 0.05). when the difference based on time of the bottom surface hardness of the samples was analyzed, all immediate hardness values of the tested materials showed a significant increase after 24 h (p 0.05). one of the most important factors affecting the clinical success of nanofiller composite resins, such as microfiller and hybrid composite resins is the materials polymerization throughout the restoration. adequate polymerization is important in terms of the ideal physical and mechanical properties. as a result of inadequate polymerization, possible microleakage at the margins of the restoration, discoloration, increased erosion, decreased mechanical strength, increased water absorption and decreased bonding strength have been reported. in addition, the residual monomers released due to inadequate polymerization may pass through dentin tubules and cause irreversible damage. measurement of surface hardness is an indirect method of evaluating the polymerization degree of composite resin. researchers have used different hardness measurement tests, such as those of vicker, barcoll and knoop. the diamond indenter used in the procedure does not deform over time and is reportedly suitable for measurement of the hardness of fragile materials and dental tissue. in the present study, the surface hardness of two methacrylate - based composite resins and one silorane - based composite was compared using vicker 's hardness measurement device. the hardness of composite resin is reportedly affected by the color and depth of the composite material used as well as by the light device, period of light application and distance between the light tip and composite resin surface. for this reason, the thickness of the samples was standardized to 2 mm in the present study. to avoid the effects of color on hardness, all three composites used in the study all samples were polymerized using the same led light device for 40 s, the light was implemented through a glass slab and the distance between the light device and the sample was anchored. on both the top and bottom surfaces in all measurement periods, the highest hardness value was obtained in the samples of the methacrylate - based nanofiller composite majesty posterior and the lowest was obtained in the samples of the silorane - based composite filtek silorane. compared the top and bottom surface hardness of methacrylate- and silorane - based composite resins and found that the bottom surface value of the silorane - based composite resin was statistically significantly higher. there was a statistically significant difference among the hardness values of the composite resins used in this study. the reason for this difference is that the monomer types, filler types and filler volume and polymerization mechanism of the composite resins used in the study differed. in methacrylate - based composites, proximity of monomers react to establish a covalent bond in the polymerization process. on the other hand, the kinetics of the initiation and polymerization begin with cleavage and opening of the ring systems through a cationic ring opening reaction. the resin matrix of one of the methacrylate - based composite resins, majesty posterior, comprises bis - gma and tegdma. filtek supreme, on the other hand, comprises bis - gma, tegdma, bis - ema and udma. the filler ratio of these materials is 82% for majesty posterior and 59.5% for filtek supreme volumetrically. the size of the filler particles is similar between the two. in the silorane - based composite filtek silorane, which showed the lowest hardness degree, this type of monomer comprises siloxane and oxirane monomers in contrast to methacrylate - based composite resins. in addition, polymerization of this composite resin is quite different from the polymerization reaction of methacrylate - based composite resins. the reaction of this chemical with tertiary amines generates free radicals. in silorane - based composites, three main photoinitiators were used. these were camphorquinone, an iodonium salt and an electron donor. camphorquinone was used because it is consistent with the emission spectra of the light used in the light devices. the electron donor, on the other hand, is come down to resolve the iodonium salt in an acidic cation and a split ring polymerization reaction is thus initiated. there were no significant differences between the top and bottom surfaces of the two methacrylate - based composites, whereas there was a significant difference between the top and bottom surface hardness of the silorane - based composite filtek silorane. previous studies showed that the difference in hardness between the top and bottom surfaces of the composite resins was caused by inadequate light reaching the deep parts of the composite material. in addition, while some of the light was absorbed by the composite material, some of it was shed. this situation prevents the light from adequately penetrating the deeper parts of the composite material. the top and bottom surface hardness values gathered from in vitro studies do not always indicate inadequate polymerization. for this reason, it is important to define a hardness ratio that states the ratio between the top and bottom hardness values in such studies. this suggests that the led light curing was adequate for polymerization of the 2 mm thick methacrylate and silorane - based composite samples. in all three composites resins used in this study, the top and bottom surface hardness values differed significantly in the 1 24 h. this is an indication that the polymerization reaction continues in the 1 24 h. previous studies have shown that microhardness values of composite resins are not constant and increase with time. researchers suppose that unreacted free - radicals in the structure lead this event by continuing to generate cross - links after light application. according to the day 7 data, the hardness values of all three composite resins decreased, followed by a relative increase. however, polymer - structured composite materials tend to absorb water and dissolve there in. studies have shown a correlation between the water absorption and dissolving characteristics in water of composite resins. pereira. analyzed the physicochemical features of restorative materials including different fillers and stated that when the filler amount increased, composite materials tended to absorb less water and exhibited lower solubility. the filler amount of the silorane - based composite resin filtek silorane was lower than that of the other two methacrylate - based composites. stated that the water absorption and water solubility of silorane - based composite resins were greater than those of the methacrylate - based composites. this might be why the silorane - based composite filtek silorane exhibited lower top and bottom surface hardness values and a lower hardening rate. although silorane - based composite resin filtek silorane showed adequate hardness ratio, the use of incremental technic during application is more important than methacrylate based composites. because both the top and bottom surface hardness values of the silorane - based composite resin filtek silorane were lower than those of the methacrylate - based composite resins and there was a significant difference between the top and bottom surface hardness values of the silorane - based composite resin filtek silorane. | objective : the aim of this study was to evaluate the change in surface hardness of silorane - based composite resin (filtek silorane) in time and compare the results with the surface hardness of two methacrylate - based resins (filtek supreme and majesty posterior).materials and methods : from each composite material, 18 wheel - shaped samples (5-mm diameter and 2-mm depth) were prepared. top and bottom surface hardness of these samples was measured using a vicker 's hardness tester. the samples were then stored at 37c and 100% humidity. after 24 h and 7, 30 and 90 days, the top and bottom surface hardness of the samples was measured. in each measurement, the rate between the hardness of the top and bottom surfaces were recorded as the hardness rate. statistical analysis was performed by one - way analysis of variance, multiple comparisons by tukey 's test and binary comparisons by t - test with a significance level of p = 0.05.results:the highest hardness values were obtained from each two surfaces of majesty posterior and the lowest from filtek silorane. both the top and bottom surface hardness of the methacrylate based composite resins was high and there was a statistically significant difference between the top and bottom hardness values of only the silorane - based composite, filtek silorane (p < 0.05). the lowest was obtained with filtek silorane. the hardness values of all test groups increased after 24 h (p < 0.05).conclusion : although silorane - based composite resin filtek silorane showed adequate hardness ratio, the use of incremental technic during application is more important than methacrylate based composites. |
currently, diabetes mellitus (dm) is more prevalent than any other hereditary metabolic diseases. it is a chronic disorder of carbohydrate, fat, and protein metabolism caused by lower amounts or absence of insulin. it can lead to several complications such as blindness, cardiac arrest, kidney failure, etc. according to the statistics issued by the world health organization (who), the prevalence of dm was 171 million, in 2000 and 285 million in 2010. the prevalence is likely to rise by more than two - third between 2010 and 2030. haemoglobin a1c (hba1c) plays a significant role in dm. the hba1c test or glycosylated hba1c test is a laboratory test that reveals the average blood glucose over a period of the previous two to three months (long - term control test). it helps assess whether patients have had optimal glycemic control and the control status between checkups. hba1c can, therefore, provide a reliable reflection of long - term blood glucose control because its value is not affected by brief or infrequent fluctuations in blood glucose levels affecting the viscosity of blood. generally, three techniques are in practice for the early detection of dm ; invasive, minimally invasive, and non - invasive. the first two methods have certain limitations such as patients preparation, reagent preparation, piercing the skin that can cause infection, need to sophisticated instruments, and skilled technicians. optical techniques come under different categories of non - invasive methods. among them, scattering changes are adopted. these scattering changes are of two types, namely spatially resolved diffuse reflectance and optical coherence tomography. out of the two, the spatially resolved diffuse method is adopted in our study. this principle also applies to light and other electromagnetic radiations. here, a laser beam is focused on the moving objects like the rbc or stationary particles like tissue structures in the skin. the shift in the frequency of the back scattered light gives a measure of the velocity. since the vessel diameter is not known because of its elasticity, the flow rate can not be measured. developing laser system using the he - ne laser system, photo detectors (as optical detectors) are arranged in an axial fashion at 19 different angles for the detection of transilluminated and scattered laser beam from the index finger. the index finger is placed in the sensing arrangement and each detector is capable of detecting the optical signal up to 10 away from the point of observation. for the surface measurement, a laser beam directed by a single optical fiber is transmitted through the index finger. the transmitting and receiving fibers are positioned in parallel with their centers separated by 1 mm and encased in epoxy resin with black painted mica housing to maintain this separation. the scattered beam is then detected by the photo detector and is stored in a personal computer through digital storage oscilloscope. these functions divide the signal information into different frequency components, without any modification of signal shape, amplitude, and frequency components. wavelets are used in the fields of electrical engineering, mathematics, medical science, quantum physics, and seismic geology. haar wavelet structure the wavelet transform can be implemented by a two channel perfect reconstruction (pr) filter bank. a filter bank is a set of filters, which are connected by sampling operators. figure 1 shows an example of a two - channel filter bank applied to a one dimensional signal. in general, figure 1 is a two channel wavelet structure which consists of an analysis and synthesis bank. two channel wavelet structure d(n) is an input signal. in the analysis bank, b0(n) is a low pass filter while b1(n) is a high pass filter. in the synthesis bank, a0(n) is the reconstruction low pass filter (lpf) and a1(n) is the reconstruction high pass filter (hpf). in this study, only the analysis bank was required and it was modified as shown in figure 2. modified haar wavelet analysis bank shifting the down sampler to the input causes reduction in the computational complexity by a factor 2. with this method, computational complexity as well as the time required for computation is reduced by a quarter of the original structure. the data for the research were obtained from 900 individuals. among them, 750 patients had dm and were undergoing treatment in government and private hospitals during 2007 - 2011, and the remaining were patients from the same hospital without dm. the data contained information on the patients personal details as well as their medical profile such as pathology, biochemistry, and lipid profile along with the corresponding developed system sensor outputs. the blood samples were collected twice the time of examining the patients in fasting and post parandial conditions. the experiments were carried out on the index finger of the patients at a room temperature of 325c. in each case, the arm was strapped in the horizontal position (at heart level) by a special arrangement to minimize any kind of movement. the probe was not in direct contact with the skin, in order to enhance the air flow and to prevent humidity condensation which could possibly modify the skin s optical properties and microcirculations. once the probe is applied, the power spectra were recorded to detect any difference in frequency and amplitude response. the measurements were repeated at least twice for each patient in order to check the reproducibility of the method. preparing data to implement neural network techniques the decomposed outputs are classified into several groups as follows : group i : 0 - 150 mg / dl, group ii : 151 - 250 mg / dl, group iii : 251 - 350 mg / dl, group iv : 351 - 450 mg / dl, and group v : 451 mg / dl. the prediction of blood glucose concentration was done using back propagation network (bpn) with gradient descent algorithm and radial basis function (rbf), with extreme learning machine. proving the validation using six sigma concept a statistical analysis chart for continuous real time process of human blood flow is used for the verification. the feasibility of the laser system technique in measuring peripheral blood glucose concentration has been reported in this present investigation. table 1 displays the statistical details of the clinical baseline characteristics of the sampled patients. mean (sd) of the clinical baseline characteristics of the patients with dm this study predicts the blood glucose concentration in the peripheral blood by using developed laser system. as demonstrated in table 2, the transillumination profiles of some subjects were taken randomly and verified with the help of coefficient of variation (cv) between the blood glucose concentration and transilluminated voltage. the cv is the ratio of the standard deviation and the mean, computed to measure the precision for the dispersion of data sets on ratio scale. coefficient of variations in transilluminated voltages in relation to the corresponding blood glucose concentration because of the anisotropy factor, the maximum scattered signal was obtained at 29 and 337 from the index finger. the scattered signals obtained are decomposed by the modified haar wavelet transform into approximation and detailed coefficient with an error rate ranging between the classical haar wavelet method and proposed as -140 db and -200 db to -260 db, respectively (figure 3). results of error rate compared with existing and proposed modified haar wavelet transform table 3 gives the prediction of blood glucose for different groups using bpn and rbf networks expressed as meansstandard error. in figure 3, the legends, + shows the approximation error and the legends, indicates the detailed error. the prediction of blood glucose concentration for different groups using bpn and rbf networks displayed as in meanstandard error with the values in mg / dl as displayed in figure 3, the notation + depicts approximation error and - shows the detail error. by trial and error process the data of 450 patients were randomly used for training, 225 for testing, and the remaining 225 for validation. these parameters render good predictive capabilities of possible relationships between dependent and independent variables. a glimpse of the foregoing tabulated data shows that the outputs from rbf radial basis function with extreme learning machine algorithm, are nearer to their clinical values than bpn, outputs. the significant variations can be seen from signals obtained from patients with and without dm. they are compared using six sigma statistical analysis chart for 200 ms (figure 4). blood flow variation chart in patients with and without dm the signals received from the patients without dm reach the centre limit line approximately at regular intervals. however, in patients with dm, the signal variations are large. it reveals that the distributions of the blood particles are not uniform in patients with dm. we showed that with the proposed non - invasive blood glucose monitoring system, the optical signals are transmitted to the index finger. the scattered signals were collected from the stratum corneum, dermis, epidermis layers, subcutaneous tissue, interstitial fluid and blood vessels in both the arterial and venous blood. using the continuous modified haar wavelet transform, then the back propagation neural network, with gradient descent algorithm and radial basis function with extreme learning machine algorithm were implemented to predict the blood glucose classification and concentration. the method presented here, shows the average efficiency of the architectures by testing the real time signal data sets obtained through indigenous laser based developed system, from the human skin and capillaries of the index finger. it provides the information about the determination of blood glucose concentration by back propagation neural network and radial basis function architectures. comparative results of other articles venkatesan and anita (2006) discussed the use of radial basis function (rbf) as a hidden layer in a supervised feed forward network. the performance was compared with the most commonly used multilayer perceptron network and classical logistic regression. they used diabetes database for empirical calculations and the results showed that rbf performed better than the other models. the correction prediction percentage was found to be 97 to 98% and it was improved to 99.24 to 99.80% in this research work by using dynamic rbf neural networks. jayalakshmi and santhakumar (2010) mooted a method that was implemented the improved form of gradient descent back propagation algorithm. this has been done to increase the accuracy of the network, and by missing data replacement, data preprocessing and introducing the performance vector (pv). it has been proved that the new method improved the system performance by more than 7%. this method is not a real time analysis but done offline on existing pima indian diabetes dataset. as depicted in table 4, venkatesan and anita using rbf algorithm determined the blood glucose concentration in the measure of average testing efficiency as 98.0% and jayalakshmi and santhakumaran determined it as 99.73% using bpn algorithm, and with the proposed method the average testing efficiency were found to be 99.136% by bpn and 99.53% using rbf algorithms. the management of dm is mainly based on the continuous analysis of blood glucose level. our results showed that the proposed non - invasive optical glucose monitoring system is able to predict the glucose concentration. the experimental outputs proved that there was a definite variation in the hematological distribution between the patients with and without dm. as the continuous monitoring of blood glucose concentration is important for the management of dm, this study proves to be clinically relevant and useful. | background : early and non - invasive determination of blood glucose level is of great importance. we aimed to present a new technique to accurately infer the blood glucose concentration in peripheral blood flow using non - invasive optical monitoring system. methods : the data for the research were obtained from 900 individuals. of them, 750 people had diabetes mellitus (dm). the system was designed using a helium neon laser source of 632.8 nm wavelength with 5mw power, photo detectors and digital storage oscilloscope. the laser beam was directed through a single optical fiber to the index finger and the scattered beams were collected by the photo detectors placed circumferentially to the transmitting fiber. the received signals were filtered using band pass filter and finally sent to a digital storage oscilloscope. these signals were then decomposed into approximation and detail coefficients using modified haar wavelet transform. back propagation neural and radial basis functions were employed for the prediction of blood glucose concentration. results : the data of 450 patients were randomly used for training, 225 for testing and the rest for validation. the data showed that outputs from radial basis function were nearer to the clinical value. significant variations could be seen from signals obtained from patients with dm and those without dm. conclusion : the proposed non - invasive optical glucose monitoring system is able to predict the glucose concentration by proving that there is a definite variation in hematological distribution between patients with dm and those without dm. |
due to its simplicity and convenience, acupuncture has become popular as a complementary therapy. in this chinese medicine, doctors have to find the traditional meridian acupuncture points before the needle is punctured into those points. because of the significant remedial effects, acupuncture has drawn a lot of attention from the western world over the past 30 years. around 2002, there were 42 states in the united states that had established statutory licensure governing more than 14,000 acupuncture practitioners. in the past decade, acupuncture had been widely practiced to treat various diseases. besides, moxibustion, which is an important auxiliary part of acupuncture, may enhance the effect of curing disease by conducting heat to certain points or areas on the surface of the body through regulation of the function of meridians and visceral organs. when performing direct moxibustion, moxa sticks are burnt at acupuncture points on the skin. on the contrary, in indirect moxibustion, the moxa cone (i zh) does not touch the skin and is burnt insulated, by some substance, against the skin. in traditional chinese medicine, moxibustion is usually used for the patients with cold pattern such as rheumatic arthritis, joint pain, diarrhea, or cold numbness limbs. warm - needling acupuncture (wna ; wn zhn ji) is classified under the category of indirect moxibustion. when practicing wna, the moxa cone is firstly put on the tail of the needle, and is then lighted up in such a way that the heat of moxa will be transmitted through the punctured needle to the correspondent acupuncture point. however, in some unexpected situations, practitioners might get burns while removing the needles after treatment. in order to prevent the practitioners from getting burns, it is necessary to study the temperature changes in some designated parts of the needles. we conducted an experiment to record the temperatures in the designated portion of the needles at some predetermined time intervals. the extreme temperatures are outlined, and 95% confidence interval of the average temperatures in different portions at different time intervals is determined. according to the outcome of the statistical analysis, we may propose a warning suggestion for the practitioners conducting wna. in section 2, the method of this study is described. in the experiment, two groups with 10 stainless steel needles (an chi handy acupuncture needle, iso 13485) and 10 pieces of cylinder - shaped moxa [figure 1 ] were used. in the first group, each moxa used in wna had a weight of 0.6 g, and in the second group each moxa was enlarged with a weight of 1.0 g. the main ingredient of the moxa is dried mugwort (artemisia argyi). the size, shape, and weight of the moxa () were designed according to the traditional literature ; for example, the base of 0.6 g moxa had a diameter of 10 mm and the height of the moxa was 10 mm. the 1.0 g moxa had a diameter of 13 mm and the height of the moxa was 15 mm. the length of the needles was 1.5 inch 32 gauge, and the needles were divided into two parts the handle and the needle body. a standard moxa for warm acupuncture the needles were punctured into polystyrene plastics in good upright positions. the handles of the needles were surrounded by the moxa cylinders in the middle part [figure 2 ]. there were 40 moxa cones used for each group. in order to estimate the mean and extreme temperatures in different parts of the needles, we measured for each needle the temperatures of the upper end of the handle, the lower end of the handle, the needle body above the polystyrene plastics, and the tip of the needle body, respectively. the distance between upper end of the handle and the moxa cone was 1.5 cm, between lower end of the handle and the moxa cone was also 1.5 cm, at the needle body above the polystyrene plastics was 2.0 cm, and between the tip of the needle body and the polystyrene plastics was also 2.0 cm. the temperature and moisture of the laboratory were controlled to be stable. after lighting up the figures and tables given in appendix illustrate the changes in temperature recorded every 30 seconds in each of the four designated parts of a needle. the experimental results show the hazardous time and positions when conducting a warm acupuncture (wn zhn). the extreme (maximal) temperature at each recording time is especially important as it might cause danger. eight groups of warming needles from tables a1 and a2, it seems that the highest temperature occurs around 430 or 530 from ignition. the two tables summarize the mean temperatures of the warm needles using 0.6 g moxa. in particular, table a2 shows the highest temperatures at the designated parts of the needles. in group 1a (temperatures taken at the upper end of the handle), the highest temperature may reach 344c and it still remains 45c at 9 min 30 seconds. in group 2a (temperatures taken at the lower end of the handle), note that in group 2a, the highest temperature may reach 320c at 5 min 30 seconds. in group 3a (temperatures taken at the needle body above the polystyrene plastic), the temperatures are lower than those in groups 1a and 2a. tables a3 and a4 show the temperatures of the warming needles using 1 g moxa. the highest temperature (375c) in group 1b is 31c higher than that (344c) in group 1a. because the moxa used in group b is more than in group a, the required cooling time also gets prolonged. it takes almost 15 min for the needles to cool down to the temperature before ignition. the time series plots of the temperatures recorded in group a are shown in figures a1a to a4a, while those in group b are illustrated in figures a1b to a4b. if the effects of energy transmission were via the needles, the bodies of the needles should not have lower temperatures as suggested by the present study. a possible explanation for the remedial effect of wna may be due to radiation or convection. while some researchers suggested that the therapeutic effect of moxibustion is correlated with the temperature, some others have shown that infrared irradiation might have the same effect. there are also some studies suggesting that indirect heating may be a possible effect of moxa. lin suggested that the effect of moxibustion is highly associated with thermal as well as the pharmacological reaction of the materials used. the treatment effects of moxibustion are not only induced by heat but also by its chemical function. also, a study shows the safety of ph level, heavy metals, and uv absorbance spectrum on the cytotoxicity and hemolysis of the needle. the radical scavenging effect of moxa or moxa - tar is measured by a chemical reaction with 1,1-diphenyl-2-picrylhydrazyl. the inhibitor effects of moxa or moxa - tar on superoxide production are caused by the radical scavenging mechanism. as the above references suggest, the chemical ingredients of moxa play an important role in the therapeutic effect. in the present study, the main difficulties arose when we measured the temperatures of four designated parts of a warming needle. there are some studies observing the temperature changes in the human body during the burning of moxa ball. but they did not check needle tip temperature. since it is impossible to measure the temperatures of the needles punctured into patients, we designed an experiment to mimic the real situations. in tcm, wna is an efficient way for the patients with cold pattern. traditional chinese physicians believed that wna can transmit heat to the meridian acupuncture points of the patients. however, when the physicians remove the needles from the patients, they might suffer burns. so far, there is no related study on the possible hazardous time when conducting wna. for the sake of medical safety, it is necessary to investigate the proper time at which the needles can be removed from the patients. when conducting acupuncture, the handle of the needles is frequently touched. in the present study, no matter which moxa we used, the most hazardous part of a warming needle is around the handle. fortunately, when conducting wna, the other parts of a needle do not cause burns. when the weight of a moxa increases from 0.6 to 1 g, the temperature of the warming needle also increases and the required cooling time also gets prolonged. we suggest that a safer timing for a tcm physician to remove the needles should not be earlier than 930 (resp. 1330) for a 0.6 g moxa (resp. 1 g moxa) as the heat source. since mugwort in this study has been made of the moxa cone, the study investigates the routine use of moxa cone and the burning time, in order to avoid the practitioner suffering from burns. future studies could focus on the influence of the production process of a. argyi leaves. | due to its simplicity and convenience, acupuncture has become popular as a complementary therapy. in this chinese medicine, doctors have to find the traditional meridian acupuncture points before puncturing the needles into them. moxibustion (i ji) is also an important part of the acupuncture remedy. treatment by acupuncture can be classified roughly into two types direct moxibustion and indirect moxibustion. warm - needling acupuncture (wn zhn ji) is classified under the method of indirect moxibustion. in the present study, 10 standard stainless steel acupuncture needles with 10 pieces of cylinder - shaped moxa cone (i zh) as the heat source of warm needles were used. in order to prevent the practitioners from getting burns, it is necessary to study the temperature changes in some designated parts of the needles. two sizes, 0.6 g and 1.0 g, of moxa cones were used for comparison of the measured temperatures. the needles are typically divided into two parts the handle part and the needle body. in our experiment, the temperatures of wna at different parts of the needles were measured. the larger the size of moxa cone is, the longer is the burning time. based on the observations we suggest that when 0.6 g moxa is used, the physicians should better pick out the needles around 9 min after ignition ; however, while using the 1 g moxa, it might be safer to pick out the needles around 13 min after ignition. |
tobacco use and its health consequences is one of the most serious public health problems worldwide. evidence is accumulating that smoking increases the risk of cancers and cardiovascular and respiratory diseases. despite proven negative health effects of smoking, it is becoming more prevalent, particularly among adolescents, in the last few decades. most researches concerned with adult smoking have reported that the majority of smokers begin to smoke early in the adolescent period, before the age of 18 years. adolescence is a critical period characterized by psychological and behavioral changes that may affect adolescents smoking behavior. this makes school years a crucial period to study not only the smoking prevalence and predictors but also the beliefs and attitudes of adolescents toward smoking during this period. the highest reported prevalence rates of smoking in saudi adolescents were 37% in jeddah, and 31% and 29% in riyadh, riyadh province, saudi arabia. the most important predictors associated with the risk of adolescent smoking revealed in those studies were parental and friends smoking behavior, teachers smoking, academic achievement, school level, age, and sex of adolescents. with the exception of one study, most published reports on the saudi population have failed to clearly address the role of the beliefs and attitudes toward smoking as risk factors for adolescent smoking behavior. investigating and knowing the adolescent beliefs and attitudes about smoking may aid in the reduction of smoking among them and in the design of appropriate and effective smoking prevention and control programs targeting this important segment of the population. the aim of this study was to identify the beliefs and attitudes toward smoking among smoker and nonsmoker male and female adolescents, and to investigate the association between belief- and attitude - related factors and the risk of adolescent smoking in madinah city, al madinah region, saudi arabia. this school - based, cross - sectional study analyzed data from 3,322 students from intermediate and secondary schools in madinah city, al madinah region, saudi arabia during the 1st semester (september to january) of the academic year 2013/2014. a multistage, stratified cluster sampling procedure was employed, in which schools in madinah city, al madinah region, saudi arabia were defined in strata according to their level (intermediate vs secondary), status (public vs private), and students sex (male vs female) with the final sample proportional to the size of the stratum. within each stratum, a cluster sampling was implemented in which the primary sampling unit was the school. finally, within each selected school, one class from each grade the primary calculated sample of this study was 780 students based on the average of the estimated smoking prevalence among school students in previous saudi studies (20 - 30%), and a precision of 0.03 and confidence interval (ci) of 95% was assumed. to obtain the same level of accuracy in both male and female students as well as in intermediate and secondary schools, bias of nonresponse and missing data were also taken into consideration to arrive at the sample size of 3,400 students. the study data were collected through valid, structured, self - administered, anonymous questionnaires. the used questionnaire was based on the global youth tobacco survey (gyts) questionnaire. the gyts is a school - based instrument consisting of 56 core questions designed and validated to gather data on the prevalence of cigarette smoking and its associated risk factors. the questionnaire addresses questions about the smoking status, smoking - related factors, sociodemographic factors, beliefs and attitudes toward smoking, and behavioral characteristics of the respondents. arabic translation of the questionnaire was done and verified by back translation performed by a different bilingual person who had not seen the original english language version. a pilot testing of the questionnaire was performed on a group of students by asking them to fill in the questionnaire and give feedback about their understanding of the questions and any ambiguity. public health and tobacco - control experts reviewed the results of the pilot testing as well as the back translation. the construct and content validity of the used arabic questionnaire was obtained from discussions with them and it exceeded 98%. the study questionnaire was explained to the students by trained public health personnel and then the students were asked to fill the questionnaire anonymously. participation in the study was voluntary and the school officials were clearly informed about the aim and scope of the study. the study was announced to parents, giving them the chance to refuse the participation of their children by informing the school officials. before the administration of the study questionnaire, the interviewer read out the consent form in order to obtain written consent. finally, the study protocol was approved by the deanship of scientific research ethics committee at taibah university, madinah city, al madinah region, saudi arabia. smoking status, the dependent variable, was assessed in the study questionnaire by the following questions : have you ever tried smoking a cigarette, even once ?, during the past 30 days, how many days did you smoke cigarettes ? followed by during the past 30 days, on the days you smoke, how many cigarettes did you usually smoke ? and how old were you when youfirst tried a cigarette ? smokers were defined as students who had smoked at least once in their lifetime while never smokers were those who had never tried smoking in their lifetime. the current smokers were defined as students who had smoked at least once in the past 30 days. the main independent variable in this study was the factors related to beliefs and attitudes toward smoking. these factors were assessed by asking the student to select one of the three response options concerning the studied belief. the adolescent 's belief about the harmful effects of smoking was assessed by the question do you think that smoking harms your healthno, responses of no and do not know were grouped as no and categorized as the reference group. other explanatory variables included in this study were : i) sociodemographic characteristics age in years (13, 14, 15, 16), sex (male vs female), school level (intermediate vs secondary), school status (public vs private), pocket money (sar300 vs > sar300), parent education (illiterate, basic education, and university or higher education) ; ii) smoking - related factors ; parental smoking (none, one parent smokes, both parents smoke), best friends smoking (none, some, most, or all), and having seen tobacco adverts in mass media (yes vs no). the data were analyzed using the statistical analysis system software package. descriptive statistics were used to estimate smoking prevalence, and chi - square test was used to compare adolescents beliefs and attitudes toward smoking by their smoking status and sex. multivariate logistic regression analyses were used to investigate the association between adolescent smoking and the studied beliefs and attitude factors while controlling the possible confounders. to avoid the confounding effects of factors known to be associated with adolescent smoking, the following potential confounding factors were considered : age, sex, school level and status, pocket money, friends and parental smoking, parental educational level, and family composition. the prevalence of smoking among the studied adolescents was 33.02% (1,097/3,322), and the majority of smokers in this sample (84%) reported to have started smoking before the age of 14 years with no significant sex difference. the prevalence of smoking was significantly high among adolescents who lived with neither parent (57%), those reported that their best friends smoked (53%) and those who reported that one or both parents smoked (45%). concerning adolescents sociodemographic characteristics, a significantly high prevalence was also detected among adolescents aged 16 years, reporting a monthly pocket money > sar300, males, secondary and private school students, and those in the illiterate parents group. prevalence of adolescent smoking by their characteristics, madinah, al madinah region, saudi arabia percentages are presented by its round, significant table 2 showed adolescents beliefs and attitudes toward smoking by their smoking status and sex. there were statistically significant differences between smoker and nonsmoker adolescents regarding most of the studied beliefs and attitudes about smoking. the percentage of adolescents who believed in the harmful effects of smoking was significantly higher among nonsmokers compared to smokers, particularly among females (95% vs 89%, p <.0001). the percentages of adolescents who believed that smokers have more friends, were more attractive, and that smoking helps people feel comfortable in social gatherings were statistically and significantly higher among smokers compared to nonsmokers. these differences also remained statistically significant when the analysis was stratified by sex, with the exception of the variable belief that boy smokers have more friends. the stratified analyses by sex revealed that nonsmoker female students had a lower approval rate about male and female smokers, compared to nonsmoker male students (9% vs 15% in case of female smokers approval and 17% vs 19% in case of male smokers approval). adolescents beliefs and attitudes toward smoking by their smoking status and sex, madinah, al madinah region, saudi arabia smokers were 1,097 among all subjects, 685 among males, and 412 among females, nonsmokers were 2,225 among all subjects, 1,078 among males, and 1,147 among females, significant table 3 presents the risk of adolescent smoking associated with beliefs and attitude factors considering adolescents sex. a significantly lower risk of smoking was detected among adolescents who believed in the harmful effects of smoking and it was more marked among females [odds ratio (or) = 0.55% ; 95% ci = 0.35 - 0.91 ] when compared to those who did not believe in the harmful effects of smoking. compared to adolescents who did not believe that smokers look more attractive, the risk of smoking was significantly increased among adolescents who believed that smokers were more attractive, with the highest risk found among females who believed that male smokers were attractive (or = 2.10 ; 95% ci = 1.65 - 2.91), and among males who believed that females smokers were attractive (or = 1.65 ; 95% ci = 1.17 - 2.10). a significant positive risk was also detected among adolescents who believed that smoking helps people feel comfortable in social gatherings when compared to those who did not believe in it with an adjusted or of 2.70 (95% ci = 2.10 - 3.27) among all studied adolescents, 2.95 (95% ci = 2.22 - 3.87) among male adolescents and 2.50 (95% ci = 1.90 - 3.40) among female adolescents. risk of smoking behavior associated with adolescents beliefs and attitudes toward smoking by their sex, madinah, al madinah region, saudi arabia reference group constituted those who did not believe in the studied factors in the corresponding category, ors are adjusted by age, sex, school level, pocket money, parental education, and family structure, or : odds ratio, ci : confidence interval our results have shown that about a third of intermediate and secondary school adolescents in madinah, al madinah region, saudi arabia smoked. although the proportion who smoked was higher among male and secondary school students, it also remained alarmingly high among female and intermediate school students. further analyses showed that in both secondary and intermediate schools, male smoking outweighed female smoking. the reported overall prevalence rates of adolescents smoking in those studies ranged 29 - 37% compared to 33.02% in this study. smoking prevalence with the same patterns of sex and level of education in adolescents was observed in a school - based study conducted in riyadh, riyadh province, saudi arabia. based on the theory of planned behavior (tpb), behavior is influenced by intention, attitude, subjective norm, and perceived behavioral control. the latter factor represents one 's beliefs about the advantages / ease and disadvantages / difficulties coupled with adopting a behavior. tpb has been widely used in designing theory - based intervention programs to reduce smoking and it was shown that perceived behavioral control exerts a powerful influence both on the intention and the behavior of smoking in a way that is presumed to be culture - specific. although several local studies have addressed a number of social and psychological risk factors associated with adolescent smoking, they have failed to address the role of adolescents beliefs and attitudes toward smoking as risk factors for adolescent smoking in this country. the belief that smoking has harmful health effects has significantly reduced the risk for adolescent smoking by more than a third. on stratified analysis, and although lower risk of smoking was demonstrated in both male and female adolescents who believed in the harmful health effects of smoking, it reached statistical significance only in female adolescents. a similar lower risk was also reported in previous studies from different cultures, where the perception of hazards and long - term consequences of smoking were negatively associated with adolescent smoking. these findings reflect the importance of disseminating the information about the health hazards of smoking and discouraging tobacco use among adolescents. the adolescents perception that smoking enhances social image and physical image our findings revealed that an overwhelming majority of the studied males as well as females, whether smoking or not, believed that smoking affected the body weight. in a previous clinical trial, students submitted to smoking cessation programs were concerned with weight gain and body image following smoking cessation. other previous reports have also revealed a positive association between weight concerns and smoking among female adolescents but not among male adolescents. however, our findings suggested that the belief that smoking affects body weight increased the likelihood of smoking among male adolescents more than it did in female adolescents. while the risk of smoking behavior in female adolescents was not associated with the belief that smokers, boys or girls, have more friends, in male adolescents, unexpectedly, the risk of smoking behavior showed a significant association with the belief that girl smokers and not boy smokers have more friends. this might indirectly reflect that male adolescent smokers expect forming a friendship with a female adolescent to be easier if she smokes. a previous study reported no significant difference between male and female adolescents regarding their belief that smokers have more boyfriends or girlfriends. in that study, the authors have not stratified the results by smoking status as this study did. however, the ability to form friendships substantially depends on the social and cultural milieu that adolescents are living in. a significant positive association was found between adolescent smoking and the belief that smoking individuals were more attractive. the risk for developing a smoking habit was high among adolescent females who believed that male smokers were more attractive and among adolescent males who believed that female smokers were more attractive. nonsmoker female students reported a lower approval rate about male or female smoking, compared to nonsmoker male students. these rates about adolescents attitude toward male and female smokers were consistent with those reported in the previous african and asian studies. a positive image of male smokers appeared to fit into the social, traditional, and cultural concepts in these communities such that smoking may be viewed as an acceptable male social behavior while being considered unusual for females. the previous findings point to two important implications on antismoking programs directed to adolescents.first, antismoking messages should be designed differently for male and female adolescents. second, addressing the image of the opposite sex in such programs seems to be important. male and female adolescent smokers alike believed more than their nonsmoker counterparts that smoking helped people feel more comfortable in social gatherings. the study findings also revealed a significant positive risk of smoking among adolescents who believed that smoking helps people to feel comfortable in social gatherings. this was concomitant with the findings from other international and local studies, in which the majority of adolescent smokers reported smoking as a mean of comfort in social gathering that was in turn reflected as a main reason for smoking among adolescents. although the previous studies have not stratified results by sex, their findings together with the result of this study support the argument that adolescents, particularly males, smoke for pleasure and to feel comfortable at celebrations, parties, and other social gatherings. the present study has a number of strengths that include being school - based with a relative large sample size, high response rate and precisely estimated risks as indicated by the observed narrowness of their cis, which supports the robustness of the study findings. furthermore, the study presented the risk of smoking behavior associated with the studied variables, separately, in the studied male and female students the data collection in the study was based on self - completion of the gyts questionnaire. the validation of self - report via biochemical tests was not feasible due to logistical and cultural constraints. however, the previous studies have reported a high reliability of results on teenager smoking when self - reported questionnaires were administered anonymously. also, the causality between the studied factors and adolescent smoking can not be determined because of the cross - sectional design of the study. in summary, this study revealed a considerably high prevalence of adolescents smoking in madinah city, al madinah region, saudi arabia. believing in the health hazards of smoking was significantly associated with a lower risk of adolescent smoking while believing that smokers were more attractive, especially if they were from the opposite sex, and the belief about more comfort in social gatherings increased the likelihood of attracting adolescents to smoking. the beliefs that smoking affects the body weight and smokers have more friends seemed to affect the risk of adolescent smoking differently in males and females. given the abovementioned limitations, the findings of this study have important implications for smoking prevention and control among adolescents. without modifying such beliefs and attitudes about smoking among adolescents and without attention to the differences between males and females, the prevalence of smoking among this important segment of the population is not expected to decline. since the majority of smokers in this study have reported initiating smoking before the age of 14 years, a logical suggestion for smoking prevention would be to work early on modifying such beliefs and attitudes by disseminating effective structured messages about smoking in the childhood period through professionals who work with children such as pediatricians and family physicians, and to include knowledge about the health hazards of smoking in the educational curriculum as early as primary education. this project was funded by the deanship of scientific research, taibah university, madinah, saudi arabia, under grant no. the authors declare that there have no competing interests regarding the publication of this manuscript. this project was funded by the deanship of scientific research, taibah university, madinah, saudi arabia, under grant no. the authors declare that there have no competing interests regarding the publication of this manuscript. | background : adolescent smoking relates to numerous risk factors, of which beliefs and attitudes toward smoking may play a role. the study aimed to investigate the association between beliefs and attitudes and the risk of adolescent smoking.materials and methods : in a school - based cross - sectional study, 3,400 students were recruited from 34 intermediate and secondary schools in madinah city, al madinah region, saudi arabia. data about sociodemographics, smoking - related factors, and beliefs and attitudes toward smoking were collected using a valid and reliable self - administered questionnaire. prevalence of smoking was estimated and the studied beliefs and attitudes were compared by smoking status and sex using appropriate statistical analyses including multivariate logistic regression.results:of the 3,322 respondents, 33.02% (38.9% males and 26.4% females) were current smokers. beliefs and attitudes toward smoking significantly differed between smokers and nonsmokers in the studied male and female students. the adjusted risk of smoking was significantly increased among female adolescents who believed that male smokers were more attractive [odds ratio (or) = 2.2 ; 95% confidence interval (ci) = 1.6 - 2.9 ] and among male smokers who believed that female smokers are more attractive (or = 1.7 ; 95% ci = 1.2 - 2.2). the risk was also increased among all adolescents who believed that smoking lent comfort in social gatherings. belief that smoking is harmful, however, was negatively associated with the risk of smoking, particularly among females (or = 0.55 ; 95% ci = 0.35 - 0.91).conclusions : the study revealed a considerable high prevalence of smoking among male and female adolescents. addressing the beliefs and knowledge about smoking during childhood is crucial in any antismoking program. |
methicillin - resistant staphylococcus aureus (mrsa) strains are well known to be hospital - associated or healthcare - associated pathogens. however, within the past two decades, the incidence of infections due to community - acquired methicillin - resistant s. aureus (ca - mrsa) strains have been rapidly increasing worldwide. similar to hospital - acquired mrsa (ha - mrsa), ca - mrsa can cause severe infections such as soft - tissue infection (including necrotizing fasciitis), necrotizing pneumonia, severe sepsis, and disseminated infection. moreover, s. aureus can produce many exotoxins such as toxic shock syndrome toxin-1 (tsst-1) and staphylococcal enterotoxin b (seb). exotoxins of s. aureus are associated with a severe illness that includes shock and multiple organ failure and is called toxic shock syndrome (tss). specifically, in women of childbearing age, an insanitary ectocervical environment (e.g., long - term tampon - use or post - partum period) is associated with bacterial growth, including the growth of s. aureus, and the risk of tss. we herein report 2 cases of tss due to ca - mrsa in japanese women. a literature review of cases from japan revealed that transvaginal invasion is a very rare portal of entry of ca - mrsa. the population of ca - mrsa in japan is relatively low compared with that in other countries. despite this fact, we assert that the initiating treatment for tss with vancomycin and clindamycin, even in japan. informed consent was obtained from the patients for the publication of their respective case reports. case 1a 46-year - old japanese woman was admitted to our hospital after a 1-day history of fever, shaking chills, and diarrhoea. she previously was seen at another clinic on the same day of admission (day 1), and fosfomycin was prescribed she did not have any relevant past history and had not been prescribed any medications. she had never been abroad, and she had not eaten any raw foods in the previous month. a 46-year - old japanese woman was admitted to our hospital after a 1-day history of fever, shaking chills, and diarrhoea. she previously was seen at another clinic on the same day of admission (day 1), and fosfomycin was prescribed she did not have any relevant past history and had not been prescribed any medications. she had never been abroad, and she had not eaten any raw foods in the previous month. her vital signs on the day of admission were as follows : blood pressure, 67/47 mmhg ; heart rate, 118 beats per minute ; body temperature, 39.4 c ; respiratory rate, 30 breaths per minute ; and peripheral capillary oxygen saturation level on room air, 98%. physical examination showed capillary refilling time of 12 s, and generalized rash on her chest and abdomen. laboratory data revealed white blood cell (wbc) count of 12,300/l, c - reactive protein (crp) level of 15.4 mg / dl, blood urea nitrogen (bun) of 26.4 mg / dl, serum creatinine (s - cre) level of 3.3 mg / dl, serum total protein level of 7.1 g / dl, serum albumin level of 3.4 g / dl, and procalcitonin level of 69.8 ng / ml. results of liver function tests were normal except for a lactate dehydrogenase (ldh) level of 328 iu / l. urine dipstick examination revealed 2 + protein and 2 + occult blood. a microscopic examination detected 510 red blood cells (rbcs) per high - power field and 510 wbcs per high - power field. at first, we diagnosed severe bacterial colitis and hypovolemic shock and started large volumes of hydration, dopamine infusion, and intravenous ciprofloxacin (300 mg, 2 times daily), we learned that she had begun menstruating 4 days prior to admission, and that she kept a tampon inserted 3 days until the day of admission. additional testing of her vaginal discharge showed the following ; polymorphonuclear leukocytes 2 +, and nugent score, 1. we suspected tss or septic shock of unknown origin and changed the antibiotics to vancomycin (1 g, 2 times daily, intravenously), clindamycin (600 mg, 4 times daily, intravenously), and meropenem (500 mg, 4 times daily, intravenously). on day 4, community - acquired s. aureus was detected from the tampon and vaginal discharge and her vital signs gradually recovered enough to stop the dopamine infusion, and her diarrhoea stopped. we diagnosed staphylococcal tss owing to tampon use, discontinued meropenem on day 11, and continued antibiotic treatment with the combination of vancomycin and clindamycin for 14 days. on day 14, the patient was found to have peeling skin at the end of her fingers (fig. the patient was discharged on day 18, without any damage to her organs or deterioration in activities of daily living.case 2a 40-year - old japanese woman was admitted to our hospital after a 5-day history of yellowish vaginal discharge, and 3-day history of fever, shaking chills, and appetite loss. she did not have any relevant past medical history and had not been prescribed any medications, including antibiotics. she had never been abroad and had not had any raw foods in the previous month. 1 a 40-year - old japanese woman was admitted to our hospital after a 5-day history of yellowish vaginal discharge, and 3-day history of fever, shaking chills, and appetite loss. she did not have any relevant past medical history and had not been prescribed any medications, including antibiotics. she had never been abroad and had not had any raw foods in the previous month. her vital signs on the day of admission were as follows : blood pressure, 86/49 mmhg ; heart rate, 121 beats per minute ; body temperature, 39.8 c ; respiratory rate, 36 breaths per minute ; and peripheral capillary oxygen saturation level on room air, 96%. results of cardiovascular, respiratory, and abdominal examinations were normal, except tenderness on the right costal - vertebral angle. laboratory data revealed wbc of 19,200/l, crp level of 14.5 mg / dl, bun level of 28.4 mg / dl, s - cre level of 1.7 mg / dl, serum total protein level of 6.0 g / dl, serum albumin level of 3.3 g / dl, and procalcitonin level of 5.4 ng / ml. results of liver function tests were normal except for an ldh level of 328 iu / l. a microscopic examination detected 5 to 10 rbcs per high - power field and more than 100 wbcs per high - power field. testing of her vaginal discharge showed the following : polymorphonuclear leukocytes, 4 + ; and nugent score, 4. gram stain of both urine and vaginal discharge showed clustered gram - positive cocci. at first, we diagnosed severe urinary tract infection and septic shock. we started large volumes of hydration, dopamine infusion, and intravenous ampicillin (2 g, 3 times daily) and gentamicin (120 mg, once daily). however, her vital signs did not recover and she experienced diarrhoea. on day 2, s. aureus was detected from urine and vaginal discharge. we suspected staphylococcal tss or septic shock of unknown origin, and changed the antibiotics to vancomycin (1 g, 2 times daily, intravenously), clindamycin (600 mg, 4 times daily, intravenously), and meropenem (1 g, 3 times daily, intravenously) for covering enteric bacteria and anaerobes like bacteroides spp. on day 3, community - acquired s. aureus was detected from both urine and vaginal discharge, and she recovered adequately enough to discontinue dopamine and large volumes of hydration. gynaecological examination revealed narrowing of the vaginal opening caused by vulvar lichen sclerosus, a chronic and progressive dermatologic disorder of genital skin that can cause vulvar pruritus, dysuria, and sexual dysfunction due to cleft narrowing. we diagnosed staphylococcal tss caused by vaginitis and discontinued meropenem on day 9 and continued vancomycin in combination with clindamycin for 14 days. on day 14, we noted peeling skin at the end of her fingers (fig. the following 13 antimicrobials were tested : ampicillin, cefazolin, imipenem, gentamicin, gentamicin, erythromycin, clindamycin, telithromycin, levofloxacin, minocycline, vancomycin, teicoplanin, arbekacin, and trimethoprim / sulfamethoxazole. according to m100-s20, published by the clinical and laboratory standards institute, the isolates from case 1 and case 2 were resistant to ampicillin, cefazolin, imipenem, and gentamicin, but were susceptible to erythromycin, clindamycin, and minocycline. the 2 isolates were tested for genes encoding selected exotoxins including tsst-1, exfoliative toxins a and b (eta and etb), staphylococcal enterotoxins a to e (enta, entb, entc, entd, and ente), and panton - valentine leukocidin (pvl) by multiplex real - time polymerase chain reaction (pcr) analysis. both isolates contained genes encoding for the toxins eta, etb, and tsst-1, but were negative for other exotoxins, including pvl. the isolates were also typed to determine their staphylococcal cassette chromosome mec (sccmec) type by multiplex pcr targeting the cassette chromosome recombinases (ccr) and mec gene complex. both isolates were identified as the group st8-mrsa with sccmec type iv, which has recently been one of a major genotype in the community in japan. the following 13 antimicrobials were tested : ampicillin, cefazolin, imipenem, gentamicin, gentamicin, erythromycin, clindamycin, telithromycin, levofloxacin, minocycline, vancomycin, teicoplanin, arbekacin, and trimethoprim / sulfamethoxazole. according to m100-s20, published by the clinical and laboratory standards institute, the isolates from case 1 and case 2 were resistant to ampicillin, cefazolin, imipenem, and gentamicin, but were susceptible to erythromycin, clindamycin, and minocycline. the 2 isolates were tested for genes encoding selected exotoxins including tsst-1, exfoliative toxins a and b (eta and etb), staphylococcal enterotoxins a to e (enta, entb, entc, entd, and ente), and panton - valentine leukocidin (pvl) by multiplex real - time polymerase chain reaction (pcr) analysis. both isolates contained genes encoding for the toxins eta, etb, and tsst-1, but were negative for other exotoxins, including pvl. the isolates were also typed to determine their staphylococcal cassette chromosome mec (sccmec) type by multiplex pcr targeting the cassette chromosome recombinases (ccr) and mec gene complex. both isolates were identified as the group st8-mrsa with sccmec type iv, which has recently been one of a major genotype in the community in japan. we described 2 cases of tss due to ca - mrsa from transvaginal invasion, which are the first case reports of this kind in japan. appropriate antibiotics, including clindamycin or linezolid, and vancomycin or teicoplanin, have to be started immediately for the patients with staphylococcal tss. however, we did not administered these antibiotics initially because of clinical misjudgment and subsequently, each case had a severe clinical course. we identified the reasons for the misjudgment in each case. in case 1, we were unable to obtain information about tampon use at admission, which is an essential clue for the diagnosis of tss. therefore, it is important to obtain a detailed history regarding tampon use in female patients with septic shock of unknown origin. in case 2, we did not consider ca - mrsa as a causative pathogen of septic shock at admission, although the gram stain of vaginal discharge samples showed clustered gram - positive cocci. ca - mrsa strains accounts a certain percentage of staphylococcus aureus strains in japan and ca - mrsa can cause tss as our cases. the prevalence of ca - mrsa isolation in japan has been reported as 4.3% overall and 3.7% among children. we have to administer appropriate combination of antibiotics to cover ca - mrsa immediately in cases of suspected staphylococcal tss even though they have no risk factors for ha - mrsa infection. learning points from the 2 cases are as follows : 1) taking a detailed history about genital symptoms and female hygiene practice is essential for the diagnosis of females with sepsis of unknown cause, 2) even in japan, we have to administer a combination of antibiotics including antibiotics with toxin - suppressing properties and anti - mrsa antibiotics to the patients with suspected staphylococcal tss. there have been no previous reports about the prevalence of tss due to ca - mrsa in japan, and only a few case reports have ever been reported in japan. we extensively reviewed the literature for past studies and case reports about tss due to ca - mrsa in japan. based on our search of medline and igaku chuo zasshi (a tool for searching japanese biomedical publications), no case series or controlled studies were identified. we could identify 8 cases (3 male and 5 female) ; 4 case reports were written in english, and 4 were written in japanese and had been cited by both japanese and english studies (age range : 1677 years ; median age : 44 years) (table 1),,,,,,,. among these cases, skin and soft tissue infections and necrotizing pneumonia were the main portal of entry. this is similar to the results of a previous study in another country other than the proportion of menstrual tss. gynaecologic ca - mrsa infections as the primary cause of tss have not been reported in japan. on the other hand, we consider that lower prevalence of menstrual tss in japan is probably related to the lower proportion of tampon use in japan. in only 1 case, bacteraemia due to ca - mrsa was detected, and in another case, the patient died. most of the ca - mrsa strains, which had been examined for staphylococcal cassette chromosome mec (sccmec) typing, had sccmec iv (5 cases in 6 evaluated cases), and tsst-1 production was confirmed in 8 of 10 cases, including the present 2 cases. according to previous data from japan, the majority of ca - mrsa strains have sccmec iv or v, and 46% of ca - mrsa strains are tsst-1-producing strains. this is the first literature review about tss due to ca - mrsa in japan, and our results are consistent with the results of previous studies regarding the epidemiology of ca - mrsa in japan.table 1literature review of cases of toxic shock syndrome caused by community - acquired mrsa reported in japan.table 1no.agesexunderlying diseasefocusoutcomeblood culturesccmecexotoxinspvlref.english publications159fnecrotizing pneumoniasurvivednatsst-1na11262mbipolar depressionnecrotizing pneumoniadiednaiitsst-112316ftype a influenzanecrotizing pneumoniasurvivedivseb+13474fdmsstisurvived+ivsecselpselltsst-114japanese publications577fra, ntmi, burnsstisurvivednatsst-1na15631mburnsstisurvivedivtsst-116727msstisurvivednanatsst-1na17821fsstisurvivednanasebna18our cases946fmenstrual tsssurvivedivetaetbtsst-1case 11040fvaginitissurvivedivetaetbtsst-1case 2abbreviations : ra = rheumatoid arthritis ; ntmi = nontuberculous mycobacterial infection ; dm = diabetes mellitus ; ssti = skin and soft tissue infections ; sec = staphylococcal enterotoxin c ; selp = staphylococcal enterotoxin - like p ; sell = staphylococcal enterotoxin - like l ; na = not available, ref literature review of cases of toxic shock syndrome caused by community - acquired mrsa reported in japan. abbreviations : ra = rheumatoid arthritis ; ntmi = nontuberculous mycobacterial infection ; dm = diabetes mellitus ; ssti = skin and soft tissue infections ; sec = staphylococcal enterotoxin c ; selp = staphylococcal enterotoxin - like p ; sell = staphylococcal enterotoxin - like l ; na = not available, ref., the authors described 2 cases of tss due to ca - mrsa associated with a vaginal portal of entry, which represent the first case reports of its kind in japan. appropriate antibiotics, including vancomycin and clindamycin, should be initiated when tss is suspected in patients. in addition, the literature review suggested that tss due to ca - mrsa in japan has similar characteristics to those reported in other countries. | community - acquired methicillin - resistant staphylococcus aureus has been spreading worldwide, including in japan. however, few cases of toxic shock syndrome caused by community - acquired methicillin - resistant staphylococcus aureus have been reported in japan. we report 2 cases, in middle - aged women, of toxic shock syndrome due to community - acquired methicillin - resistant staphylococcus aureus via a vaginal portal of entry. the first patient had used a tampon and the second patient had vaginitis due to a cleft narrowing associated with vulvar lichen sclerosus. both patients were admitted to our hospital with septic shock and severe acute kidney injury and subsequently recovered with appropriate antibiotic treatment. in our review of the literature, 8 cases of toxic shock syndrome caused by community - acquired methicillin - resistant staphylococcus aureus were reported in japan. in these 8 cases, the main portals of entry were the skin and respiratory tract ; however, the portal of entry of community - acquired methicillin - resistant staphylococcus aureus from a vaginal lesion has not been reported in japan previously. |
biliary obstruction is preferentially managed by endoscopic retrograde cholangiopancreatography (ercp). however, after ercp failed, alternatives include percutaneous transhepatic drainage, surgery and more recently, endoscopic ultrasonography (eus)-guided hepaticogastrostomy. the limitation of this technique is that the drainage is restricted to the left side. the aim of this study is to describe a new method of drainage of both hepatic ductal systems by hepaticogastrostomy in patients with hilar obstruction. nine prospectively patients were included, all with hilar obstruction (metastasis of a pancreatic adenocarcinoma n = 4, cholangiocarcinoma n = 1, gallbladder cancer n = 2 and metastasis from a pancreatic neuroendocrine tumor n = 2). a total of four patients had previously whipple surgery and the others five had duodenal involvement by the tumor. the topography of the stenosis varied from bismuth type 2 (n = 7) and hilar infiltration in the others two. the first one consisted in a transgastric eus - guided puncture of the left - side bile duct with a 19 gauge needle, insertion of a 0.0035 inch guide wire which was positioned at the right biliary tree crossing the bile bifurcation. after a dilatation with 6 fr cystotome, a non - covered self - expandable metal stent was placed communicating the right and left biliary ducts. finally, a second stent, partially covered, was inserted at the left biliary duct, with the distal part inside the previously stent and the proximal edge positioned at the stomach. successful drainage was observed in seven patients, two of them presented abdominal pain during the first 72 h. one patient developed sepsis and death 7 days after the procedure and the other one had drainage failure. this pilot study shows the feasibility of this new technique to drain the right biliary duct in patients with hilar obstruction, with few major complications rates. | introduction : biliary obstruction is preferentially managed by endoscopic retrograde cholangiopancreatography (ercp). however, after ercp failed, alternatives include percutaneous transhepatic drainage, surgery and more recently, endoscopic ultrasonography (eus)-guided hepaticogastrostomy. the limitation of this technique is that the drainage is restricted to the left side. the aim of this study is to describe a new method of drainage of both hepatic ductal systems by hepaticogastrostomy in patients with hilar obstruction.results : nine prospectively patients were included, all with hilar obstruction (metastasis of a pancreatic adenocarcinoma n = 4, cholangiocarcinoma n = 1, gallbladder cancer n = 2 and metastasis from a pancreatic neuroendocrine tumor n = 2). a total of four patients had previously whipple surgery and the others five had duodenal involvement by the tumor. the topography of the stenosis varied from bismuth type 2 (n = 7) and hilar infiltration in the others two. all of them were submitted a three - step drainage. the first one consisted in a transgastric eus - guided puncture of the left - side bile duct with a 19 gauge needle, insertion of a 0.0035 inch guide wire which was positioned at the right biliary tree crossing the bile bifurcation. after a dilatation with 6 fr cystotome, a non - covered self - expandable metal stent was placed communicating the right and left biliary ducts. finally, a second stent, partially covered, was inserted at the left biliary duct, with the distal part inside the previously stent and the proximal edge positioned at the stomach. successful drainage was observed in seven patients, two of them presented abdominal pain during the first 72 h. one patient developed sepsis and death 7 days after the procedure and the other one had drainage failure. jaundice was reduced significatively in seven patients and a chemotherapy was started in 6/7 patientsconclusion : this pilot study shows the feasibility of this new technique to drain the right biliary duct in patients with hilar obstruction, with few major complications rates. |
paleoparasitology is the study of parasites found in ancient remains and the tracing, on that basis, of the history of host - parasite relationships. it began in the early 20th century in egypt, where sir marc armand ruffer developed a special rehydration technique applicable to mummified tissues and prepared histological slides suitable for their examination. over the succeeding several decades, paleoparasitology continued to develop all over the world, as experts examined sediments or coprolites obtained from archaeological sites, discovering therein ancient parasite eggs by conventional or molecular techniques. in korea, such samples are now the focus of interdisciplinary collaboration, most notably between archaeologists and parasitologists seeking to trace and delineate past parasitic infection patterns. some of the fruits of this labor will be reviewed briefly here. the first paleoparasitological investigation in korea was carried out on ascaris and trichuris eggs discovered in wetland soil samples obtained in shinchang - dong. although this was not a full - scale survey, it was nonetheless meaningful for being the first study of its kind in korea. several years later, another korean paleoparasitological investigation was conducted, this one on archaeological soil samples dating to the bronze age (2000 - 1000 bc), the three - kingdoms period (100 bc-650 ad), the unified silla dynasty (668 - 935 ad) and the joseon dynasty (1392 - 1910). as in the shinchang - dong case, nematode (i.e. this study was remarkable, though, for representing the first actual korean archaeological - parasitological collaboration by which parasitic infection patterns for a different historical era could be revealed. over time, mummies discovered in tombs dating to the joseon dynasty stood out as especially relevant and valuable to paleoparasitology. these tombs in fact had been constructed in significant numbers throughout most of the korean peninsula, mainly during the latter half of the period (16th-18th century). according to historical texts, in fact,... since lime, sand, and red clay (filled around the coffin) became hardened like a stone after a long while, the coffin could be protected from the possible intrusions of insects or even grave robbers.... fortunately enough for archaeologists, these joseon tombs allowed for superb preservation of the cultural artifacts and human remains they contained, though the exact mechanism of preservation was not fully, and remains imperfectly, understood. the former has proved instrumental in revealing detailed aspects of joseon society. as for the latter, there have been some exceptionally well preserved joseon mummies that have yielded particularly valuable evidence to investigators (fig. coprolites for example, generally shown on computed tomography (ct) images as well - demarcated masses in the intestinal cavity, have been collected from joseon mummies in the course of endoscopic examinations or by direct dissection, and have proved ideal for the broad purposes of paleoparasitological studies in korea (fig. these findings, indeed, have begun to paint pictures of parasitic infection patterns prevailing during the joseon dynasty. in the course of sample collection, every effort has been made to minimize false - positivity due to contamination : investigators wore protective gloves, head cap, gown, and mask (fig. 2a - c), and surface soils from the given archaeological site were always applied as negative controls. as for findings, our own data accumulated over the past several years, and especially our accurate predictions of the presence of parasite eggs in a number of sample sets, indicates a close relationship between the preservation status of certain types of artifacts or remains (e.g., clothes, hair, or brain tissue) and that of ancient parasite eggs discovered in the same tombs. microscopic examinations of samples (typically rehydrated in 0.5% trisodium phosphate solution) have been the standard investigative modality. what has really captured the imagination of scientists, even of archaeologists, over the past several decades, however, is dna analysis. dna studies of ancient parasites including trypanosoma cruzi, plasmodium sp., ascaris sp., t. trichiura, enterobius vermicularis, and clonorchis sinensis have been numerously and widely reported. inspired by these successes, our research team has performed ancient dna (adna) analyses on many of the parasite egg specimens we have obtained from joseon mummies. the availability of coprolite samples from joseon dynasty mummies has proved very fortunate indeed. due to their great preservation quality, we were able to collect invaluable data on the patterns of parasitic infections affecting certain joseon societies (fig. the first - ever paleoparasitological study on a joseon population was based on a child mummy discovered in 2001 (fig. 2d) in a 16th to 17th century joseon tomb unearthed in yangju, gyonggi - do (fig. endoscopy revealed the presence of coprolites, samples of which showed, under scanning electron microscopy (sem), t. trichiura, a. lumbricoides and c. sinensis eggs still perfectly intact even after several hundreds of years (fig. 15), the sample 's preservation was so perfect that even larvae of various species, in addition to eggs, were detected. in the course of our paleoparasitological study series, we have found ancient parasite eggs and larvae representing a. lumbricoides, t. trichiura, c. sinensis, paragonimus westermani, metagonimus yokogawai, gymnophalloides seoi, strongyloides stercoralis, and trichostrongylus spp. these and other, related data have answered many questions concerning historical parasitic infection patterns in korea. among our most noteworthy findings was that the infection prevalence of nematode parasites in joseon samples does not differ so much from those of mid-20th century korea. in fact, the overall positive rates for a. lumbricoides and t. trichiura in our joseon data were 50.0% (9/18) and 77.8% (14/18), respectively, compared with 1969 national survey results of 58.2% (a. lumbricoides) and 74.5% (t. trichiura). notwithstanding this general consistency with the national survey data of 1969, there were some unique findings represented in our data. the joseon samples contained some parasite species not readily identifiable, such as e. vermicularis, hookworms, and taenia spp. to date, there have been only 2 reports of e. vermicularis eggs in archaeological samples : one on roman latrines, and the other on an egyptian mummy. ours, then, is the first such report for any east asian country. in feces remnant in the colon of a female joseon mummy from dangjin - gun (fig. 8), e. vermicularis along with p. westermani and a. lumbricoides eggs were identified under microscopy. its infection prevalence among koreans reportedly was only 1.9% in 1971, and it had not been found in any archaeologically obtained korean samples other than our 17th century joseon case. we will need to examine additional samples before we are able to confirm this rarity or, if confirmed, to determine the corresponding infection route it is relatable to the dietary habits of the joseon people. in joseon mummy coprolites we have observed many trematode eggs (table 1), among which those of c. sinensis eggs have been very common. considering that c. sinensis infection can be transmitted via ingestion of raw or undercooked freshwater fish harboring the infectious metacercariae, we speculated that this korean culinary tradition might in fact have been practiced by pre-20th century koreans more than that had previously been supposed. these many cases of p. westermani eggs show that this type of infection might not have been rare in joseon populations. infected individuals might have ingested crabs or crayfish in a raw or undercooked state, especially considering the fact that freshwater crustaceans are the second intermediate hosts of paragonimus spp. among our cases of paragonimiasis, the most impressive and, incidentally, the first - ever archaeological - sample - based case of ectopic paragonimiasis reported anywhere in the world, is a ca 17th century female mummy discovered in 2009 (fig. 4). according to the extant historical texts, she was likely to have lived in hadong sometime between the late 1500s and early 1600s. on microscopic examinations, many paragonimus eggs were found in the liver, lung, intestinal tissues, and feces (fig. other very interesting findings were g. seoi eggs in the coprolites of another female mummy discovered in hadong (fig. this was a very unexpected and illuminating discovery, as there had been no previous reports of g. seoi eggs anywhere in hadong - gun (county), the area in which the mummified individual had lived. the first report of g. seoi infection was made in 1993, concerning a korean woman residing on an island off the southwest coast of korea ; and in fact, a 2001 nationwide survey established that human g. seoi infection was confined to that and other small islands or counties in the southwestern corner of the country. in this light, we suspected that g. seoi infection might have contracted from what had been a wider coastal distribution to a much more restricted region of the korean peninsula (fig. this, our contraction theory of g. seoi infection, though convincing to us, required more than just a single mummy case from a non - endemic area to be considered dispositive. fortunately enough, we did discover, in sapgyo, a 16th century joseon mummy (fig. 3) in which, along with c. sinensis, m. yokogawai, and t. trichiura, g. seoi eggs were evident. in one of our other analyses, this one of ancient ascaris eggs from another joseon specimen, we were successful in extracting and sequencing ascaris adna. however, the ascaris 18s rrna gene sequence in our study was more similar to a. suum than to a. lumbricoides, though the samples were collected from joseon human individual remains during anthropological investigations. as in the previous relevant studies, we speculated, therefore, that adna analysis of ascaris is inadequate for confirmation of a. lumbricoides. in our pcr - based adna analysis of the t. trichiura ssu rrna gene, we could obtain a sequence 100% homologous to that of t. trichiura, which is also distinct from that of t. suis (97%) and t. muris (91%) (table 2). we have applied adna analysis also to the study of trematode eggs, specifically paragonimus eggs collected from a 17th century korean female mummy (fig. the extracted its 2 gene sequence showed 100% homology to those of modern p. westermani dna reported from korea and japan, which collective results constituted a genetic cluster distinct from other south asian p. westermani. although ancient c. sinensis eggs have frequently been observed in korean archaeological samples, reported analyses of c. sinensis adna have been very scarce. in one of our studies on a joseon mummy, the presence of c. sinensis eggs was confirmed by microscopic observations, and c. sinensis adna was successfully extracted. a pcr - based adna analysis was then performed using primer sets for nuclear ribosomal internal transcribed spacers 1 (its1) and 2 (its2), and mitochondrial cytochrome c oxidase 1 (co1) genes. the obtained sequences, significantly, were 100% homologous to the contemporary c. sinensis gene sequences reported from korea and other east asian countries (table 2). for the past several years, we have studied korean mummies and their paleoparasitological samples, which subsequently have been maintained in the joseon dynasty human remains collection (jdhrc) of seoul national university as the fruit of our collaboration with archaeologists. although our paleoparasitological studies in korea have provided important information on parasitic infection patterns, we must admit that most of the credit is owed to the discovery of well - preserved joseon mummies. our estimations of parasite prevalence, moreover, are based on samples representing relatively limited time, regional, and socio - economic spans. for instance, we should acknowledge that most of the joseon mummies we have examined were individuals who belonged to the ruling classes of society. according to historians, the unique tombs (hoegwakmyo tombs) in which korean mummies have been discovered this means that the mummies are likely to have been landowners, scholars, or courtiers. in a sense then, the coprolites of korean mummies have helped to reveal the parasitic infection status not so much of a certain joseon society in general but rather of the highest social strata of that society. in future studies, therefore, our scope will be extended to types of paleoparasitological samples other than those from korean mummies. a great deal of data from various origins will likely prove fertile soil for the development of comprehensive theories on past parasitic infection patterns. the answering and resolving of which will occupy current work and guide subsequent future research. in our upcoming studies, we must try to find some types of helminth eggs that heretofore have not been considered. as already explained, some parasite species, with very few exceptions, have not been found in coprolite samples from joseon mummies, despite their high reported prevalence in recent national survey reports. examples of these are e. vermicularis, hookworms, and taenia. as for protozoan cysts, many intensive and detailed studies are still needed in order to identify certain parasite eggs, larvae, cysts, and oocysts rarely discovered in previous studies. also, whereas our previous adna studies served to confirm the academic significance of combining pcr - based molecular data with microscopic findings on ancient parasite eggs, such analysis will have to be extended to much deeper levels of inquiry and detail. we admit, for example, that adna study focusing more on phylogenetic analysis of ancient parasites over wider geographical and temporal ranges is required in order to broaden our understanding of the genetic differences among specific parasite species. in conclusion, paleoparasitology is now on the advance, and growing as a field worldwide. expanding cooperation among parasitologists, archaeologists and, more recently, palaeontologists, provides the comprehensive context in which the parasitic infection patterns of the past and, thereby, the origins and evolution of infectious diseases worldwide, can be fully understood. in korea, paleoparasitological approaches to the study of archaeological samples are really just beginning. even so, we expect that our forthcoming analyses finally will move even closer to the goal of revealing the parasitological infection patterns of joseon society. indeed, interpreting our data outcomes from the global parasitological perspectives will help to reconstruct a history of pre - modern parasitic infections for the entire asian continent. | paleoparasitology is the application of conventional or molecular investigative techniques to archeological samples in order to reveal parasitic infection patterns among past populations. although pioneering studies already have reported key paleoparasitological findings around the world, the same sorts of studies had not, until very recently, been conducted in sufficient numbers in korea. mummified remains of individuals dating to the korean joseon dynasty actually have proved very meaningful to concerned researchers, owing particularly to their superb preservation status, which makes them ideal subjects for paleoparasitological studies. over the past several years, our study series on korean mummies has yielded very pertinent data on parasitic infection patterns prevailing among certain joseon dynasty populations. in this short review, we summarized the findings and achievements of our recent paleoparasitological examinations of joseon mummies and discussed about the prospects for future research in this vein. |
their radiological features suggest malignant neoplasms, whereas they are not ct and mr imaging are the most used tools in their evaluation inflammatory myofibroblastic tumours (imts) constitute a rare group of neoplasms composed of a mixture of spindle - shaped myofibroblasts or fibroblasts and a variable amount of inflammatory cells (eosinophils, plasma cells and lymphocytes). many different terms have been used to refer to these tumours : plasma cell granuloma, inflammatory myofibrohistiocytic proliferation, fibroxanthoma, xanthogranuloma. the most frequently affected organs are lung and orbit, but they have been described in nearly every organ. different aetiologies have been proposed for imt, being different chronic infections, autoimmune diseases and trauma the most accepted. specific inflammatory diseases, such as igg4 disease, have also been recently associated [4, 5 ]. an aggressive behaviour with metastases has been described [3, 6, 7 ]. clinical presentation of imts depends on the organ in which they arise, but they frequently associate general inflammatory symptoms as fever or malaise. radiological appearance of imts is unspecific and they are often misdiagnosed as malignant neoplasms. many of them are incidentally discovered when an imaging technique (computed tomography [ct ], ultrasonography [us ] or magnetic resonance imaging [mri ]) is performed for any other reason. their most common radiological presentation is as solid, irregular, well - defined masses. immunohistochemical studies of t- and b - cell subpopulations, cd34, cd117, s-100 and c - kit may be helpful in distinguishing imts from other soft - tissue neoplasms [8, 9 ]. at the molecular level, approximately half of imts contain a clonal cytogenetic aberration that activates the anaplastic lymphoma kinase (alk-). positive immunohistochemical staining of alk is in approximately 40100 % of imts, depending on the anatomical sites where they arise [810 ]. furthermore, alk expression could be a prognostic factor of aggressiveness for imt. according to an update based on the new world health organisation (who) classification, imts are considered as neoplasms which may recur or metastasise in as many as 5 % of cases. the objective of this pictorial review is to present the most important characteristics of imts arising in different locations of the body with their histological correlation., these tumours can appear as hypoechoic or hyperechoic masses with ill - defined or well - circumscribed edges and a variable doppler appearance with increased vascularity. us is usually the first imaging technique when imts appear in specific locations, such as testicles or neck. in other locations on contrast - enhanced ct, imts can appear as homogeneous or heterogeneous lesions, with variable enhancement on delayed acquisitions due to the presence of fibrosis. these findings are also present on gadolinium contrast - enhanced mri. on t1-weighted and t2-weighted sequences, imts usually show low signal intensity reflecting also the presence of fibrotic tissue (table 1).table 1symptoms, radiological findings and differential diagnosis of the main imts of the bodysymptomsimaging findingsdifferential diagnosisorbitpain, redness, oedema, proptosis, ptosis, oculomotor deficits, diplopia, swelling, mass - effectsolid and heterogeneous enhancing. associated retrobulbar fat or oedemagranulomatous diseases, primary infection, sarcoid, sjgren disease, primary and secondary tumours of the orbit, lymphoma and connective tissue diseaseslungcough, chest pain, dyspnea, haemoptysis, fever, malaise and weight loss.solitary, well - circumscribed, peripheral, predominance for the lower lobes, unusual calcifications. low intensity on t2-weighted imagesmalignant lung masses : primary bronchogenic carcinomascrotumlump in the scrotumsolid and heterogeneous masses with internal vascularity on us. small calcifications can be associatedscrotum primary neoplasmhepaticunspecific, depending on the sizeheterogeneous or peripheral enhancement during the arterial phase. or homogeneous enhancement during the arterial phase and washout during the delayed phase on ct. t1 hypointense and t2 hyperintense with heterogeneous enhancementhccsplenicleft upper quadrant or epigastric painlow - density pattern. low - intensity lesion in the early phase after gadolinium injection and high - intensity in the delayed phasesplenic haemangioma and other primary splenic neoplasms as lymphoma or splenic angiosarcomagrastrointestinal tract and mesenteryabdominal pain, intestinal obstruction, dysphagia, anaemiahypodense, heterogeneous, well - delimitedgist, inflammatory fibroid polyp, smooth muscle neoplasm, peripheral nerve sheath tumour, solitary fibrous tumour, fibromatosis, the follicular dendritic cell sarcoma, lymphoma and adenocarcinomamusculoskeletalpain, oedemanon - homogeneous, solidmalignant lesions : rabdomyosarcoma symptoms, radiological findings and differential diagnosis of the main imts of the body imts have been reported in various sites in the head and neck such as bucal space, maxillary sinus, nasal cavity, parotid gland, nasopharynx and larynx (fig. 1pathology : imt in the left supraglottic space (arrow) with paraglottic space involvement indicated by replacement of the paraglottic fat with soft tissue. a axial reformation of contrast - enhanced neck ct. a solid, well - defined, normal tissue was present in the peripheral zone () pathology : imt in the left supraglottic space (arrow) with paraglottic space involvement indicated by replacement of the paraglottic fat with soft tissue. a axial reformation of contrast - enhanced neck ct. a solid, well - defined, little - enhancing nodule in the supraglottic larynx was observed (yellow arrow). b microscopic study obtained after surgical removal of the lesion. the sample showed proliferation of fusiform cells mixed with macrophages and giant multinucleated cells. normal tissue was present in the peripheral zone () however, the orbit is the most common location of imts. they account for approximately 10 % of orbital masses, being the third most common cause of orbital masses. their aetiology is unknown, but they have been associated with inmunological disorders, different infections and history of trauma. recently, they have been strongly associated with the igg4-related sclerosing systemic inflammatory disease and with chronic infection by epstein - barr virus. the clinical presentation and symptoms may vary with the location, extension and aggressiveness of the tumour. patients with imts of the orbit can present local symptoms as pain, redness, oedema, proptosis, ptosis, oculomotor deficits, diplopia, lid swelling or mass - effect and systemic symptoms due to the general inflammatory disorders they are frequently associated with. mri is superior to ct in the evaluation of inflammation of the nerves and muscles. they usually appear as solid and heterogeneous enhancing masses, which can affect retro bulbar fat, cause bone destruction, and present intracranial extension. when they show low intensity on t2-weighted images prominent, fibrotic composition is present (fig. a, b axial and coronal mr reformations of the same patient on t2-weighted sequences showed a well - delimited, hyperintense intraorbital mass, in the intraconal compartment, in the medial aspect. c, d t1-weighted images demonstrated a hypointense, homogeneous, well - delimited mass with peripheral enhancement after gadolinium injection. f microscopic sample obtained after surgery demonstrated an epithelial tumour composed of fusiform cells mixed with an extensive chronic inflammatory infiltrate of plasmatic cells, lymphocytes and macrophages. in the peripheral of the sample a, b axial and coronal mr reformations of the same patient on t2-weighted sequences showed a well - delimited, hyperintense intraorbital mass, in the intraconal compartment, in the medial aspect. c, d t1-weighted images demonstrated a hypointense, homogeneous, well - delimited mass with peripheral enhancement after gadolinium injection. f microscopic sample obtained after surgery demonstrated an epithelial tumour composed of fusiform cells mixed with an extensive chronic inflammatory infiltrate of plasmatic cells, lymphocytes and macrophages. in the peripheral of the sample striated muscle cells were also observed pathologically, imts of the head and neck consist of many fibroblastic cells, fibroblastic cells and inflammatory cells, which include plasma cells and eosinophils. the differential diagnosis includes granulomatous diseases, primary infection, sarcoid, sjgren disease, primary and secondary tumours of the orbit, lymphoma and connective tissue diseases. the administration of corticosteroids an inmunosupresors usually leads to a decrease of size of the mass but also radiotherapy or surgery may be indicated. they account for around the 50 % of benign pulmonary masses in children, representing only about 0.7 % of all tumours of the lung in general population. as in other organs sometimes, imts are also related with antecedents of lung surgery and they can arise in surgical lung scars. common clinical characteristics include unspecific respiratory symptoms, such as cough, chest pain, dyspnea, haemoptysis and unspecific inflammatory symptoms as fever, malaise and weight loss. they usually appear as solitary, well - circumscribed peripheral lung masses, with predominance for the lower lobes (fig. on ct with intravenous contrast, they present a variable heterogeneous or homogeneous degree of enhancement pattern [3, 21 ]. it has been described an aggressive behaviour with invasion of the adjacent structures. on mri because of their similar radiological appearance to malignant lung masses, biopsy is necessary to obtain the diagnosis.fig. a 55-year - old man with a cough and haemoptysis.a axial reformation of non - contrast ct with mediastinum window. two solid, ill - defined nodules in right lower lobe are observed (yellow arrows). b axial reformations with lung window of the same patient where the lung nodules are shown (white arrows). c partial lobectomy of the right lower lobe specimen. note the presence of the nodules (). d microscopic study of the resected lung sample where fusiform cells with an associated inflammatory infiltrate of lymphocytes, plasmatic cells and histiocytes was found pathology : imt of the lung. a 55-year - old man with a cough and haemoptysis.a axial reformation of non - contrast ct with mediastinum window. two solid, ill - defined nodules in right lower lobe are observed (yellow arrows). b axial reformations with lung window of the same patient where the lung nodules are shown (white arrows). c partial lobectomy of the right lower lobe specimen. note the presence of the nodules (). d microscopic study of the resected lung sample where fusiform cells with an associated inflammatory infiltrate of lymphocytes, plasmatic cells and histiocytes was found the treatment of choice of lung imts is surgery to exclude malignancy and to achieve the cure. although spontaneous regression may occur, local expansion may cause significant morbidity and occasional death. the prognosis after complete surgical resection is excellent, with a low rate of recurrence. in these cases, alternative treatments (radiotherapy, corticoids or chemotherapy) to surgery could be also used. only a few cases of imts in the spermatic cord have been reported. the most common presentation of epididymal (fig. 4) and paratesticular imts is a lump in the scrotum.fig. 4pathology : imt of the epididymus. a 40-year - old man with a lump in the scrotum. a a hypoechoic a diffuse infiltrate composed of fibroblastic and fusiform cells mixed with inflammatory cells (mainly lymphocytes and macrophages) was observed pathology : imt of the epididymus. a 40-year - old man with a lump in the scrotum. a a hypoechoic, well - delimited epididymal lesion in the right scrotum was detected. a diffuse infiltrate composed of fibroblastic and fusiform cells mixed with inflammatory cells (mainly lymphocytes and macrophages) was observed in the evaluation of scrotal masses, us is the most accurate imaging technique, helping to distinguish intratesticular from extratesticular lesions and solid from cystic lesions. on ct, they appear as well - defined, hypodense masses with little enhancement after intravenous contrast injection. in some cases, a 34-year - old man with no relevant medical history came to our hospital with a lump in the scrotum. a us showed an isoechoic, solid, paratesticular nodule with fine calcifications (yellow arrow). b axial reformation of contrast - enhanced ct on portal phase demonstrated a well - delimited, heterogeneous, little - enhancing mass in the right scrotum. d microscopic study of the lesion demonstrated a well - delimited lesion constituted of fibroblasts, lymphocytes and plasmatic cells with associated lymphoid follicle. a 34-year - old man with no relevant medical history came to our hospital with a lump in the scrotum. a us showed an isoechoic, solid, paratesticular nodule with fine calcifications (yellow arrow). b axial reformation of contrast - enhanced ct on portal phase demonstrated a well - delimited, heterogeneous, little - enhancing mass in the right scrotum. d microscopic study of the lesion demonstrated a well - delimited lesion constituted of fibroblasts, lymphocytes and plasmatic cells with associated lymphoid follicle. there was no evidence of adjacent testicle invasion surgical resection without orchiectomy is the ideal treatment, but in some cases the testicle must be removed due to the size of the mass or when the mass is attached to the testis. imts of the penis are extremely rare. to our knowledge, no imts at this location have been reported. on ct, this tumour appeared as a well - defined, little - enhancing and non - aggressive mass surrounding the penile urethra and the corpora cavernosa with no evidence of invasion of them.fig. a 46-year - old man with a mass in the penis came to our hospital. he related more than 6 years of slow growth of the mass with no suspicious associated symptoms. c, d axial and coronal reformations of enhanced - ct after the injection of intravenous contrast on portal phase. macroscopic view showed an heterogeneous aspect due to the mixture of tissues (solid tumour, lipoid component and areas of haemorrhage). f microscopic study confirmed a mesenchymal tumour composed of fusiform cells, some of them multinucleated, with round or elongate nucleus. a 46-year - old man with a mass in the penis came to our hospital. he related more than 6 years of slow growth of the mass with no suspicious associated symptoms. c, d axial and coronal reformations of enhanced - ct after the injection of intravenous contrast on portal phase. macroscopic view showed an heterogeneous aspect due to the mixture of tissues (solid tumour, lipoid component and areas of haemorrhage). f microscopic study confirmed a mesenchymal tumour composed of fusiform cells, some of them multinucleated, with round or elongate nucleus. no mitotic figures were observed surgical treatment was performed due to the size and non - conclusive radiological features of the tumour. hepatic imts constitute an extremely rare group of hepatic tumours, accounting only for the 0.7 % of all the hepatic lesions. they have been associated with autoimmune diseases, immunological disorders and viral infections, such as igg4 disease, sclerosing cholangitis, crohns disease, hepatic viral infections and epstein - barr virus. symptoms of hepatic imts depend on their size. on contrast - enhanced ct, they usually show heterogeneous or peripheral enhancement during the arterial phase. despite this, some of them present homogeneous enhancement during the arterial phase and washout during the delayed phase, which can lead to misdiagnosing them as hepatocarcinomas (hccs) (fig. 7). on mri, these lesions usually are t1 hypointense and t2 hyperintense with heterogeneous enhancement (fig. 8). hepatobiliary contrast (gadoxetate disodium, primovist ; bayer healthcare, leverkusen, germany) could be of help to differentiate hcc from imts of the liver. on the hepatobiliary phase, high signal intensity in the centre of the lesion has been described, suggesting a benign lesion.fig. well - defined, hypointense lesion on t1-weighted images (a) which presented weak enhancement in the arterial phase (b) and wash - out on portal and equilibrium phases (c-, d) located in hepatic left lobe. f the lesion demostrated an expansive chronic inflammatory infiltrate of plasmatic cells, lymphocytes and macrophages. a abdominal ultrasound where a slightly hypoechoic, solid, heterogeneous mass arising in the left hepatic lobe was observed. axial t1-weighted unenhanced (b) and contrast - enhanced mr images (c, d) showed an ill - defined with heterogeneous enhancement mass located in lateral hepatic segments. mesenchymal tumour composed of fusiform cells, lymphocytes and lipid - filled macrophages can be observed. normal hepatocytes with large and small fatty droplet change are also present pathology : imt of the liver. well - defined, hypointense lesion on t1-weighted images (a) which presented weak enhancement in the arterial phase (b) and wash - out on portal and equilibrium phases (c-, d) located in hepatic left lobe. e macroscopic view of partial hepatectomy obtained after surgical excision of the lesion. a solid, well - defined and yellowing appearance mass is showed. f the lesion demostrated an expansive chronic inflammatory infiltrate of plasmatic cells, lymphocytes and macrophages. a abdominal ultrasound where a slightly hypoechoic, solid, heterogeneous mass arising in the left hepatic lobe was observed. axial t1-weighted unenhanced (b) and contrast - enhanced mr images (c, d) showed an ill - defined with heterogeneous enhancement mass located in lateral hepatic segments. mesenchymal tumour composed of fusiform cells, lymphocytes and lipid - filled macrophages can be observed. normal hepatocytes with large and small fatty droplet change are also present surgery is the most accepted treatment. if there is a biopsy - proven diagnosis of imts that excludes malignancy, medical treatment with no steroidal anti - inflammatory drugs in patients with peripheral hepatic imts could be prescribed. patients with spleen imts frequently associate unspecific symptoms as left upper quadrant or epigastric pain. splenomegaly is common and some patients can present fever, anaemia and signs of hypersplenism. different radiological imaging modalities have been proposed for their evaluation. on all of them, they are depicted as low - density masses in both non - enhanced and enhanced acquisitions (fig. 9). on mri studies, they present low - intensity mass on both the t1- and t2-weighted images. their typical enhancement pattern after gadolinium injection is as a low - intensity lesion in the early phase, which changes into a high - intensity one in the delayed phase.fig. 9pathology : imt of the spleen. a axial reformation of contrast - enhanced ct on portal phase. prominent cellularity composed of an irregular, weakly basophil set of mioepithelial - fusiform cells. numerous inflammatory cells (plasmatic, lymphocytes and few neutrophils and eosinophils) with isolated macrophages were also found. no atypia or mitotic figures were present pathology : imt of the spleen. a axial reformation of contrast - enhanced ct on portal phase. prominent cellularity composed of an irregular, weakly basophil set of mioepithelial - fusiform cells. numerous inflammatory cells (plasmatic, lymphocytes and few neutrophils and eosinophils) with isolated macrophages were also found. no atypia or mitotic figures were present the differential diagnosis includes splenic haemangioma and other primary splenic neoplasms such as lymphoma or splenic angiosarcoma. when a suspicious spleen mass is found, the treatment of choice is splenectomy due to the high risk of bleeding associated to splenic biopsy [8, 31 ]. they can arise anywhere, the stomach and small intestine being the most common locations [3, 9 ]. clinical symptoms may vary from none to abdominal pain, intestinal obstruction, dysphagia or anaemia, depending on the location and size of the mass. contrast - enhanced ct is the imaging technique, which give us more information about of imts of the gastrointestinal tract. they are usually found as hypodense, heterogeneous and well - delimited masses (figs. axial reformation of contrast - enhanced ct on arterial (a), portal (b) and 3 min after injection delayed (c) acquisitions. a well - defined, heterogeneous nodule with moderate enhancement on portal phase arising from the gastric fundus was found (yellow arrows). d microscopic studies performed after surgical removal of the lesion demonstrated high amount of mesenchymal fusiform cells combined with vascular structures. axial and coronal reformations of contrast - enhanced ct on portal (a, b) and 8 min after injection delayed acquisitions (c, d). pararectal solid and well - defined mass with moderated peripheral enhancement adjacent to the rectum and left seminal vesicle was detected. mesenchymal neoplasm composed of polygonal cells with clear cytoplasm and round normochromatic nuclei with abundant vessels and occasional scattered inflammatory cells was observed. mixed within the neoplasm cells, there was a chronic inflammatory infiltrate of lymphoid cells. 12pathology : metastatic imt of the appendix. a 63-year - old man with antecedents of left hemicolectomy due to colon cancer. a axial reformation of contrast - enhanced ct on portal phase acquired seven years after partial colectomy. an appendicular, irregular, solid mass with slight enhancement was identified (yellow arrow). due to its radiological features, b axial reformation of contrast - enhanced ct on portal phase performed 8 months later showed a significant growth of the appendicular mass with extension and infiltration of the adjacent small bowel loops. axial reformation of contrast - enhanced ct on arterial (a), portal (b) and 3 min after injection delayed (c) acquisitions. a well - defined, heterogeneous nodule with moderate enhancement on portal phase arising from the gastric fundus was found (yellow arrows). d microscopic studies performed after surgical removal of the lesion demonstrated high amount of mesenchymal fusiform cells combined with vascular structures. the lesion showed well - defined contours and an expansive growth pattern pathology : pararectal imt. axial and coronal reformations of contrast - enhanced ct on portal (a, b) and 8 min after injection delayed acquisitions (c, d). pararectal solid and well - defined mass with moderated peripheral enhancement adjacent to the rectum and left seminal vesicle was detected. mesenchymal neoplasm composed of polygonal cells with clear cytoplasm and round normochromatic nuclei with abundant vessels and occasional scattered inflammatory cells was observed. mixed within the neoplasm cells, there was a chronic inflammatory infiltrate of lymphoid cells. there were no foci of necrosis or mitotic figures pathology : metastatic imt of the appendix. a 63-year - old man with antecedents of left hemicolectomy due to colon cancer. a axial reformation of contrast - enhanced ct on portal phase acquired seven years after partial colectomy. an appendicular, irregular, solid mass with slight enhancement was identified (yellow arrow). due to its radiological features, b axial reformation of contrast - enhanced ct on portal phase performed 8 months later showed a significant growth of the appendicular mass with extension and infiltration of the adjacent small bowel loops. c distant lymphadenopathy and liver metastases were present the differential diagnosis of these tumours includes gist, inflammatory fibroid polyp, smooth muscle neoplasm, peripheral nerve sheath tumour, solitary fibrous tumour, fibromatosis, the follicular dendritic cell sarcoma, lymphoma and adenocarcinomas. in most of these cases imts of the gastrointestinal tract present a higher recurrence rate than in other locations of the body. other treatments, such as steroids, non - steroidal anti - inflammatory drugs and thalidomide, have been used in these tumours. radiotherapy may have a place as adjunctive therapy in local recurrence and incomplete surgical removal. imts of the appendix are extremely rare, with few cases reported in the literature [9, 33, 34 ]. chronic infections, antecedents of trauma or surgery have been proposed as possible aetiological factors. extremely rare cases of imts of the appendix showing local aggressiveness and metastases have been reported (fig. many different aetiological factors have been proposed for mesenteric imts [37, 38 ], such as chronic infections, autoimmune diseases and trauma. on contrast - enhanced ct they usually appear as solid masses with heterogeneous enhancement pattern and ill- or well - defined margins. they are often associated with a general inflammatory response manifested with fever, weight loss and other symptoms that are related to the compressive effect of the tumour, such as abdominal pain and vomiting. rapidly growing tumours or symptomatic ones needing treatment may follow from small tumours which are not encroaching any nearby structures. different treatments have been proposed : non - steroidal anti - inflammatories, anti - oestrogens, chemotherapeutic agents and surgery. surgery remains as a useful modality although quite a high local recurrence rate has been described for tumours [3, 37, 38 ]. the symptoms that most often appear include pain, haematuria, dysuria or urinary tract obstruction. the radiological features do not help to distinguish them from malignant neoplasms, such as rabdomyosarcomas (fig., fine - needle biopsy may fail to yield a sufficient volume of tumour tissue to achieve diagnosis.fig. a axial and b coronal reformations of contrast - enhanced ct where an ill - defined and heterogeneous mass encompassing iliopsoas muscle and external iliac vessels, mimicking malignant sarcomatous neoplasm was observed. proliferation of fibroblast with a diffuse infiltration of lymphocytes, eosinophils and macrophages were found. the mass was well - defined and no signs of invasiveness were found pathology : imt of the iliopsoas muscle. a axial and b coronal reformations of contrast - enhanced ct where an ill - defined and heterogeneous mass encompassing iliopsoas muscle and external iliac vessels, mimicking malignant sarcomatous neoplasm was observed. proliferation of fibroblast with a diffuse infiltration of lymphocytes, eosinophils and macrophages were found. no mitotic figures or cellular atypia were present. imts have been reported in various sites in the head and neck such as bucal space, maxillary sinus, nasal cavity, parotid gland, nasopharynx and larynx (fig. 1pathology : imt in the left supraglottic space (arrow) with paraglottic space involvement indicated by replacement of the paraglottic fat with soft tissue. a axial reformation of contrast - enhanced neck ct. a solid, well - defined, normal tissue was present in the peripheral zone () pathology : imt in the left supraglottic space (arrow) with paraglottic space involvement indicated by replacement of the paraglottic fat with soft tissue. a axial reformation of contrast - enhanced neck ct. a solid, well - defined, little - enhancing nodule in the supraglottic larynx was observed (yellow arrow). b microscopic study obtained after surgical removal of the lesion. the sample showed proliferation of fusiform cells mixed with macrophages and giant multinucleated cells. normal tissue was present in the peripheral zone () however, the orbit is the most common location of imts. they account for approximately 10 % of orbital masses, being the third most common cause of orbital masses. their aetiology is unknown, but they have been associated with inmunological disorders, different infections and history of trauma. recently, they have been strongly associated with the igg4-related sclerosing systemic inflammatory disease and with chronic infection by epstein - barr virus. the clinical presentation and symptoms may vary with the location, extension and aggressiveness of the tumour. patients with imts of the orbit can present local symptoms as pain, redness, oedema, proptosis, ptosis, oculomotor deficits, diplopia, lid swelling or mass - effect and systemic symptoms due to the general inflammatory disorders they are frequently associated with. mri is superior to ct in the evaluation of inflammation of the nerves and muscles. they usually appear as solid and heterogeneous enhancing masses, which can affect retro bulbar fat, cause bone destruction, and present intracranial extension. when they show low intensity on t2-weighted images prominent, fibrotic composition is present (fig. a, b axial and coronal mr reformations of the same patient on t2-weighted sequences showed a well - delimited, hyperintense intraorbital mass, in the intraconal compartment, in the medial aspect. c, d t1-weighted images demonstrated a hypointense, homogeneous, well - delimited mass with peripheral enhancement after gadolinium injection. f microscopic sample obtained after surgery demonstrated an epithelial tumour composed of fusiform cells mixed with an extensive chronic inflammatory infiltrate of plasmatic cells, lymphocytes and macrophages. in the peripheral of the sample a, b axial and coronal mr reformations of the same patient on t2-weighted sequences showed a well - delimited, hyperintense intraorbital mass, in the intraconal compartment, in the medial aspect. c, d t1-weighted images demonstrated a hypointense, homogeneous, well - delimited mass with peripheral enhancement after gadolinium injection. f microscopic sample obtained after surgery demonstrated an epithelial tumour composed of fusiform cells mixed with an extensive chronic inflammatory infiltrate of plasmatic cells, lymphocytes and macrophages. in the peripheral of the sample striated muscle cells were also observed pathologically, imts of the head and neck consist of many fibroblastic cells, fibroblastic cells and inflammatory cells, which include plasma cells and eosinophils. the differential diagnosis includes granulomatous diseases, primary infection, sarcoid, sjgren disease, primary and secondary tumours of the orbit, lymphoma and connective tissue diseases. the administration of corticosteroids an inmunosupresors usually leads to a decrease of size of the mass but also radiotherapy or surgery may be indicated.. they account for around the 50 % of benign pulmonary masses in children, representing only about 0.7 % of all tumours of the lung in general population. as in other organs, they are also associated with immunological disorders and chronic infections. sometimes, imts are also related with antecedents of lung surgery and they can arise in surgical lung scars. common clinical characteristics include unspecific respiratory symptoms, such as cough, chest pain, dyspnea, haemoptysis and unspecific inflammatory symptoms as fever, malaise and weight loss. they usually appear as solitary, well - circumscribed peripheral lung masses, with predominance for the lower lobes (fig. on ct with intravenous contrast, they present a variable heterogeneous or homogeneous degree of enhancement pattern [3, 21 ]. it has been described an aggressive behaviour with invasion of the adjacent structures. on mri because of their similar radiological appearance to malignant lung masses, biopsy is necessary to obtain the diagnosis.fig. a 55-year - old man with a cough and haemoptysis.a axial reformation of non - contrast ct with mediastinum window. two solid, ill - defined nodules in right lower lobe are observed (yellow arrows). b axial reformations with lung window of the same patient where the lung nodules are shown (white arrows). c partial lobectomy of the right lower lobe specimen. note the presence of the nodules (). d microscopic study of the resected lung sample where fusiform cells with an associated inflammatory infiltrate of lymphocytes, plasmatic cells and histiocytes was found pathology : imt of the lung. a 55-year - old man with a cough and haemoptysis.a axial reformation of non - contrast ct with mediastinum window. two solid, ill - defined nodules in right lower lobe are observed (yellow arrows). note the air brocogram observed in the biggest mass. b axial reformations with lung window of the same patient where the lung nodules are shown (white arrows). d microscopic study of the resected lung sample where fusiform cells with an associated inflammatory infiltrate of lymphocytes, plasmatic cells and histiocytes was found the treatment of choice of lung imts is surgery to exclude malignancy and to achieve the cure. although spontaneous regression may occur, local expansion may cause significant morbidity and occasional death. the prognosis after complete surgical resection is excellent, with a low rate of recurrence. in these cases, alternative treatments (radiotherapy, corticoids or chemotherapy) to surgery could be also used. the most common presentation of epididymal (fig. 4) and paratesticular imts is a lump in the scrotum.fig. 4pathology : imt of the epididymus. a 40-year - old man with a lump in the scrotum. a a hypoechoic a diffuse infiltrate composed of fibroblastic and fusiform cells mixed with inflammatory cells (mainly lymphocytes and macrophages) was observed pathology : imt of the epididymus. a 40-year - old man with a lump in the scrotum. a a hypoechoic, well - delimited epididymal lesion in the right scrotum was detected. a diffuse infiltrate composed of fibroblastic and fusiform cells mixed with inflammatory cells (mainly lymphocytes and macrophages) was observed in the evaluation of scrotal masses, us is the most accurate imaging technique, helping to distinguish intratesticular from extratesticular lesions and solid from cystic lesions. on ct, they appear as well - defined, hypodense masses with little enhancement after intravenous contrast injection. in some cases, a 34-year - old man with no relevant medical history came to our hospital with a lump in the scrotum. a us showed an isoechoic, solid, paratesticular nodule with fine calcifications (yellow arrow). b axial reformation of contrast - enhanced ct on portal phase demonstrated a well - delimited, heterogeneous, little - enhancing mass in the right scrotum. d microscopic study of the lesion demonstrated a well - delimited lesion constituted of fibroblasts, lymphocytes and plasmatic cells with associated lymphoid follicle. a 34-year - old man with no relevant medical history came to our hospital with a lump in the scrotum. a us showed an isoechoic, solid, paratesticular nodule with fine calcifications (yellow arrow). b axial reformation of contrast - enhanced ct on portal phase demonstrated a well - delimited, heterogeneous, little - enhancing mass in the right scrotum. d microscopic study of the lesion demonstrated a well - delimited lesion constituted of fibroblasts, lymphocytes and plasmatic cells with associated lymphoid follicle. there was no evidence of adjacent testicle invasion surgical resection without orchiectomy is the ideal treatment, but in some cases the testicle must be removed due to the size of the mass or when the mass is attached to the testis. imts of the penis are extremely rare. to our knowledge, no imts at this location have been reported. we have recorded only one case in our institution (fig. 6). on ct, this tumour appeared as a well - defined, little - enhancing and non - aggressive mass surrounding the penile urethra and the corpora cavernosa with no evidence of invasion of them.fig. a 46-year - old man with a mass in the penis came to our hospital. he related more than 6 years of slow growth of the mass with no suspicious associated symptoms. c, d axial and coronal reformations of enhanced - ct after the injection of intravenous contrast on portal phase. macroscopic view showed an heterogeneous aspect due to the mixture of tissues (solid tumour, lipoid component and areas of haemorrhage). f microscopic study confirmed a mesenchymal tumour composed of fusiform cells, some of them multinucleated, with round or elongate nucleus. a 46-year - old man with a mass in the penis came to our hospital. he related more than 6 years of slow growth of the mass with no suspicious associated symptoms. c, d axial and coronal reformations of enhanced - ct after the injection of intravenous contrast on portal phase. macroscopic view showed an heterogeneous aspect due to the mixture of tissues (solid tumour, lipoid component and areas of haemorrhage). f microscopic study confirmed a mesenchymal tumour composed of fusiform cells, some of them multinucleated, with round or elongate nucleus. no mitotic figures were observed surgical treatment was performed due to the size and non - conclusive radiological features of the tumour. hepatic imts constitute an extremely rare group of hepatic tumours, accounting only for the 0.7 % of all the hepatic lesions. they have been associated with autoimmune diseases, immunological disorders and viral infections, such as igg4 disease, sclerosing cholangitis, crohns disease, hepatic viral infections and epstein - barr virus. on contrast - enhanced ct, they usually show heterogeneous or peripheral enhancement during the arterial phase. despite this, some of them present homogeneous enhancement during the arterial phase and washout during the delayed phase, which can lead to misdiagnosing them as hepatocarcinomas (hccs) (fig. 7). on mri, these lesions usually are t1 hypointense and t2 hyperintense with heterogeneous enhancement (fig. 8). hepatobiliary contrast (gadoxetate disodium, primovist ; bayer healthcare, leverkusen, germany) could be of help to differentiate hcc from imts of the liver. on the hepatobiliary phase, high signal intensity in the centre of the lesion has been described, suggesting a benign lesion.fig. well - defined, hypointense lesion on t1-weighted images (a) which presented weak enhancement in the arterial phase (b) and wash - out on portal and equilibrium phases (c-, d) located in hepatic left lobe. f the lesion demostrated an expansive chronic inflammatory infiltrate of plasmatic cells, lymphocytes and macrophages. a abdominal ultrasound where a slightly hypoechoic, solid, heterogeneous mass arising in the left hepatic lobe was observed. axial t1-weighted unenhanced (b) and contrast - enhanced mr images (c, d) showed an ill - defined with heterogeneous enhancement mass located in lateral hepatic segments. mesenchymal tumour composed of fusiform cells, lymphocytes and lipid - filled macrophages can be observed. normal hepatocytes with large and small fatty droplet change are also present pathology : imt of the liver. well - defined, hypointense lesion on t1-weighted images (a) which presented weak enhancement in the arterial phase (b) and wash - out on portal and equilibrium phases (c-, d) located in hepatic left lobe. e macroscopic view of partial hepatectomy obtained after surgical excision of the lesion. a solid, well - defined and yellowing appearance mass is showed. f the lesion demostrated an expansive chronic inflammatory infiltrate of plasmatic cells, lymphocytes and macrophages. a abdominal ultrasound where a slightly hypoechoic, solid, heterogeneous mass arising in the left hepatic lobe was observed. axial t1-weighted unenhanced (b) and contrast - enhanced mr images (c, d) showed an ill - defined with heterogeneous enhancement mass located in lateral hepatic segments. mesenchymal tumour composed of fusiform cells, lymphocytes and lipid - filled macrophages can be observed. normal hepatocytes with large and small fatty droplet change are also present surgery is the most accepted treatment. if there is a biopsy - proven diagnosis of imts that excludes malignancy, medical treatment with no steroidal anti - inflammatory drugs in patients with peripheral hepatic imts could be prescribed. patients with spleen imts frequently associate unspecific symptoms as left upper quadrant or epigastric pain. splenomegaly is common and some patients can present fever, anaemia and signs of hypersplenism. different radiological imaging modalities have been proposed for their evaluation. on all of them, they are depicted as low - density masses in both non - enhanced and enhanced acquisitions (fig. their typical enhancement pattern after gadolinium injection is as a low - intensity lesion in the early phase, which changes into a high - intensity one in the delayed phase.fig. 9pathology : imt of the spleen. a axial reformation of contrast - enhanced ct on portal phase. prominent cellularity composed of an irregular, weakly basophil set of mioepithelial - fusiform cells. numerous inflammatory cells (plasmatic, lymphocytes and few neutrophils and eosinophils) with isolated macrophages were also found. no atypia or mitotic figures were present pathology : imt of the spleen. a axial reformation of contrast - enhanced ct on portal phase. prominent cellularity composed of an irregular, weakly basophil set of mioepithelial - fusiform cells. numerous inflammatory cells (plasmatic, lymphocytes and few neutrophils and eosinophils) with isolated macrophages were also found. no atypia or mitotic figures were present the differential diagnosis includes splenic haemangioma and other primary splenic neoplasms such as lymphoma or splenic angiosarcoma. when a suspicious spleen mass is found, the treatment of choice is splenectomy due to the high risk of bleeding associated to splenic biopsy [8, 31 ]. they can arise anywhere, the stomach and small intestine being the most common locations [3, 9 ]. clinical symptoms may vary from none to abdominal pain, intestinal obstruction, dysphagia or anaemia, depending on the location and size of the mass. contrast - enhanced ct is the imaging technique, which give us more information about of imts of the gastrointestinal tract. they are usually found as hypodense, heterogeneous and well - delimited masses (figs. axial reformation of contrast - enhanced ct on arterial (a), portal (b) and 3 min after injection delayed (c) acquisitions. a well - defined, heterogeneous nodule with moderate enhancement on portal phase arising from the gastric fundus was found (yellow arrows). d microscopic studies performed after surgical removal of the lesion demonstrated high amount of mesenchymal fusiform cells combined with vascular structures. axial and coronal reformations of contrast - enhanced ct on portal (a, b) and 8 min after injection delayed acquisitions (c, d). pararectal solid and well - defined mass with moderated peripheral enhancement adjacent to the rectum and left seminal vesicle was detected. e macroscopic view of the tumour after surgical resection. f histological sample obtained after biopsy. mesenchymal neoplasm composed of polygonal cells with clear cytoplasm and round normochromatic nuclei with abundant vessels and occasional scattered inflammatory cells was observed. mixed within the neoplasm cells, there was a chronic inflammatory infiltrate of lymphoid cells. 12pathology : metastatic imt of the appendix. a 63-year - old man with antecedents of left hemicolectomy due to colon cancer. a axial reformation of contrast - enhanced ct on portal phase acquired seven years after partial colectomy. an appendicular, irregular, solid mass with slight enhancement was identified (yellow arrow). due to its radiological features, b axial reformation of contrast - enhanced ct on portal phase performed 8 months later showed a significant growth of the appendicular mass with extension and infiltration of the adjacent small bowel loops. axial reformation of contrast - enhanced ct on arterial (a), portal (b) and 3 min after injection delayed (c) acquisitions. a well - defined, heterogeneous nodule with moderate enhancement on portal phase arising from the gastric fundus was found (yellow arrows). d microscopic studies performed after surgical removal of the lesion demonstrated high amount of mesenchymal fusiform cells combined with vascular structures. the lesion showed well - defined contours and an expansive growth pattern pathology : pararectal imt. axial and coronal reformations of contrast - enhanced ct on portal (a, b) and 8 min after injection delayed acquisitions (c, d). pararectal solid and well - defined mass with moderated peripheral enhancement adjacent to the rectum and left seminal vesicle was detected. mesenchymal neoplasm composed of polygonal cells with clear cytoplasm and round normochromatic nuclei with abundant vessels and occasional scattered inflammatory cells was observed. mixed within the neoplasm cells, there was a chronic inflammatory infiltrate of lymphoid cells. there were no foci of necrosis or mitotic figures pathology : metastatic imt of the appendix. a 63-year - old man with antecedents of left hemicolectomy due to colon cancer. a axial reformation of contrast - enhanced ct on portal phase acquired seven years after partial colectomy. an appendicular, irregular, solid mass with slight enhancement was identified (yellow arrow). due to its radiological features, b axial reformation of contrast - enhanced ct on portal phase performed 8 months later showed a significant growth of the appendicular mass with extension and infiltration of the adjacent small bowel loops. c distant lymphadenopathy and liver metastases were present the differential diagnosis of these tumours includes gist, inflammatory fibroid polyp, smooth muscle neoplasm, peripheral nerve sheath tumour, solitary fibrous tumour, fibromatosis, the follicular dendritic cell sarcoma, lymphoma and adenocarcinomas. in most of these cases imaging features are not enough to diagnose imts and biopsy or surgery are needed. imts of the gastrointestinal tract present a higher recurrence rate than in other locations of the body. other treatments, such as steroids, non - steroidal anti - inflammatory drugs and thalidomide, have been used in these tumours. radiotherapy may have a place as adjunctive therapy in local recurrence and incomplete surgical removal. imts of the appendix are extremely rare, with few cases reported in the literature [9, 33, 34 ]. chronic infections, antecedents of trauma or surgery have been proposed as possible aetiological factors. extremely rare cases of imts of the appendix showing local aggressiveness and metastases have been reported (fig. many different aetiological factors have been proposed for mesenteric imts [37, 38 ], such as chronic infections, autoimmune diseases and trauma. on contrast - enhanced ct they usually appear as solid masses with heterogeneous enhancement pattern and ill- or well - defined margins. they are often associated with a general inflammatory response manifested with fever, weight loss and other symptoms that are related to the compressive effect of the tumour, such as abdominal pain and vomiting. rapidly growing tumours or symptomatic ones needing treatment may follow from small tumours which are not encroaching any nearby structures. different treatments have been proposed : non - steroidal anti - inflammatories, anti - oestrogens, chemotherapeutic agents and surgery. surgery remains as a useful modality although quite a high local recurrence rate has been described for tumours [3, 37, 38 ]. the symptoms that most often appear include pain, haematuria, dysuria or urinary tract obstruction. the radiological features do not help to distinguish them from malignant neoplasms, such as rabdomyosarcomas (fig., fine - needle biopsy may fail to yield a sufficient volume of tumour tissue to achieve diagnosis.fig. a axial and b coronal reformations of contrast - enhanced ct where an ill - defined and heterogeneous mass encompassing iliopsoas muscle and external iliac vessels, mimicking malignant sarcomatous neoplasm was observed. proliferation of fibroblast with a diffuse infiltration of lymphocytes, eosinophils and macrophages were found. no mitotic figures or cellular the mass was well - defined and no signs of invasiveness were found pathology : imt of the iliopsoas muscle. a axial and b coronal reformations of contrast - enhanced ct where an ill - defined and heterogeneous mass encompassing iliopsoas muscle and external iliac vessels, mimicking malignant sarcomatous neoplasm was observed. proliferation of fibroblast with a diffuse infiltration of lymphocytes, eosinophils and macrophages were found. no mitotic figures or cellular although imts in some organs are not uncommon, they are not usually included in the differential diagnosis of nodules and masses. consequently, this is an underdiagnosed entity and only after an histological exam could definitive diagnosis be achieved. it is fundamental for radiologists to know about the existence of imts, as they frequently have better treatment options and prognosis than the malignant neoplasms they are confused with. | objectivesto present the most important characteristics of inflammatory myofibroblastic tumours (imts) arising in different locations of the body with histological correlation.methodsto review the symptoms and main radiological findings of imts. on ultrasonography (us), these tumours can appear as hypoechoic or hyperechoic masses and a variable doppler appearance with increased vascularity. computed tomography (ct) and magnetic resonance (mr) are the most used imaging tools in their evaluation. on contrast - enhanced ct, imts can appear as homogeneous or heterogeneous lesions, with variable enhancement on delayed acquisitions due to fibrosis. these findings are also present on gadolinium contrast - enhanced mr. on t1-weighted and t2-weighted sequences, imts usually show low signal intensity reflecting also the presence of fibrotic tissue.resultsto show the main clinical symptoms and radiological features of imts in different locations : head and neck, lung, genitourinary, hepatic, splenic, gastrointestinal tract, mesenteric, muskuloskeletal.conclusionsalthough imts in some organs are not uncommon, they are not usually included in the differential diagnosis of masses. their radiological features suggest malignant neoplasms, whereas they are not. consequently, this is an underdiagnosed entity and only after an histological exam could a definitive diagnosis be achieved.teaching points their radiological features suggest malignant neoplasms, whereas they are not ct and mr imaging are the most used tools in their evaluation imt is an underdiagnosed entity the definitive diagnosis is only after histological exam |
the cyanine - dye labeled nucleotide triphosphates cy3- and cy5-dctp are among the standard labeling reagents used in microarray and other multicolor hybridization applications due to their bright, photostable and spectrally resolved fluorescence. however, cy3- and cy5-dctp (figure 1b) proved exceedingly difficult polymerase substrates at full substitution, and initial attempts at selection from previously described polymerase repertoires were unsuccessful (not shown). b - family polymerases had previously been identified as having favorable properties with respect to the incorporation of substituted nucleotides. we chose the polb polymerase from pyrococcus furiosus (pfu),(36) which is widely used in pcr applications as it proved relatively straightforward to clone and express in e. coli k12 strains with only mild toxicity (unlike, e.g., other polbs such as vent or 9n (not shown)). due to the relative ease of high efficiency transformation of k12 strains compared to b - strains (like the commonly used bl21), pfu also displayed favorable properties in model csr selections but only after inclusion of several additives (dtt, bsa, formamide (fa), rnasea, see the supporting information, materials & methods) to improve compatibility with classical span80-based w / o - emulsions. presumably these serve i.a. to prevent oxidation of free cysteine thiols in the pfu structure by peroxide contaminants present in the span80 surfactant (dtt),(37) to enhance replication and reduce interference from bulk cellular nucleic acids (fa, rnase) and to buffer interaction of the polymerase with the hydrophobic surfactant shell (bsa). to enhance incorporation of unnatural nucleotides and increase processivity we also disabled the 3-5 exonuclease-(38) and uracil - stalling functions(39) yielding pfu variant (v93q, d141a, e143a), henceforth referred to as pfuexo-, as a scaffold for library construction and spcsr seicction. we anticipated that accommodation of the bulky cy - labeled nucleotide analogues would require remodeling of the polymerase active site. in order to improve accommodation of the dye - labeled substrate we initially targeted the a - motif(40) (and adjacent residues), a conserved sequence motif in the polymerase active site (figure 2a) involved in both catalysis and recognition of the incoming deoxinucleotide triphosphate (dntp) substrate. we diversified the sequence region of e399-i415 using a combination of random mutagenesis (avoiding the catalytic aspartate d405) of the central a - motif and synthetic shuffling(41) (i.e., encoding phylogenetic diversity) of the immediate flanking sequences (figure 2b). polymerases were selected for self - replication with complete replacement of dctp with cy5-dctp by spcsr, requiring the replication of 460 nucleotides (nts) for each strand and the incorporation and replication of 191 cy5-dcs in round 1. (a) structural model of the active site of a polb - family polymerase (rb69, pdb : 1ig9(42)). the polypeptide chain is shown as a ribbon diagram overlaid with a transparent surface model. primer and template strands are shown in orange and purple, incoming nucleotide triphosphate in elemental colors, and gray spheres represent the two catalytic mg ions. regions corresponding to the a - motif are colored yellow, and those for the c - motif cyan. (b) for round 1 selection, diversity was focused on the a - motif and vicinity (399415) comprising random mutation spike at the core of the a - motif (lime) as well as phylogenetic diversity in the adjacent sequences (purple). for round 2, successful clones from round 1 (e.g., 15) were diversified in the c - motif region (cyan), and selected for replication of a- and c - motif (399546) yielding polymerase e10 (selected mutations in red). 380 selected variants from round 1 were screened by polymerase - elisa(34) and ranked for their ability to incorporate 4 consecutive cy5-dctps or cy3-dctps. polymerase - elisa identified 4 mutant polymerases with significantly enhanced ability to incorporate either cy3-dctps or cy5-dctps compared with wild - type pfuexo- : a23 (n400d, i401l, r407i), ah12 (e399d, n400 g, i401l, v402a, r407i, q572h), 55 (n400 g, r407i) and in particular 15 (v337i, e399d, n400 g, r407i) (figure 3a). (a) polymerase elisa. shown are the hairpin substrate and activities of round 1 clones (15, a23, 55, ah12) and round 2 clones (9, 10, e10, 23). synthesis through the g8 template stretch was detected by incorporation of digoxigenin-11-dutp.(34) (b) page gel of a 0.4 kb pcr amplification (70% gc) with complete replacement of dctp with either cy5- or cy3-dctp comparing selected clones to wild - type pfuexo- (pfu). only e10 (and to a lesser extent a3) are able to perform pcr amplification with both dyes. visible spectrum absorption by the 287 cyanine dyes incorporated (on both strands) colors the dna fragment brightly pink (cy3) or blue (cy5). these four clones were diversified in another region of the polymerase active site in proximity to the incoming dntp substrate (v537y546) comprising the conserved c - motif region,(40) which together with the a - motif harbors the catalytic triad of aspartates (d405, d541, d543), which are essential for catalysis and substrate binding (figure 2). we again used a combination of spiked random mutation of conserved residues (excluding the catalytically important aspartates (d541, d543)) and synthetic shuffling(41) of phylogenetically diverse flanking sequences to optimize the percentage of active clones in our library (figure 2b). for round 2, polymerase variants were selected for the ability to amplify a much larger, more challenging 780bp fragment encompassing both a- and c - motif diversity and requiring the incorporation and replication of 312 cy5-dcs on both strands. selected clones were ranked by polymerase - elisa for their ability to incorporate 8 consecutive cy5-dctps or cy3-dctps. these were found to be highly uniform, with mutations at a single residue (y546) predominating (either y546h or y546l (20/20)). in both elisas we not only screened for the ability to incorporate cy5-dctp or cy3-dctp but also for the ability to use them both with comparable efficiency. as with all directed evolution experiments, spcsr also yielded a significant number of specialists, i.e. polymerases that had selectively adapted only to the selection substrate cy5-dctp and were unable to incorporate cy3-dctp with comparable efficiency (not shown). only clones fulfilling these criteria (e.g., 9, 10, e10, 23, figure 3a) were expressed, purified, and screened for the ability to pcr amplify dna fragments with full replacement of dctp by cy3- or cy5-dctp in the pcr mix. the best clones were challenged with the amplification of a 70% gc 0.4 kb fragment comprising 289 fluorophore dyes on both strands and resulting products were resolved by polyacrylamide gel electrophoresis (page) (figure 3b). this screen identified pfu variant e10 (v337i, e399d, n400d, r407i, y546h) (figure 2b) as the most promising polymerase capable of processive synthesis of brightly colored, highly fluorescent dna in pcr, in which every dc is replaced by cy3- or cy5-dc (henceforth called cydna). expansion of the substrate spectrum of a polymerase can be accompanied by reduced fidelity, in particular when it involves the accommodation of a bulky, distorting substrate in the active site.(43) we therefore determined the fidelity of e10 in pcr both with standard dntps as well as with complete replacement of dctp by either cy3- or cy5-dctp by sequencing of amplification products and compared it to the fidelity of the parent pfuexo- enzyme (table 1). corrected for the number of doublings (pcr cycles). as determined by a lacz reversion assay.(44) in the absence of additives (1% formamide, 10% glycerol, 10 g sequencing of amplification products from both e10 and pfuexo- revealed that the enhanced incorporation and replication of bulky fluorescent dye substituted nucleotides by e10 does not appear to significantly impair fidelity with standard dntps. the error rate of e10 (4 10 per base - pair (bp)) was comparable to that measured for pfuexo- (1.1 10 per bp). similar results were independently obtained using a well - established lacz forward mutation assay (m. arana, ph, t. kunkel, unpublished results). however, unlike pfuexo-, the e10 error rate was dependent on the inclusion of the additives (formamide, glycerol, rnase, dtt) used in the selection of e10 and increased more than 6-fold when they were omitted. replacement of dctp by cy3- or cy5-dctp in pcr resulted in an error rate (4.9 10 per bp (cy5-dctp)) approximately 2-fold higher than that of pfuexo- (2.4 10 per bp) utilizing standard dntps under the same conditions (with dc da or dt mutations predominating (not shown)). when corrected for the number of total pcr cycles, the error rates for both pfuexo- and e10 for pcr with standard dntps and for e10 for pcr with replacement dctp by cy3- or cy5-dctp are in excellent agreement with previously obtained measurements for the error rate of pfuexo- using a laci reversion assay (table 1).(44) these mild effects on polymerase fidelity are not unexpected as csr inherently selects for polymerase fidelity. all self - replication must proceed with a minimal fidelity, as first recognized by manfred eigen and formulated as the error threshold, defining the minimal level of replication fidelity below which genetic information would be irretrievably corrupted.(45) in other words, polymerases that are too error - prone will introduce too many mutations into their own gene during csr with mostly deleterious effects on the fitness of their offspring. it is clear that this error threshold is to some degree context dependent. in the present case, where self - replication includes regions in the polymerase active site that are absolutely essential for catalysis, particularly stringent selection for fidelity would be expected. high - density display of the large cyanine dye heterocycles on the dna scaffold significantly altered the physicochemical properties of resulting dna (cydna). while cydna could still be by resolved by gel electrophoresis (figure 3b), unlike normal dna it displayed almost no binding to silica resins,(46) did not stain with ethidium bromide, and partitioned to the organic phase in a phenol extraction (figure 4a). (a) organic phase partitioning of cydna is shown for cy3-dna (left) and cy5-dna (right). essentially 100% partioning occurs in the presence of 150 mm nacl (the yellow color of the phenol phase is due to addition of 8-hydroxyquinoline to prevent oxidation). both cy3- and cy5-dna are resistant to cleavage by the restriction endonuclease ddei but are cut by msei. cy5-dna displays no fluorescence despite ethidium bromide staining, while cy3-dna fluorescence is due to uv excitation (and seen to the same extent in ethidium bromide free gels (not shown)). we therefore wondered if cydna would still adopt a canonical double helical structure or if the presence of the heterocyclic dyes had promoted a structural transition. to probe cydna structure we initially used restriction endonucleases, which are sensitive probes of noncanonical dna conformations such as those which occur under torsional strain.(47) indeed, some like ddei (ctnag) failed to cut cydna, presumably due to steric hindrance by bulky cyanine dyes, while others, notably msei (ttaa), cut cydna efficiently (figure 4b). this indicates that at least the local regions of at - sequence in cy - dna adopt a canonical b - form conformation. to get a more global picture of cydna conformation, we used atomic force microscopy (afm) imaging of single cydna molecules. afm unequivocally revealed contours consistent with a double - stranded conformation over the whole length of the cydna fragments with only minor deviations (< 7%) in both average contour length and stiffness (persistence length) compared to canonical b - form dna (figure 5, supporting information, table 1). however, afm also revealed a substantially increased contour height of cydna that was increased by up to 40% compared to native dna for cy5-dna (figure 5), presumably due to dense packing of fluorophore heterocycles on the outside of the dna helix. atomic force microscopy (afm) imaging of mica - adsorbed identical dna (left), cy3-dna (middle), and cy5-dna (right) fragments. a putative dna or cydna molecule can be seen at the center of each image. histogram of afm contour heights for n = 40 molecules for dna (black), cy3-dna (red), and cy5-dna (green). contour heights (width) of cy3-dnas or cy5-dnas were 20% or 40% respectively larger than dna, while contour lengths were unchanged. high - density fluorophore labeling of dna probes should translate into high photon yields, increasing the sensitivity and expanding the dynamic range in fluorescent hybridization applications. having established a methodology for the synthesis of cydna displaying a fluorophore at every dc base, we went on to assess whether such high - density fluorophore labeling of dna probes was still compatible with specific target hybridization. we first performed model microarray experiments with randomly spotted targets interspersed with nonspecific targets (salmon - sperm dna). these experiments revealed highly specific binding to target dna with bright fluorescence and no nonspecific binding to either the glass surface or salmon - sperm dna (figure 6). specific binding of cydna measured by a model array comprising a dilution series (20012.5 ng/l) of probe molecules (pfu sequence and sheared salmon sperm dna. specific hybridization of cydna is evident with no binding to the slide surface or salmon genomic dna. next we examined if the high - density labeling of dna probes using e10 would translate into a higher fluorescence signal. when we compared fluorescent cy - probes synthesized by e10 with those synthesized by pfuexo- with 10% cy3 and cy5-dctp (the maximum tolerated by pfuexo-), we obtained an up to 4.5-fold increased fluorescence signal from the e10 probes. surprisingly, we found that fluorescence was affected by labeling densities and was, in fact, improved by reducing labeling densities from 100% to 50% for cy5-dctp (figure 7) and by white - light excitation (not shown). the effect is likely to be due to reabsorption and re - emission effects, causing red - shifting of the excitation and emission maxima in cydna probes. scatter plot of fluorescence signals from cohybridization of dna fragments labeled with cy3- or cy5-dctp using pfuexo- (red) or e10 (blue, green) on the model array (figure 6). fragments labeled using e10 display an up to 4.5-fold higher fluorescence signal for cy3 (blue) or up to 2.5-fold higher fluorescence signal for cy5 (green). high - density labeling (100%) quenches cy5 (but not cy3) fluorescence and reverses signal gains. melting temperature analysis of cydna revealed a slightly lower tm (0.1 kb cydna fragment (70% gc) 70 cy - dyes) : cy3-dna : 90 c, cy5-dna : 88 c) compared to native dna (dna : 91 c)as previously observed with highly cy3-du modified oligonucleotide probes(48)as well as slightly less cooperative melting behavior (supporting information figure 1). we therefore modified hybridization temperatures and wash stringencies for future microarray experiments accordingly (see materials and methods). when we compared fluorescence signals of state - of - the art commercial microarray probes (generated by random priming with klenow polymerase) with cydna probes generated using e10, we discovered probe length as another critical parameter affecting fluorescence signals. standard labeling protocols using klenow yield very short probe fragments (< 0.15 kb) due to aborted elongation at homopolymeric dc runs. in contrast, due to its proficiency at incorporating cy labeled dntps, e10 yields cydna probes up to 3 kb in length (supporting information, figure 2a). to determine the influence of probe length on relative fluorescence signal, we prepared dna probes of defined length and labeled them with cy3- and cy5-dctp using either klenow or e10. then we compared them in cohybridization experiments, whereby equivalent amounts of the same length cy3-klenow and cy5-e10 probes (and vice versa) are hybridized to the same array targets. indeed, we found that for short fragments e10 labeled probes gave on average an up to 32-fold higher fluorescence signal than klenow labeled probes, whereas for longer probes e10 yielded only a 24-fold gain (figure 8). the fluorescence signal of e10 labeled cydna (blue / teal) was measured relative to klenow labeled dna (lime / red) prepared from templates of the same length (270bp/1.3 kb) for cy3 (left panel) and cy5 (right panel). e10270 (teal) yielded a 34-fold higher cy3 signal (or 20-fold higher cy5 signal than klenow270 (red)) whereas the longer e101300 fragment (blue) resulted in only a 3.6-fold higher cy3 signal (2.5-fold higher cy5 signal) than klenow1300 (lime). boxes describe the interquartile range, lines across the box the median and central cross the average. error bars define the signal spread. while e10 labeled probes can provide enormous signal gains, fragment length is a crucial parameter. to realize more of the potential signal gains of cydna for microarray applications we therefore developed an alternative enzymatic fragmentation method based on sporadic dutp (10%) incorporation, followed by uracil excision using uracil dna glycosylase (udg) and strand cleavage using human apurinic / apyrmidinic endonuclease i (ape-1), which cleaves the phosphodiester backbone of dna immediately 5 to the abasic site generated by udg. although still larger than klenow probes, the resulting cydna fragments were now consistently smaller than 200 nts and showed 68 fold higher fluorescence (compared to standard klenow labeled probes) in a model microarray experiment (figure 9a). (a) model array showing cohybridization of fragmented cydna (e10) with klenow labeled dna (k) : k cy3/e10 cy5 (left) ; k cy3/k cy5 (middle) ; e10 cy3/k cy5 (right). e10 yields 8- (cy3) or 6-fold (cy5) higher signals as evident by the red (e10 cy5 (left)) and green ((b) comparative genome hybridization array (acgh) of human genomic dna (m (male) vs f (female)). average fluorescence signal of acgh probes for cy3 m vs cy5f dna as well as dye - swap control (cy3f vs cy5 m) for klenow labeling, (klenow, blue), klenow or e10 labeling of adaptor linked dna (white (k), orange (e10)). next we applied our combined strategies to a 30000 feature comparative genome hybridization (cgh) array experiment. fragmentation of human genomic dna labeled with e10 proved more challenging and yielded larger probes (average ca. 300 nts (supporting information, figure 2b)) and consequently smaller yet still significant signal gains of up to 4-fold in the cgh array compared to state - of - the - art klenow labeling (figure 9b, supporting information, tables 2 and 3, and si figure 5). we anticipate that further optimization of cydna fragmentation protocols together with customized filter optics and light sources (see above) should realize significant further signal gains in microarray applications. we also attempted to observe single cydna molecules in motion within a microfluidic flowcell device calibrated using single - particle detection of quantum dots. cydna pcr products (0.27 kb) labeled with either 100% cy5-dctp (102 cy5) or 50% cy3-dctp (51 cy3), at near single molecule dilution, were coinjected into a fused silica capillary and imaged at a fixed point inside the lumen of the capillary. the bright fluorescence of cydna allowed us to observe cydna molecules as discrete peaks of cy3 and cy5 fluorescence, comparable in signal to that observed from single quantum dots (supporting information figure 3). while we can not completely exclude the possibility that we observe the simultaneous detection of multiple cydna molecules or aggregates thereof, we note that peaks with mixed cy3/cy5 intensity ratios were not observed. we further note that signal intensities were in accordance with the detection of molecules carrying the expected number of cy fluorophores consistent with major peaks corresponding to single cydna molecules in motion. dna modifications in which substituents are placed at the 5-position of either du or dc are well tolerated by polymerases. this places the substituent in the mayor groove of the double helix, where interference with helix structure or minor groove hydrogen bonding to the polymerase is comparatively minimal. indeed, the majority of the known natural dna modifications occur at the 5-position of the pyrimidine bases ranging from the well - known epigenetic markers 5-methylcytosine (and the recently discovered 5-hydroxymethylcytosine(49)) to bases bearing sugar, amino, or carbamoyl substituents (reviewed in ref (50)). they mostly occur sporadically but, remarkably, can completely replace the canonical bases in the case of some viral genomes, such as the t - even bacteriophages (t2, t4, t6) of e. coli, where dc is completely replaced by hexosylated 5-hydroxymethylcytosine, or some b. subtilis phages (e.g., sp8, spo1, 25), where dt is completely replaced by 5-hydroxymethyluracil. some of these modifications (e.g., -putrescinylthymine) are thought to improve packaging of the phage dna (phage w14, p. acidovorans(51)) into viral capsids by reducing charge density, in analogy to unnatural modifications such as zwitterionic dna.(52) others may serve to allow preferential replication of the modified genome by viral replicases,(50) but most alter the physicochemical properties of the viral dna and protect it from nuclease degradation. by analogy, cydna also displays altered properties, which render is resistant to cleavage by some endonucleases including bovine pancreatic dnase i. chemists have studied a great variety of unnatural c5-substituted nucleotides and many have been successfully incorporated into dna at low density. high - density incorporation, however, especially of deoxinucleotides bearing large (and charged) substituents, has remained challenging. to the latter group belong fluorescent dye - labeled deoxinucleotide triphosphates, which are essential components of several core technologies of modern biology including sequencing, microarrays, and fluorescent in situ hybridization (fish). despite systematic efforts to optimize substituent and linker chemistry, many dye labeled nucleotides have remained poor polymerase substrates, presumably due to steric incompatibility of the large substituents with the polymerase active site and/or primer - template duplex binding surface. indeed, while high - density fluorophore labeling of dna by polymerases has been reported in primer extension reactions, yields have generally been poor. a further challenge is presented by the replication of such polymers as the bulky substituents are now also present in the template strand, which can cause additional steric strain resulting in pausing or abortive synthesis.(16) in contrast, replication of nucleotide analogues functionalized with smaller substituents appears comparatively straightforward. the most advanced of these systems was described by jger and famulok, who, through rigorous optimization of reaction conditions, achieved successful pcr amplification of short (< 100bp) fragments in which all nucleotide analogues were functionalized with a range of small nonfluorescent groups. we substituted only one base (dc) but with a large, heterocyclic, charged cyanine dye substituent (cy3-, cy5-dc), which turned out to be a challenging substrate at full substitution. nevertheless, two rounds of directed evolution of polymerase function by spcsr, targeting the conserved a- and c - motif regions in the polymerase active site, yielded e10 (pfuexo- : v93q, v337i, e399d, n400d, r407i, y546h), a variant polymerase with a substantially enhanced capacity to utilize cy3- and cy5-dctp as substrates. e10 is able to pcr amplify most sequences up to 1 kb with complete substitution of dctp by cy3- or cy5-dctp. in the resulting cydna all dcs on both strands bear a large, hydrophobic heterocycle as substituent. the resulting display of hundreds of aromatic cyanine dyes on the dna scaffold altered the physicochemical properties of cydna substantially and caused organic phase partitioning in phenol extractions commonly observed for proteins and lipids. unlike naked dna, but in analogy to many proteins, cydna is taken up by cells via the endocytic pathway (not shown). although denatured dna can also partition to the phenol / water interphase, we found no evidence that cydna is substantially denatured at ambient temperatures. on the contrary, restriction enzyme digests and, in particular, afm strongly suggest a canonical b - form double - stranded configuration throughout. we therefore favor an alternative explanation, whereby the presence of the hydrophobic cyanine heterocycles in the major groove disrupt hydration and dna solvation, facilitating organic phase partitioning and the observed salting - out effect. the dense packing of fluorophores on the outside of the dna helix rather than any unusual (non - b - form) conformation of cydna would also seem to be the most likely cause for the apparent increased diameter observed in afm. the resulting shielding of the major groove by the organic heterocycles would be consistent with loss of binding to silica resins and of intercalating dyes such as ethidium bromide and with the protection against nuclease digestion. -arrays and clusters of organic chromophores and fluorophores have previously been examined because of their potential application in molecular devices and biosensors.(59) the spatial arrangement of the dye molecules on the dna scaffold allows for novel photophysical interactions such as excimer fluorescence enhancement. in a striking example, kool and colleagues examined a combinatorial array of chromophores displayed on a dna backbone as c - nucleosides uncovering a wide range of emission colors.(60) we also observed significant interactions between fluorophores in cydna noticeable as significant red shifting of excitation and emission maxima (not shown) and a quenching of cy5 (and to a lesser degree cy3) fluorescence at 100% substitution. such effects clearly depend on the precise arrangement of fluorophores in space and thus are a function of both labeling density and sequence.(61) it might therefore be possible to evolve optimal sequences for cy - dye display on dna to maximize photon yields. such sequences might display brighter fluorescence than the cydna fragments analyzed herein, which, as single molecules displayed fluorescence yields comparable to those of single quantum dots. fluorescence beacons, e.g., for attachment to antibodies for fluorescence microscopy or other imaging applications. in the context of microarrays, we found that shorter cydna probes exhibited a distinct signal advantage in cohybridization experiments. for one, the bulkier cydna probes display slower diffusion and hybridization kinetics than the shorter, less heavily labeled klenow probes, thus competing ineffectively for target binding. second, once bound, long probes provide further hybridization sites for the shorter probes directed against the same target therefore boosting their relative signal yield in cohybridization experiments (supporting information, figure 4). given the increased bulk of the cydna helix, it is surprising that only four mutations (e399d, n400d, r407i, y546h) are sufficient to give rise to the e10 phenotype (v337i does not contribute to the phenotype (not shown)). inspection of the available polb structures indicates that residues e399 and e400 reside at the surface of the polymerase domain, in a surface loop that connects to the polymerase active site via a beta - strand also comprising r407. in previous polymerase evolution experiments e.g. for increased incorporation of ribonucleotide triphosphates,(34) distal mutations in the same loop and beta - strand motif in taq, a pola - family polymerase, were found to fine - tune catalytic activities. mutations at taq s612 (pola equivalent of polb pfu r407) in the same region were found to enhance incorporation of fitc-12-datp,(62) a fluorescent dye - labeled nucleotide triphosphate, in which the fluorophore is attached to n7 of adenine, which also projects into the major groove. the functional consequence of the y546h mutation is less clear but is observed in phylogeny, for example, in the closely related polb - family polymerase 9n, which displays a wide - ranging capacity to incorporate, extend and replicate unnatural nucleotide analogues, in particular in conjunction with the a485l mutation(20) (commercially available as therminator). however, neither y546h, nor the combined a - motif mutations (e399d, n400d, r407i) by themselves provide a significant improvement in the synthesis and replication of cy - dye labeled dna (figure 10a). to determine the relative phenotypic contributions of the selected mutations of pfu e10 (v337i, e399d, n400 g, r407i, y546h), we determined activities of either wild - type pfuexo- polymerase or pfu variants comprising only a - motif mutations (a : pfuexo- e399d, n400 g, r407i)) or c - motif mutations (c : pfuexo- y546h) in (a) polymerase elisa with cy3- or cy5-dctp and (b) pcr with unmodified dntps. both (a) cy3- and cy5-dctp polymerase - elisa and (b) pcr reveal that there is a strong synergistic epistasis between a- and c - motif mutations for the incorporation of cy3- or cy5-dctp, which also results in an unexpected shortening of extension times in standard pcr compared to wild - type pfuexo- polymerase. only their combination gives rise to the striking e10 phenotype allowing cydna synthesis and replication. thus they display a strong and unusual epistatic interaction, whereby the selected mutations do not alter the polymerase phenotype independently but through a crucial functional interaction between the four residues during the polymerase catalytic cycle, which enables the synthesis and replication of cy - dye labeled dna. the same interaction also gives rise to a significant shortening of extension times in pcr amplification using standard dntps (figure 10b). the iterative nature of directed evolution experiments, which has also been likened to a continuous uphill walk on the protein fitness landscape, commonly favors the acquisition of mutations that contribute to the phenotype in an incrementally additive way. thus, epistatic interactions, whereby the phenotypic contribution of one mutation is significantly altered by the presence of another are usually not selected for. an exception are compensatory mutations that buffer some of the undesirable side - effects of adaptive mutations (e.g., loss of protein stability), which have been observed multiple times. synergistic mutations, on the other hand, are very unusual and, to our knowledge, have not previously been observed in directed evolution experiments. in conclusion, spcsr(34) allowed the evolution of a polymerase with a striking proficiency for the synthesis and replication of highly fluorophore - substituted dna. high - density substitution of dna with fluorophores is a potentially enabling feature for a number of applications in molecular genetics. we foresee many uses for cydna for example in hybridization applications such as microarrays or as fluorescent tags attached to antibodies or other proteins. e10 (or its descendants) should find applications outside fluorophore labeling such as the replication and evolution of dna displaying a broad spectrum of chemical groups, which previously were poorly incorporated because of their bulky nature such as large organic heterocycles. we anticipate a variety of such dna - based polymers tailored to display a wide range of such chemical functionalities on the nucleic acid scaffold. cydna offers a glimpse of the potential range of physicochemical and photophysical properties such polymers may exhibit. | dna not only transmits genetic information but can also serve as a versatile supramolecular scaffold. here we describe a strategy for the synthesis and replication of dna displaying hundreds of substituents using directed evolution of polymerase function by short - patch compartmentalized self - replication (spcsr) and the widely used fluorescent dye labeled deoxinucleotide triphosphates cy3-dctp and cy5-dctp as substrates. in just two rounds of spcsr selection, we have isolated a polymerase that allows the pcr amplification of double stranded dna fragments up to 1 kb, in which all dc bases are substituted by its fluorescent dye - labeled equivalent cy3- or cy5-dc. the resulting cydna displays hundreds of aromatic heterocycles on the outside of the dna helix and is brightly colored and highly fluorescent. cydna also exhibits significantly altered physicochemical properties compared to standard b - form dna, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. cydna also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. cydna represents a step toward a long - term goal of the encoded synthesis of dna - based polymers of programmable and evolvable sequence and properties. |
chromium exits in two stable oxidation states, namely, the trivalent (iii) and hexavalent (vi) forms. hexavalent cr is more toxic than trivalent cr and the toxicological impact is due to its oxidizing ability and high solubility [14 ]. chromium (vi) is capable of damaging the skin due to its high penetration power and ability to form free radicals. printing workers operated by five separate and distinct processes, lithography, letterpress, flexography, gravure, and screen printing [5, 6 ]. the international agency for research on cancer (iarc) classified occupational exposure in the printing industry as possibly carcinogenic to humans. the toxicity of chromium within the cell may result from damage to cellular components during the hexavalent to trivalent chromium reduction process, by generation of free radicals, including dna damage [8, 9 ]. printing workers are potentially exposed to hexavalent cr when involved in the production use of chromate pigments, chromate paints, and printing inks. studies of workers in the chromium pigment, chrome plating, and ferrochromium industries showed a statistically significant association between worker exposure to cr(vi) and lung cancer and nasal and sinus cancer [812 ]. more recent studies also disclosed excess risk of lung cancer death resulting from occupational exposure to cr(vi) compounds [13, 14 ]. direct contact of cr(vi) compounds with intact skin can induce chromium dermatitis or sensitization. in the case of preexisting small skin lesions, impact of hexavalent chromium compounds will lead to slowly healing chromium ulcers. the objectives of this study were to determine and evaluate the level of urinary chromium and level of serum chromium among printing workers exposed to chromium from printing factories and compare them with the nonexposed group and to describe worker behaviors and evaluate them in terms of their possible role in worker contamination and transfer of chromium to the body and to determine any correlation between level of urinary chromium and level of serum chromium and airborne chromium levels. data for this cross - sectional study were collected by sampling from 75 printing workers (49 males ; 26 females) in southern thailand from january to september 2014. seventy - five printing workers were recruited from 16 printing factories. in this study, the process of printing production included prepress process (digitization, preflight, imposition, digital proof ting, process film making, plating making, and plate proofing), press process (print preparation and printing) and postpress process (surface decoration, coating, hot stamping, embossing, forming, book making packing). inclusion criteria for the study subjects were as follows : printing work, aged 2054 years, in direct contact with heavy metals, and working in printing factories for at least one year prior to the study ; persons who had been in occupational contact with chromium dust during printing production ; persons who agreed to participate in the study and who signed the informed consent form. the nonexposed group (75 persons) matched to exposed subjects by age and sex were recruited from the printing workers who worked at the same printing factories but had not had occupational contact chromium (35 office workers, 25 drivers, and 15 cleaners, resp.). after two hours, stay blood samples were centrifuged at 3500 rpm for 10 min and separated sera were put in closed plastic laboratory vessels and kept at 18c in the fridge. blood serum samples, obtained from subjects of participation, were used to determine the chromium levels by the analytical procedure. the 150 subjects (75 exposed and 75 unexposed) were interviewed using structured questionnaire interviews. spot urine samples (30 ml) were collected, which extends from the time the subjects goes to bed through the first urination of the morning. the urine samples were kept in polypropylene sampling vessels and stored at 20c prior to analysis. in the questionnaire interviews, detailed descriptive information was collected, including personal descriptive characteristics, occupational life styles, working positions, working environment, and personal hygiene. direct observations were also made and recorded to confirm the questionnaire interviews. at the end of shifts, september 2014. regarding the instrument for air sampling, personal pumps (model 224-pcxr8 ; skc inc., eighty four, pa, usa) were calibrated at 2 l / min before and after sampling, containing a mixed cellulose ester membrane filter (pore size : 0.8). sampling was carried out for the regular work duration of 8 h. the air sampling equipment was fitted to the subject at the start of the day, removed, or switched off during the break and finally removed at the end of the day. air sample filters and field blanks were subjected to slow wet acid digestion in accordance with the niosh standard analytical method 7024. each sample solution was diluted with 0.1 m nitric acid to 10 ml in a volumetric flask prior to chemical analysis. the concentrations of cr in the digested breathing zone air were determined using faas following the niosh method 7024. working standards for cr were diluted appropriately in 0.01 m nitric acid (1% (v / v) as a stabilizer). aqueous standard solutions with concentrations of cr were run, which gave the required standard calibration curves. the concentrations of cr in the digested breathing zone air samples were assayed in triplicates using faas at optimized operational conditions. this was subsequently obtained directly from the standard calibration graphs after correction of the absorbance for the signals from appropriate reagent blanks. airborne levels of cr were expressed as micrograms (g) per cubic meter (m) of air over an 8-hour time - weighted average (twa). this method of serum chromium determination was modified from that of randall and gibson, 1987. total chromium (cr) standards were prepared from the commercial stock solution of 1000 g / ml in 0.01 m nitric acid by successive dilution with distilled and deionized water. similarly for cr analysis, aliquots of 10 l of the diluted urine samples were introduced directly into a pyrolytically coated graphite furnace tube and, with an equal volume of 10 l matrix modifier mixture (0.6% palladium nitrate and 0.15% m / v magnesium nitrate in 0.01 m nitric acid), were automatically injected sequentially. the concentrations of cr were obtained directly from the calibration graphs after automatic correction of the absorbance of the signal from appropriate reagent blanks. creatinine levels were analyzed in all spot urine samples using the alkaline picrate method, which was based on a modified jaffe reaction. all samples were analyzed using adequate quality control procedure to ascertain reliability of the results. the reagents that were used through the analytical procedure were of high purity analytical grade. the main instrument parameter for graphite flame atomic absorption spectrometry (gfass) and flame atomic absorption spectrometry (fass) was optimized separately for each metal. a stock stand containing 1000 mgl of chromium was obtained from sigma chemical co. (st. louis, mo). quality control was further ascertained by inter laboratory comparison of the levels of chromium in five sets of representative breathing zone air, serum, and urinary samples. the rage of linearity was also determinate checking the linear regression coefficient (r) of calibration values. the validity of the method was further assured by method cross check and replication analysis. all of the participants received a clear explanation of the purpose of this study and agreed to participate using signed consent forms. descriptive statistics were used to present the airborne, serum, and urine concentration results. the independent t - test was used to compare the means of continuous variables. most of the exposed subjects (65.3%) were male and 53.3% were aged between 2034 years. most of the exposed subjects had bachelor 's degree (41.3%), similar to the control subjects (36.4%). the mean airborne chromium level was 7.20 2.86 g / m (range : 112 g / m) where 82.67% exceeded the standard of the occupational safety and health administration (osha) level of 5 g / m as cr(vi) g / m and pel for chromic acid and chromates and (8-hour twa) as air workplace. the mean serum chromium levels of the exposed and control subjects were significantly different at p value of 5 years had significantly higher serum chromium levels and urinary chromium levels than those who had worked 5 years (p 8 hours per day and > 6 days per week had significantly higher serum chromium levels and urinary chromium levels than those who had worked 8 hours per day and 6 days per week (p = 0.012 and p 5 years had significantly higher serum and urinary chromium levels than those who had worked 5 years. in addition, for hours workers per day and days worked per week, mean serum chromium levels and urinary chromium levels differed significantly ; printing workers who had worked > 8 hours per day and > 6 days per week had significantly higher serum and urinary chromium levels than those who had worked 8 hours per day and 6 days per week (p < 0.001 and p < 0.001, resp.). this may be due to printing workers ' long term exposure to chromium, leading to its accumulation in their bodies, due to the lack of appropriate prevention measures. printing workers were considered informal workers ; some used their work areas during their breaks. therefore, personal working habits and the conditions at the workplace area seem to affect the exposure and cause difference. printing workers who used masks and gloves had significantly lower serum and urinary chromium levels than those who did not. however, the personal hygienic practices were not appropriate for field work in these printing factories. in addition, fume of chromium and other chemicals (voc) may penetrate though a cotton mask and gain access to a printing worker 's airway. the results have indicated that chromium is a risk factor where there are inadequate engineering controls, industrial hygiene, and work practice, particularly in occupational risk. the present study supported by chuang. and tawichascri. has indicated that poor behavior plated a role in accumulation of airborne chromium among workers. thus the combinations of poor work practices and safety behavior among workers, the limited training and education on safety procedures, may have immensely contributed to elevated levels of chromium in both the breathing zone air and urinary that were measured in these facilities. (1993), who reported that, in addition to individual difference in hygiene behavior, general hygiene condition also has an impact on uptake of chromium. that showed a significant impact of hygiene behavior on the variance in urine chromium levels (r = 0.94, p < 0.001). from observations of work areas, contamination of hands, cloths, hand tool, and working surface with chemical was clearly evident at each work site. printing workers who always ate snacks or drank water while working had significantly higher serum and urinary chromium levels than those who only did so sometimes. printing workers who always washed their hand before lunch had significantly lower serum and urinary chromium levels than those who sometimes did. in addition, printing workers who always washed their hand after worked had significantly lower serum and urinary chromium levels than those who sometimes did. however, the factor of smoking cigarettes in this study has not demonstrated increased risk for cancer. in addition sparse data precluded the control of tobacco smoke as a confounder in analyses of the us cohort. [36, 37 ] have reported that examining lung cancer mortality among chromate production workers in the us and in germany was subsequent to significant process changes and enhanced industrial hygiene controls. cr(vi) compounds may be used as pigments in dyes, paints, inks, and plastics. it also may be used as an anticorrosive agent added to paints, primers, and other surface coatings as a known human carcinogen. thus, this study demonstrated that urinary chromium levels and serum chromium levels were associated with airborne chromium levels and hygiene behaviors of printing workers. this study recommends conducting education and training about personal hygiene to minimize occupational chromium exposure. in addition, engineering controls are also recommended to reduce chromium or other chemical exposures and to reduce the printing worker 's cancer hazard to minimal levels. in addition, the limitation in this study was able to determine total chromium in both breathing zone, serum, and urinary sample and we found it difficult to analyse chromium (vi). however, we recommended that further research should distinguish chromium (iii) from chromium (vi) because of greater toxicity of chromium (vi). | objectives. the main objective of this study was to assess the chromium exposure levels in printing workers. the study evaluated the airborne, serum, and urinary chromium levels and determines any correlation between level of chromium in specimen and airborne chromium levels. material and methods. a cross - sectional study was conducted with 75 exposed and 75 matched nonexposed subjects. air breathing zone was measured by furnace atomic absorption spectrophotometer. serum and urine samples were collected to determine chromium levels by graphite furnaces atomic absorption spectrometer chromium analyzer. results and discussion. the printing workers ' urinary chromium levels (6.86 1.93 g / g creatinine) and serum chromium levels (1.24 1.13 g / l) were significantly higher than the control group (p < 0.001 and p < 0.001). work position, duration of work, personal protective equipment (ppe), and personal hygiene were significantly associated with urinary chromium level and serum chromium levels (p < 0.001 and p < 0.001). this study found a correlation between airborne chromium levels and urinary chromium levels (r = 0.247, p = 0.032). a multiple regression model was constructed. significant predictors of urinary and serum chromium levels were shown in this study. conclusion. improvements in working conditions, occupational health training, and ppe use are recommended to reduce chromium exposure. |
nasal septal deviation is a common nasal deformity. it can be a congenital disorder or a consequence of nasal trauma. deviation of the bony or cartilaginous component of the nasal septum from the midline leads to its deviation. this results in external nasal deformity, internal nasal obstruction due to nasal airway constriction, or a combination [13 ]. presently, septal deviation classification has largely been descriptive, based on nasal septal geometry and relationships between the bony and cartilaginous septa [47 ]. jang. presented a simplified classification of nasal deviation and the associated treatment outcome into five types based on the orientation of the bony pyramid and the cartilaginous vault. jin. presented a four - category classification of septal deviation based on the morphology, site, severity, and its influence on the external nose. buyukertan. reported a morphometric study of nasal septal deviation by separating the nasal septum into 10 segments. they concluded that the system would constitute a new, objective, simple, and practical classification system. i. baumann and h. baumann argued that the existing nomenclatures of septal deviation only dealt with nasal septum deformation exclusively and were rarely used in routine clinical work. they instead presented a method for the classification of septal deviations based upon the anatomical structures of the nasal septum and common clinical concepts. however, the most observable nasal septal deviation classification system was proposed by rohrich.. therefore, for simplicity, nasal septal deviations will be classified according to that proposed by rohrich.. in order to improve the clinical outcome of septoplasty, a greater understanding of the etiopathogenesis of nasal septal deviation is necessary. we aim to apply incremental force to a computer - generated septal model using structural modal analysis, which has also been utilized by laura., who previously described a simple method for determining the fundamental mode of a vibrating ulna to approximate its dynamic response. the objective of this study is to identify areas of high - stress and septal deformation patterns. clinically, this may assist surgeons in the delineation of key areas for septal realignment and reconstruction. cranial ct scans were obtained from a patient who possessed normal features a straight nasal septum, normal occlusion and a perceivably symmetric face (figure 1(a)). this study was performed in accordance with the guidelines of the institutional review board (irb) and conforms to the helsinki 's declaration. the patient had not previously undergone septoplasty or rhinoplasty, nor subject to nasal injury. superposition of the ct images to create a three - dimensional (3d) model was conducted with mimics software (materialise technologies, leuven, belgium). an idealized model (figure 1(b)) and a patient - specific finite element model were generated for the study. from the ct scans we chose to base the idealized model on the middle slice (figure 1(a)) and measured the significant features of the nasal septum. we then utilized these measurements to create an idealized model (figure 1(b)) in the finite element analysis software, abaqus (dassault systmes technologies, providence, ri, united states of america (usa)), where the idealized model was, subsequently, meshed. however, to simplify analyses and gain an estimate of nasal deformation, models were prescribed with a uniform thickness of 2 mm, which is an approximate average septum thickness, as reported previously [1113 ]. to ensure mesh accuracy, convergence studies were carried out on the model. to create a more realistic representation of the septum, which incorporated thickness variation, a 3d patient - specific model was created from the same ct scan utilizing mimics software (materialise technologies, leuven, belgium) and meshed with hypermesh (altair hyperworks, troy, mi). nonhomogenous, anisotropic, nonlinear, viscoelastic behaviour. for deformations below 20%, however, no significant changes occur within the cartilage, and it is therefore sufficiently accurate to model cartilage as a homogenous, linearly elastic material in our analyses. utilized a similar homogenous, linear elastic material property to simulate septal l - strut deformation. to define the linear elastic model of the cartilage, the young 's modulus, e, and poisson 's ratio, v, are required. however, the tensile and compressive young 's moduli are vastly different due to the structure of cartilage. according to lee., the tensile modulus ranges from 2.62 mpa to 10.6 mpa, the compressive modulus ranges from 0.40 mpa to 0.83 mpa, and the poisson 's ratio ranges from 0.26 to 0.38. cartilage is approximately 75% water, while the other 25% consists mainly of type - two collagen fibrils and proteoglycan molecules. the density of water is 1000 kg / m, while the other components are highly dense structures. therefore, the density of cartilage was estimated to be 2000 kg / m. as the relative displacement within the septum is the main area of concern in this analysis, and since material properties affect the absolute and not the relative displacement of the septum, the average values of the elastic modulus and poisson 's ratio and an estimated value of the density were used. the elastic modulus was assigned a value of 5 mpa, poisson 's ratio was 0.32, and the density was 2000 kg / m. as the bony interfaces with the nasal septum ethmoidal, vomer, hard palate, and nasal bone interfaces (figure 1(a))are much stiffer than the septal cartilage, most of any applied force will be absorbed by the cartilaginous septum, leaving the bony septum uninjured. the nasal bone length overlapping the cartilaginous septum may affect the degree of nasal deformation and normally ranges from 3 to 15 mm. however, to simplify analyses, a candidate that displayed a length that fell within this range in this case, 14 mm was considered, so that a typical deformation pattern could be observed. in vivo, the nasal tip lies anterior to the anterior septal angle (asa) where the lower lateral cartilages (llcs) meet, although this may vary. however, due to the small distance between the asa and the nasal tip, and for simplification in this analysis, the asa was assumed to be the nasal tip. according to lee, the nasal tip cartilages may be thought of as a spring and a cantilever, as they exhibit deformation recoil and elasticity. a cantilever is a result of the unequal stability in the tripod formed by the medial crura and paired llcs, and a spring results from the llcs, which produce an upward force that is in the form of stored elastic potential energy. the spring - stiffness constant, k, may be defined by (1), and a spring - stiffness constant of 20 kn / m is applied in the three orthogonal axes. a free nasal tip was prescribed as a preliminary step. a spring - supported nasal tip boundary condition, where the spring was connected between the two orange points on the nasal tip (figure 2), the dorsal and caudal septa were prescribed a free boundary condition. as frontal force to the septum causes damage ranging from simple fracture of the nasal bones to severe flattening of the nasal bones and the septum, two forms of frontal loading were applied the force and pressure applied are estimates and are inconsequential to the relative displacements of the septum. as the present intention is to determine the eigen modes, or the most likely deformed patterns of the septum, only the possible in - plane loading which will affect the resulting eigen modes will be considered. in the case of anteroposterior loading (figure 2), a couple of forces of 1n each in both the vertical and horizontal axes were applied to the nasal tip to simulate a direct frontal punch at an angle such that the forces on the nasal tip are the most significant. in the case of dorsal and caudal septa in - plane loading (figure 2), a uniform pressure of 2000 pa was applied to both the dorsal and caudal septa. this was to simulate a frontal punch at an angle such that both the dorsal and caudal septa components are equally significant. every object, including the septum, has a set of eigen modes, depending on its structure and composition. in each mode, all parts of the system vibrate with the same distinct frequency, which is referred to as the system 's eigen value at that mode. since lower modes have lower frequencies and energies, they are more likely to occur. hence, only the first 10 modes of the nasal septum were analyzed. abaqus (dassault systmes technologies, providence, ri) was used to obtain the eigen mode shapes of the septum under the various loading conditions. a general, static step is created, in which one of the two loading conditions is applied. thereafter, a linear perturbation, frequency step is created, in which the natural frequency and the corresponding mode shape will be extracted. the patterns of nasal septal deviation were similar to those described by rohrich.. in our study, the deviation patterns were therefore classified into three groups, each with its specific sites of high stress and dislocation and possible surgical corrective procedures (table 1). through observation of all deformation patterns, we were also able to identify the intrinsic points of fatigue within the cartilaginous septum the bc junction, anterior nasal spine (ans), vomer - ethmoidal cartilage junction (vej), and a single or couple of cracks in the quadrangular cartilage that lead to c - shaped and s - shaped nasal deformations, respectively. these points could lead to the septum levering off the vomerine groove and, in the latter two cases, a shortening of the septum (figure 3). for an idealized model, the slanted (figure 4(a)), c - shaped (figure 4(b)), and s - shaped (figure 5(a)) deviation patterns were all observed (table 2). in some modes, the system vibrates in - plane and therefore lacks a resultant deformation shape. in such cases, a dash is indicated. however, due to the lack of restriction on the nasal tip, it moves relatively freely, which may not represent in the vivo conditions. in the following idealized model, the nasal tip is now constrained by a spring. while displaying similar patterns of deviation with a free nasal tip model, the spring - supported nasal tip model exhibits decreased displacement due to its prescribed restriction. a patient - specific model was then analysed. by observation of the previous idealized models the patient - specific model exhibited similar deformation patterns as the idealized nasal septal models (table 2 and figure 5). the nasal septum is of utmost importance in the support of the distal nose and for the maintenance of the bilateral nasal airway. a straight septum exists where there is force equilibrium, which may be disturbed in fracture, resulting in warping of the septal cartilage [18, 26 ]. depending on the sustained trauma, the septum may deform in a myriad of patterns. presently, however, studies have reported that septal deformation patterns may be categorized in a number of broad categories, regardless of the trauma and/or injuries sustained. guyuron., rohrich., and rhee. categorized nasal deformities broadly into a septal tilt, anteroposterior or cephalocaudal c - shaped and an s- or reverse - s - shaped deformities. unfortunately, these studies have not correlated these deviation patterns with degrees of force. through the correlation of septum deformation patterns with increasing degrees of force, as well as with areas of dislocation and fracture, preoperative planning and septoplasty may be improved. the prompt identification and management of septal fractures, we were able to identify clinically observable nasal septal deviations and the aforementioned high - stress areas that would require stress - relief and the possible dislocation sites (figure 3). we observed that regardless of force direction, with increasing force, the septum first tilts (type i) and then crumples into a c - shape (type ii) and finally into an s - shape (type iii). this was observed through the prevalence of the tilted septum in lower modes, while the c - shaped followed by the therefore, the lower the mode in which the deviation pattern is observed, the smaller the force required to cause this deformation, and consequently, the greater the probability of observing this pattern., in a sample size of 93 patients who had undergone primary septoplasty, 40% had a septal tilt, 32% had a c - shape anteroposterior septum, 4% had a c - shape cephalocaudal septum, 9% had an s - shape anteroposterior septum, and 1% had an s - shape cephalocaudal septum. in type i, when a tilted septum is observed, the highest stress concentration occurs at the bc junction and ans. this suggests that with a low to moderate force, the septum dislocates en - mass from the midline vomerine groove (figure 3) and levers off the bc junction to a tilted position. this may be observed on ct and mri scans, and naso - endoscopy, where posterior buckling is frequently observed at the vej. through submucous resection, the septum may be repositioned onto the groove, with prior resection of the cartilage tongue in the nasal floor. the septum may then be reset to the ans (table 1). with a higher moderate force, a deformation is likely due to a central line of stress in the septum, bending it into two pieces. the line of high stress may run through the anteroposterior (figure 4(a)) or cephalocaudal (figure 4(b)) directions. we propose that with significant loading, intrinsic septal fractures occur by breaking the cartilaginous septum into two, leading to the clinical morphology of a c - shaped nose and shortening of the septum. in addition, the septum will be displaced off its vomerine groove and/or the ans and will likely buckle at the vej (figure 3). this is clinically significant as it can not be observed on ct or mri scans due to the invisibility of the septum in such modalities. hence, a clinical observation of a c - shaped septum may be the only indication. in addition to the corrective procedures mentioned previously, spreader grafts may be required on both sides of the septum, to assist in its straightening by providing the necessary nasal support that was relinquished when the septum fractured, thereby allowing the septum time to heal (table 1). with a force of higher magnitude, an deformation may result due to multiple lines of stress leading to a septal concertina and shortening of the septum into a minimum of three overlapping pieces. this is due to two lines of stress running in the anteroposterior direction (figure 5). in addition to being shortened, the septum might be displaced from the vomerine groove and ans (figure 3). as with a c - shaped deformed septum, in addition to the aforementioned corrective procedures, longer spreader grafts will be required to brace both deformed sites to support the septum and allow it to heal (table 1). therefore, regardless of the deviation pattern, by relieving stress in these specific strips of concavity, in combination with the aforementioned surgical procedures, we propose that a more stable, straight septum may be achieved. despite different loading conditions, the nasal septum deviates in a relatively constant pattern of a septal tilt, c- and s - shaped deviations with insignificant differences between the resultant modal shapes. the free nasal tip and spring - supported nasal tip models responded differently to the loading conditions, specifically in mode three. a septal tilt is observed in the free nasal tip model, while a c - shaped deformation is observed in the latter model. as the c - shape deviation is noted to occur with higher energy and septal tilt deviation with lower energy, this finding suggests that the spring of the llcs acts to insulate and constrain the nasal tip and septum against deformation. the protective interrelationship of the llcs to the nasal septum should be preserved during surgery. the prevalence of modal shapes in patient - specific and idealized septal models, subject to frontal point - loading, is almost identical (table 1). slight deviations, such as those in mode three, are expected, due to the difference in shape between the models. despite the patient - specific model exhibiting greater relative movement than the idealized model, this difference is insignificant as the basic modal shape remains (figure 5). the similarities observed between these two models are a testament to the accuracy of the idealized model. it is imperative to note that nasal septal deviations are secondary to the bony vault and cartilaginous changes. for the purpose of this study, future research aims to combine the study of the deformations of the bony and cartilaginous septa. due to the inherent collagen fibrils and the consequent anisotropy within the cartilaginous septum, we recognize that the prescription of a linearly elastic material model to the nasal septum material properties may not be fully representative of in vivo cartilage. in spite of this, an understanding of the relative displacement that occurs within the different models in different eigen modes remains beneficial in aiding surgeons to correct a deviated nasal septum. no physical model was mechanically tested to validate the computational model in this preliminary study, which means that absolute stresses and relative stress patterns should be considered cautiously. such an experimental validation study would typically make use of strain gages, but also infrared thermography, and global stiffness measurements [29, 30 ]. the purpose of this study was to gain a greater understanding of the septal deformation biomechanics. we found that despite different loading directions, the septum deformed consistently into only three shapes a tilted position, a c - shaped septum, and an s - shaped septum. the tilted septum is seen with the least force, c shape with moderate force, and s shape with high force. this suggests an intrinsic fracture of the septum into increased number of overlapping fragments with escalating force. clinically, this is important information that provides insight into predictable patterns of internal septal fractures that need to be realigned and reconstructed to create a straight septum. | background. with the current lack of clinically relevant classification methods of septal deviation, computer - generated models are important, as septal cartilage is indistinguishable on current imaging methods, making preoperative planning difficult. methods. three - dimensional models of the septum were created from a ct scan, and incremental forces were applied. results. regardless of the force direction, with increasing force, the septum first tilts (type i) and then crumples into a c shape (type ii) and finally into an s shape (type iii). in type i, it is important to address the dislocation in the vomer - ethmoid cartilage junction and vomerine groove, where stress is concentrated. in types ii and iii, there is intrinsic fracture and shortening of the nasal septum, which may be dislocated off the anterior nasal spine. surgery aims to relieve the posterior buckling and dislocation, with realignment of the septum to the ans and possible spreader grafts to buttress the fracture sites. conclusion. by identifying clinically observable septal deviations and the areas of stress concentration and dislocation, a straighter, more stable septum may be achieved. |
post - extraction bone tissue atrophy in the alveolar ridge was found both, in vertical and horizontal dimensions (chackartchi and stabholz 2013). the systematic reviews demonstrated that the alveolar ridge underwent mean horizontal reduction in width of 3.8 mm and mean vertical reduction in height of 1.24 mm within this time (hmmerle. the bone loss in the maxilla was more significant than in the mandible and in both cases atrophy on the vestibular side prevailed, which is associated with its reduced bone width within this location (irinakis 2006). the process occurs mostly during the first 56 weeks following tooth extraction (amler 1993) but a complete rebuilding process lasts approximately 6 months. combined with traumatic extraction or chronic inflammations of the periodontium surrounding teeth qualified for extraction, the dimensions of the remnant alveolar ridge may be too small to allow implantation. reconstruction of the alveolar ridge most often is performed through autografting or alternatively by the use of allografts or bone replacement materials (saravanan. autografting from the ilium constitutes a burden for the patient, requires general anaesthesia and subjects them to additional complications related to the donor site (barone and covani 2007). bone distraction requires additional surgical procedure as well as time for recovery (zwetyenga 2012). the third solution is the use of allogeneic bone blocks, the availability of which combined with their nearly unlimited dimensions, easy processing and adjusting to the recipient site gathers an increasing number of followers (wallace. although allografts with unlimited size seem to be the best option, the risk of infection transmission from the donor to the recipient has to be taken into account (eastlund 2006). therefore, donor screening based on medical and social history, donor physical examination and autopsy results (if applicable), as well as biological examination of blood, are required for proper evaluation of donors (pruss. additionally the introduction of secondary sterilisation reduces the abovementioned risk related to tissue allografts (loty. however, it was shown, that the use of irradiation as a sterilisation method may impair bone allograft properties (cornu. the patient reported that the last teeth were extracted several years before and he used a complete removable prosthesis which he did not approve. the patient reported enormous discomfort due to insufficient retention of the prosthesis and unsatisfactory adhesion to the gingiva as well as taste disturbances. the intraoral examination revealed a narrow, atrophic alveolar ridge and high palatal vault (gothic palatal arch). computed tomography imaging showed extensive bone loss throughout the entire maxillary alveolar ridge (fig. 1ct before grafting within location of tooth 16 ct before grafting within location of tooth 16 the following four treatment modalities were discussed : complete acrylic prosthesis supported by mucous membrane.complete acrylic prosthesis supported by two, three or four implants.mixed prosthesis consisting of an implant - based fixed prosthesis in the front (33) and removable prosthesis supported by crowns fixed on implants in lateral segments.fixed prosthesis supported by six implants. mixed prosthesis consisting of an implant - based fixed prosthesis in the front (33) and removable prosthesis supported by crowns fixed on implants in lateral segments. fixed prosthesis supported by six implants. in view of previous experience of the patient, modalities, which involved a removable prosthesis, were excluded. moreover, the patient wished for a fixed prosthesis, which would not have to be removed from his mouth. therefore, a fixed prosthesis supported by six implants was chosen. the implant - prosthetic plan provided for restoration of 12 teeth (1626). the required bone graft involving the entire length of the alveolar ridge due to its considerable atrophy was discussed with the patient. since the necessary amount of material exceeded the capacity of the intraoral donor site, autologous iliac bone grafting was considered, which was rejected by the patient. allogeneic bone blocks were processed from iliac ala of deceased donors and subsequently radiation - sterilised with a dose of 35 kgy on dry ice using an electron beam accelerator (lae-10 ; 10 mev). the patient accepted a graft consisting of four independent bone blocks, filled in and signed required documents. the mucoperiosteal flap was detached. reduced width of the maxillary alveolar ridge was confirmed intraoperatively (fig. 2), which was consistent with prior ct finding. the width of the ridge ranged from 1 to 2 mm and the height from 8 to 15 mm along the entire ridge.fig. 2narrow alveolar process narrow alveolar process based on clinical and radiological (ct) findings four bone blocks (compact - trabecular bone) were ordered from the tissue bank. two of them were 20 10 10 mm and two were 10 10 10 mm (fig. the bone blocks were harvested from the iliac ala and radio - sterilised with a dose of 35 kgy.fig. 320 10 10 mm frozen, radiation - sterilised bone block 20 10 10 mm frozen, radiation - sterilised bone block the bone block was aligned so the compact lamella was the external layer located towards the vestibule and the trabecular layer of the block (internal layer) was shaped so it could precisely adhere to the recipient site in the maxilla and then the sharp bone edges were smoothed. due to anatomic features the longer block was fixed within the area of teeth 1614 and the smaller bone block was located within the area of tooth 12 having considered the curve of the alveolar arch. 4). shavings formed as a result of shaping of the bone blocks were placed in the space between the donor and recipient sites as well as on each side of the bone block. the operational site was covered with platelet rich plasma membrane and with a free mucous membrane flap. the wound was sutured with 4.0 safil hr 22 in a way preventing excessive stretching of the flap. on the left side bone grafting 4right - side location of oral allografts right - side location of oral allografts a 7-day per os antibiotic therapy was prescribed to the patient as well as analgesics and antibacterial mouth rinse to follow the procedure. due to considerable widening of the ridge a new complete upper prosthesis was made for temporary use, which did not apply any pressure to the bone grafting sites. a follow - up ct imaging (fig. the result indicated normal bone union and slight bone atrophy at the margins of the grafts. the intraoral examination showed normal mucous membrane and a wide alveolar ridge of optimal height (fig. 6healed bone block grafts ; bone bed ready for implantation ct after grafting, the same location healed bone block grafts ; bone bed ready for implantation the second part of the plan was commenced and under local anaesthesia with 4 % ubistesin forte six implants were embedded within the area of teeth 16, 14, 12, 22, 24, 26. after the mucoperiosteal flap was detached, normal union of the patient s bone and the homogenic bone was found. the graft - securing screws were removed and six biomet 3i implants (16-nint 485, 14-nint 410, 12-nint 3211, 22-nint 3211, 24-nint 3210, 26-nint 3210) (fig. (the implants were embedded within the grafted area only) bleeding was observed, which indicated normal revascularisation of the graft. after normal primary stability was determined unequivocally, the closing screws were fixed and the wound was sutured.fig. 7implants embedded in the bone implants embedded in the bone just as it was in case of the first procedure, the patient underwent 7-day antibiotic therapy and was recommended to use analgesics and antibacterial mouth rinse. the sutures were removed after 14 days and at the same time the prosthesis was slightly corrected so it did not apply any pressure on implants. following 6 months of osseointegration the implants were uncovered under local anaesthesia. after normal secondary stability was determined, healing screws were fixed for a period of 2 weeks. later, at subsequent visits the height of occlusion was established and full arch bridge impressions were taken to restore the defects of teeth 1626. at the last visit a porcelain - fused - to - metal bridge was cemented, which completed the 13-month treatment period. during the 38-month follow - up period secondary stabilisation was maintained in all the grafts, the double porosity surface contributed to improved integration with the bone tissue under reconstruction. 8) did not demonstrate gingival recession or bone atrophy around the implants, which confirmed the long - term follow - up efficacy and stability of the treatment.fig. the procedure was performed under local anaesthesia with 4 % ubistesin forte. an incision in the mucous membrane the mucoperiosteal flap was detached. reduced width of the maxillary alveolar ridge was confirmed intraoperatively (fig. 2), which was consistent with prior ct finding. the width of the ridge ranged from 1 to 2 mm and the height from 8 to 15 mm along the entire ridge.fig. 2narrow alveolar process narrow alveolar process based on clinical and radiological (ct) findings four bone blocks (compact - trabecular bone) were ordered from the tissue bank. two of them were 20 10 10 mm and two were 10 10 10 mm (fig. 3). the bone blocks were harvested from the iliac ala and radio - sterilised with a dose of 35 kgy.fig. 320 10 10 mm frozen, radiation - sterilised bone block 20 10 10 mm frozen, radiation - sterilised bone block the bone block was aligned so the compact lamella was the external layer located towards the vestibule and the trabecular layer of the block (internal layer) was shaped so it could precisely adhere to the recipient site in the maxilla and then the sharp bone edges were smoothed. due to anatomic features the longer block was fixed within the area of teeth 1614 and the smaller bone block was located within the area of tooth 12 having considered the curve of the alveolar arch. shavings formed as a result of shaping of the bone blocks were placed in the space between the donor and recipient sites as well as on each side of the bone block. the operational site was covered with platelet rich plasma membrane and with a free mucous membrane flap. the wound was sutured with 4.0 safil hr 22 in a way preventing excessive stretching of the flap. on the left side bone grafting 4right - side location of oral allografts right - side location of oral allografts a 7-day per os antibiotic therapy was prescribed to the patient as well as analgesics and antibacterial mouth rinse to follow the procedure. the patient was referred to monthly surgical follow - up. due to considerable widening of the ridge a new complete upper prosthesis was made for temporary use, which did not apply any pressure to the bone grafting sites. a follow - up ct imaging (fig. the result indicated normal bone union and slight bone atrophy at the margins of the grafts. the intraoral examination showed normal mucous membrane and a wide alveolar ridge of optimal height (fig. 6healed bone block grafts ; bone bed ready for implantation ct after grafting, the same location healed bone block grafts ; bone bed ready for implantation the second part of the plan was commenced and under local anaesthesia with 4 % ubistesin forte six implants were embedded within the area of teeth 16, 14, 12, 22, 24, 26. after the mucoperiosteal flap was detached, normal union of the patient s bone and the homogenic bone was found. the graft - securing screws were removed and six biomet 3i implants (16-nint 485, 14-nint 410, 12-nint 3211, 22-nint 3211, 24-nint 3210, 26-nint 3210) (fig. (the implants were embedded within the grafted area only) bleeding was observed, which indicated normal revascularisation of the graft. after normal primary stability was determined unequivocally, the closing screws were fixed and the wound was sutured.fig. 7implants embedded in the bone implants embedded in the bone just as it was in case of the first procedure, the patient underwent 7-day antibiotic therapy and was recommended to use analgesics and antibacterial mouth rinse. the sutures were removed after 14 days and at the same time the prosthesis was slightly corrected so it did not apply any pressure on implants. after normal secondary stability was determined, healing screws were fixed for a period of 2 weeks. later, at subsequent visits the height of occlusion was established and full arch bridge impressions were taken to restore the defects of teeth 1626. balanced occlusion was obtained following correction of contact points of opposing teeth. at the last visit a porcelain - fused - to - metal bridge was cemented, which completed the 13-month treatment period. during the 38-month follow - up period no case of a lost implant was found. secondary stabilisation was maintained in all the grafts, the double porosity surface contributed to improved integration with the bone tissue under reconstruction. clinical assessment and intraoral examination and opg (fig. 8) did not demonstrate gingival recession or bone atrophy around the implants, which confirmed the long - term follow - up efficacy and stability of the treatment.fig. many scientists and dental practitioners as well as the authors of this case report, believe that the best grafting material is the autograft (acocella. 2010 ; hyeon - jung. it eliminates the risk of spreading infectious diseases such as hiv infection as well as prions between the donor and the recipient, there is no rejection reaction and the highest efficacy and predictability of the procedure are provided (sutherland. this procedure was also considered in the discussed case. however, in view of the large amount of material required, the number of donor sites was limited. moreover, considering the complications of an additional procedure of harvesting a bone graft from a donor site, such as hindered walking, delayed healing or the risk of the procedure itself, finally a frozen, radiation - sterilised, compact - trabecular bone allograft was chosen. the advantages of this decision were almost unlimited availability of grafted tissues as well as possibility to freely modify the size and shape of them (kim. allogeneic bone block provides a predictable reconstruction in both faciolingual and vertical directions (schwartz - arad. there have been several reports published describing the transmission of bacterial (eastlund 2006) and viral infections (eastlund 2005), including human immunodeficiency virus type 1hiv-1, hepatitis c virus (conrad. 1995 ; cdc 2011) with bone allografts procured and processed under aseptic conditions and preserved by freezing. moreover, there have been publications proving that durability of such bone grafts deteriorates over the preparation process and, above all, during radiation - sterilisation (pelker. 2012a, b), as well as the presented case report, proved that the concerns are groundless. frozen bone grafts radiation - sterilised even with as high dose as 35 kgy were entirely safe for the recipient s health and its structure provided excellent scaffold for the new bone formed in the process of osteoinduction. this is only one of possible grafting materials for reconstruction of extremely atrophic alveolar ridge. due to the lack of the permission of the bioethics committee it was impossible to perform the bone biopsy at the area of implantation. marked bleeding observed during implant embedment indicating revascularisation was the only unbiased proof of the graft incorporation and remodelling. authors own experience implied that the size of the grafted block should be only slightly bigger than necessary to embed implants, because bone atrophy was found only at the margin of the allograft and around the securing screws. the material may be ordered in any shape and size and adjusted to the form of the recipient site, which provides an advantage over autograft material and seems to possess comparable biological properties. the authors would like to emphasise the need of obtaining normal stability of the graft, i.e. durable fixation with screws, sealing the donor / recipient site borderline with bone shavings as well as covering the operational site with prf membranes, which are a concentrate of growth factors such as : three pro - inflammatory cytokines (il-1beta, il-6, and tnf - alpha), an anti - inflammatory cytokine (il-4), and a key growth promoter of angiogenesis (vegf) and hence accelerate reorganisation of the graft (dohan. it is also important to cover the wound with an unstretched mucous membrane to avoid its abrasion and exposing the allograft to the oral environment. | due to atrophy of the tissue within the alveolar ridge, implantation must sometimes be preceded by bone regeneration. the use of allogeneic material allows the surgeon to prepare grafts of any shape and amount ; therefore it is a good alternative to autograft reconstruction in patients with extensive atrophy of the alveolar ridge. the patient with maxillary anodontia showed insufficient width of the ridge along its entire length, which prevented implantation. therefore, alveolar ridge reconstruction was planned. four frozen, radiation - sterilised bone blocks processed in the tissue bank in warsaw were used for reconstruction of the alveolar ridge. the blocks were grafted to the area of molars, premolars and lateral incisors bilaterally. three months after surgery a normal union of transplants with the recipient site was achieved. six implants were embedded and following the 6-month integration period a permanent prosthetic restoration was successfully performed. during a 38-month follow - up none of the implants were lost and the aesthetic or functional condition of the prosthetic restoration did not deteriorate. frozen allogeneic radiation - sterilised bone blocks constitute good, efficient and safe material used in reconstruction of the alveolar ridge in extensive bone atrophy. this is only one of possible grafting materials for reconstruction of extremely atrophic alveolar ridge. |
lipids and their simplest structural elements, the fatty acids, provide myriad functions at all levels of cellular life. nutritional scientists are still wrestling to develop a rudimentary understanding of the roles that dietary lipids exert. lipids as simple fats are the most concentrated energy source in the diet. until recently, this fact alone made lipids a valuable food component ; however, a global epidemic of caloric imbalance and obesity has undermined this one aspect of lipid nutrition. nonetheless, dietary lipids are well recognized as providing the essential fatty acids and to dissolve and aid in the absorption of fat - soluble vitamins. fats also produce a broad range of effects to whole body metabolism when consumed in foods. these effects, although not yet fully understood, are a complex consequence of the absolute and relative fat content, the fatty acid composition, the structure of other components in the foods, the timing of consumption and individual variations among those consuming them. once ingested, lipids provide a diverse range of molecular functions and actions within cells and tissues beyond providing simple energy. fatty acids are required for membrane synthesis, modifications of proteins and carbohydrates, construction of various structural elements in cells and tissues, production of signaling compounds and for oxidative fuel. the ability of lipids to associate spontaneously into multi - molecular structures of non - polar substituents provides a unique domain structure to biology (vesicles, globules, lipoproteins). these structures solubilize a variety of non - polar and poorly soluble cellular and extracellular constituents and transport such molecules within and between cells and tissues. given these various roles, why would saturated fats be so poorly thought of nutritionally ? in one sense, saturated fats in the diet are unnecessary. all organisms, including humans, are fully capable of synthesizing saturated fatty acids. in the absence of sufficient dietary fat, the body is apparently capable of synthesizing all of the saturated fatty acids that it needs from the ubiquitous precursor building block acetate. in fact, cells produce a remarkable diversity of saturated fatty acids under particular conditions, and although not all of their functions are known, they are clearly not simply interchangeable. compositional analyses reveal remarkable specificities for particular saturated fatty acids in different lipid classes, cellular compartments and tissues. stubbs and smith reviewed studies aimed at understanding the requirement of membranes for specific fatty acid compositions. interestingly, although composition is sensitive to polyunsaturated fatty acids, the content of saturated fatty acids in rat tissue membrane phospholipids is relatively constant at ~40% regardless of dietary fat source, indicating a control mechanism at some level. the de novo synthesis of saturated fatty acids is inhibited by feeding a high - fat diet, and membrane fragility resulting from feeding a low dietary saturated fatty acid diet is overcome by feeding a diet rich in fat. the complexity of structure and the diversity of functions of fatty acids, both unsaturated and saturated, remain poorly understood, and in only a few biological situations have distinct actions of fatty acids been described. the majority of research on fatty acids consumed in the diet has focused principally on their role in lipoprotein metabolism. authors of a recent meta - analysis of prospective studies on dairy food consumption and incident vascular disease and type 2 diabetes concluded that it is not possible to estimate quantitative relationships with disease incidence with any confidence. the authors also point out that apart from the effects of dairy foods on plasma lipids and on blood pressure, very little is known about the biological mechanisms underlying such relationships. even for lipoprotein metabolism, for which literally billions of dollars have been invested in research, little is actually known. for example, only in 2005 was the basic mechanistic link between saturated fatty acids and cholesterol metabolism revealed. this relationship between saturated fat in the diet and cholesterol metabolism was one of the most baffling scientific challenges of the twentieth century. how could such a ubiquitous, non - essential component of diets and tissues saturated fat cause an increase in the accumulation of cholesterol - rich ldl in blood ? as scientific research on cholesterol metabolism proceeded through the twentieth century, the question became even more perplexing. brown and goldstein won the nobel prize for identifying the ldl receptor on the liver as being what controlled the concentration of serum cholesterol. what then regulates the expression of the ldl receptor on the surface of liver cells ? cellular cholesterol levels within the liver cell simultaneously control cholesterol synthesis and uptake by regulating the expression of the genes for the proteins that make cholesterol in the cell and for the proteins that take up cholesterol from blood as ldl. not surprisingly, when cholesterol levels in the cell are adequate, the genes are not turned on. however, when cholesterol levels are low, all of these genes are turned on using the identical transcription factor protein although these studies made sense of cellular cholesterol regulation if the cell needs more cholesterol, it simultaneously turns on the genes to make more via cholesterol biosynthetic enzymes and takes more from blood via the ldl receptor they failed to explain the role of diet in these processes. if the same transcription factor turns on both cholesterol biosynthesis and the ldl receptor, how can saturated fat uncouple these two processes, simultaneously making more cholesterol and yet shutting down the receptor ? puigserver and spiegelman found that the liver contains an additional gene control system, the peroxisome proliferator activating receptor (ppar), and it is in turn controlled by a higher order protein complex termed (logically) the ppar gene transcription coactivator (pgc-1). this transcription factor coactivator family recruits entire complexes of proteins into transcriptional regulatory units controlling such multi - faceted properties as mitochondrial biogenesis. in a striking result, lin. discovered that, when exposed to high levels of saturated fatty acids, liver cells both in vivo and in vitro actively turned on pgc-1b, and even more astonishingly, this coactivator simultaneously turned on cholesterol biosynthesis and turned off the ldl receptor. thus, in one bold study, the basic target linking dietary saturated fat and serum cholesterol was revealed. this mechanism would account for the tendency of diets very high in saturated fat to raise total cholesterol in blood. for some individuals, such a response could raise one of the risk factors for heart disease if they consumed diets high in saturated fat. an obvious question is what are the benefits to this biochemical response that would have caused it to be selected through evolution ? scientists are only now beginning to address the basic biological value of this regulatory control system. nonetheless, diets very high in saturated fat would not seem prudent, as would any diet high in any food component. recommendations that the population decrease their intake of saturated fats was based not on a mechanistic understanding, but on years of observational evidence that dietary saturated fats generally increase blood cholesterol concentrations in animals and humans. this alteration of risk factors does not necessarily lead to increases in heart disease and is certainly not universally true in all populations studied. some studies of human populations evaluating the effects of saturated fat diets do not show the predicted elevation in heart disease ; in fact, some studies see the reverse effect. recent studies are beginning to assign genetic or physiological explanations to these varying outcomes ; for example, low birth weight appears to have an effect on subsequent responses to dietary fat. beare - rogers suggested that the saturated fatty acid requirements are also related to the stage of development. for example, saturated fatty acids appear to be essential for the newborn, the young and during rapid growth as they are required for the synthesis of membranes and lipoprotein. is the justification for broad recommendations to lower total fat intakes in all individuals supported by scientific evidence ? in 1977, the us population was first recommended to reduce the intake of fat, with some recommendations being to reduce total fat to below 30% of calories. the american heart association recommended that the percentage of calories be 28.6 and 25.3% total fat, respectively, and 9 and 6.1% saturated fat, respectively, in step 1 and step 2 diets for treatment of high blood cholesterol. framingham heart study data showed that people with high triacylglycerol concentrations (> 1.7 mmol / l) and low hdl cholesterol concentrations (1.7 mmol / l) and low hdl cholesterol concentrations (< 1.03 the long - term health benefits of consuming a low - fat diet particularly taking into account the variation in human responses have not been proven and, to the contrary, some individuals move their risk profile, even for heart disease, in an adverse direction [17, 18 ]. in one study, healthy, non - diabetic volunteers consumed diets that contained, as a percentage of total calories, either 60% carbohydrate, 25% fat and 15% protein, or 40% carbohydrate, 45% fat and 15% protein. those consuming the 60% carbohydrate diet had higher fasting plasma triacylglycerol, remnant lipoprotein and remnant lipoprotein triacylglycerol, and lower hdl cholesterol without changing ldl cholesterol concentration. the low - fat diet lowered hdl cholesterol and caused a persistent elevation in remnant lipoproteins, both factors that are increasingly recognized to be important independent risk factors for heart disease and other metabolic diseases. these findings led the investigators to publish the question whether it is wise to recommend that all americans replace dietary saturated fat with carbohydrate. it is important to point out that dietary carbohydrates have been associated with dyslipidemia, and lipoprotein risk factors are similar whether diets are high in fat and saturated fat or low in fat and high in sugar. elevated triglyceride concentrations are related to increased hepatic secretion and impaired clearance of vldl lipoprotein [21, 22 ]. triglyceride response to dietary sugar may vary with the amount of sugar and the presence of other nutrients. stanhope and havel reported that a high - fructose diet led to visceral adiposity, dyslipidemia and insulin resistance, and insulin resistance upregulates vldl production. perhaps the study most devastating to the basic principle that a lower fat diet improves the health of the entire population was a prospective study (the women s health initiative randomized controlled dietary modification trial) that selected approximately 49,000 women to compare a group of women consuming low - fat diets and increased fruit and vegetable consumption with a group receiving only diet - related education materials. two possible interpretations could be drawn from this study : first, that lower fat intakes have no effect on any women, and second, that individuals vary in their response to fat and some women are benefitted and some are adversely affected and the net numbers of each are relatively close, leading to a conclusion in this trial of no effect of a low - fat diet. ordovas reviewed key factors in lipid metabolism and obesity that indicate an interplay among genes, gender, and environmental factors that modulate disease susceptibility. studies of response to dietary fats have found variation among individuals and differences between men and women in their response to dietary fat changes. studies of individual sensitivity to changes in dietary saturated fats showed groups of consistent hyper - responders and minimal responders within a population of hypercholesterolaemic individuals. measurements of serum cholesterol in response to a decrease in dietary saturated fat showed that total cholesterol decreased to a greater extent in men than in women. a series of studies showed that very - low - fat (10%), high - carbohydrate diets enriched in simple sugars increased the synthesis of fatty acids, especially palmitate, and that the individual differences in increased blood triglyceride concentrations varied considerably. these fluctuations observed time after time have given impetus to the field of nutrigenomics, and scientists are now pursuing more detailed analyses of individuals, their responses to diet and the mechanistic basis for variations in diet and health risk. controversy still remains high as to the roles that dietary fat and cholesterol play in the risk of heart disease, and the wealth of confounding factors demonstrate that saturated fat is not an overwhelming input variable for any population studied to date. dietary saturated fats are not the only variables associated with heart disease the causes are multi - factorial. the results of studies on the etiology of heart disease are inclusive and sometimes contradictory. it is time to take a broader view to the multiple actions and functions of each of the different saturated fats and a more individual view to assessment of diet and risk. the overwhelming emphasis on the role of saturated fats in the diet and risk of coronary heart disease has distracted investigators from studying other effects that individual saturated fatty acids may have in the body. this omission is perhaps important considering the abundance in mammalian milks of a wide range of saturated fatty acids with different chain lengths. in the context of evolution and the obvious natural selective pressure on the development of milk and all of its constituents, how do saturated fatty acids affect growth, development and survival of mammalian offspring ? fatty acids are present in all body tissues, where they are a major part of the phospholipid component of the cell membrane. they contribute to the structural diversity within the membrane, which is now recognized to be a key aspect of membrane functions. fatty acids anchor proteins to particular regions of cell membranes, participate in signaling activities, transport cellular components and provide fuel. saturated fatty acids have been suggested as being the preferred fuel for the heart. in the absence of sufficient fat from the diet, an interesting observation has been made that in adult rat liver, erucic acid (22:1)a fatty acid that has been associated with heart disease and that is present in rapeseed oil is rapidly converted to 18:0, demonstrating the conservation of carbons by chain - shortening of a monounsaturated fatty acid to an unsaturated fatty acid. even though all fatty acids present in the diet can be broken down and resynthesized into saturated fats, they have discrete effects. different structures of fatty acids appear to have differing effects on a variety of metabolic and physiological processes when they are ingested. short - chain fatty acids are hydrolyzed preferentially from triacylglycerols and absorbed from the intestine into the portal circulation without resynthesis of triacylglycerols. butyric acid (4:0) is the shortest saturated fatty acid and is present in ruminant milk fat at 25% by weight, which on a molar basis is about one - third the amount of palmitic acid (16:0). no other common food fat contains this fatty acid directly ; however, the consumption of a wide range of fermentable carbohydrates can lead to the synthesis of butyric acid by endogenous microflora in the lower intestine. butyrate is a well - known modulator of genetic regulation, and its ability to promote differentiation has led various investigators to pursue this mechanism as a means to alter the risk and development of cancer [35, 36 ]. this fatty acid also lowers processes of inflammation in the intestine, acting through short - chain fatty acid - binding receptors. in bovine and human milk, caproic acid (6:0). 1 and 0.1%, respectively, and caprylic acid (8:0) and capric acid (10:0) are present at ca. 0.3 and 1.2%, respectively, of the milk fat. not surprisingly from its nomenclature, goat milk contains the highest percentage of caprylic acid, at 2.7% of milk fat. studies to date have documented that these three fatty acids have similar biological activities when tested as antimicrobial agents. the monoglyceride form, monocaprin, has been shown in vivo in animals to possess antiviral activity against retrovirus infection. studies have shown antiviral and antibacterial activities of lauric acid [40, 41 ]. release of lauric acid in the stomach may have direct antimicrobial activities towards helicobacter pylori, either as the fatty acid or monoacylglycerols produced by lingual lipase(s) acting on the triacylglycerols present milk fat [42, 43 ]. these antibacterial actions of lauric acid have been proposed to provide anticaries and antiplaque activities. the overall antimicrobial effects of the medium - chain saturated fatty acids and their monoacylglycerol derivatives on various microorganisms, including bacteria, yeast, fungi and enveloped viruses, were originally suggested to be acting through the lipid membranes of the organisms. monolaurin released from milk lipids by lipases may account for milk s anti - protozoal activities. the biological activity of laurate has been interesting in other aspects not related to the diet. for example, a remarkable experiment showed that monolaurate provided considerable protection from hiv infection when used topically on reproductive tissues in primates. bovine milk fat contains 814% myristic acid (14:0) and in human milk, it averages 8.6% of the milk fat. human epidemiological studies have shown that myristic acid and lauric acid were the saturated fatty acids most strongly related to the average serum cholesterol concentrations in humans. nonetheless, several studies have shown that myristic acid increases hdl cholesterol at least as much as ldl cholesterol, and further studies have demonstrated that the unique positional distribution of myristic acid in the sn-2 position of triglycerides in milk fat is responsible for its tendency to raise hdl. palmitic acid is present in human and bovine milk at 22.6 and 26.3%, exclusively esterified at the sn-2 position of the triglyceride. human infants consuming a formula containing triacylglycerides similar to those in human milk (16% palmitic acid esterified predominantly to the sn-2 position) have improved intestinal absorption not just of the palmitic acid but calcium as well [51, 52 ]. recently, speigelman s group showed that palmitic acid stimulated the expression and activities of the transcription coactivator pgc-1b and by so doing promoted the transcriptional regulation of biosynthesis of lipoproteins from the liver. this finding places a mechanistic understanding of the cellular actions of saturated fatty acids, particularly palmitic acid. in the context of milk, this mechanism implies that palmitic acid may have an important role in promoting successful lipoprotein metabolism in infants. finally, pgc-1b was shown to increase biogenesis of mitochondria in neurons, further implying that this mechanism could well be involved in diverse aspects of metabolic regulation and its saturated fatty acid - appropriate development throughout the body in infants. it is not known if the presence of palmitic acid in human milk is important in the coordinate regulation and activity of pgc-1b in any of these activities. stearic acid (18:0) is an abundant fatty acid in milk, present in human and bovine milk fat at 7.7 and 13.2% of fat, respectively. stearic acid is synthesized from palmitate via the elongase enzyme, either in the mammary gland by the same enzyme that is active in liver, coded by gene elov5 [55, 56 ], or by elov1, whose expression is induced in the mammary gland during lactation [57, 58 ]. both of these genes are regulated by diet, hence the net production of stearic acid is under various aspects of metabolic control. surprisingly, little research has pursued the specific actions of stearic acid when consumed in milk by infants, in spite of its abundance and obvious regulation within the mammary gland during fat synthesis. in adults, stearic acid does not appear to raise serum cholesterol, hence it is considered neutral to heart disease risk. this fatty acid may exert other effects also consistent with protection from heart disease via separate mechanisms. g / d) for 4 weeks exhibited beneficial effects on thrombogenic and atherogenic risk factors as compared with the effects of dietary palmitic acid. fat - soluble nutrients include the essential nutrients vitamins a, d, e and k, carotenoids as vitamin a precursors, essential polyunsaturated fatty acids, and non - essential nutrients such as various tocopherols, phenolics, carotenoids (e.g., lycopene, lutein and zeaxanthin) and conjugated linoleic acid isomers that can not be made by humans. fat - soluble nutrients are increasingly recognized as pleotrophic nutrients with several discrete actions in addition to the direct functions for which their essentiality has been established. as a result, consumption of these components is considered to have biological activities beyond the simple prevention of deficiency and is consistent with many aspects of health [6062 ]. in epidemiological studies, the abundance of fat - soluble nutrients in tissues is frequently reported to be inversely correlated with a variety of chronic and degenerative diseases, including cancers [63, 64 ], cardiovascular diseases [65, 66 ], diabetes and specific tissue degeneration such as macular degeneration [58, 59 ]. with the recognition that there are potential health values associated with the presence of fat - soluble nutrients in tissues non - polar molecules are poorly absorbed, and it is not certain that the presence of a component in a food means that it will be absorbed and delivered to particular tissues in which it might be active. the lipid - soluble components of milk appear to be well absorbed into and accumulated in tissues, although the basic mechanisms by which such a net delivery is accomplished are not known [70, 71 ]. fat - soluble nutrients include the essential nutrients vitamins a, d, e and k, carotenoids as vitamin a precursors, essential polyunsaturated fatty acids, and non - essential nutrients such as various tocopherols, phenolics, carotenoids (e.g., lycopene, lutein and zeaxanthin) and conjugated linoleic acid isomers that can not be made by humans. fat - soluble nutrients are increasingly recognized as pleotrophic nutrients with several discrete actions in addition to the direct functions for which their essentiality has been established. as a result, consumption of these components is considered to have biological activities beyond the simple prevention of deficiency and is consistent with many aspects of health [6062 ]. in epidemiological studies, the abundance of fat - soluble nutrients in tissues is frequently reported to be inversely correlated with a variety of chronic and degenerative diseases, including cancers [63, 64 ], cardiovascular diseases [65, 66 ], diabetes and specific tissue degeneration such as macular degeneration [58, 59 ]. with the recognition that there are potential health values associated with the presence of fat - soluble nutrients in tissues non - polar molecules are poorly absorbed, and it is not certain that the presence of a component in a food means that it will be absorbed and delivered to particular tissues in which it might be active. the lipid - soluble components of milk appear to be well absorbed into and accumulated in tissues, although the basic mechanisms by which such a net delivery is accomplished are not known [70, 71 ]. breast feeding stimulates the production of serum lipids and lipoproteins [72, 73 ]. interestingly, this increase in serum lipids in infancy is reversed in adulthood. nonetheless, throughout life, when compared with either carbohydrates or polyunsaturated fatty acids, the consumption of bovine milk fat results in the elevation of circulating hdl cholesterol. decades of research have documented that blood hdl cholesterol concentrations are a very strong and independent predictor of heart disease. this relationship has not been as successfully exploited therapeutically as lowering ldl, however, because hdl concentrations are not as responsive to diet and drugs as those of ldl. a major pharmacologic effort has pursued an increase in hdl cholesterol concentrations in humans as a means to reduce cardiovascular risk. this research is based on the extensive evidence of the associations of high hdl with protection from heart disease, even in the face of elevated ldl. there is also evidence of an opposite relationship, that low hdl is associated with increased risk, with or without elevated triglycerides. however, it has not been possible to assign independent variables to hdl differences, and studies have largely been based on hdl concentrations that are presumably high or low based on genetic rather than dietary determinants [76, 77 ]. hdl exert beneficial effects on overall health by myriad mechanisms, including binding and eliminating toxins, delivering bioactive compounds, protecting various cells and lipoproteins from damage and participating in their repair [7880 ]. hdl is particularly important in the successful response to infection by binding and clearing bacterial endotoxin or lipopolysaccharide (lps). lps is the major glycolipid component of gram - negative bacterial outer membranes and is responsible for pathophysiological symptoms characteristic of infection. a wide variety of studies have documented that lps is associated with plasma lipoproteins, suggesting that sequestering of lps by lipid particles may form an integral part of a humoral detoxification mechanism [81, 82 ]. the binding of lps to lipoproteins is highly specific under simulated physiological conditions, and hdl has the highest binding capacity for lps [83, 84 ]. this basic protection mechanism may be particularly important for children and for intestinally derived endotoxin. lipoprotein complexes may be part of a local defense mechanism of the intestine against translocated bacterial toxin. because milk fats enhance hdl concentrations, they are of potential importance in protection against bacterial lps toxicity. the genes and biochemical processes of lactation that produce milk fat evolved under the constant selective pressure of nourishing mammalian infants. the composition and structures of lipids in milk provide bioactive components that, although not identified as essential nutrients by standard definitions, none - the - less serve important functions as structural building blocks, fuels, transport systems, anti - inflammatory, anti - bacterial and antiviral agents in the intestine. these lipids include triacylglycerols which are metabolized to monoacyl- and diacylcerides and fatty acids and phospholipids such as sphingomyelin. the lipids in milk are also carriers of important fat - soluble vitamins such as vitamin e, vitamin a and vitamin d. the absolute quantities and proportionate balance of the various macronutrients in human diets remains the subject of both scientific research and public health speculation. although unquestionably evolved to nourish infants, detailed examinations of milk and lactation in humans and other mammals are revealing new insights into structures and functions of different components in the diet, including fat. the gene set responsible for the production of lipids is a conspicuously retained subset of the genome throughout mammalian lactation, implying that milk is, in many respects, a lipid delivery system. saturated fatty acids are a significant component of all mammalian milks examined, including human milk. thus, whereas diets inordinately high in any component are likely to be net deleterious, finite quantities of saturated fatty acids may provide distinct mechanistic benefits to various metabolic processes. recognizing that different humans with different lifestyles respond differently to fat intakes and compositions in such a future, finite intakes of specific saturated fats may actually be recommended. | for recommendations of specific targets for the absolute amount of saturated fat intake, we need to know what dietary intake is most appropriate ? changing agricultural production and processing to lower the relative quantities of macronutrients requires years to accomplish. changes can have unintended consequences on diets and the health of subsets of the population. hence, what are the appropriate absolute amounts of saturated fat in our diets ? is the scientific evidence consistent with an optimal intake of zero ? if not, is it also possible that a finite intake of saturated fats is beneficial to overall health, at least to a subset of the population ? conclusive evidence from prospective human trials is not available, hence other sources of information must be considered. one approach is to examine the evolution of lactation, and the composition of milks that developed through millennia of natural selective pressure and natural selection processes. mammalian milks, including human milk, contain 50% of their total fatty acids as saturated fatty acids. the biochemical formation of a single double bond converting a saturated to a monounsaturated fatty acid is a pathway that exists in all eukaryotic organisms and is active within the mammary gland. in the face of selective pressure, mammary lipid synthesis in all mammals continues to release a significant content of saturated fatty acids into milk. is it possible that evolution of the mammary gland reveals benefits to saturated fatty acids that current recommendations do not consider ? |
as a common side effect of treatment for diabetes, hypoglycemia is a constant threat and can have far - reaching and potentially devastating consequences, from irreversible coma and infarction to cognitive decline and dementia, although the relationship between treatment - related hypoglycemia and cognition remains unclear. one of the priority research areas defined by a recent workgroup of the american diabetes association and the endocrine society is to better understand the mechanisms of the associations between hypoglycemia and long - term outcomes, such as cognitive dysfunction (1). previous studies have reported a relationship between diabetes and dementia and between a history of severe hypoglycemia and impaired cognitive function in people with diabetes (2,3). severe hypoglycemic episodes, with or without coma, were associated with impaired cognitive function in children and young adults with type 1 diabetes and a greater risk of dementia among older patients with type 2 diabetes mellitus (t2 dm) (49). whether these effects are due to the hypoglycemia or to some related factor, however, remains unclear. in contrast, the diabetes control and complications trial (dcct) study found no significant correlation between diabetes treatment related hypoglycemic events and neuropsychological function (10). in a recent american diabetes association sponsored research symposium, diabetes and the brain, several hypotheses were proposed regarding how diabetes could affect learning and memory (11). an area that has not been well studied is the relationship between severe hypoglycemia and chronic anatomical changes in the brain that are known to correlate with cognitive decline. specifically, it is unclear whether hypoglycemia in patients with t2 dm is associated with chronic brain changes such as atrophy and increased abnormal white matter (awm). brain atrophy, whether measured by total brain volume (tbv) or regionally, is a sensitive and powerful correlate of cognitive function and decline (1216). awm tissue volume is indicative of diffuse and focal ischemic, demyelinating, and inflammatory processes related to small vessel disease, and increased awm tissue volume is associated with diabetes and impaired cognition (17,18). thus, the specific aim of our study was to determine whether severe symptomatic hypoglycemic events in treated t2 dm patients are associated with loss of tbv and/or increase of awm. we conducted a cohort analysis of the memory in diabetes (mind) study (19), which was done in 51 clinical sites in north america as part of action to control cardiovascular risk in diabetes (accord), a randomized trial of standard compared with intensive management of glycemia in people with t2 dm. participants aged 4579 years with t2 dm and who had a current glycosylated hemoglobin (hba1c) level at 7.511.0% (5897 mmol / mol) and were at high risk for cardiovascular disease events suggested by atherosclerosis, left ventricular hypertrophy, albuminuria, or at least two additional cardiovascular risk factors, were enrolled between 21 august 2003 (34 months after the start of accord) and 16 december 2005. participants were randomly assigned to an intensive strategy, aiming to achieve an hba1c 2 or any lower - extremity amputation. letters, 75 equals visual acuity of 20/30. there were 28 participants who reported having at least one episode of ha before the 40-month mri exam, and 6 of them reported coma during the ha episode. among the 28 participants with ha, 19 (67.86%) had one episode, 5 (17.86%) had two, 2 (7.14%) had three, and 2 (7.14%) had five. the mean time between ha and the 40-month mri scans was 25.5 11.7 months, with the median at 28 months (min 3.29, max 41.2). compared with participants without ha, participants who developed ha were significantly more likely to be older (66.82 6.37 vs. 61.91 5.50 years, p 5 years (39). our study also found that hypoglycemia was significantly more likely to occur in older patients who had insulin, intensive glycemic control, longer duration of t2 dm, more neuropathy and worse visual acuity scores. these findings are consistent with the accord study and comparable with the studies of other t2 dm populations (22,40,41). the intensive glycemia control intervention of the accord trial was stopped early because of higher mortality in this study arm (42). however, although hypoglycemia was associated with an increased risk of death, no differences in glycemia - related mortality risk between participants in the two treatment arms were noted (22). the mechanism for the increased mortality in the intensive glycemia control arm of accord therefore remains unknown. a cerebral mechanism is not supported by our study, which shows no incremental adverse effect of hypoglycemic events on tbv and awm. this would be consistent with, but not proof of, the relative resilience of the brain to hypoglycemic insult ; however, these brain mri variables reflect only structure, not function. first, even though this appears to be the largest mri study of brain structural changes in hypoglycemic patients, the number of hypoglycemic events was relatively few in our study, and future studies with larger sample sizes may provide further evidence to support our findings. second, not every t2 dm patient had 40-month follow - up mri scans, and thus, the factors associated with missing mri scans were used in a multiple imputation procedure to explore the sensitivity of results to missing outcomes. in conclusion, results from this small sample of events show that severe symptomatic hypoglycemic events in treated t2 dm patients are not associated with accentuated loss of tbv and/or increase of awm compared with t2 dm patients without hypoglycemia. although the undesirable effects of treatment - induced hypoglycemia are of increasing clinical concern, particularly when more aggressive management of hyperglycemia is contemplated, the lack of associated changes in gross brain structure suggests that there may be some fortuitous resilience of this organ. further studies with more details of hypoglycemic events and longitudinal mri scans before and after hypoglycemia can help to better specify whether the severity of hypoglycemia affects the longitudinal changes of mri brain structure. | objectivethe effect of hypoglycemia related to treatment of type 2 diabetes mellitus (t2 dm) on brain structure remains unclear. we aimed to assess whether symptomatic severe hypoglycemia is associated with brain atrophy and/or white matter abnormalities.research design and methodswe included t2 dm participants with brain mri from the action to control cardiovascular risk in diabetes - memory in diabetes (accord - mind) trial. symptomatic severe hypoglycemia was defined as blood glucose < 2.8 mmol / l or symptoms resolved with treatments that required the assistance of another person or medical assistance (hypoglycemia requiring assistance [ha ]). standardized brain mri was performed at baseline and at 40 months. total brain volume (tbv) and abnormal white matter (awm) volume were calculated using an automated computer algorithm. brain mri scans of hypoglycemic participants were also reviewed for local disease.resultsof the 503 t2 dm participants (mean age, 62 years) with successful baseline and 40-month brain mri, 28 had at least one ha episode during the 40-month follow - up. compared with participants without ha, those with ha had marginally significant less atrophy (less decrease in tbv) from baseline to 40 months (9.55 [95% ci 15.21, 3.90 ] vs. 15.38 [95% ci 16.64, 14.12 ], p = 0.051), and no significant increase of awm volume (2.06 [95% ci 1.71, 2.49 ] vs. 1.84 [95% ci 1.76, 1.91 ], p = 0.247). in addition, no unexpected local signal changes or volume loss were seen on hypoglycemic participants brain mri scans.conclusionsour study suggests that hypoglycemia related to t2 dm treatment may not accentuate brain pathology, specifically brain atrophy or white matter abnormalities. |
the concept of cancer stem cells (csc) arose from the discovery that a majority of cells from some human leukemia (acute myeloid leukemia), at different stages of differentiation, originated from transformed undifferentiated pluripotent stem cell. this led to the hypothesis, then the theory, that as in the normal somatic stem cells (ssc) and their derived tissues, a small population of cells, the cancer stem cells (csc) would reproduce ad infinitum and generate the very diverse, limited lifespan, multi - lineage differentiated majority of cells in a cancer, called the derived population cells (dc) (fig. 1). this was the concept of an aberrant stem cell system, a system gone awry. in agreement with such a scheme, csc were assumed to originate from somatic stem cells (ssc) and to represent a minor, qualitatively distinct, eternal population, transforming deterministically and irreversibly in a limited lifespan, more or less differentiated hierarchy of derived cells that would constitute the bulk of phenotypically diverse cancer cell populations. of course clinicians, frustrated by the only partial and transient success of their therapies, liked the idea. if the concept was valid, they would have to deal with one well - defined, but difficult to identify and study, population of cells responsible for a cancer. this would allow them to solve the problem of a cancer with one therapy well aimed at these cells. for industry the definition of different causal populations of cells in different patients would also allow proposing combinations of diagnoses and treatments for each patient. the experimental support of the concept was essentially that in xenotransplant experiments in immunologically deficient mice, only a minor fraction of the injected cells would generate tumors : the cancer stem cells. the origin of the csc in the transformation of ssc seemed logical. in cell - shedding tissues, like the skin and mucosae, it makes sense, as only the ssc would have the time to accumulate the range of mutations necessary for cell transformation. however, if the first oncogenic event is to confer an increase in loco of the lifespan to the cells, this would not be necessary. in fact, in experiments in various models in which an oncogene was expressed all during the cells reproducing and differentiating sequence, the transformation took place at the stage of pluripotency as well as of unipotency and even later (for review, see ref., although, to our knowledge, it was not formally expressed, the concept of a minor population of cells, responsible for the progression and evolution of a cancer cast a doubt about the validity and interest of the work on epigenetics and transcription expression performed in bulk tumors, in which most of the cells would be degenerate or terminally differentiated. the fundamental results of such studies in our knowledge of the biology of cancer and on prognostic diagnosis disagree with this concept : for instance gene expression allows to clarify and treat specifically breast and other cancers. the notion of csc as a qualitatively distinct population of cells has been implicit and sometimes expressed. it is not supported by the various experimental tests applied to the cells (transplants, sphere formation in vitro, cell sorting with biomarkers, etc.) which always show overlaps with other cells (dc). on the other hand, if properly transplanted in fully immune - compromised mice, more than one - third of human melanoma cells generate tumors, i.e., would be csc. even in acute lymphoid leukemia, intratumor heterogeneity also bears against the concept of one population of cells generating all the cells in a cancer. for example, the intratumor heterogeneity of human glioblastomas and other tumors suggests a multiclonal evolution compatible with the stochastic but not the csc model. another implied property of csc is, as for ssc, the irreversibility of the transformation in more differentiated cells, dc. this property, in fact, is now questioned for ssc and embryonic stem cells (esc), since the expression of just 4 transcription factors has now been shown to be sufficient to convert fully differentiated cells to an esc state, and progenitor cells spontaneously revert to ssc upon crypt cloning. as for csc dc cell interconvertibility, the constant conversion of a few csc cells in many multiplying dc is certainly easier to demonstrate than a rare reversion of these numerous dc into a tiny minority of csc. the irreversibility is not valid in the case of human melanoma and is disproved by the regeneration over time in culture of a diversified cell population from purified dc and by the generation of cancer cells with stemness properties spontaneously following oncogene expression in primary p53-null mouse cells or metastasis or following emt induction in cell lines. conversely, csc, emt, chemoresistance characteristics are acquired by lung cancer cell lines after treatment with anti egfr drugs. linked to the concepts of irreversibility and qualitative nature of csc to dc transformation the deterministic character of the conversion was even related to the semiconservative character of dna replication, an asymmetric cell division, of one csc to one conserved csc and one derived cell. this deterministic character opposes all the evidence in favor of stochastic mechanisms of cancer cell diversity. of course, again, if these properties applied only to csc, the therapeutic problem would also converge on this cell population. however if csc multiply fast (vide infra), it should be related to another attribute. indeed abcg drug transporters and enhanced dna repair have been observed in some cancer cells. however, again, the congruence of these properties with the other proposed csc properties has not been demonstrated. the analogy between ssc and csc has even been extended to the concept of the niche. in fact, rapidly dividing supposed csc are often found in preferential sites of the tumor, e.g., near capillaries. however, it is a farfetched extrapolation to interpret these favorable spaces as preordained sites, the niche. it seems simpler, even if less spectacular, to assume that an oxygen - rich environment is more favorable for proliferation. the use of biomarkers, allowing purifying, to some extent, csc by cell sorting, has greatly contributed to the expansion of the field (e.g., cd44, cd133, ald4). however, the validity of these biomarkers is cast in doubt by one disturbing fact. although the question was asked several times in reviews, sequential cell sorting purifying csc having all the properties and biomarkers of csc has, to our knowledge, never been reported. in fact, the fraction of cells expressing one biomarker in breast csc was not expressing another biomarker. with this and other methodologies, the separation of a proportion of cells by any criterion (whether seeding capacity, sphere formation, biomarker - based cell sorting, etc.) assumes that the property used is stable in tissue, i.e., cells in a negative phase of any fluctuating property (e.g., epigenetic, transcriptional, feedback linked control) will be missed. of course this would not apply to a qualitatively irreversibly well - defined csc population ! in fact, any criterion used to define a cell population applies to those cells that answer it at the moment it is used. in fact some of them are good markers for normal tissue adjacent to a tumor. in the csc literature, there has always been an ambiguity with regard to the proliferative or quiescent state of the csc cells. in fact, the normal stem cell models have opposite characteristics : while esc are strongly proliferative, ssc were, until recently, considered as rarely dividing cells. this was even used, for some ssc, as a criterion to identify them by the remnants of their dna labeling. however, even for some ssc, it is now accepted that there may be 2 interconverting populations of ssc, one quiescent, one proliferating. the concept of quiescent csc, while useful to explain the continued presence of cancer cells after therapy, does not fit in with other proposed properties of the supposed csc : e.g., the spheroid formation assay, which allows purification to some extent csc by selective growth, the sp cells which extrude drugs and which would soon disappear if they reproduced less than other cancer cells. moreover, this concept is even more incompatible with the existence of csc in cell lines : they would soon disappear. with the progressive abandonment of claimed ssc and esc properties, the csc concept has evolved toward the existence of very competitive cancer cells continuously generating a majority of cells, less active, degenerating, and with limited reproduction capacity and even survival potential. it explained the important discrepancy between cell proliferation rates, evaluated by tritiated thymidine labeling of biopsies, and the measured in vivo tumor growth rate in breast cancers. this has been confirmed and visualized more directly by the more sophisticated method of linear tracing in experimental models in which the expression of an oncogene is induced at a chosen time and the consequences examined in the cell of origin of the tumor and in its successive descendants. the hierarchy of progressively more diversified tumor phenotypes, in fact, reproduces the well - known behavior of cells in developing metastases. it is easily explained by the fact that most genetic (and probably epigenetic and others) events, when not neutral, lead to loss of competitiveness and much more rarely to an increase of competitiveness. in the case of cancer cells, as in esc, the default state of the cell is differentiation, which may be due to loss of the stem cell program. in this framework, the different characteristics of so - called csc do not necessarily coexist in the same cells. in fact, in several cases, cells that exhibit the biochemical markers of csc are precisely not those that have the functional characteristics of sphere formation and tumorigenicity. the progressive simplification and evolution of the definition of csc has been called euphemistically a paradigm shift and described as the plasticity of csc, plasticity and clonal diversity of csc, stemness as a flexible quality of cancer cell, this constantly evolving definition has of course made the concept an elusive target for critical analysis. reprogramming stemness with nanog and lin28 may even reverse back the cancer character of sarcoma cells and, as shown more than 30 y ago, the oncogenic potential of teratocarcinoma cells after grafting in blastocysts was reversed. is stemness not rather a property or phenotype that cancer cells may have, lose, and even acquire, like the other hallmarks of cancer, rather than the definition of a distinct population of cells ? in the present stage of uncertainty, the use of a precise well - defined terminology based on operational definitions would certainly clarify a literature confused by the use of the variously defined, constantly changing, fuzzy concept of csc. the literature proposes such definitions : the cell of origin of a tumor (cot) is the one in which the original oncogenic event(s) occurred. the tumor initiating cell (tic) is the cell that first showed the signs of transformation and initiated the tumor. it may not necessarily be the cell of origin : for instance, in linear tracing experiments the expression of the oncogene begins at the time chosen by the experimenter, but the transformation may occur at a later stage of the cell evolution (e.g., pluripotent cell). cell of origin and tic identity may bear on the phenotype of the resulting tumor. the tumor propagating cell (tpc) or cancer repopulating cell is the already committed cancer cell that is able to generate a tumor ; for instance, in xenotranplants the cancer cell transplant does not initiate a cancer, it merely propagates it in a new site. xenotransplants can be orthoptic (implanted at the site of the naturally occurring tumor) or ectopic (other sites). in situ and metastatic propagations should be distinguished. cancer stemloids would be the rapidly dividing, evolving cells of the cancer that generate the slower dividing, more differentiated derived cells. this is a phenotype adopted for example by disseminated tumor cells, which at some time may explode in metastatic growth. in some cases, (e.g., cd44++ cd24- cells, cd133 + cells, aldehyde dehydrogenase - expressing cells adh, etc.) should be called by their operational property, e.g., cd133 cells etc. cancer cells expressing an embryonic stem cell program, essential for maintaining dedifferentiation, and thus repressing the expression of the various differentiation programs that constitute the hierarchical descendants of these cells, are just cells expressing the stemness esc program or pluripotency. such a program may totally or partially overlap with other programs developed at some time by cancer cells, such as the epithelial the 3 programs stemcellness, emt, and radioresistance can be induced together by emt inducing transcription factors (e.g., zeb) in some cell lines. thus, stemness would be a hallmark or a phenotype of cancer, probably linked to dedifferentiation, characterizing in a cancer some cells at some time. there would not be a separate population of csc, just as there is no population of cancer emt cells ! the stemness could be a functional (transplant, hierarchy, etc.) or / and a biochemical (expression of esc inducing transcription factors) property or hallmark. in fact, the state of any given cell in a tumor results from the programs expressed, at the time of the investigation, by this cell. this is more or less reversible depending of the mechanisms involved, genetic, epigenetic, or resulting from a signaling equilibrium. presumably, a large majority of tumor cells with a differentiated phenotype and a limited lifespan will never revert to active dedifferentiated phenotype. of course, if to treat a cancer one had to target one population of well - defined csc, the aim of therapeutic research should be to find drugs for this target. our and other analyses bear against this approach. on the other hand, if, as the evidence suggests, we have to deal with a constantly evolving diversified population of cells, the best therapies should then, as in the case of hiv, be simultaneous multi - target therapies : hit most of the cells at the same time, not allowing cells already resistant to one therapy to survive and permit some of them to acquire later resistance to another therapy. | the cancer stem cells (csc) hypothesis represents a pathological extrapolation of the physiological concept of embryonic and somatic stem cells. in its initial definition, it encompassed the hypothesis of a qualitatively distinct population of immortal cancer cells originating from somatic stem cells, which generate in xenotransplants by a deterministic irreversible process, the hierarchy of more differentiated finite lifespan derived cells, which constitute, themselves, the bulk of the cancer. these csc would express specific biomarkers and gene expressions related to chemo- and radioresistance, stemness, epithelial mesenchymal transition, etc. no convincing congruence of several of these properties in one cell population has been demonstrated. the concept has greatly evolved with time and with different authors (the plasticity of cancer stem cells), leading to a minimal definition of cells generating a hierarchy of derived cells. in this article these concepts are analyzed. it is proposed that stemness is a property, more or less reversible, a hallmark of some cells at some time in a cancer cell population, as immortality, dormancy, chemo- or radioresistance, epithelial mesenchymal transition etc. these phenotypic properties represent the result of independent, linked, or more or less congruent, genetic, epigenetic, or signaling programs. |
cirrhosis of the liver the only cure for which is liver transplant is associated with several serious complications, including ascites, spontaneous bacterial peritonitis, variceal bleeding, and hepatic encephalopathy (he). guidelines established by the american association for the study of liver diseases currently recommend referring patients with cirrhosis for liver transplant when their model for end - stage liver disease (meld) score is 10 and their child - turcotte - pugh (ctp) score is 7 or when they experience their first major complication (e.g., he, ascites, or variceal bleeding). however, the current united network for organ sharing allocation system only uses the meld score for prioritizing adults for liver transplant. the meld scoring system evaluates a patient 's short - term prognosis based on 3 common laboratory test results : serum bilirubin, international normalized ratio, and serum creatinine levels. however, this scoring system does not take into account several serious complications of cirrhosis, such as he, when prioritizing patients for liver transplant. this may have negative ramifications for patient care, as the development of he may be associated with substantial morbidity, mortality, and cost. he imposes a significant burden on patients, their families, and health care resources [5, 6 ]. he is characterized by alterations in behavior, cognitive abilities, consciousness, and neuromuscular function. it negatively affects patient quality of life (qol), and patients may be unable to drive, work, or adequately care for themselves because of its effects [811 ]. patients may be less compliant with all prescribed medications, and hospitalizations related to he may increase patient exposure to opportunistic infections and be associated with substantial costs. furthermore, he may be an independent predictor of mortality in patients with chronic liver disease. he occurring before liver transplant can also have a substantial negative impact on posttransplant outcomes [1322 ]. this paper will review the potential consequences of pretransplant he on posttransplant outcomes and therapeutic strategies for the pretransplant management of he. he may occur in patients with acute liver failure (type a he), in patients with portosystemic shunting but no intrinsic hepatocellular disease (type b he), or, as in the majority of he cases, in patients with cirrhosis and cirrhosis - related portosystemic shunting (type c he). because he is a progressive neuropsychiatric condition, he may be graded or scored based on the severity of the clinical manifestations, which may range from subtle neurologic abnormalities in mild cases to coma in severe cases. minimal he (sometimes referred to as covert he), which may occur in nearly 70% of patients with cirrhosis, is not associated with any clinical signs of brain dysfunction, but patients experience cognitive abnormalities that can lead to qol impairment [8, 24, 25 ]. unlike minimal he, overt he can manifest as a wide spectrum of symptoms that can be observed clinically, including those related to motor and neuropsychologic functions. overt he has been shown to occur in nearly half of patients with cirrhosis and, as with minimal he, also has a substantial negative impact on qol [710 ]. although research is ongoing, the pathogenesis of he is believed to primarily involve the exposure of the brain to elevated neurotoxin levels, particularly ammonia and other gut - derived toxins, leading to cellular morphologic changes (e.g., astrocyte swelling) and the development of a variety of neurochemical, neurotransmitter, and neuroinflammatory changes [27, 28 ]. many factors that can precipitate he (e.g., hypokalemia, infection, and gastrointestinal bleeding) serve to increase the production of ammonia or other gut - derived toxins or to reduce toxin metabolism by the liver (e.g., dehydration, anemia). although the exact pathophysiology of he is unclear, data are accumulating to suggest that the cascade of neuropsychiatric and neuromuscular sequelae of he may have a longer term or more permanent negative impact on patients with chronic liver disease than originally suspected. neurologic complications are common following liver transplant and may include alterations in mental status, seizures, and focal motor deficits. the majority (75%) of these complications are observed within the first month after liver transplant, suggesting a possible relationship between preoperative status and liver transplant rather than the effect of immunosuppression [13, 19, 31 ]. however, neurologic complications may be observed in the long term, even 1 year after transplant [19, 32 ]. of the neurologic complications, encephalopathy is most commonly observed, although the reported incidence has varied widely from 12% to 84% of patients at some point postoperatively [19, 20, 31, 3336 ]. a variety of factors may cause neurologic complications, including encephalopathy, infection (e.g., sepsis), perioperative complications, persistence of major portosystemic shunts, and immunosuppressant - associated toxicity. neurologic complications have also been associated with a greater risk of patient mortality [13, 37 ]. thus, encephalopathy can be seen as a neurologic complication in itself and as a potential cause of neurologic complications. preoperative history of he is a significant predictor of posttransplant neurologic complications. in a prospective analysis of 84 patients with chronic liver disease who had undergone a liver transplant, the presence of an abnormal neurologic exam suggestive of he before transplant was an independent risk factor for developing in - hospital central nervous system complications after transplant (p = 0.007). in a retrospective study of 101 patients who had undergone a liver transplant, a history of he was strongly associated with neurologic complications after transplant (univariate odds ratio (or), 2.6 ; 95% confidence interval (ci), 1.16.4 ; p = 0.03). furthermore, using a multivariate analysis, he in the immediate preoperative period was associated with posttransplant neurologic complications (adjusted or, 10.7 ; 95% ci, 3.829.9 ; p < 0.013). data continue to accumulate suggesting that even with resolution of prior episodes of overt he, patients may continue to have cognitive deficits after transplant. patients who had undergone a liver transplant, on average 17 to 19 months previously, received a battery of cognitive tests (e.g., psychometric hepatic encephalopathy score (phes) and repeatable battery for the assessment of neuropsychological status (rbans ; pearson education inc., san antonio, tx)) to determine if the presence of he before liver transplant was associated with more substantial neurocognitive abnormalities within about 1.5 years after transplant. patients with a history of he before transplant (n = 25) had significantly lower scores for 3 of 6 phes domains compared with healthy individuals (n = 20) and for 2 of 6 phes domains (attention domains) compared with patients without he before transplant (n = 14 ; figure 1). the investigators of this study did not determine the total phes score because of a lack of available normative values. for rbans, patients with a history of he before transplant had significantly lower scores compared with healthy individuals in total rbans score and 4 of the 5 rbans subscores (p < 0.05). although the total rbans score, immediate memory, delayed memory, and attention subscores were lower for patients with he before transplant than for patients without he before transplant, no significant differences were observed. in cross - sectional (n = 226) and prospective assessments (n = 59) of patients with cirrhosis, patients who had experienced overt he had greater cognitive dysfunction compared with patients without overt he. patients who had an episode of overt he had persistent impairment in cognitive function despite normalization of mental status on lactulose therapy, and the severity of impairment increased with the number of overt he episodes. thus, patients who experience overt he may have persistent and cumulative deficits in working memory, response inhibitor, and learning that are chronic, cumulative, and not readily reversible (i.e., permanent). although not studied in patients who had eventually undergone a liver transplant, persistent cognitive impairment after overt he was also supported by a study in 106 patients with cirrhosis currently without overt he who were examined on 2 occasions within a 3-day period for the presence of mild cognitive impairment (phes). among 45 patients (42%) without a history of overt he and 34 patients (32%) without a history of overt he but with a current diagnosis of minimal he, phes results improved significantly from the first to the second exam (p = 0.04 and p = 0.016, resp.), suggesting a learning capacity for taking the tests involved in phes. however, there was no significant improvement in phes results at the second exam for the 27 patients (25%) who had experienced at least 1 prior episode of overt he, indicating a lack of learning capacity in patients with a history of overt he. therefore, patients with a history of overt he may have persistent cognitive impairment despite having a normal mental status and, in some cases, even in the presence of normal cognitive test results (phes), which further supports the hypothesis that he is not a fully reversible condition. evidence also exists for the posttransplant persistence of cognitive dysfunction or radiologic abnormalities in patients exhibiting minimal he before transplant [1418 ]. in a small prospective study, 14 patients with minimal he underwent liver transplant and were assessed for visuomotor function (average time of assessment, 21 months after transplant). improvement of visuomotor and visuoconstructive skills (e.g., trail making tests, reconstruction of drawing, or picture) was observed in some patients after transplant, but worsening was observed in others. of note, no significant improvement in posttransplant visuomotor and visuoconstructive performance was noted compared with pretreatment performance, with 50% of patients showing deterioration in performance. in addition, mean posttransplant results for the 14 patients with minimal he were significantly lower than for 22 age - matched healthy individuals (p = 0.04). in another study of patients with minimal he before transplant (n = 23), most assessed cognitive functions improved at 6 months after transplant, with some cognitive functions improving only 18 months after transplant (e.g., verbal short - term memory). data support the hypothesis that patients with a history of he before transplant can have more pronounced cognitive dysfunction after transplant than patients without a history of he. however, results are confounded by some studies that suggest almost complete normalization of radiologic findings after transplant, including gradual normalization of glutamine / glutamate and choline signals in a majority of patients as measured by magnetic resonance spectroscopy [40, 41 ]. in addition, the mechanisms by which more pronounced posttransplant cognitive impairment occurs in patients with pretransplant histories of he are unclear, especially the lack of clearly defined variables that may play a role in short - term or long - term cognitive dysfunction. in a prospective study of 52 patients with cirrhosis who had a liver transplant (54% with minimal he and 0% with overt he), cognitive function significantly improved from pretransplant values for memory, attention, executive function, motor function, and visuospatial domains of the battery of tests administered (p < 0.05). however, 13% of patients still had global cognitive impairment 6 to 12 months after transplant. in addition, after liver transplant, cognitive function in patients with cirrhosis of alcoholic etiology, diabetes mellitus, and prior he was more severely impaired compared with patients without these factors. posttransplant patients with alcohol - induced cirrhosis had memory decline, patients with diabetes mellitus exhibited attention impairment, and patients with histories of he had impaired motor function (figure 2). a multivariate analysis indicated that prior he, diabetes mellitus, and cirrhosis of alcoholic etiology were considered risk factors for poor cognitive function that persists after transplant. more research is necessary to gain a clearer understanding of the key demographics and disease characteristics that are involved to better identify patient subpopulations that are at the greatest risk for posttransplant neurologic complications. the development of minimal or overt he before liver transplant may affect posttransplant outcomes, and he is a probable risk factor for neuropsychiatric symptoms after transplantation. therefore, although the cause of neuropsychiatric symptoms following a liver transplant is likely multifactorial, improving patient he status before transplant may improve posttransplant outcomes. current pharmacologic therapies for he are primarily directed at reducing the systemic levels of ammonia and other toxins produced in the gastrointestinal tract, thereby reducing cerebral exposure. patients may be treated for minimal he, overt he, or the prevention of he relapse. however, most patients with minimal he do not currently receive treatment outside the context of clinical trials. in all of these cases, the mainstays of therapy are nonabsorbable disaccharides and antibiotics. nonabsorbable disaccharides, such as lactulose and lactitol (not available in the united states), are metabolized in the colon by intestinal bacteria, resulting in a reduction in colonic ph. the acidic environment promotes uptake of ammonia by colonic bacteria, facilitates diffusion of ammonia from the blood into the intestine, and may reduce the survival of urease - producing bacteria. nonabsorbable disaccharides also increase the osmotic pressure of the intestinal lumen, which induces catharsis and elimination of potential sources of gut - derived toxins from the body. a 2004 meta - analysis of 22 randomized studies was conducted to evaluate the efficacy of nonabsorbable disaccharides compared with no treatment, placebo, or antibiotics in patients with acute, chronic, or minimal he. nonabsorbable disaccharides appeared to improve he (i.e., reduced the risk of no improvement) when compared with no intervention or placebo (p = 0.002). however, when studies of poor methodologic quality were removed from the analysis, no significant effect was observed in the few high - quality trials that had been conducted. in addition, nonabsorbable disaccharides had no significant effect on mortality compared with no treatment or placebo intervention. the authors concluded that there was insufficient evidence to support or contest the use of lactulose or lactitol for the treatment of he. a 2011 meta - analysis of 5 studies specifically evaluated nonabsorbable disaccharides for the treatment of minimal he and concluded that compared with placebo these agents significantly improved minimal he (i.e., reduced the risk of no improvement) (p < 0.0001). another 2011 meta - analysis of 9 studies evaluating lactulose for the treatment of minimal he confirmed that lactulose prevented the progression to overt he, compared with either placebo or no intervention. subsequent to this analysis, an open - label randomized study concluded lactulose to be effective for the primary prophylaxis of overt he in patients with cirrhosis. twenty (19%) of 105 patients followed for 12 months developed an episode of overt he, six (11%) in the lactulose group and 14 (28%) in the nonlactulose treated group (p = 0.02). however, consistent with other studies, no significant difference in mortality was observed (p = 0.16). in the 2004 meta - analysis, nonabsorbable disaccharides were significantly less effective than antibiotics in improving he (i.e., they were associated with a higher risk of no he improvement ; p = 0.03) and did not have a significantly different impact on mortality. patients with he who received nonabsorbable disaccharides also had higher blood ammonia levels after treatment compared with patients who received antibiotics. however, a separate meta - analysis reported similar efficacy between antibiotics and nonabsorbable disaccharides in improving he. in addition, a few studies have evaluated nonabsorbable disaccharides for the prevention of he recurrence (i.e., secondary prophylaxis) [4851 ]. in 1 randomized, and unblinded, placebo - controlled study, 140 patients with cirrhosis who had recovered from a previous he episode were randomly assigned within 1 week of recovery to receive either lactulose 30 to 60 ml / d (n = 70) or placebo (n = 70). thirteen patients (9%) were lost to follow - up ; 61 patients in the lactulose group and 64 patients in the placebo group were followed for a median of 14 months (range, 120 months). lactulose significantly reduced the percentage of patients who experienced overt he recurrence compared with placebo (20% versus 47%, resp. ; however, no significant difference in the median time of he recurrence was observed (7.5 months (range, 113 months) versus 6.0 months (range, 215 months), resp.). in addition, no significant differences between the 2 groups were reported in admissions to the liver intensive care unit for conditions other than he or deaths during the study. adverse events (aes) associated with nonabsorbable disaccharides are commonly gastrointestinal - related and include abdominal pain, diarrhea, flatulence, and nausea. diarrhea can also lead to secondary complications, including dehydration, hypokalemia, and hypernatremia. anorexia and vomiting have also been reported as aes occurring with use of nonabsorbable disaccharides (rate of 2% for each). one additional concern with lactulose is that administration can cause abdominal distention, which may result in technical difficulties during liver transplant [54, 55 ]. nutritional status before liver transplant has also been shown to correlate with posttransplant survival and is independently associated with the number of infection episodes after transplant. malnutrition, assessed by a subjective global nutritional assessment exam, was found to be an independent risk factor for the length of stay in the intensive care unit and the total number of days spent in the hospital after transplant. furthermore, alterations in specific laboratory measures (e.g., albumin, sodium, and potassium) [5860 ], which may be negatively impacted by gastrointestinal aes, have also been identified as risk factors for surgical complications in patients who received a liver transplant, and serum sodium levels are a prognostic factor for survival in patients awaiting liver transplant [61, 62 ]. thus, gastrointestinal aes may increase patient risk, particularly for patients who are prone to malnutrition because of various comorbid variables or conditions (e.g., dietary restrictions or gastroparesis). it is possible that administration of nonabsorbable disaccharides such as lactulose may exacerbate pretransplant nutritional deficits, thereby contributing to poor posttransplant outcomes. antibiotics are administered to reduce systemic levels of ammonia and other gut - derived toxins by targeting gastrointestinal bacteria. because of the risk for systemic aes and bacterial antibiotic resistance with systemic antibiotics, nonsystemic antibiotics are preferred agents. rifaximin is a nonsystemic gut - selective antibiotic and more than 20 studies have evaluated rifaximin for the treatment of overt he (see review by lawrence and klee) or minimal he [6668 ]. in 2010, rifaximin was approved by the us food and drug administration for the maintenance of overt he remission in adults. in a randomized, double - blind, phase 3 trial of rifaximin for the maintenance of he remission, patients in remission from he were treated with rifaximin 1100 mg / d (n = 140) or placebo (n = 159) for up to 6 months. concomitant lactulose administration was permitted during the study : 91% of patients in each group received concomitant lactulose. only 22% of patients in the rifaximin group experienced a breakthrough he episode compared with 46% of patients in the placebo group. furthermore, rifaximin significantly reduced the risk of he breakthrough by 58% compared with placebo during the 6 months of treatment (hazard ratio (hr), 0.42 ; 95% ci, 0.280.64 ; p < 0.001 ; figure 4). data indicated that the number needed to treat (nnt) was 4 (i.e., for every 4 patients treated with rifaximin for 6 months, 1 episode of breakthrough he would be prevented). in addition, 14% of patients in the rifaximin group reported an he - related hospitalization compared with 23% of patients in the placebo group. rifaximin significantly (p = 0.01) reduced the risk of he - related hospitalizations by 50% compared with placebo (hr, 0.50 ; 95% ci, 0.290.87). data indicated that the nnt was 9 (i.e., for every 9 patients treated with rifaximin for 6 months, 1 episode of he - related hospitalization would be prevented). health - related qol in the phase 3 trial was assessed using the chronic liver disease questionnaire (cldq), which was administered every 4 weeks, and the time to he breakthrough recorded [70, 71 ]. a significant (p = 0.0087 to 0.0436) improvement with rifaximin treatment was noted in the overall cldq scores and in each domain score compared with placebo treatment, and scores were significantly (p < 0.0001) lower in patients who experienced he breakthrough compared with those who remained in remission. in the phase 3 trial, rifaximin was well tolerated, with a similar incidence of aes reported in both groups. the most common aes with rifaximin and placebo were nausea (14.3% versus 13.2%), diarrhea (10.7% versus 13.2%), fatigue two cases of clostridium difficile infection (cdi) were reported during the double - blind portion of the trial, both in the rifaximin group. the authors noted that these 2 patients had multiple risk factors for cdi, including repeated hospitalizations during which they received multiple courses of antibiotic therapy, advanced age, and pantoprazole use. the impact of long - term rifaximin therapy on gut flora, including a risk of bacterial antibiotic resistance, is largely unknown. however, the drug appeared to have a protective effect against infections within 90 days after transplant (p = 0.026) in patients treated with rifaximin for he during liver transplant candidacy and was not associated with a higher risk of multidrug - resistant bacterial infections. conventional antibiotics, neomycin and metronidazole, are also administered for the treatment of he. however, strong clinical data supporting their efficacy in the treatment of he are lacking. one randomized, double - blind study failed to demonstrate a benefit with neomycin (n = 20) compared with placebo (n = 19), with no significant differences observed for time to resolution of he symptoms or for 5-day, 30-day, or 12-month mortality. two small, randomized, double - blind studies (n = 33 and n = 45) and 1 randomized, unblinded trial (n = 173) have suggested that neomycin and lactulose may have similar efficacy in the treatment of he [7476 ]. two randomized studies (n = 35 and n = 49) have compared neomycin with rifaximin and suggested that they have similar efficacy in the treatment of he, although in one of the studies, patients who received rifaximin showed improvements sooner than those who received neomycin (3 versus 5 days, resp.) [77, 78 ]. for metronidazole, 1 small study (n = 18) comparing metronidazole with neomycin suggested that both antibiotics improved mental state, reduced asterixis, and improved electroencephalogram measures. however, the risk of aes associated with neomycin and metronidazole may limit their use in patients with he and suggest that they might not be an ideal pretransplant choice of treatment. intestinal malabsorption and diarrhea have been observed with neomycin therapy and thus could impact pretransplant nutritional status of the patients. although neomycin is poorly absorbed, prolonged administration may result in cumulative systemic concentrations sufficient to increase the risk of serious aes, such as ototoxicity and nephrotoxicity. because the meld scoring system includes measures of renal dysfunction and neomycin may cause renal damage, neomycin may not be an ideal choice for patients, particularly those with high meld scores. metronidazole has been associated with peripheral neurotoxicity and requires dosing adjustments in patients with severe liver disease because of impaired drug clearance. metronidazole is a systemic antibiotic frequently administered for the treatment of cdi, and the potential risk of c. difficile resistance to metronidazole warrants judicious use of this agent. compared with nonabsorbable disaccharides, neomycin, and metronidazole, rifaximin exhibits a more favorable safety and tolerability profile [47, 65, 82 ]. rifaximin has not been associated with gastrointestinal aes such as diarrhea or nausea in clinical studies and would be unlikely to increase the risk of dehydration, weight loss, abdominal distention, malnutrition, or intestinal malabsorption in patients awaiting transplant, thereby minimizing the possible negative consequences of he therapy on patient nutritional status. a 2012 meta - analysis, incorporating data from 12 randomized controlled active comparator trials for the treatment of patients with he, assessed the efficacy and psychometric outcomes of rifaximin compared with other oral therapies (including disaccharides and other antibiotics). however, more favorable effects were observed with rifaximin with regard to psychometric parameters and serum ammonia levels. a tolerability analysis, which included he prevention trial data, indicated that rifaximin was also associated with fewer adverse effects. he is a common complication of cirrhosis that substantially affects patient morbidity and mortality. furthermore, he can have a detrimental impact on posttransplant outcomes, including patient survival. data continue to emerge demonstrating the potential persistence of cognitive deficits associated with he, even after liver transplant. therefore, prevention of he in patients with cirrhosis may improve pretransplant health status and thus improve posttransplant outcomes. commonly prescribed therapies include nonabsorbable disaccharides (e.g., lactulose) and nonsystemic antibiotics (e.g., rifaximin), and their various risks and benefits should be taken into consideration when deciding the most appropriate he management algorithm in patients awaiting liver transplant. further studies to evaluate currently available therapies in preventing he and improving posttransplant outcomes are warranted. | patients with cirrhosis commonly experience hepatic encephalopathy (he), a condition associated with alterations in behavior, cognitive function, consciousness, and neuromuscular function of varying severity. he occurring before liver transplant can have a substantial negative impact on posttransplant outcomes, and preoperative history of he may be a predictor of posttransplant neurologic complications. even with resolution of previous episodes of overt or minimal he, some patients continue to experience cognitive deficits after transplant. because he is one of the most frequent pretransplant complications, improving patient he status before transplant may improve outcomes. current pharmacologic therapies for he, whether for the treatment of minimal or overt he or for prevention of he relapse, are primarily directed at reducing cerebral exposure to systemic levels of gut - derived toxins (e.g., ammonia). the current mainstays of he therapy are nonabsorbable disaccharides and antibiotics. the various impacts of adverse effects (such as diarrhea, abdominal distention, and dehydration) on patient 's health and nutritional status should be taken into consideration when deciding the most appropriate he management strategy in patients awaiting liver transplant. this paper reviews the potential consequences of pretransplant he on posttransplant outcomes and therapeutic strategies for the pretransplant management of he. |
colorectal cancer is one of the most common malignancies in western countries and its incidence has also risen in asian countries. currently it is the second most common cancer and second leading cause of cancer death in hong kong. however, the operation is a major undertaking and is associated with significant morbidity, especially is elderly patients with concomitant medical conditions. laparoscopic resection has been reported to improve the short - term outcomes in terms of less postoperative pain and analgesic requirement, quicker recovery and a shorter hospital stay [36 ]. data from randomized trials comparing open and laparoscopic colon resection also demonstrated that survival after laparoscopic resection was not inferior to open resection [710 ]. whether the advantage of fewer complications and better short - term outcomes can be translated to a better survival in patients with cancer is controversial. more favorable survival in patients who underwent laparoscopic resection has been demonstrated in a randomized trial and a population study. we previously also reported better overall survival in patients who underwent laparoscopic colon and rectal resection in series of smaller number of patients [12, 13 ]. in this study, we would like to confirm the findings with a cohort of larger number of patients with longer follow - up. the current study aimed to evaluate the outcomes including survival of consecutive patients who underwent laparoscopic resection for colorectal malignancy in a high volume tertiary center. comparison of the outcomes with those patients who underwent open resection performed during the same period of time was performed. laparoscopic resection has become widely applied in the authors department since 2000. during the study period from 2000 to 2009, the choice of surgical approach was decided mainly by the surgeons experience and patients preference with all the risk of both approaches discussed with the patients. since 2008, when the senior author was in charge of the division of colorectal surgery, laparoscopic resection was offered to all suitable patients who planned to have elective surgery for colorectal cancer unless contraindicated. the majority of patients underwent laparoscopic assisted resection with three to five ports and the retrieval of the specimen was performed through an abdominal incision. in those patients who underwent laparoscopic abdominoperineal resection or low anterior resection with coloanal anastomosis, the specimen would be retrieved through the perineum or anus. transvaginal retrieval of the specimen was performed in selected female patients who underwent commitment hysterectomy. from 2008, some selected patients were operated on with robotic - assisted resection or single incision laparoscopic surgery. during the study period, the patients who underwent laparoscopic or open resection had similar preoperative workup and preparation for surgery. the postoperative management and the policies of adjuvant therapy were similar in all the patients, regardless of the surgical approach. all the operations were performed or supervised by specialists in the division of colorectal surgery. in the early period, two staff surgeons performed or supervised the majority of laparoscopic operations and they also performed the open operations. from 2004, with the departure of one laparoscopic surgeon, the senior surgeon supervised and performed the majority of the laparoscopic resection until the other staff members were trained to perform laparoscopic colectomy in a standardized technique. all the laparoscopic rectal resections are still performed or under the supervision by the senior author. operative mortality was defined as deaths that occurred within 30 days following the primary operation. operative morbidities were defined as complications that contributed to prolonged hospital stay or led to additional interventions or procedures. conversion was defined as the need for prematurely making the abdominal incision for bowel mobilization and/or vascular control. the necessity for an abdominal incision to deal with any intra - operative complication was also considered conversion. data on the patients demographics, medical comorbidities, locations of the tumors, operative details, postoperative outcomes, and follow - up status were collected prospectively and entered into a database for colorectal malignancy. in the comparison of data on patients with laparoscopic and open resection, comparison of the categorical or ordinal variables was performed using chi - square test or fisher s exact test where appropriate. survival analysis was performed after excluding patients who died within 30 days after the surgery and who had stage iv disease. survival was analyzed using kaplan meier method and comparison of variables was performed with log rank test. multivariate analysis was performed with cox regression using variables found to be statistically significant in univariate analysis. operative mortality was defined as deaths that occurred within 30 days following the primary operation. operative morbidities were defined as complications that contributed to prolonged hospital stay or led to additional interventions or procedures. conversion was defined as the need for prematurely making the abdominal incision for bowel mobilization and/or vascular control. the necessity for an abdominal incision to deal with any intra - operative complication was also considered conversion. data on the patients demographics, medical comorbidities, locations of the tumors, operative details, postoperative outcomes, and follow - up status were collected prospectively and entered into a database for colorectal malignancy. in the comparison of data on patients with laparoscopic and open resection, comparison of the categorical or ordinal variables was performed using chi - square test or fisher s exact test where appropriate. survival analysis was performed after excluding patients who died within 30 days after the surgery and who had stage iv disease. survival was analyzed using kaplan meier method and comparison of variables was performed with log rank test. multivariate analysis was performed with cox regression using variables found to be statistically significant in univariate analysis. after excluding those patients who underwent emergency surgery, operations without resection and local excision of rectal cancer, 2,011 patients were included in the current study. there were 1,157 men (57.5%) and the median age was 71 years (range, 2296 years). in 911 patients (45.3%), the tumors were located at the rectum or rectosigmoid. all operations were performed on an elective setting and patients with emergency operations were excluded. thirty - nine patients, who had obstructing left - sided colorectal cancer, were included. twenty - seven had metallic stent insertion prior to resection and 12 had prior colostomy for rectal cancer so that resection can be performed on an elective setting, usually after neoadjuvant therapy. the operative mortality and morbidity rates of all the patients were 1.3% and 23.7%, respectively. laparoscopic resection was performed in 814 patients (40.5%). in those patients with laparoscopic resection, 58 required conversion and the conversion rate was 7.1%. the 30-day mortality was 0.5% and the complication rate was 17.3% in those patients who had laparoscopic resection. conversion was associated with significantly more blood loss, higher complication rate, and a longer hospital stay when compared with successful laparoscopic procedures. laparoscopic resection was associated with significantly less blood loss, a lower incidence of postoperative complications and mortality as well a shorter hospital stay. the cardiac, pulmonary complications as well as postoperative ileus were also significantly fewer in patients who underwent laparoscopic resection (table 2).table 1comparison of patient with open and laparoscopic resection for colorectal canceropenlaparoscopicp valuesn = 1197n = 814male / female702:495455:3590.232median age (years)71 (6177)70 (6178)0.472colon631 (52.7%)468 (57.5%)0.036rectum566 (47.3%)346 (42.5%)presence of medical diseases687 (57.4%)473 (58.1%)0.748asa class 35261(21.8%)153 (18.8%)0.081operating time (min)130 (105169)180 (141218) 7063.3% 701.72<0.0011.3992.110presence of medical diseases1.100.3570.8971.351stage of disease1.56<0.0011.3351.832differentiation1.260.0950.9611.651perineural invasion1.550.0031.1612.075lymphovascular invasion1.230.0790.9761.548postoperative complication1.60<0.0011.2991.969table 5multivariate analysis of factors affecting cancer specific survivalrisk ratiop value95% confidence intervalopen operation1.320.0481.0051.738stage of disease1.83<0.0011.4892.246differentiation1.660.0021.2072.288perineural invasion1.89<0.0011.3722.611lymphovascular invasion1.370.0241.0441.811postoperative complication1.570.0011.2152.041rectal cancer1.280.0481.0031.635 univariate analysis of overall survival and cancer specific survival in patients with colorectal resection multivariate analysis of factors affecting overall survival multivariate analysis of factors affecting cancer specific survival colorectal cancer is a common malignancy, which usually occurs in the elderly age group. many of the patients have significant medical co - morbidities, which affect the operative outcomes. laparoscopic resection revolutionized the treatment of colorectal malignancy in recent years. with the introduction of laparoscopic resection, favorable operative outcomes in terms of less pain, less analgesic requirement, quick recovery of the gastrointestinal tract, and a shorter hospital stay were demonstrated in most randomized controlled trials [36 ]. the current study showed that laparoscopic surgery not only improved the short - term outcomes of patients with colorectal cancer, but it also led to lower postoperative morbidity and mortality as well as improvement in both overall and cancer specific survivals in a center with a high volume of cases. despite improvement in many parameters, which assessed postoperative outcomes, nevertheless, a significant reduction in mortality rate and a trend towards a lower morbidity could be demonstrated in a meta - analysis. moreover, a lower complication rate was shown in population studies which included a large number of patients. the inability to demonstrate improvement in most randomized trials might be due to the fact that the trials were not powered to show the difference. in clinical practice, both in the community as well as individual center with a large number of patients, laparoscopic surgery helped to reduce the complication rate. another reason for not able to demonstrate a lower complication rate following laparoscopic surgery is likely due to the high conversion rates most of the published trials. we believe that a low conversion rate is important to generate the benefit of laparoscopic surgery as most of the studies are analyzed with the intention to treat principle. there are controversies on whether conversion is associated with poor outcome. in the clasicc trial, other studies showed that the outcome was not worse with open operation [18, 19 ]. however, in cases of conversion, the outcome would at best be similar to open resection and the benefit of operation regarding the cardiopulmonary complications, ileus and wound complication of laparoscopic surgery can not be derived. lacy. demonstrated fewer complications and better survival in patients with laparoscopic resection and the presence of a low conversion rate is important to obtain the beneficial results. the survival is the most important outcome to assess treatment success for malignant disease. in colorectal cancer, both the overall survival and cancer specific survival are important as many patients are elderly and death might be due to other medical diseases, which might be related to the operation. we demonstrated both a significantly better overall and cancer specific survivals in patients who underwent laparoscopic resection. the approach of surgery was shown to be an independent significant factor of both overall and cancer specific survival. this confirmed our previous findings on the improved survival in patients with laparoscopic colon and rectal resection with a larger patient population. the improvement occurred mainly both stage ii and stage iii cancer and occurred both in colon and rectal cancer. this is different from multicenter randomized studies [79 ], which showed equivalent survival in patients with open and laparoscopic resection. we postulated that most of these randomized trials such as the cost trial were initially planned as non - inferiority studies and were not powered to show the difference in survival. lacy. reported better survival in laparoscopic resection in a single center randomized trial and the better survival was mainly in the group of patients with stage iii cancer. capussotti. also found that in patients with stage iii colon cancer laparoscopic resection was associated with a significantly disease free and cancer related survival. we also demonstrated better overall survival in stage ii and stage iii cancer. in the study by bilimora. using the national cancer data, better survival was found in the patients with laparoscopic surgery. in case of rectal cancer, laurant. also demonstrated better survival in patients with laparoscopic resection, but there was no difference in cancer free survival. one of the reasons accounting for the better survival might be the better immunological response in patients who underwent laparoscopic surgery. this has been demonstrated in many studies on the inflammatory markers after laparoscopic surgery [22, 23 ]. the association of cytokines such as interleukin 6 and vegf, which was produced significantly more after open surgery, with tumor recurrence has been demonstrated in animal models. the less release of cytokines such as interleukin 6 and vegf in the postoperative period has been demonstrated in laparoscopic surgery. moreover, the lower complication rate associated with laparoscopic resection might also contribute to the better survival. khuri and colleagues showed with the nsqip data that the presence of postoperative complications adversely affected the long - term survival of patients in eight operations, which included colorectal resection. the presence of postoperative complications has also been demonstrated to affect the survival of patients who underwent resection for colorectal, esophageal and liver cancer [2629 ]. thus the lower complication rate associated with laparoscopic resection might be the reason accounting for the better survival. this is exemplified by lacy.s trial in which a significant lower complication rate as well as better survival were demonstrated in the patients with laparoscopic resection. admittedly, the study is not a randomized trial and biases in the selection of patients for laparoscopic procedures were unavoidable. it could be shown that there were more patients with earlier cancer in the laparoscopic group. the size of the tumor was also larger in the open group. however, other parameters including the distal resection margin as well as the number of lymph nodes examined were not inferior in laparoscopic resection. moreover, the difference in survival between the laparoscopic and open resection could be demonstrated in analysis according to the stage of the disease. this study also highlighted the impact of laparoscopic resection on the outcomes of treatment of colorectal cancer in a high volume center. with the wider application of laparoscopic resection for colorectal malignancy worldwide, it is expected that more data from large volume centers or from population studies can give more information on the impact of survival with the shift to laparoscopic surgery. in the current study, laparoscopic resection for colorectal cancer was shown not only to be associated with better short - term results, but the overall and disease specific survivals were also better when compared to open resection. this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. | backgroundthis study aimed to compare the overall and disease specific survivals of patients who underwent laparoscopic and open resection of colorectal cancer in a high volume tertiary center.methodsconsecutive patients who underwent elective resection for colorectal cancer (open resection, n = 1,197 ; laparoscopic resection, n = 814) from january 2000 to december 2009 were included. the operative details, postoperative complications, postoperative outcomes, and survival data were collected prospectively. comparison was made between patients who had laparoscopic and open surgery.resultsthe age, gender, medical morbidity, and american society of anesthesiologists status were similar in the two groups. laparoscopic resection was associated with significantly less blood loss and a shorter hospital stay. the operating mortality and morbidity were significantly lower in the laparoscopic group. the qualities of the specimens in terms of the distal resection margin and the number of lymph nodes examined were not inferior in the laparoscopic group. with the median follow - up of 40.3 months, the 5-year overall survival (74.1% vs. 65.5%, p < 0.001) and disease specific survival (81.9% vs. 75.2%, p = 0.002) were significantly better in patients with non - disseminated disease in the laparoscopic group. the operative approach was an independent prognostic factor in the overall (risk ratio 1.36, 95% ci 1.0931.700, p = 0.006) and disease specific (risk ratio 1.32, 95% ci 1.0051.738, p = 0.048) survivals in multivariate analysis.conclusionlaparoscopic resection for colorectal cancer is associated with more favorable overall and disease specific survivals when compared with open resection in a high volume tertiary center. |
a 52-year - old korean female visited seoul veterans hospital with a complaint of acute pain and gingival swelling on the right mandibular molar region. on palpation, bilateral buccal bony expansion was noted on the posterior mandible and maxilla (fig. 1). she had a complete denture, however her denture was out of use due to the gingival swelling and pain. panoramic radiograph showed diffuse, lobular, and irregularly shaped radiopacities or cotton - wool appearance throughout the alveolar process of both quadrants of the maxilla and mandible. multiple sclerotic masses with radiolucent borders were found in the maxilla and mandible, confined within the alveoli at the level corresponding to the roots of the teeth, above the inferior alveolar canal (fig. cone beam computed tomography (cbct) (kavo 3d exam, kavo, biberach, germany) images also revealed large radiopaque shadow extending into the mandible and maxilla. the bucco - lingual aspects of the lesions could be visualized on the cbct images, which demonstrated the relationship of the bony lesions to the cortical plates in the bucco - lingual dimension. some lesions appeared larger and connected with the buccal and lingual cortical plates. on the parasagittal reformatted images at this level, the radiographic appearance varied from radiolucent to mixed lesions or rather radiopaque masses. under local anesthesia, incision and surgical curettage was performed on the right mandibular posterior area. irregular bony defects and inflammatory fibrous tissue were seen in the operation field. histological finding of this lesion showed the formations of dense sclerotic calcified cementum - like masses. the lesion was composed of cementum - like substances characterized by islands of calcified deposits and areas of loose fibro - collagenous stroma, which showed the evidence of proliferation. the patient of the present case has been followed up over the last 12 month and fcod has remained asymptomatic. exuberant fibro - osseous lesions occurring in multi - quadrants of the jaws were designated as gigantiform cementomas or familial multiple cementomas in the first edition of the world health organization 's histological typing of odontogenic tumor, jaw cysts and allied lesions.11 however, the etiopathogenesis has not been clear. our case was diagnosed with fcod based on the clinical, histologic, and radiographic features. fcod should be differentiated from paget 's disease, chronic diffuse osteomyelitis, and gardner 's syndrome. paget 's disease is polyostotic and shows the raised alkaline phosphatase level which is not a consistent feature of fcod. it is a primary inflammatory condition of mandible with cyclic episodes of unilateral pain and swelling. fcod is a reactive, non - neoplastic process confined to tooth - bearing areas of the jaws that is found most frequently in middle - aged and older black women. melrose reported a study of 34 cases of such lesions, of which 32 were black women (in a predominantly caucasian population) with mean age of 42 years.12 the definite female gender predilection of the condition is unclear.2,3 in the present case, the patient was a 52-year - old korean female ; it was rare in regard to the race and gender. fcod may be familial with an autosomal dominant inheritance pattern. however, there were only few examples in the literatures in which the familial pattern had been confirmed.10,13,14 in this case, the patient 's mother and twin daughters had similar lesions in the jaw, and they were diagnosed with familial gigantiform cementoma in other clinic, however the lesions were focal without pain and any other symptom. conventional radiograph, which exhibit multi - quadrant diffuse radiopaque masses in the tooth - bearing areas of the jaws, plays an important role in the diagnosis for asymptomatic fcod patients. the ct findings of the lesion have been previously reported.10 axial ct images clearly showed the location and extent of the lesion, especially in the maxilla. the expansion of the cortical bone was clearly evaluated on the ct images even though it was slight. extraction is not recommended due to the poor socket healing from the impaired blood circulation in the affected area of the bone. extensive surgical resection and saucerization are proposed as treatment options when lesions become extensive and symptomatic.15,16 in this case, surgical resection and saucerization were performed due to the symptoms of the pain and gingival swelling on the edentulous area. | cemento - osseous dysplasias are a group of disorders known to originate from periodontal ligament tissue and involve, essentially, the same pathological process. they are usually classified into three main groups : periapical, florid, and focal cemental dysplasias depending on their extent and radiographic appearances. radiographically, florid cementoosseous dysplasia (fcod) appears as dense, lobulated masses, often symmetrically located in various regions of the jaws. the best management for the asymptomatic fcod patient consists of regular recall examinations with prophylaxis. the management of the symptomatic patient is more difficult. a case of fcod occurring in a 52-year - old edentulous korean female is reported which is rare with regard to race and sex. |
while violence remains the third most common cause of spinal cord injury (sci) in the united states, only 1% of scis occurs from stab wounds.1 non - missile injuries, namely stabbings, are more common in developing countries where access to firearms is limited.2,3 in a large series from south africa, 25% of scis were reported to result from penetrating injuries not due to firearms, of which 84.2% were caused by stabbing.4 commonly used weapons include knives, ice picks, screwdrivers, and bicycle spokes. to our knowledge, no case of sci caused by a fall onto a nail has been reported in the english - language literature. here we present a patient who developed complete sci after his thoracic spinal cord was transected by a nail. the causative mechanism, diagnostic tools, and review of the literature on non - missile sci are discussed. a detailed history and physical examination were performed along with a careful review of the patient 's medical records. plain x - rays and magnetic resonance imaging (mri) were performed to help determine the etiology of the patient 's injury. a literature review was also conducted to assess the incidence of similar mechanism of sci. a 6-year - old boy was initially evaluated in the emergency department in mulago hospital, kampala, uganda, after he fell from a tree. at the scene of the fall it was then discovered that he had landed on a nail, which was upright in a piece of wood. the nail penetrated his body at about the t8 vertebra and was found buried in the soft tissue ; it was removed shortly after. the patient was then admitted to the spine ward at mulago hospital where he was examined 12 days later by a team of visiting surgeons. on examination the patient had 0/5 motor strength in the lower extremities, symmetrical areflexia, and hypoesthesia below the t8 level. clinically, his american spinal injury association (asia) score was a with a t8 level. after the initial neurological assessment, the team believed further imaging to evaluate the cord integrity and rule out a surgically correctable cause of paralysis would be in order. an mri scan of the thoracic spine revealed a complete transection of the spinal cord at the t8 vertebra (fig. 3). there was no evidence of infection, tumor, or epidural abnormalities that could have otherwise accounted for the patient 's symptoms. the mri confirmed the diagnosis that the boy suffered a spinal cord transection from the penetrating injury, namely the nail. a detailed discussion was conducted with the patient and his family concerning the prognosis and the fact that it would be unlikely for him to independently walk again. also discussed was the role of nonoperative management, namely aggressive rehabilitation, bowel, and bladder care. ap x - rays of the thoracic spine. t8 vertebra (arrow). digital photograph of the patient back highlighting the healed nail puncture wound. t8 vertebra (arrow). a t2 mri scan of the thoracic spine. the white arrow indicates the nail tract and the resultant spinal cord (black arrow) transection. the mechanism of sci differs between developed and developing countries. in the united states scis are typically due to motor vehicle injuries (42.1%), falls (26.7%), violence (15.1%), sporting injuries (7.6%), and unknown events (8.6%).1 the latter is more common in the pediatric population and when this occurs it is called sci without radiographic abnormality (sciwora).5 in addition, scis in juvenile patients are most often a result of motor vehicle injuries and falls from heights.6 in developing countries, including south africa, where there is a lack of access to firearms, the primary cause of sci was a penetrating injury from a sharp object of which 84.2% resulted from stabbing. most injuries caused hemi - section of the spinal cord with incomplete neurological deficits.4 there are numerous case reports of sci caused by nonviolent penetrating injuries. a recent case described sci as a result of penetration from an epidural needle.7 another case report documented sci from a glass fragment.8 most pencil injuries have been reported to occur in or around the orbital region,9,10 with the exception of one report of sci from pencil impalement.11 although craniofacial and both upper and lower extremities injury caused by nail impalement have been reported,12 to the best of our knowledge, no case of sci, especially complete transection has been described in the english - language literature. in this case the sci was believed to occur in the following manner : the nail transected the spinal cord via a path through skin, thoracolumbar fascia, either lateral to the spinal process or directly through the t8/t9 interspinous ligaments ; then through the lamina or interlaminar space, and finally through the spinal cord. it is also postulated that the nail interrupted the posterior blood supply to the cord as well as damaging the artery of adamkiewicz, which generally enters the spinal cord through t8 and t12 levels to supply the anterior part of the spinal cord.13 in cases with suspected sci due to impalement, plain x - rays are recommended to detect the level of lesion and penetration into the spinal canal. an mri is recommended for further evaluation of the spinal cord and surrounding soft tissue and the relation between the object and the spinal cord. the surprising aspect of this case was the absence of any other morbidity, such as infection or cerebrospinal fluid leak. in summary, we report a rare case of a complete sci caused by cord transaction by a nail. to our knowledge the editors have selected this case to continue the discussion on spinal cord injury (sci) in emerging countries with limited medical infrastructure. we have received diverse opinions on our question of how much and what type of spine reconstructive surgery to do in developing countries as delivered by short - term visiting surgeons. so many opinions, in fact, that we have collected these and plan to create a special focus issue on this topic in the future. there is little doubt about the acute care needs of this patient, both the commentator and the presenting authors agree. the much bigger questions arise regarding the long - term prospects of this t8 asia a paraplegic boy. what form of rehabilitation support is the patient receiving, what bowel / bladder care program will he be a candidate for fusion surgery when he develops the expected neuromuscular paralytic curve ? what does his decubitus prophylaxis look like ? what is he doing scholastically and what is his eventual life expectancy ? in developed countries, patients with mid - thoracic sci can achieve near normal life expectancy at reasonable annual healthcare costs. societal protections, such as afforded by the americans with disabilities act, are priceless for the life quality of many patients, especially those with sci. this again raises the question of the responsibility of visiting surgeons. aside from bringing surgical expertise to the developing countries are they engaged in creating a rehabilitation medicine community or knowledge transfer, for instance, through the use of internet communications ? should this be part of a visiting spine surgeon 's portfolio, or should this be left to others to care for ? chief of spine surgery seattle children 's hospital the authors present a case in which a 6-year - old child in uganda fell from a tree and suffered a complete spinal cord injury (sci). a nail was found embedded in the soft tissue directly over the t8 midline and had been removed before presentation. the mri showed complete cord transection at the t8 level, which was believed to have occurred from penetrating injury at this level from the nail. the mri is consistent with the proposed mechanism, and not with a transection due to sciwora, fracture, or dislocation from the fall. the combination of lack of soft- tissue coverage in this child, and larger interlaminar space in children may have contributed to this occurrence. in developed countries, pediatric scis are most commonly caused by motor vehicle injuries, falls, sports injuries, or penetrating trauma from firearms. in developing countries where firearms are less readily available, penetrating injuries from stabbings with sharp instruments are more common causes, and they most often result in incomplete sci syndromes, such as brown - sequard.1 neither the authors nor this reviewer could find a report of sci caused by nail penetration in the english - language literature. although many other implements have been reported including acupuncture needles, broken glass fragments, chopsticks, migrated k - wires from clavicle fracture fixation, stingray spine, and a senegalese tiger tooth. local wound debridement and prophylactic antibiotics for 24 hours are appropriate.2 indications for surgery from a non - missile penetrating injury include evidence of progressive neurological deficit, spinal cord compression from hematoma or bony fragment, or persistent spinal fluid leak. late instability is rare, and treatment should focus as it did on appropriate rehabilitation. | study design : case report.objective : to describe a case of spinal cord transection in a 6-year - old child.background information : non - missile injury of the spinal cord is not common and its incidence varies according to the country. in addition, to our knowledge, there are no published reports of spinal cord injury (sci) from a penetrating nail. here, we report the case of a child who developed complete sci because of cord transection by a nail.methods : a detailed history and physical examination were performed along with careful review of the patient 's medical records. in addition, a review of the literature was conducted to assess the incidence and treatment of similar injuries.case description : a 6-year - old boy was admitted to the hospital after falling from a tree and landing on a nail. his physical examination revealed an emaciated child with multiple decubitus ulcers, lying on his side in bed. visible was a well - healed posterior puncture wound at the t8 vertebral level. on neurological examination, the patient had 0/5 muscle strength in his lower extremities, symmetrical areflexia, and hypoesthesia below the t8 level. plain x - ray of the thoracolumbar spine was normal. magnetic resonance imaging revealed a transected spinal cord at the t8 vertebra, consistent with his nail puncture wound.discussion : this report describes an unusual case of a complete sci in a pediatric patient caused by penetrating trauma from a nail. to our knowledge, this is the first case to report on complete sci due to trauma from a nail. |
an otherwise healthy mother had a routine ultrasound at 20 weeks, which showed giant omphalocele with liver included in the omphalocele sac. karyotyping of the amniotic fluid cells showed a normal female karyotype (46, xx), -fetoprotein (afp) in amniotic fluid was raised (38.7 mg / l ; 3 mom = 20.1 mg / l).these findings were discussed with the parents and it was decided to continue the pregnancy. the baby was born at 38 weeks and 2 days of gestation by elective cesarean delivery. the birth weight was 3,500 g and apgar scores were 8/9 at 1 and 5 minutes, respectively. the omphalocele contained a big part of the liver, almost the whole bowel, and the spleen. the omphalocele was covered with a sterile petrolatum (vaseline, smith & nephew, kruisweg, hoofddorp, netherlands) gauze dressing, which was changed every other day. the baby expelled meconium shortly after birth and also upon arrival in the neonatal intensive care unit. feeding was started via nasogastric tube and was well tolerated during the first days of life. on the 6th day after birth,, we resected the omphalocele sac (which was vulnerable by manipulation) and performed adhesiolysis to appropriately position the spleen and bowel in the abdominal cavity. the liver remained outside the abdominal cavity. a silo bag (sb06, 60-mm ; medicina ltd., bolton, united kingdom) was used to temporarily cover the defect and central venous access was obtained in the left subclavian vein (supplementary fig. the biggest silo bag available (60-mm) was not big enough to accommodate the liver and was therefore enlarged by attaching a triangular patch made of silastic mesh. because the use of a silo is not optimal for treatment when the liver is situated outside the abdominal cavity, alternative treatment approaches had to be considered. on the 14th day after birth, we removed the silo and placed non cross - linked intact porcine - derived acellular dermal matrix (padm ; strattice reconstructive tissue matrix, lifecell corp., branchburg, new jersey, united states) at the fascia and sutured it with prolene 30 sutures (ethicon, somerville, new jersey, united states) (fig. 1). a vacuum dressing system was used to cover the padm with a negative pressure of 75 mm hg (supplementary fig. the vacuum system was changed in the operation theater on postoperative days 3 (supplementary fig. s3 in the online version of the article), 9, and 12 and on the pediatric ward thereafter. amoxicillin / potassium clavulanate and gentamicin were initiated for prophylaxis against infection and then changed to meropenem. s4 in the online version of the article). completed suturing of the fascia to the porcine - derived acellular dermal matrix (14th day after birth). on the 26th day of life, the patient developed sepsis from the central venous catheter infection. central venous system blood cultures were positive and the patient experienced nephritis and renal insufficiency that subsided spontaneously. enteral feeding was suspended during the sepsis ; however, the patient was tolerating full enteral nutrition by 45 days postnatal age. on the 41st day of life the result of the postnatal array - cgh (agilent technologies, santa clara, california, united states) was returned, it showed a maternally inherited 780 kb deletion on chromosome band 3q28 containing part of the tp63 gene, and four more genes that cause autosomal recessive syndromes. the patient was discharged home at age 2 months and 10 days with the vacuum therapy, which was discontinued 1 week later. after removing the vacuum therapy, the patient was bathed daily by her parents and had a sterile petrolatum gauze dressing in place until the wound closed completely (supplementary fig. ventral hernia repair was performed when the patient was 2 years and 3 months old (supplementary fig. the padm had become integrated into the surrounding fascia and was used to close the abdominal wall (supplementary fig. a sample of this padm taken during ventral hernia repair was sent for histological examination. s8 in the online version of the article) and experienced no complications through 6 months of follow - up (fig. omphaloceles are often associated with other malformations and chromosomal anomalies.1 2 the small chromosome 3 deletion in this case (1) is to our best knowledge (based on queries in pubmed database and decipher database) not known to be associated with omphalocele, (2) contains no genes explicitly associated with omphalocele,3 and (3) was inherited from a healthy mother.4 therefore, this deletion was considered to be most likely of neutral effect. the raised amniotic fluid afp was associated with the omphalocele.5 giant omphaloceles are not common, which makes it difficult for a single pediatric surgery department to gain extensive treatment experience. furthermore, most of the very large omphaloceles can be treated conservatively with sterile petrolatum dressing until the epithelialization of the defect results in a large ventral hernia that can be subsequently closed when there is adequate space in the abdominal cavity. in cases where the omphalocele sac has to be removed or when a rupture occurs the use of a combination of a biological matrix graft and vacuum therapy for treating abdominal wall defects has been previously reported in the medical literature6 7 8 ; however, the use of padm for this purpose has not yet been described in our knowledge. strattice is a non cross - linked porcine, acellular dermal matrix for use in xenograft transplantation, serving as a biological scaffold to support tissue regeneration.9 10 it is derived from porcine dermis and processed in a manner to remove the cells and porcine antigens which would provoke an immune response in humans, while retaining all its extracellular matrix (ecm) components, its three - dimensional structure and its biomechanical strength. as such when implanted, it acts as a scaffold to support tissue ingrowth. its ecm components attract in cells and vascularity from the patients adjacent tissue, and over time it is replaced by the patients ' own tissue with all components of the porcine matrix removed, in a regenerative tissue healing process.11 12 in this patient, the use of padm was successful and showed good long - term biological compatibility, as the histological examination of the collagen showed no difference with the human collagen 2 years after suturing the patch to the fascia (supplementary fig. the microscopic examination showed dissections of tissue fragments, irregular collagen bundles containing numerous small vessels, and signs of very mild chronic infection. based on the histology examination, it was not possible to determine if the sample consisted of porcine collagen tissue or human collagen, even after applying sirius red and verhoeff van gieson staining. the padm became integrated with the fascia and was strong enough to be partially used to strengthen closure of the abdominal wall defect. however, management can be challenging if the omphalocele is ruptured or the sac has to be removed. we present padm with vacuum therapy as a good alternative in the treatment of giant complicated omphaloceles. | the management of giant omphaloceles at our department is primarily conservative. however, management can be challenging if the omphalocele is ruptured or the sac has to be removed. we report a case in which a giant omphalocele in a newborn female patient was managed by covering the abdominal defect with non cross - linked intact porcine - derived acellular dermal matrix (strattice reconstructive tissue matrix, lifecell corp., branchburg, new jersey, united states) sutured to the fascia combined with vacuum therapy. |
the prickly pear cactus, opuntia ficus - indica, is well adapted for cultivation in semi - arid regions.1 the cladodes, also known as cactus pads, of the spineless cultivars can be used as livestock feed, but because of their low crude protein content of about 40 g kg they should be regarded as a cheap energy source rather than as a balanced fodder crop.13 for instance, a ration for non - reproductive sheep should contain at least 70 g crude protein kg ; this necessitates the supplementation of cactus cladodes with some form of crude protein for use as a balanced animal feed.1,4 because of the high annual productivity of 1040 tonnes (dry weight) of cladode biomass per hectare, the cladodes have potential as a lignocellulosic feedstock to produce a protein - enriched biomass product for use as an animal feed. feedstocks such as molasses, cheese whey and hydrocarbons are the major carbon sources used for single - cell protein (scp) production and, more recently, lignocellulosic feedstocks have received attention.5,6 yeasts are suitable for scp production because of their high protein and low nucleic acid content. candida utilis has been widely used, but kluyveromyces marxianus has potential for scp production owing to its greater temperature tolerance (resulting in a bioprocess with lower cooling costs and which is less prone to microbial contamination) and ability to utilize a broader range of carbon substrates than c. utilis.710 whereas galactose and arabinose are poorly utilized by c. utilis, the ability of k. marxianus to utilize these sugars confers an advantage when grown on lignocellulosic hydrolysates. furthermore, like c. utilis, k. marxianus has gras (generally recognized as safe) status and is regarded as one of the most crabtree - negative yeasts (i.e. does not produce ethanol under aerobic conditions), which is advantageous for scp production since under aerobic conditions yeast biomass production from carbohydrates would be maximized because no carbon and energy are lost due to ethanol production.8 there are several contradictory reports on the composition of o. ficus - indica cladodes2,3,11,12 that may be attributed to variable factors such as plant age, climate and geographical location. the dried and milled cladodes used in this study had a total carbohydrate content of 420 g kg on a dry weight basis,13 which was substantially less than other conventional lignocellulosic feedstocks such as sugar cane bagasse (666 g kg) and corn stover (650 g kg),14,15 mainly due to the lower glucan and xylan content of the o. ficus - indica cladodes. we investigated the feasibility of enriching the protein content and quality of an enzymatic cladode hydrolysate of an o. ficus - indica cultivar by aerobic cultivation of k. marxianus and c. utilis, including a comparative evaluation of the performance of these two yeasts. fresh cladodes of opuntia ficus - indica (cultivar ' algerian ') were harvested in may from a cactus plantation outside bloemfontein, south africa. the cladodes, having a high water content of about 880950 g kg, were cut into strips using a mechanical shredder, sun dried and subsequently hammer milled to a particle size of 1 mm. the resulting cladode meal, having a dry matter content of 963 1 g kg, was thoroughly mixed to ensure representative samples and stored in a sealed container at room temperature. the pretreatment conditions had previously been optimized by means of a central composite response surface statistical design using dilute sulfuric acid and pretreatment time as variables at a fixed temperature of 120 c.13 a stock hydrolysate of the cladode meal was prepared in a 15 l stainless steel biostat c bioreactor (sartorius stedim biotech, gttingen, germany) by mixing 2.5 kg dry meal in 8.4 l h2so4 (15 g kg) to obtain a solids loading of 300 g l. the bioreactor was sealed and left overnight at 24 c with slow stirring. subsequenty, the contents were autoclaved in situ at 120 c for 45 min, cooled to 50 c and adjusted to ph 4.8 by automatic titration with 3 mol l koh or 1.5 mol l h2so4 with slow stirring at 300 rev min. a mixture of commercial enzymes consisting of 15 fpu cellulase (spezyme cp, genencor, leiden, netherlands), 15 iu -glucosidase (novozym 188, novozymes a / s, bagsvaerd, denmark) and 100 iu pectinase (pectinex ultra sp - l, novozymes a / s) per gram of dry cladode meal was added directly to the slurry and maintained at 50 c with slow stirring for 48 h to allow hydrolysis. samples were withdrawn at intervals for sugar analysis and the final hydrolysate was collected in sterile 1 l bottles, which were stored at 20 c. kluyveromyces marxianus uofs y2791, isolated from a local agave plant (agave americana), and candida utilis nrrl y1084 were obtained from the university of the free state mircen yeast culture collection. these cultures were maintained on gpy agar slants containing (per litre) 40 g glucose, 5 g peptone, 5 g yeast extract and 20 g agar, stored at 4 c and subcultured every 2 months. pre - cultures of k. marxianus and c. utilis were grown in a sterile medium containing (per litre) 5 g glucose, 0.25 g citric acid, 3 g yeast extract (merck, darmstadt, germany), 5 g (nh4)2so4, 9.61 g kh2po4, 0.76 g k2hpo4, 0.75 g mgso4.7h2o, 0.05 g cacl2.2h2o, 0.1 g nacl and 1 ml of a trace elements stock solution, adjusted to ph 5.5 with 3 mol l koh prior to autoclaving. the trace elements stock solution contained (per litre) 3.5 g feso4.7h2o, 0.7 g mnso4.7h2o, 1.1 g znso4.7h2o, 0.1 g cuso4.5h2o, 0.2 g coso4.6h2o, 0.13 g na2moo4.2h2o, 0.2 g h3bo3, 0.04 g ki and 0.16 g al2(so4)3.18h2o.16 a 500 ml erlenmyer side - arm flask containing 50 ml of the above medium was inoculated with a loopful of cells from a 24 h agar slant of k. marxianus or c. utilis and incubated at 40 or 35 c, respectively, on an orbital shaker at 200 rev min to late exponential phase. these temperatures were the upper limit of the optimum temperature range obtained from temperature profiles previously determined. a 1 ml volume was subsequently transferred to a second shake flask containing the same medium, which was similarly incubated and immediately used to inoculate the bioreactor vessel. batch cultivations were carried out with k. marxianus and c. utilis at 40 and 35 c, respectively, in a 1.6 l biostat b - plus stirred tank reactor fitted with an exhaust gas condenser cooled to 1 c and using a 1 l culture volume. the ph was maintained at ph 5.0 by automatic titration with either 3 mol l koh or 1.5 mol l h2so4. the bioreactor vessel containing 950 ml medium was inoculated with 50 ml of the shake flask culture. the dissolved oxygen tension (dot) was monitored with a polarographic po2 electrode (mettler toledo, halstead, uk) and controlled above 30% of saturation by cascade control of the stirrer speed and the aeration rate in the range 1.03.0 l min. foaming was controlled by automatic addition of dow corning 1510 silicone antifoam (bdh laboratory supplies, poole, uk). when used as carbon substrate, the frozen cladode hydrolysate was thawed and an 800 ml volume sterilized in the bioreactor vessel at 110 c for 10 min, cooled to the desired cultivation temperature, supplemented with a 150 ml concentrate of a mineral salts solution (below) that had been sterilized by autoclaving at 121 c for 20 min, which also served as diluent to obtain a more miscible slurry, and adjusted to ph 5.0. mineral salts were added to all culture media at the following final concentrations (per liter) : 0.5 g citric acid, 10 g nh4cl, 5 g kh2po4, 0.5 g k2hpo4, 1 g mgso4.7h2o, 0.1 g cacl2.2h2o and 2 ml of the trace elements stock solution described above. in the case of k. marxianus cultures, a filter - sterilized vitamin stock solution that contained (per liter) 0.025 g biotin, 0.5 g nicotinic acid, 0.5 g pyridoxine hydrochloride, 0.5 g thiamine hydrochloride (sigma - aldrich, steinheim, germany), 0.5 g calcium pantothenate (merck), 0.1 g p - aminobenzoic acid (hopkin & williams ltd, chadwell heath, uk) and 12.5 g m - inositol (bdh laboratory supplies)17 was added to the sterile basal medium. two chemically defined media were also used, namely a simulated hydrolysate medium containing hexose and pentose sugars at similar concentrations as present in the enzymatic cladode hydrolysate, and a similar glucose - limited medium that contained glucose as the only sugar at a final concentration of 10 or 50 g l. in each case 150 ml of the sterile concentrated sugar solution was added to 800 ml of the above mineral salts basal medium in the bioreactor vessel, which had been adjusted to ph 5.0 with 3 mol l koh and autoclaved at 121 c for 20 min. cellulase and -glucosidase activities were determined according to iupac guidelines,18 and pectinase activity as described elsewhere.19 cell concentrations were monitored by measuring culture turbidity against a medium blank with a photolab s6 spectrophotometer (wtw, weilheim, germany) at 690 nm. dry cell weight was gravimetrically determined using duplicate 10 ml samples that were centrifuged, washed with distilled water and dried overnight at 105 c. the co2 and o2 content of the bioreactor exhaust gas was continuously monitored using an uras 10e infrared and a magnos 6 g paramagnetic gas analyser (hartman & braun, frankfurt, germany) and the gas exchange rates were calculated by means of a nitrogen balance. during some of the k. marxianus cultivations, ethyl acetate in the exhaust gas was trapped with orbo desorption tubes (sigma - aldrich, deisenhofen, germany) containing activated coconut charcoal as adsorbent and the ethyl acetate subsequently extracted from these tubes with dichloromethane (sigma - aldrich, steinheim),20 followed by analysis by gas chromatography (gc) as described below. samples collected for determining residual sugars and metabolic products were immediately cooled in ice before centrifugation at 10 600 g and 4 c using an eppendorf 5430 r centrifuge (eppendorf ag, hamburg, germany) and the supernatants filtered through a 0.45 m acetate membrane filter (pall, port washington, the concentrations of glucose, xylose, galactose, arabinose and fructose were determined with a waters high - performance liquid chromatography (hplc) instrument (waters corp., milford, ma, usa) using an aminex hpx-87p column (bio - rad, hercules, ca, usa) at 85 c with milliq water at a flow rate of 0.4 ml min as eluent, or with a rezex rpm - monosaccharide pb cation exchange column (phenomenex, torrance, ca, usa), the latter giving a better peak resolution. ethanol, acetaldehyde and ethyl acetate were determined with a shimadzu gc 2010 gas chromatograph (shimadzu scientific instruments, columbia, md, usa) equipped with a zb wax column (phenomenex) and a flame ionization detector with hydrogen as carrier gas at a linear velocity of 35 cm s, using a 0.6 l injection volume at a 50:1 split ratio. the oven temperature was 80 c for 2.5 min, ramped at 25 c min to 180 c with a 2 min isothermal period. for determination of the ethyl acetate recovered from the bioreactor exhaust gas, an oven temperature of 40 c for 5 min, ramped at 10 c min to 150 c with a 5 min isothermal period was used, with a 1.0 l injection volume at a 10:1 split ratio and injector and detector temperatures of 150 and 280 c, respectively. a refractive index detector (waters) the protein content of the cells was determined the by biuret method21 using bovine serum albumin, fraction v (sigma - aldrich, st louis, mo, usa) as protein standard. amino acids were determined by transferring a washed biomass suspension equivalent to 0.5 mg dry mass into vacuum reaction hydrolysis tubes with norleucine as internal standard. following liquid phase acid hydrolysis at 108110 c for 24 h to access the proteinogenic amino acids, these were derivatized by incubating with a 1:1 volumetric ratio of n-(tert - butyldimethylsilyl)-n - methyltrifluoroacetamide (mtbstfa) (merck, hohenbrunn, germany) and n, n-dimethylformamide (dmf) (merck, darmstadt, germany) for 60 min at 60 c.22 the derivatized amino acids were analysed by gas chromatography mass spectrometry (gc - ms) using a gc trace ultra instrument with a dsq quadrupole mass spectrometer (thermo finnigan, san jose, ca, usa). fresh cladodes of opuntia ficus - indica (cultivar ' algerian ') were harvested in may from a cactus plantation outside bloemfontein, south africa. the cladodes, having a high water content of about 880950 g kg, were cut into strips using a mechanical shredder, sun dried and subsequently hammer milled to a particle size of 1 mm. the resulting cladode meal, having a dry matter content of 963 1 g kg, was thoroughly mixed to ensure representative samples and stored in a sealed container at room temperature. the pretreatment conditions had previously been optimized by means of a central composite response surface statistical design using dilute sulfuric acid and pretreatment time as variables at a fixed temperature of 120 c.13 a stock hydrolysate of the cladode meal was prepared in a 15 l stainless steel biostat c bioreactor (sartorius stedim biotech, gttingen, germany) by mixing 2.5 kg dry meal in 8.4 l h2so4 (15 g kg) to obtain a solids loading of 300 g l. the bioreactor was sealed and left overnight at 24 c with slow stirring. subsequenty, the contents were autoclaved in situ at 120 c for 45 min, cooled to 50 c and adjusted to ph 4.8 by automatic titration with 3 mol l koh or 1.5 mol l h2so4 with slow stirring at 300 rev min. a mixture of commercial enzymes consisting of 15 fpu cellulase (spezyme cp, genencor, leiden, netherlands), 15 iu -glucosidase (novozym 188, novozymes a / s, bagsvaerd, denmark) and 100 iu pectinase (pectinex ultra sp - l, novozymes a / s) per gram of dry cladode meal was added directly to the slurry and maintained at 50 c with slow stirring for 48 h to allow hydrolysis. samples were withdrawn at intervals for sugar analysis and the final hydrolysate was collected in sterile 1 l bottles, which were stored at 20 c. kluyveromyces marxianus uofs y2791, isolated from a local agave plant (agave americana), and candida utilis nrrl y1084 were obtained from the university of the free state mircen yeast culture collection. these cultures were maintained on gpy agar slants containing (per litre) 40 g glucose, 5 g peptone, 5 g yeast extract and 20 g agar, stored at 4 c and subcultured every 2 months. pre - cultures of k. marxianus and c. utilis were grown in a sterile medium containing (per litre) 5 g glucose, 0.25 g citric acid, 3 g yeast extract (merck, darmstadt, germany), 5 g (nh4)2so4, 9.61 g kh2po4, 0.76 g k2hpo4, 0.75 g mgso4.7h2o, 0.05 g cacl2.2h2o, 0.1 g nacl and 1 ml of a trace elements stock solution, adjusted to ph 5.5 with 3 mol l koh prior to autoclaving. the trace elements stock solution contained (per litre) 3.5 g feso4.7h2o, 0.7 g mnso4.7h2o, 1.1 g znso4.7h2o, 0.1 g cuso4.5h2o, 0.2 g coso4.6h2o, 0.13 g na2moo4.2h2o, 0.2 g h3bo3, 0.04 g ki and 0.16 g al2(so4)3.18h2o.16 a 500 ml erlenmyer side - arm flask containing 50 ml of the above medium was inoculated with a loopful of cells from a 24 h agar slant of k. marxianus or c. utilis and incubated at 40 or 35 c, respectively, on an orbital shaker at 200 rev min to late exponential phase. these temperatures were the upper limit of the optimum temperature range obtained from temperature profiles previously determined. a 1 ml volume was subsequently transferred to a second shake flask containing the same medium, which was similarly incubated and immediately used to inoculate the bioreactor vessel. batch cultivations were carried out with k. marxianus and c. utilis at 40 and 35 c, respectively, in a 1.6 l biostat b - plus stirred tank reactor fitted with an exhaust gas condenser cooled to 1 c and using a 1 l culture volume. the ph was maintained at ph 5.0 by automatic titration with either 3 mol l koh or 1.5 mol l h2so4. the bioreactor vessel containing 950 ml medium was inoculated with 50 ml of the shake flask culture. the dissolved oxygen tension (dot) was monitored with a polarographic po2 electrode (mettler toledo, halstead, uk) and controlled above 30% of saturation by cascade control of the stirrer speed and the aeration rate in the range 1.03.0 l min. foaming was controlled by automatic addition of dow corning 1510 silicone antifoam (bdh laboratory supplies, poole, uk). when used as carbon substrate, the frozen cladode hydrolysate was thawed and an 800 ml volume sterilized in the bioreactor vessel at 110 c for 10 min, cooled to the desired cultivation temperature, supplemented with a 150 ml concentrate of a mineral salts solution (below) that had been sterilized by autoclaving at 121 c for 20 min, which also served as diluent to obtain a more miscible slurry, and adjusted to ph 5.0. mineral salts were added to all culture media at the following final concentrations (per liter) : 0.5 g citric acid, 10 g nh4cl, 5 g kh2po4, 0.5 g k2hpo4, 1 g mgso4.7h2o, 0.1 g cacl2.2h2o and 2 ml of the trace elements stock solution described above. in the case of k. marxianus cultures, a filter - sterilized vitamin stock solution that contained (per liter) 0.025 g biotin, 0.5 g nicotinic acid, 0.5 g pyridoxine hydrochloride, 0.5 g thiamine hydrochloride (sigma - aldrich, steinheim, germany), 0.5 g calcium pantothenate (merck), 0.1 g p - aminobenzoic acid (hopkin & williams ltd, chadwell heath, uk) and 12.5 g m - inositol (bdh laboratory supplies)17 was added to the sterile basal medium. two chemically defined media were also used, namely a simulated hydrolysate medium containing hexose and pentose sugars at similar concentrations as present in the enzymatic cladode hydrolysate, and a similar glucose - limited medium that contained glucose as the only sugar at a final concentration of 10 or 50 g l. in each case 150 ml of the sterile concentrated sugar solution was added to 800 ml of the above mineral salts basal medium in the bioreactor vessel, which had been adjusted to ph 5.0 with 3 mol l koh and autoclaved at 121 c for 20 min. batch cultivations were carried out with k. marxianus and c. utilis at 40 and 35 c, respectively, in a 1.6 l biostat b - plus stirred tank reactor fitted with an exhaust gas condenser cooled to 1 c and using a 1 l culture volume. the ph was maintained at ph 5.0 by automatic titration with either 3 mol l koh or 1.5 mol l h2so4. the bioreactor vessel containing 950 ml medium was inoculated with 50 ml of the shake flask culture. the dissolved oxygen tension (dot) was monitored with a polarographic po2 electrode (mettler toledo, halstead, uk) and controlled above 30% of saturation by cascade control of the stirrer speed and the aeration rate in the range 1.03.0 l min. foaming was controlled by automatic addition of dow corning 1510 silicone antifoam (bdh laboratory supplies, poole, uk). when used as carbon substrate, the frozen cladode hydrolysate was thawed and an 800 ml volume sterilized in the bioreactor vessel at 110 c for 10 min, cooled to the desired cultivation temperature, supplemented with a 150 ml concentrate of a mineral salts solution (below) that had been sterilized by autoclaving at 121 c for 20 min, which also served as diluent to obtain a more miscible slurry, and adjusted to ph 5.0. mineral salts were added to all culture media at the following final concentrations (per liter) : 0.5 g citric acid, 10 g nh4cl, 5 g kh2po4, 0.5 g k2hpo4, 1 g mgso4.7h2o, 0.1 g cacl2.2h2o and 2 ml of the trace elements stock solution described above. in the case of k. marxianus cultures, a filter - sterilized vitamin stock solution that contained (per liter) 0.025 g biotin, 0.5 g nicotinic acid, 0.5 g pyridoxine hydrochloride, 0.5 g thiamine hydrochloride (sigma - aldrich, steinheim, germany), 0.5 g calcium pantothenate (merck), 0.1 g p - aminobenzoic acid (hopkin & williams ltd, chadwell heath, uk) and 12.5 g m - inositol (bdh laboratory supplies)17 was added to the sterile basal medium. two chemically defined media were also used, namely a simulated hydrolysate medium containing hexose and pentose sugars at similar concentrations as present in the enzymatic cladode hydrolysate, and a similar glucose - limited medium that contained glucose as the only sugar at a final concentration of 10 or 50 g l. in each case 150 ml of the sterile concentrated sugar solution was added to 800 ml of the above mineral salts basal medium in the bioreactor vessel, which had been adjusted to ph 5.0 with 3 mol l koh and autoclaved at 121 c for 20 min. cellulase and -glucosidase activities were determined according to iupac guidelines,18 and pectinase activity as described elsewhere.19 cell concentrations were monitored by measuring culture turbidity against a medium blank with a photolab s6 spectrophotometer (wtw, weilheim, germany) at 690 nm. dry cell weight was gravimetrically determined using duplicate 10 ml samples that were centrifuged, washed with distilled water and dried overnight at 105 c. the co2 and o2 content of the bioreactor exhaust gas was continuously monitored using an uras 10e infrared and a magnos 6 g paramagnetic gas analyser (hartman & braun, frankfurt, germany) and the gas exchange rates were calculated by means of a nitrogen balance. during some of the k. marxianus cultivations, ethyl acetate in the exhaust gas was trapped with orbo desorption tubes (sigma - aldrich, deisenhofen, germany) containing activated coconut charcoal as adsorbent and the ethyl acetate subsequently extracted from these tubes with dichloromethane (sigma - aldrich, steinheim),20 followed by analysis by gas chromatography (gc) as described below. samples collected for determining residual sugars and metabolic products were immediately cooled in ice before centrifugation at 10 600 g and 4 c using an eppendorf 5430 r centrifuge (eppendorf ag, hamburg, germany) and the supernatants filtered through a 0.45 m acetate membrane filter (pall, port washington, ny, usa) prior to analysis. the concentrations of glucose, xylose, galactose, arabinose and fructose were determined with a waters high - performance liquid chromatography (hplc) instrument (waters corp., milford, ma, usa) using an aminex hpx-87p column (bio - rad, hercules, ca, usa) at 85 c with milliq water at a flow rate of 0.4 ml min as eluent, or with a rezex rpm - monosaccharide pb cation exchange column (phenomenex, torrance, ca, usa), the latter giving a better peak resolution. ethanol, acetaldehyde and ethyl acetate were determined with a shimadzu gc 2010 gas chromatograph (shimadzu scientific instruments, columbia, md, usa) equipped with a zb wax column (phenomenex) and a flame ionization detector with hydrogen as carrier gas at a linear velocity of 35 cm s, using a 0.6 l injection volume at a 50:1 split ratio. the oven temperature was 80 c for 2.5 min, ramped at 25 c min to 180 c with a 2 min isothermal period. for determination of the ethyl acetate recovered from the bioreactor exhaust gas, an oven temperature of 40 c for 5 min, ramped at 10 c min to 150 c with a 5 min isothermal period was used, with a 1.0 l injection volume at a 10:1 split ratio and injector and detector temperatures of 150 and 280 c, respectively. a refractive index detector (waters) the protein content of the cells was determined the by biuret method21 using bovine serum albumin, fraction v (sigma - aldrich, st louis, mo, usa) as protein standard. amino acids were determined by transferring a washed biomass suspension equivalent to 0.5 mg dry mass into vacuum reaction hydrolysis tubes with norleucine as internal standard. following liquid phase acid hydrolysis at 108110 c for 24 h to access the proteinogenic amino acids, these were derivatized by incubating with a 1:1 volumetric ratio of n-(tert - butyldimethylsilyl)-n - methyltrifluoroacetamide (mtbstfa) (merck, hohenbrunn, germany) and n, n-dimethylformamide (dmf) (merck, darmstadt, germany) for 60 min at 60 c.22 the derivatized amino acids were analysed by gas chromatography mass spectrometry (gc - ms) using a gc trace ultra instrument with a dsq quadrupole mass spectrometer (thermo finnigan, san jose, ca, usa). the sugar concentrations in the slurry resulting from acid pretreatment of the o. ficus - indica cladode dried meal were (per litre) 7.4 g glucose, 3.9 g xylose, 2.3 g galactose, 4.0 g arabinose and 5.0 g fructose. subsequent hydrolysis with cellulase, -glucosidase and pectinase released 78% of the theoretical sugar content based on previous analysis of this cladode biomass in our research group,13 resulting in a hydrolysate with a carbohydrate composition as shown in table 1 with a total sugar concentration of 81.3 g l. these results served as a basis for formulating a chemically defined simulated hydrolysate culture medium. sugar composition of o. ficus - indica cladode biomass after enzymatic hydrolysis based on a cladode composition of 182 g glucose, 25 g xylose, 58 g galactose, 26 g arabinose and 38 g fructose per kilogram dry biomass.13 in the hydrolysate medium k. marxianus and c. utilis preferentially utilized glucose followed by fructose uptake (fig. 1). after 50 h of cultivation k. marxianus had consumed 97% and c. utilis only 74% of the total sugars. k. marxianus utilized galactose, arabinose and xylose mainly after exhaustion of the glucose and, with the exception of arabinose, the sugars were almost completely consumed within 30 h. candida utilis consumed glucose, fructose and xylose within 35 h, but at the end of the cultivation only 32% of the galactose and 11% of the arabinose had been consumed (fig. the volumetric rate of total sugar uptake by k. marxianus was 2.63 g l h, which was substantially faster than c. utilis (table 2). candida utilis produced small amounts of glycerol and acetic acid, whereas k. marxianus produced up to 2.9 g ethanol l as well as low concentrations of acetic acid and ethyl acetate, despite the aerobic culture conditions (table 2). cultivation profiles of c. utilis (a) and k. marxianus (b) with the dot controlled above 30% of saturation in an enzymatic hydrolysate of o. ficus - indica cladode biomass supplemented with mineral salts. growth parameters of c. utilis and k. marxianus in an enzymatic hydrolysate of o. ficus - indica cladode biomass supplemented with mineral salts (mean values are shown) yx / s, biomass yield coefficient on total sugars assimilated (estimated from biuret protein assays) ; yp / s, ethanol yield coefficient on total sugars assimilated ; qs, maximum volumetric rate of total sugars uptake, calculated from the slope of the curve of total sugar concentration versus time ; nd, not detected. the cellular protein content of c. utilis, grown in a chemicaly defined medium and determined by the biuret method, was 530 g kg on a dry weight basis, whereas that of k. marxianus was 443 g kg. the yeast dry biomass concentration in the cladode hydrolysate could not directly be determined owing to the viscosity and presence of particulate matter. therefore the yeast biomass was indirectly estimated from biuret protein assays of the hydrolysate culture broth, correcting for the initial content of plant protein. these estimates indicated that k. marxianus produced a dry biomass concentration of 11.1 g l, whereas the c. utilis biomass was slightly higher at 12.2 the biomass yields of both yeasts were lower than expected, especially that of k. marxianus (table 2). cultivation of k. marxianus and c. utilis increased the total protein content of the hydrolysate, on a dry weight basis, from an initial value of 80 g kg to 180 and 220 g kg, respectively (table 2). to serve as a benchmark for the performance of yeasts in the cladode hydrolysate and to investigate the low biomass yield found with k. marxianus, experiments were conducted using a chemically defined medium containing a sugar mixture simulating the composition of the cladode hydrolysate to facilitate carbon and degree of reduction balances, using a biomass composition of ch1.8o0.5 n0.2. in this medium k. marxianus had a high maximum specific growth rate (max) of 0.71 h, whereas c. utilis had a max value of 0.40 h and a biomass yield coefficient of 0.42. by contrast, the biomass yield coefficient of k. marxianus remained low at 0.24 (table 3). the sugar utilization profiles were similar to those in the actual cladode hydrolysate medium (fig. although arabinose utilization by k. marxianus appeared slightly slower than in the actual hydrolysate, the volumetric rate of total sugar uptake by both yeasts was about 1.5-fold higher than in the hydrolysate. after 30 h of cultivation, k. marxianus had utilized about 88% of the total sugars and c. utilis 79%. kluyveromyces marxianus again produced up to 6.4 g ethanol l as well as ethyl acetate, acetaldehyde and acetic acid, whereas c. utilis produced glycerol and acetic acid (fig. 2 and table 3). in this medium both yeasts produced a substantially higher acetic acid concentration than in the actual hydrolysate. the carbon and degree of reduction balances for c. utilis cultivation closed to 97% and 94%, respectively, with a respiratory quotient (rq) of 1.12. however, in the case of k. marxianus the carbon and degree of reduction balances closed to only 65% and 72%, respectively, and the rq of 1.86 indicated a respiro - fermentative metabolism. growth parameters of c. utilis and k. marxianus in a chemically defined medium containing a sugar mixture simulating an enzymatic hydrolysate of cladode biomass (mean values are shown) max, maximum specific growth rate ; yx / s, biomass yield coefficient on total sugars assimilated ; yp / s, ethanol yield coefficient on total sugars assimilated ; qs, maximum volumetric rate of total sugars uptake, calculated from the slope of the curve of product concentration versus time ; nd, not detected. cultivation profiles of c. utilis (a) and k. marxianus (b) with the dot controlled above 30% of saturation in a chemically defined medium containing a sugar mixture simulating an enzymatic hydrolysate of cladode biomass. to further investigate the low biomass yields of k. marxianus and the failure of the balances of its cultivation to close, the yeasts were grown aerobically in a chemically defined glucose - limited medium. using an initial glucose concentration of 10 g l, k. marxianus and c. utilis nrrl y-1084 exhibited max values of 0.78 and 0.42 h, respectively, with corresponding biomass yield coefficients of 0.45 and 0.50 (fig. kluyveromyces marxianus produced a minimal amount of ethanol (0.23 g l) under these conditions. however, with an initial glucose concentration of 50 g l, the biomass yield coefficient of k. marxianus decreased to 0.15, while ethanol production increased to 8.4 g l with the accumulation of lower concentrations of glycerol (1.3 g l), acetic acid (0.75 g l), acetaldehyde (0.11 g l) and ethyl acetate (0.25 g l). by contrast, the biomass yield of c. utilis remained close to the theoretical maximum, irrespective of the glucose concentration, with the production of less than 0.05 g l glycerol and acetic acid but no ethanol (fig. 3). thus, although k. marxianus is regarded as strongly crabtree negative, the above observations suggested a shift from a respiratory to a respiro - fermentative metabolism at the higher sugar concentration, as indicated by aerobic ethanol production and a concomitant increase in the rq to 1.98. effect of glucose concentration on the cultivation profiles of k. marxianus (a, b) and c. utilis (c, d) in a chemically defined medium maintained at a dot of above 30% of saturation and containing a glucose concentration of 10 (a, c) or 50 g l (b, d). symbols : biomass () ; glucose () ; ethanol (). whereas the carbon and degree of reduction balances, based on gas exchange values and metabolites in the culture broth, of k. marxianus grown in a medium containing 10 g glucose l closed to within 96% and 98%, respectively, in the medium containing 50 g glucose l the balances failed to close by a large margin. increasing the vitamin supplementation twofold or supplementing the medium with yeast extract at 3 however, in cultivations with the dot maintained at above 50% of saturation, its max increased slightly to 0.80 h, with a substantial increase in the biomass yield coefficient of up to 0.31 and ethanol production decreasing to below 2.13 g l, but still with a poor recovery of carbon and reducing equivalents in the balances (data not shown). it was subsequently discovered that the failure of the balances to close was due to the stripping of highly volatile ethyl acetate from the culture broth by aeration, despite an exhaust gas condenser cooled to 1 c on the culture vessel. the amount of ethyl acetate recovered by fitting an activated charcoal column on the exhaust gas condenser corresponded to a concentration of 12.05 g l in the culture broth, while the actual ethyl acetate in the culture broth amounted to only 0.51 g l. by including both these amounts, the carbon and degree of reduction balances resulted in recoveries of 108% and 103%, respectively. table 4 shows the amino acid profiles except for tryptophan, asparagine and glutamine, which were destroyed during acid hydrolysis, of the cladode hydrolysate and the biomass product resulting from the cultivation of c. utilis and k. marxianus in the hydrolysate. yeast cultivation effected a two- to threefold increase in the content of almost all the amino acids, with the lysine content increasing about fivefold and the total amino acid content increasing almost 2.9-fold. amino acid profiles of o. ficus - indica cladode hydrolysate and of the biomass product (g 100 g dry weight) obtained by cultivation of c. utilis and k. marxianus. the mean values of triplicate determinations and the standard deviation of the mean are shown the essential amino acid profile of the yeast - enriched hydrolysate protein compared quite favourably with the fao / who scoring pattern,23 with the exception of the sulfur - containing amino acids and lysine (table 5). even though yeast cultivation greatly increased the lysine content of the cladode hydrolysate (table 4), the ratio of the lysine content of the biomass protein to the value in the fao / who scoring pattern was only 0.49 to 0.5 (table 5). owing to the low content of methionine and cystine, the chemical score of the protein of the biomass product obtained by cultivation of c. utilis and k. marxianus was 49% and 47%, respectively. essential amino acid composition (mg g protein) of the protein in the biomass product obtained by cultivation of c. utilis and k. marxianus ratio of the amino acid content of the biomass protein to the essential amino acid in the fao / who scoring pattern. the sugar concentrations in the slurry resulting from acid pretreatment of the o. ficus - indica cladode dried meal were (per litre) 7.4 g glucose, 3.9 g xylose, 2.3 g galactose, 4.0 g arabinose and 5.0 g fructose. subsequent hydrolysis with cellulase, -glucosidase and pectinase released 78% of the theoretical sugar content based on previous analysis of this cladode biomass in our research group,13 resulting in a hydrolysate with a carbohydrate composition as shown in table 1 with a total sugar concentration of 81.3 g l. these results served as a basis for formulating a chemically defined simulated hydrolysate culture medium. sugar composition of o. ficus - indica cladode biomass after enzymatic hydrolysis based on a cladode composition of 182 g glucose, 25 g xylose, 58 g galactose, 26 g arabinose and 38 g fructose per kilogram dry biomass.13 in the hydrolysate medium k. marxianus and c. utilis preferentially utilized glucose followed by fructose uptake (fig. 1). after 50 h of cultivation k. marxianus had consumed 97% and c. utilis only 74% of the total sugars. k. marxianus utilized galactose, arabinose and xylose mainly after exhaustion of the glucose and, with the exception of arabinose, the sugars were almost completely consumed within 30 h. candida utilis consumed glucose, fructose and xylose within 35 h, but at the end of the cultivation only 32% of the galactose and 11% of the arabinose had been consumed (fig. candida utilis produced small amounts of glycerol and acetic acid, whereas k. marxianus produced up to 2.9 g ethanol l as well as low concentrations of acetic acid and ethyl acetate, despite the aerobic culture conditions (table 2). cultivation profiles of c. utilis (a) and k. marxianus (b) with the dot controlled above 30% of saturation in an enzymatic hydrolysate of o. ficus - indica cladode biomass supplemented with mineral salts. growth parameters of c. utilis and k. marxianus in an enzymatic hydrolysate of o. ficus - indica cladode biomass supplemented with mineral salts (mean values are shown) yx / s, biomass yield coefficient on total sugars assimilated (estimated from biuret protein assays) ; yp / s, ethanol yield coefficient on total sugars assimilated ; qs, maximum volumetric rate of total sugars uptake, calculated from the slope of the curve of total sugar concentration versus time ; nd, not detected. expressed in terms of total dry weight of washed solids. the cellular protein content of c. utilis, grown in a chemicaly defined medium and determined by the biuret method, was 530 g kg on a dry weight basis, whereas that of k. marxianus was 443 g kg. the yeast dry biomass concentration in the cladode hydrolysate could not directly be determined owing to the viscosity and presence of particulate matter. therefore the yeast biomass was indirectly estimated from biuret protein assays of the hydrolysate culture broth, correcting for the initial content of plant protein. these estimates indicated that k. marxianus produced a dry biomass concentration of 11.1 g l, whereas the c. utilis biomass was slightly higher at 12.2 the biomass yields of both yeasts were lower than expected, especially that of k. marxianus (table 2). cultivation of k. marxianus and c. utilis increased the total protein content of the hydrolysate, on a dry weight basis, from an initial value of 80 g kg to 180 and 220 g kg, respectively (table 2). to serve as a benchmark for the performance of yeasts in the cladode hydrolysate and to investigate the low biomass yield found with k. marxianus, experiments were conducted using a chemically defined medium containing a sugar mixture simulating the composition of the cladode hydrolysate to facilitate carbon and degree of reduction balances, using a biomass composition of ch1.8o0.5 n0.2. in this medium k. marxianus had a high maximum specific growth rate (max) of 0.71 h, whereas c. utilis had a max value of 0.40 h and a biomass yield coefficient of 0.42. by contrast, the biomass yield coefficient of k. marxianus remained low at 0.24 (table 3). the sugar utilization profiles were similar to those in the actual cladode hydrolysate medium (fig. although arabinose utilization by k. marxianus appeared slightly slower than in the actual hydrolysate, the volumetric rate of total sugar uptake by both yeasts was about 1.5-fold higher than in the hydrolysate. after 30 h of cultivation, k. marxianus had utilized about 88% of the total sugars and c. utilis 79%. kluyveromyces marxianus again produced up to 6.4 g ethanol l as well as ethyl acetate, acetaldehyde and acetic acid, whereas c. utilis produced glycerol and acetic acid (fig. 2 and table 3). in this medium both yeasts produced a substantially higher acetic acid concentration than in the actual hydrolysate. the carbon and degree of reduction balances for c. utilis cultivation closed to 97% and 94%, respectively, with a respiratory quotient (rq) of 1.12. however, in the case of k. marxianus the carbon and degree of reduction balances closed to only 65% and 72%, respectively, and the rq of 1.86 indicated a respiro - fermentative metabolism. growth parameters of c. utilis and k. marxianus in a chemically defined medium containing a sugar mixture simulating an enzymatic hydrolysate of cladode biomass (mean values are shown) max, maximum specific growth rate ; yx / s, biomass yield coefficient on total sugars assimilated ; yp / s, ethanol yield coefficient on total sugars assimilated ; qs, maximum volumetric rate of total sugars uptake, calculated from the slope of the curve of product concentration versus time ; nd, not detected. cultivation profiles of c. utilis (a) and k. marxianus (b) with the dot controlled above 30% of saturation in a chemically defined medium containing a sugar mixture simulating an enzymatic hydrolysate of cladode biomass. to further investigate the low biomass yields of k. marxianus and the failure of the balances of its cultivation to close, the yeasts were grown aerobically in a chemically defined glucose - limited medium. using an initial glucose concentration of 10 g l, k. marxianus and c. utilis nrrl y-1084 exhibited max values of 0.78 and 0.42 h, respectively, with corresponding biomass yield coefficients of 0.45 and 0.50 (fig. 3). kluyveromyces marxianus produced a minimal amount of ethanol (0.23 g l) under these conditions. however, with an initial glucose concentration of 50 g l, the biomass yield coefficient of k. marxianus decreased to 0.15, while ethanol production increased to 8.4 g l with the accumulation of lower concentrations of glycerol (1.3 g l), acetic acid (0.75 g l), acetaldehyde (0.11 g l) and ethyl acetate (0.25 g l). by contrast, the biomass yield of c. utilis remained close to the theoretical maximum, irrespective of the glucose concentration, with the production of less than 0.05 g l glycerol and acetic acid but no ethanol (fig. 3). thus, although k. marxianus is regarded as strongly crabtree negative, the above observations suggested a shift from a respiratory to a respiro - fermentative metabolism at the higher sugar concentration, as indicated by aerobic ethanol production and a concomitant increase in the rq to 1.98. effect of glucose concentration on the cultivation profiles of k. marxianus (a, b) and c. utilis (c, d) in a chemically defined medium maintained at a dot of above 30% of saturation and containing a glucose concentration of 10 (a, c) or 50 g l (b, d). symbols : biomass () ; glucose () ; ethanol (). whereas the carbon and degree of reduction balances, based on gas exchange values and metabolites in the culture broth, of k. marxianus grown in a medium containing 10 g glucose l closed to within 96% and 98%, respectively, in the medium containing 50 g glucose l the balances failed to close by a large margin. increasing the vitamin supplementation twofold or supplementing the medium with yeast extract at 3 however, in cultivations with the dot maintained at above 50% of saturation, its max increased slightly to 0.80 h, with a substantial increase in the biomass yield coefficient of up to 0.31 and ethanol production decreasing to below 2.13 g l, but still with a poor recovery of carbon and reducing equivalents in the balances (data not shown). it was subsequently discovered that the failure of the balances to close was due to the stripping of highly volatile ethyl acetate from the culture broth by aeration, despite an exhaust gas condenser cooled to 1 c on the culture vessel. the amount of ethyl acetate recovered by fitting an activated charcoal column on the exhaust gas condenser corresponded to a concentration of 12.05 g l in the culture broth, while the actual ethyl acetate in the culture broth amounted to only 0.51 g l. by including both these amounts, the carbon and degree of reduction balances resulted in recoveries of 108% and 103%, respectively. table 4 shows the amino acid profiles except for tryptophan, asparagine and glutamine, which were destroyed during acid hydrolysis, of the cladode hydrolysate and the biomass product resulting from the cultivation of c. utilis and k. marxianus in the hydrolysate. yeast cultivation effected a two- to threefold increase in the content of almost all the amino acids, with the lysine content increasing about fivefold and the total amino acid content increasing almost 2.9-fold. amino acid profiles of o. ficus - indica cladode hydrolysate and of the biomass product (g 100 g dry weight) obtained by cultivation of c. utilis and k. marxianus. the mean values of triplicate determinations and the standard deviation of the mean are shown the essential amino acid profile of the yeast - enriched hydrolysate protein compared quite favourably with the fao / who scoring pattern,23 with the exception of the sulfur - containing amino acids and lysine (table 5). even though yeast cultivation greatly increased the lysine content of the cladode hydrolysate (table 4), the ratio of the lysine content of the biomass protein to the value in the fao / who scoring pattern was only 0.49 to 0.5 (table 5). owing to the low content of methionine and cystine, the chemical score of the protein of the biomass product obtained by cultivation of c. utilis and k. marxianus was 49% and 47%, respectively. essential amino acid composition (mg g protein) of the protein in the biomass product obtained by cultivation of c. utilis and k. marxianus ratio of the amino acid content of the biomass protein to the essential amino acid in the fao / who scoring pattern. the main drawback in using the cladode biomass as feedstock for yeast cultivation to enhance its protein content and amino acid profile was the very high water content of the cladodes. this problem was addressed by sun - drying and milling the fresh cladodes, giving a more concentrated feedstock as well as facilitating easier storage and handling. the total sugar content of the cladode hydrolysate, 81.3 g l, was less than found in conventional lignocellulosic hydrolysates,14,24,25 mainly due to the lower glucan and xylan content of the cladodes. although regarded as strongly crabtree negative,8 the biomass yield of k. marxianus was substantially less than that of c. utilis due to the formation of by - products such as ethanol, despite fully aerobic cultivation conditions. this indicated that this strain of k. marxianus was in fact weakly crabtree positive, exhibiting a degree of respiro - fermentative metabolism. there are differing reports regarding the crabtree characteristic of k. marxianus and these variations were apparently strain dependent.2628 for example, it was reported that k. marxianus dsm 5420 produced 10 g ethanol l from 120 g lactose l under strict aerobic conditions.10 a shift to ethanol production could be a consequence of an imbalance between glycolysis and the respiratory chain, or to an oxygen, thiamine or iron limitation.8,29,30 a respiro - fermentative metabolism was observed in a continuous culture of a k. lactis strain when the dilution rate was increased to above 0.4 h ; in this case ethanol production was attributed to a nicotinamide limitation.31 all k. marxianus strains have an absolute nicotinamide requirement.10,32 however, we found that increasing the vitamin supplementation and even adding yeast extract at 3 g l had little effect on aerobic ethanol production by our strain of k. marxianus (data not shown), making it unlikely that ethanol production in our cultures was triggered by a nutrient or growth factor limitation. some k. marxianus strains have a strong tendency to produce pyruvate and acetate when exposed to excess sugar.33 we detected also substantial production of ethyl acetate by k. marxianus, as has previously been found with some strains grown under an iron limitation.29 in our cultivations ethyl acetate production was unexpected, since the iron content of the medium was in excess and no trace element limitation was likely. the proposed mechanism of ethyl acetate synthesis by k. marxianus is either from ethanol and acetyl - coa catalyzed by an alcohol acetyltransferase, or from ethanol and acetate catalyzed by an esterase with oxygen as an obligatory requirement for either of these reactions.29,34 ethyl acetate is more volatile than ethanol and rapid stripping of this ester from the culture broth occurs during aerobic cultivation. this, together with ethanol production, accounted for the low biomass yield obtained with k. marxianus and the failure of the carbon and degree of reduction balances to close until we could account for the ethyl acetate lost in the bioreactor exhaust gas. this could have been due to experimental error and/or the ' standard ' elemental biomass composition used in these calculations differing from the actual composition of k. marxianus. the protein content of c. utilis, 530 g kg (dry weight), as determined by the biuret assay, was comparable to values previously reported for this yeast,35,36 whereas the protein content of k. marxianus was only 443 g kg. however, despite its lower protein content, cultivation of k. marxianus in the cladode hydrolysate increased the total protein concentration to 5.4 g l, which was slightly less than the 6 g l obtained with c. utilis, probably because k. marxianus was capable of utilizing more of the hydrolysate sugars. the relatively high content of lysine and threonine in yeast biomass renders the latter well suited for supplementation of plant - based feeds.10 the lysine content of c. utilis and k. marxianus (grown in a simulated cladode hydrolysate) was 26.8 and 32.1 g kg dry cell mass, respectively (data not shown). although their cultivation in the cladode hydrolysate increased the lysine content about fivefold (table 4), the lysine content of the final biomass product amounted to only 49% of the value of the fao / who scoring pattern (table 5). similarly, the initial content of methionine plus cystine in the cladode hydrolysate was increased more than twofold, but these sulfur - containing amino acids remained limiting, giving the protein of the biomass product a chemical score of 47% (table 5). nevertheless, the profile of essential amino acids in the protein obtained by enrichment of the cladode hydrolysate through yeast cultivation compared favourably with cereals such as sorghum grains (chemical score of 37%) and millet grains (chemical score ranging from 31% to 63%).37 in addition to the chemical score, the essential amino acid index (eaai) may be calculated from the essential amino acid profile to predict the nutritional value of a protein.38 again using the fao / who scoring pattern but without tryptophan, which we did not determine, the eaai values for the biomass product obtained using c. utilis and k. marxianus, respectively, were 68.8% and 70.1%. these compared favourably with the eaai values reported for wheat flour (7484%) and peanut flour (7386%).39 the cladodes of o. ficus - indica, a plant well adapted for cultivation in semi - arid regions with a high annual productivity, have potential as a lignocellulosic feedstock for scp production by yeast cultivation. the very high water content of the cladodes and the high viscosity of the enzymatic hydrolysate of the cladode biomass are constraints as to its use, however. using k. marxianus in a bioprocess has the advantages of a higher growth rate, a higher temperature tolerance and utilisation of a broader carbohydrate substrate range than possible with most other yeasts. these advantages were offset, however, by the unexpected low biomass yield of k. marxianus under certain cultivation conditions due to the production of ethanol and ethyl acetate. aerobic ethanol production in yeasts is a complex phenomenon that has not yet been thoroughly elucidated in k. marxianus ; the physiology of this as well as of ethyl acetate production warrant further investigation. it may be worthwhile to screen for strains with minimal production of these by - products to maximise biomass production, considering the favourable results obtained with this strain of k. marxianus. cultivation of c. utilis and k. marxianus in the cladode hydrolysate improved the total protein content (i.e. plant plus yeast protein) of the biomass product from an initial value of 80 g kg to 220 and 180 g kg (dry weight), respectively, which had an amino acid profile superior to that of cereals, with the exception of the sulfur - containing amino acids. although the lysine content was increased by about fivefold, it was the second limiting essential amino acid. animal feeding trials are required to ascertain the nutritional value of the biomass product, also taking into account digestibility. | backgroundthe cladodes of opuntia ficus - indica (prickly pear cactus) have a low protein content ; for use as a balanced feed, supplementation with other protein sources is therefore desirable. we investigated protein enrichment by cultivation of the yeasts candida utilis and kluyveromyces marxianus in an enzymatic hydrolysate of the cladode biomass.resultsdilute acid pretreatment and enzymatic hydrolysis of sun - dried cladodes resulted in a hydrolysate containing (per litre) 45.5 g glucose, 6.3 g xylose, 9.1 g galactose, 10.8 g arabinose and 9.6 g fructose. even though k. marxianus had a much higher growth rate and utilized l - arabinose and d - galactose more completely than c. utilis, its biomass yield coefficient was lower due to ethanol and ethyl acetate production despite aerobic cultivation. yeast cultivation more than doubled the protein content of the hydrolysate, with an essential amino acid profile superior to sorghum and millet grains.conclusionsthis k. marxianus strain was weakly crabtree positive. despite its low biomass yield, its performance compared well with c. utilis. this is the first report showing that the protein content and quality of o. ficus - indica cladode biomass could substantially be improved by yeast cultivation, including a comparative evaluation of c. utilis and k. marxianus. 2014 the authors. journal of the science of food and agriculture published by john wiley & sons ltd on behalf of society of chemical industry. |
urinary tract infection (uti) is a significant cause of morbiditiy among children, and may result in permanent parenchymal damage with the possible development of late complications such as arterial hypertension or chronic renal failure. the likelihood of developing these late complications in utis is the greatest in the first year of life, but gradually decreases in older children. following the treatment of a full dose of antibiotics for symptomatic uti the aim of ltap is to prevent a recurring urinary infection and the potential for permanent tissue damage. recurrent uti with or without vesico ureteral reflux in children is the most frequent indication for ltap. the idea of ltap is to provide small levels of antimicrobial agents (aa) in the urinary system, sufficient to prevent bacterial multiplication. aside from some controversies regarding the use of ltap, widely accepted indications for ltap are as follows : vesico ureteral reflux, recurrent uti with or without associated anomalies, febrile uti in the first year of life, prenatal hydronephrosis due to obstruction or vesico ureteral reflux, and uti associated with voiding disturbances or the weakness of the immune system. ltap is usually administered in a single daily dose equivalent to a quarter or half of the conventional therapeutic dose. the most commonly prescribed medication is nitrofurantoin in children after the first year of life and trimethoprim / sulfamethoxazole (tmp / smx) in children older than 3 months. for newborns and infants less than 3 months of age, prescribed antibiotics for ltap. limitations of ltap include relative ineffectiveness, increasing microbial resistance rates that are proportionate to the duration of treatment, adverse effects, poor compliance and cost. some authors reported a growing bacterial resistance to common aas used in ltap [611 ]. other authors have reported an increasing rate of bacterial resistance to aas in patients who had anatomic or functional abnormalities of the us, as well as those patients who were repeatedly hospitalized for uti. nowadays, some authors do not recommend prophylaxis after a single uti in infancy [1315 ], a surprising position in light of the fact that in the first year of life the frequency of febrile uti is highest, as well as the susceptibility of permanent parenchymal damage. in cases of prescribed ltap a variety of guidelines have been proposed regarding the age of patients, grade of vesico ureteral reflux, any co - existing anatomic anomalies of urinary tract, the frequency of uti and size of renal scarring. furthermore, careful monitoring of each patient is recommended in order to detect uti as early as possible, as well as treating the patient with the proper antibiotic and changing the antibiotic in the case of a recurrent uti. although prophylactic use of antibiotics in pediatric nephrology has been commonly accepted and used all over the world, some recent studies question the effectiveness of ltap in the prevention of uti and renal scarring, stressing the great need for new well - designed studies, whose results would give new insight in antibiotic use in ltap. the american academy of pediatrics (1999) and matoo (2007) proposed several medications and their doses for ltap. we suspected that some of the proposed medications would have a high percentage of bacterial resistance in our local community ; therefore we decided to undertake our own study in order to assess the situation regarding bacterial resistance in university hospital in split. we will also give some recommendations regarding the choice of drugs for ltap in our region. in addition, we investigated the prevalence of uropathogens isolated from urine cultures in children hospitalized with uti at the department of pediatrics during the 7-year study period. the purpose of this study was to evaluate changes in bacterial resistance patterns among 3 specific patient populations : patients on / off ltap, patients with multiple / single hospitalization and patients with / without functional or anatomic abnormalities of the urinary system. finally, we compared our results with those from other studies and deduced our own recommendations for antibiotic use. we retrospectively reviewed the charts of all 1158 patients hospitalized for uti at university hospital in split between 2000 and 2007. uti was confirmed by a positive urine culture of 10 or more colony - forming units of a single organism per milliliter of urine. the initial identification of a pure bacterial culture was made by using convential biochemical tests and was confirmed by vitek 2 systems (biomerieux, marc marcyletoile, france). routine susceptibility testing to antibiotics was made by disk - diffusion tests, according to clsi recommendations. we recorded the patient s pertinent medical history, including the number of hospitalizations for uti, possible treatment of ltap in the last 6 months before hospitalization, the presence of acute or chronic infection, and the presence of any anatomic or functional anomalies of the urinary system such as vesico ureteral reflux, neurogenic bladder, or other anatomic abnormalities. in this study, acute uti was defined as the patient s first uti with no signs of permanent damage of the urinary system. at the same time, chronic uti was defined as permanent or recurrent uti with definite damage to the urinary system. functional anomaly of the urinary system was defined as a change in function of the lower urinary system, resulting in dysfunctional voiding. in the statistical analysis of data we used a test with p<0.05 as a statistically significant value and spearman s correlation test. in the statistical analysis of data we used a test with p<0.05 as a statistically significant value and spearman s correlation test. from the group of 1158 children with uti, hospitalized at our department, 1355 positive urine cultures were isolated. e. coli was isolated in 917 cases (67.7%), enterococcus in 164 isolates (12.1%), klebsiella sp. in 110 isolates (8.1%), proteus sp. in 67 isolates (4.9%), pseudomonas sp. in 64 isolates (4.6%), and other bacteria were isolated in the remaining 33 cases (2.3%). the mean resistance rates of the most prevalent e. coli were as follows : 69.5% to ampicillin, 3.5% to amoxicillin / clavulonic acid, 6.6% to cephalexin, 27.5% to tmp / smx, and 0.4% to nitrofurantoin. during the study period, follow - up of the trends in resistance rates of e. coli to antimicrobial agents disclosed a statisticaly significant positive trend to tmp / smx (=0.762 ; p=0.028), a statisticaly significant negative trend to cephalexin (=0.786 ; p=0.021), while no significant trends were detected to ampicillin (=0.120 ; p=0.776) and nitrofurantoin (r=0.192 ; p=0.648). we also proved 93% probability of a negative trend in the resistance rate of e. coli to amoxicillin / clavulonic acid (r=0.66 ; p=0.075) during the study period (figure 1). the mean resistance rates of all isolated uropathogens to common aas were the following : 64.3% to ampicillin, 5.8% to amoxicillin / clavulonic acid, 10.5% to cephalexin, 21.3% to tmp / smx, and 7.9% to nitrofurantoin. follow - up of the trends in resistence rates of all uropathogens to antimicrobial agents during the study period disclosed statisticaly significant negative trends to cephalexin (=0.934 ; p=0.001) and to amoxicillin / clavulonic acid (=0.802 ; p=0.017), while no significant trends were detected to ampicillin (=0.143 ; p=0.736) and tmp / smx (=0.419 ; p=0.301). for nitrofurantoin, we found a 90% probability of a negative trend in resistance rates for all uropathogens during the study period (=0.618 ; p=0.102). however, resistance to tmp / smx increased from 2004 to 2006, but not during the entire study period. enterococcus were not included in these results due to the fact that they have not been tested by all the aas. among the group of 324 children with functional or anatomic abnormalities, there were 27 cases of functional anomalies of the lower urinary system, 47 cases of hydronephrosis, 35 cases of duplex collecting system, 11 cases of urolithiasis, 116 cases of vesico ureteral reflux, and 29 cases of other anatomic anomalies of the urinary system. pseudomonas was 3 times more common while enterococcus was 2 times less common in patients with functional or anatomic anomalies of the urinary system than in patients with normal urinary systems (figure 2). in the group of children with normal urinary tracts, 113 cases of enterococcus sp. were found to be resistant to 5 tested aas (37%), but were susceptible in 196 cases (63%). while in the group of children who had abnormalities of the urinary tract, 20 isolated strains of enterococcus sp. were found to be resistant to the 5 tested aas (24%), but were susceptible in 63 cases (76%). this means that the isolated strains of enterococcus sp. were 1.5 times more resistant to the tested aas in the group of children who had normal urinary systems than the group of children who had anatomic or functional abnormalities of the urinary system (=4.5 ; p=0.033). in the group of patients with a normal urinary system, isolated strains of klebsiella sp. were resistant to 45 of the tested aas (29%) and were susceptible to 109 (71%), while in the group of patients who had abnormalities of urinary tract, isolated strains of klebsiella sp. were resistant to 36 of the tested aas (40%) and were susceptible to 53 of those aas (60%). this means that the isolated strains of klebsiella sp. were 1.4 times more resistant to aas in patients with anatomical or functional anomalies of urinary system than in those with a normal urinary system (=3.2 ; study the prevalence of uropathogens in terms of the number of hospitalizations showed that patients hospitalized more than once had a lower chance of having isolated e. coli. however, they had approximately a 3 times greater frequency of uti caused by pseudomonas sp. and a 2 times greater frequency of uti caused by klebsiella sp. than patients who were hospitalized only once (figure 3). in the group of children who were hospitalized only once, isolated strains of enterococcus sp. were resistant to 115 of 5 tested aas (37%) and were susceptible to 193 of those aas (63%), while in the group of patients who had multiple hospitalizations, the isolated strain of enterococcus sp. was resistant to 18 of 5 tested aas (21%) and were susceptible to 66 (79%). sp. were 1.7 times more resistant to aas in patients who were admitted to the hospital only once than in those who were hospitalized more than once (=7.45 ; p=0.006). as opposed to the results obtained in enterococcus sp., the isolated strains of esbl - producing klebsiella sp. in children with multiple hospitalizations were 1.4 times more resistant to antibiotics than the strains isolated in the group of children who were admitted only once. the isolated strains of esbl - producing klebsiella sp. in the children upon their first hospitalization were resistant to 107 of the 5 tested aas (53%) and were susceptible to 95 (47%), while in the group of children with repeated hospitalizations isolated esbl - producing klebsiella sp. were resistant to 61 of the 5 tested aas (68%), and were susceptible to 28 (32%), =6,14, p=0.013 out of all the patients who were admitted to the pediatric department for an uti, a group of 175 children received various aas for ltap prior to hospitalization at the primary health care level (table 1). the resistance rates of e. coli to some aas were significantly different between the groups of children who received ltap and those who did not receive ltap. we found an approximately 2 times greater resistance rate in patients who received ltap prior to hospitalization than in those who did not receive ltap at all. moreover, resistance to nitrofurantoin in both groups was very low (table 2). the probability that e. coli would be more resistant in patients who had previously undergone ltap than in those who did not was proven significant for ampicillin (=3.83 ; p=0.05), for amoxicillin / clavulonic acid (=4.29 ; p=0.038) and to tmp / smx (=23.4 ; p<0.01). e. coli was generally found to be the most frequent cause of uti in hospitalized (4090%), as well as in non - hospitalized (6378%) children [810,12,1921 ]. the mean prevalence of isolated e. coli in our study during the observed period (20002007) was 67.5% ; the highest being in year 2002 (73.7%), but the lowest in year 2007 (60.4%). as also reported in previous studies that the incidence of e. coli was lower in the cases of multiple hospitalizations, in patients previously treated with ltap, as well as in cases with coexisting urinary tract anomalies that allowed the increasing growth of some less common uropathogens. in our study the incidence of e.coli was slightly less in patients with urinary tract anomalies (61.9%) (figure 2), as well as in patients with multiple hospitalizations (52.6%) (figure 3), as opposed to patients with normal urinary tracts and those who were hospitalized only once for uti. the incidence of enterococcus also decreased in patients with urinary tract anomalies. on the contrary, throughout the entire study period, the incidence of pseudomonas sp. was 3 times greater in children with urinary tract anomalies as well as in those with multiple hospitalizations. in addition, isolated klebsiella sp. were 2 times more frequent in the group of patients with multiple hospitalizations than in patients hospitalized only once (figure 3). in the american study by lutter. (2005), performed during 19972001, the resistance rates of all isolates ranged from 48% to ampicillin, 17% to tmp / smx and 7% to nitrofurantoin. in taiwan, tseng. (2008) reported 77.4% resistance to ampicillin, 44.6% to tmp / smx, 27.2% to cephalotin, and 8.4% to nitrofurantoin, with growing resistance rates to ampicillin, tmp / smx, and cephalosporins in the first generation during the 14-year study period. in accordance with the above - mentioned studies, our results showed a similarly high resistance rate to ampicillin (4562%), and a growing resistance to tmp / smx (from 15% in 2000 to 35% in 2006). during the same period, the resistance of all uropathogens to nitrofurantoin remained low, while the resistance of e. coli to nitrofurantoin was even lower (figure 1). besides the relatively high resistance rate to ampicillin and tmp / smx, resistance rates of all isolates, except pseudomonas sp. and enterococcus sp., showed a decreasing trend in first - generation cephalosporins (cephalexin) and amoxicilin - clavulonic acid. these changes in resistance could be explained by the extensive consumption of antibiotics, especially azitromicyn and third - generation cephalosporins, which were over the previous years widely prescribed for various indications in our region locally as well as in the entire country [13 ]. regarding resistance of e. coli, our study showed that the resistance rate to ampicillin was continuously high (a mean of 69.5%), with the highest rate being in year 2001 (78.6%). during the same period of time, resistance rates to amoxicilin - clavulonic acid and cephalexin decreased, similar to the trends observed in all the other isolates. however, the increasing resistance rate of e. coli to tmp / smx was higher than the resistance to all other isolated uropathogens, first showing increasing trends until year 2004, but later showing a decrease in year 2007 (figure 1). this increasing resistance rate might be explained by the widespread prescription of tmp / smx for treatment of uti and providing ltap in croatia. increasing resistance rates to tmp / smx was not uncommon in other countries all over the world [13 ]. on the contrary, permanently low resistance rates to nitrofurantoin, with an average value of 0.4%, could be associated with the infrequent prescription of this drug. our results show a decreasing trend of resistance of e. coli to amoxicillin / clavulonic acid and cephalexin, but a low resistance to nitrofurantoin, in accordance with trends in other parts of croatia, as reported by the committee for monitoring bacterial resistance rates of the croatian academy of medical sciences. similar results of antibiotic resistance were reported in canada and brazil. permanently high resistance to antibiotics with increasing resistance rates of e. coli to ampicillin and tmp / smx were shown in studies from the uk, austria, spain and brasil [9,11,12,19 23 ]. because of that, some authors concluded that ampicillin, trimetoprim, and tmp / smx were insufficient for the monotherapy of uti, as recommended by wolff (2007). 2008) warned that the use of cephalosporins in ltap could increase the number of utis caused by esbl - producing bacteria or multidrug - resistant uropathogens other than e. coli, therefore they suggested keeping tmp / smx for use in ltap. in children with functional or anatomic urinary tract abnormalities, we expected to find more resistant pathogens, presuming that they probably had frequent exacerbations of uti and received long and frequent antibiotic treatment. (probability of 97% to be more resistant to aas), while enterococcus showed that patients had significantly more resistant strains of that uropathogen than if they had normal urinary systems. however, other isolated uropathogens in that group of patients were not significantly more resistant to aas as compared to patients with normal urinary systems. in contrast to our results, ladhani and grandsen (2003) found higher resistance rates of e. coli to ampicillin, amoxicillin / clavulonic acid, nitrofurantoin, and tmp / smx in the group of patients with underlying renal problems compared to patients with normal urinary systems. a study made by allen. (1999) proved that children with urinary abnormalities had a 2.4 times higher chance of acquiring a uti caused by e. coli that was resistant to tmp / smx (11). although we expected that some uropathogens from our study would disclose higher resistance rates to aas in patients who were repeatedly hospitalized for uti than those hospitalized only once, we confirmed those expectations in esbl - producing klebsiella sp. alone. our results contradicted allen s study, claiming that children with 2 or more hospital admissions were 4 times more likely to have a resistant isolate of e.coli than those who had no admissions in the previous year. moreover, we found that enterococcus was 1.7 times more resistant to tested aas in patients who were admitted to the hospital only once. our study also showed that resistance rates of e. coli to aas could be altered if patients had been receiving ltap for up to 4 weeks within the last 6 months prior to hospitalization. the resistance rates of e. coli to tmp / smx, ampicillin and amoxicillin / clavulonic acid were significantly higher in the group of children in this study who had received ltap compared to those who had not. this is in accordance with allen s study (1999) showing a 23 times greater chance of getting a uti caused by e. coli resistant to tmp / smx. lutter s study (2005) showed the same result in patients affected by all kinds of uropathogens. contrary to those results, resistance to nitrofurantoin in both groups from our study retained very low levels. unexpectedly, the resistance rate of e. coli to cephalexin in children who received ltap was even lower (4.9%) than in children who did not receive ltap (6.8%). some studies claimed that ltap with tmp / smx and ampicillin was ineffective in the prevention of uti and kidney damage, as well as its connection to the increased risk of utis caused by resistant uropathogens in some categories of patients. the use of low - dose prophylactic antibiotics to prevent recurrent uti and kidney damage has been the standard care for many children with urinary tract anomalies, especially vur. in the last few years, some studies have expressed serious doubts concerning the efficiency of ltap, but without providing convincing evidence against ltap so far. therefore, ltap is still recommended with customarily prescribed tmp / smx, nitrofurantoin, cephalexin, ampicillin, and amoxicillin [13,6 ]. in our study, approximately 40% of the children received tmp / smx for ltap for different causes, alone or with other aas. this percentage is lower in comparison to some other studies that used tmp / smx 61100% of cases. apart from tmp / smx, our patients received nitrofurantoin in 27.3% of cases and cephalosporins in 25% alone or alternating with another aa (table 1). in comparison to other studies, nitrofurantoin was used in only 018%, and cephalosporins in 03% of cases receiving ltap. in our study the incidence of e.coli was slightly less in patients with urinary tract anomalies (61.9%) (figure 2), as well as in patients with multiple hospitalizations (52.6%) (figure 3), as opposed to patients with normal urinary tracts and those who were hospitalized only once for uti. the incidence of enterococcus also decreased in patients with urinary tract anomalies. on the contrary, throughout the entire study period, the incidence of pseudomonas sp. was 3 times greater in children with urinary tract anomalies as well as in those with multiple hospitalizations. in addition, isolated klebsiella sp. were 2 times more frequent in the group of patients with multiple hospitalizations than in patients hospitalized only once (figure 3). (2005), performed during 19972001, the resistance rates of all isolates ranged from 48% to ampicillin, 17% to tmp / smx and 7% to nitrofurantoin. in taiwan, tseng. (2008) reported 77.4% resistance to ampicillin, 44.6% to tmp / smx, 27.2% to cephalotin, and 8.4% to nitrofurantoin, with growing resistance rates to ampicillin, tmp / smx, and cephalosporins in the first generation during the 14-year study period. in accordance with the above - mentioned studies, our results showed a similarly high resistance rate to ampicillin (4562%), and a growing resistance to tmp / smx (from 15% in 2000 to 35% in 2006). during the same period, the resistance of all uropathogens to nitrofurantoin remained low, while the resistance of e. coli to nitrofurantoin was even lower (figure 1). besides the relatively high resistance rate to ampicillin and tmp / smx, resistance rates of all isolates, except pseudomonas sp. and enterococcus sp., showed a decreasing trend in first - generation cephalosporins (cephalexin) and amoxicilin - clavulonic acid. these changes in resistance could be explained by the extensive consumption of antibiotics, especially azitromicyn and third - generation cephalosporins, which were over the previous years widely prescribed for various indications in our region locally as well as in the entire country [13 ]. regarding resistance of e. coli, our study showed that the resistance rate to ampicillin was continuously high (a mean of 69.5%), with the highest rate being in year 2001 (78.6%). during the same period of time, resistance rates to amoxicilin - clavulonic acid and cephalexin decreased, similar to the trends observed in all the other isolates. however, the increasing resistance rate of e. coli to tmp / smx was higher than the resistance to all other isolated uropathogens, first showing increasing trends until year 2004, but later showing a decrease in year 2007 (figure 1). this increasing resistance rate might be explained by the widespread prescription of tmp / smx for treatment of uti and providing ltap in croatia. increasing resistance rates to tmp / smx was not uncommon in other countries all over the world [13 ]. on the contrary, permanently low resistance rates to nitrofurantoin, with an average value of 0.4%, could be associated with the infrequent prescription of this drug. our results show a decreasing trend of resistance of e. coli to amoxicillin / clavulonic acid and cephalexin, but a low resistance to nitrofurantoin, in accordance with trends in other parts of croatia, as reported by the committee for monitoring bacterial resistance rates of the croatian academy of medical sciences. similar results of antibiotic resistance were reported in canada and brazil. permanently high resistance to antibiotics with increasing resistance rates of e. coli to ampicillin and tmp / smx were shown in studies from the uk, austria, spain and brasil [9,11,12,19 23 ]. because of that, some authors concluded that ampicillin, trimetoprim, and tmp / smx were insufficient for the monotherapy of uti, as recommended by wolff (2007). 2008) warned that the use of cephalosporins in ltap could increase the number of utis caused by esbl - producing bacteria or multidrug - resistant uropathogens other than e. coli, therefore they suggested keeping tmp / smx for use in ltap. in children with functional or anatomic urinary tract abnormalities, we expected to find more resistant pathogens, presuming that they probably had frequent exacerbations of uti and received long and frequent antibiotic treatment. (probability of 97% to be more resistant to aas), while enterococcus showed that patients had significantly more resistant strains of that uropathogen than if they had normal urinary systems. however, other isolated uropathogens in that group of patients were not significantly more resistant to aas as compared to patients with normal urinary systems. in contrast to our results, ladhani and grandsen (2003) found higher resistance rates of e. coli to ampicillin, amoxicillin / clavulonic acid, nitrofurantoin, and tmp / smx in the group of patients with underlying renal problems compared to patients with normal urinary systems. a study made by allen. (1999) proved that children with urinary abnormalities had a 2.4 times higher chance of acquiring a uti caused by e. coli that was resistant to tmp / smx (11). although we expected that some uropathogens from our study would disclose higher resistance rates to aas in patients who were repeatedly hospitalized for uti than those hospitalized only once, we confirmed those expectations in esbl - producing klebsiella sp. alone. our results contradicted allen s study, claiming that children with 2 or more hospital admissions were 4 times more likely to have a resistant isolate of e.coli than those who had no admissions in the previous year. moreover, we found that enterococcus was 1.7 times more resistant to tested aas in patients who were admitted to the hospital only once. our study also showed that resistance rates of e. coli to aas could be altered if patients had been receiving ltap for up to 4 weeks within the last 6 months prior to hospitalization. the resistance rates of e. coli to tmp / smx, ampicillin and amoxicillin / clavulonic acid were significantly higher in the group of children in this study who had received ltap compared to those who had not. this is in accordance with allen s study (1999) showing a 23 times greater chance of getting a uti caused by e. coli resistant to tmp / smx. lutter s study (2005) showed the same result in patients affected by all kinds of uropathogens. contrary to those results, resistance to nitrofurantoin in both groups from our study retained very low levels. unexpectedly, the resistance rate of e. coli to cephalexin in children who received ltap was even lower (4.9%) than in children who did not receive ltap (6.8%). some studies claimed that ltap with tmp / smx and ampicillin was ineffective in the prevention of uti and kidney damage, as well as its connection to the increased risk of utis caused by resistant uropathogens in some categories of patients. the use of low - dose prophylactic antibiotics to prevent recurrent uti and kidney damage has been the standard care for many children with urinary tract anomalies, especially vur. in the last few years, some studies have expressed serious doubts concerning the efficiency of ltap, but without providing convincing evidence against ltap so far. therefore, ltap is still recommended with customarily prescribed tmp / smx, nitrofurantoin, cephalexin, ampicillin, and amoxicillin [13,6 ]. in our study, approximately 40% of the children received tmp / smx for ltap for different causes, alone or with other aas. this percentage is lower in comparison to some other studies that used tmp / smx 61100% of cases. apart from tmp / smx, our patients received nitrofurantoin in 27.3% of cases and cephalosporins in 25% alone or alternating with another aa (table 1). in comparison to other studies, nitrofurantoin was used in only 018%, and cephalosporins in 03% of cases receiving ltap. we believe that, based on our own data regarding resistance patterns of uropathogens in our local community, we should influence primary care doctors by encouraging them to prescribe more effective drugs in order to achieve better effects of ltap. in cases where ltap is undoubtedly prescribed, it should be administered according to the resistance patterns on a local level. nevertheless, local studies on resistance patterns of uropathogens should be encouraged in the future. we believe that there is still room for conducting efficient ltap if we change aas according to the local results of resistant patterns, otherwise we are forced to follow the general recommendations for ltap, which are not always appropriate and effective in specific local communities. in that sense, our recommendation is to exclude ampicillin from further ltap and reconsider the use of tmp / smx. in addition, we recommend the use of amoxicillin / clavulonic acid and/or cephalexin in the first 3 months of life, and nitrofurantoin after the first year of life, whenever possible. the duration of ltap is another not yet resolved issue. in conclusion, we are strongly convinced that if doctors at the primary medical care level were encouraged to carry out our recommendations, results of ltap at the local level would greatly improve. | summarybackgroundwe assessed prevalence and resistance of uropathogens on antimicrobial agents (aa) from urine cultures (uc) in children hospitalized with urinary tract infections (uti) at university hospital in split.material/methodsduring the 7-year period, children hospitalized only once with uti alone were compared to those repeatedly hospitalized, and who received long - term antimicrobial prophylaxis (ltap), as well as those with associated anomalies of the urinary system (us).resultse. coli was the most frequent isolate (67.7%) with resistance to ampicillin by 69.5%, amoxicillin / clavulonic acid by 3.5%, cephalexin by 6.6%, trimethoprim / sulfamethoxazole (tmp - smx) by 27.5%, and nitrofurantoin by 0.4%. for other uropathogens, aa resistance rates were the following : 64.3%, 5.8%, 10.5%, 21.3%, and 7.9%. the high or increasing resistance to tmp - smx is characterized by all uropathogens. patients with anomalies of us showed a lower prevalence of e. coli and enterococcus sp., but a higher prevalence of pseudomonas sp., esbl - producing e. coli and klebsiella sp. than those without us anomalies. repeatedly hospitalized patients showed a lower prevalence of e. coli, but a higher prevalence of pseudomonas sp. and klebsiella sp. than patients hospitalized only once. both groups displayed significantly less resistance of enterococcus sp. in patients receiving ltap before hospitalization, e. coli was significantly more resistant to ampicillin, amoxicillin / clavulonic acid and tmp / smx than in those without ltap.conclusionsbased on our results, we recommend excluding ampicillin altogether, and reconsideration of further use of tmp - smx, as well as use of nitrofurantoin, cephalexin and amoxicillin / clavulonic acid for ltap in our region. |
unlike common long bones, mandible reconstruction presents many difficulties due to its unique shape and relation with dental occlusion and the temporomandibular joint (tmj). reconstruction plate or non - vascularized autogenous bone graft were used, but were prone to infection and radiation exposure, often resulting in exposure of metal plate and resorption of grafted bone [13 ]. however, there were still difficulties in handling soft tissue and lack of blood supply to grafted bone. with the development of microvascular surgery, free tissue transplantation (i.e., deep circumflex iliac artery [dcia ], fibular free flap [fff ]) transferred sufficient bone and soft tissue with localized blood flow, with greater success in patients previously treated with surgery or radiation. dcia flap offers large volume and quality of bone tissue for mandibular segmental defects, excellent esthetic shape for reestablishing mandibular continuity, and sufficient height for implant placement. dcia could be the first choice for reconstruction of segmental mandibular defects of intermediate length ; however, both mandibular resection and iliac crest osteotomy are dependent on the surgeon s experience, intraoperative judgment and technical speed. achieving satisfactory results for contouring the grafted bone and recovering the occlusion and tmj function has challenged the use of free tissue transfer. to overcome these problems, three - dimensional (3d) computed tomography (ct) and computer image processing software are being used for virtual surgical planning (vsp) with resection of lesion and reconstruction of defects, in conjunction with stereolithographic fabrication of surgical guides [710 ]. the majority of computer simulations are used to help restore the neomandible in mandibular reconstruction with fff. however, the technique is seldom applied in mandibular reconstruction with vascularized iliac crest flap. the literature is sparse on application of surgery planning and stereo - modeling with a dcia flap, and assessing the postoperative results. thus, a consistent protocol that approaches the challenging limitations encountered by either by young or experienced surgeons has not been established. herein, the authors suggest a practical protocol for mandible reconstruction based on literature review and our experiences in acquiring satisfactory results using vsp and stereo - lithographic surgical guides. we achieved satisfactory results with dcia flap with virtual surgery planning and stereolithographic surgical guides. three patients underwent resection of mandible and accurate mandibular reconstruction with dcia flap using computer simulation and stereomodeling between april 2013 to august 2013 at a single institution. these patients were three men with an average age of 50 years (range 3956 years). aesthetic contour, functional outcomes were assessed by dental occlusion, postoperative imaging, and clinical examination. planning commenced with preoperative high - resolution ct scans from both maxillofacial and pelvic area. the data was stored as digital imaging communication in medicine format and imported to an image processing software (mimics software ; materialise, leuven, belgium) for segmentation of ct images and 3d visualization and planning of lesion safe resection margins, segmental mandibulectomy and design of the reconstructed mandible (neomandible) within the iliac bone (fig. separately, the dcia were 3-dimensionally reconstructed, analyzed with mevislab (mevis research, bremen, germany) and used as surgical reference (fig. 2). computer - aided design was used to plan the mandibular resection and iliac bone cutting guides with the negative form of the mandibular inferior border (including the angle area) and the lateral aspect of the iliac crest (including the anterior superior iliac spine, asis) without considering fixation with screws. the resulting data was saved in stl format for stereolithographic fabrication of the cutting guides and neomandible (fig. 1c1f), which was used to preoperatively bend the reconstruction plate (fig. 3a) and assist the double plating technique for preservation of both occlusion and condyle position during surgery. after adequate mandibular exposure, the reconstruction plate was temporarily positioned in the native mandible to maintain the occlusion and condyles in position. as the mandibular resection guide was secured in place, osteotomies were performed following the cutting slots, replicating the virtually planned mandibular resection. the iliac crest was dissected through a lateral approach and a dcia flap was successfully harvested after securing the cutting guide to the lateral aspect of the iliac crest (fig. the iliac bone segment was fixed into the mandible defect while the neomandible shape and contour was confirmed. three patients underwent resection of mandible and accurate mandibular reconstruction with dcia flap using computer simulation and stereomodeling between april 2013 to august 2013 at a single institution. these patients were three men with an average age of 50 years (range 3956 years). aesthetic contour, functional outcomes were assessed by dental occlusion, postoperative imaging, and clinical examination. planning commenced with preoperative high - resolution ct scans from both maxillofacial and pelvic area. the data was stored as digital imaging communication in medicine format and imported to an image processing software (mimics software ; materialise, leuven, belgium) for segmentation of ct images and 3d visualization and planning of lesion safe resection margins, segmental mandibulectomy and design of the reconstructed mandible (neomandible) within the iliac bone (fig. 1). separately, the dcia were 3-dimensionally reconstructed, analyzed with mevislab (mevis research, bremen, germany) and used as surgical reference (fig. computer - aided design was used to plan the mandibular resection and iliac bone cutting guides with the negative form of the mandibular inferior border (including the angle area) and the lateral aspect of the iliac crest (including the anterior superior iliac spine, asis) without considering fixation with screws. the resulting data was saved in stl format for stereolithographic fabrication of the cutting guides and neomandible (fig. 1c1f), which was used to preoperatively bend the reconstruction plate (fig. 3a) and assist the double plating technique for preservation of both occlusion and condyle position during surgery., the reconstruction plate was temporarily positioned in the native mandible to maintain the occlusion and condyles in position. as the mandibular resection guide was secured in place, osteotomies were performed following the cutting slots, replicating the virtually planned mandibular resection. the iliac crest was dissected through a lateral approach and a dcia flap was successfully harvested after securing the cutting guide to the lateral aspect of the iliac crest (fig. 3). the iliac bone segment was fixed into the mandible defect while the neomandible shape and contour was confirmed. using mandibular resection and iliac cutting guides, the need for intraoperative measurement was eliminated, allowing exact duplication and positioning of the neomandible with minimal adjustments. postoperative 3d computed tomographic scans and orthopantography demonstrate faithful replication of preoperative vsp (fig. no patients had difficulty in deglutition and speech after surgery and all three are under continuing observation. osteotomy of dcia flap, introduced by taylor and watson in 1978, requires a long operative time to obtain the desired curvature of neomandible and increases the risk of injury to the vascular pedicle, especially for surgeons without much experience. mandibular reconstruction using virtual surgery planning and stereolithographic surgical guides is a recent introduction, especially in fibula free flaps [79,12 ], but, there are few reports with dcia flap. for example, although a virtual surgery planning is successfully implemented and a pre - bent reconstruction plate is prepared for the neomandible, to make the 3d shape of the neomandible using fibular or iliac bone flap is dependent on the surgeon s experience or the mirror image of the opposite side of the mandible without surgical guides, not considering individual characteristics. otherwise the reconstruction plate is pre - bent following the native mandible, rather than the neomandible, so it is difficult to achieve close contact between the reconstruction plate and bone flap. if the screws are tightened for fixation, segments are moving outside and away from each other. considering these problems, necessary components of success in exact mandibular reconstruction with minimal errors include accurate vsp, a mandibular resection guide, a donor site cutting guide, and precise fixation of the pre - bent reconstruction plate to neomandible. repeated simulation of the vsp helped to plan optimal osteotomy planes, and to decide which side of the iliac is used and location of vascular pedicle. the mandibular resection guide yields a reproducible adaptation and stable location without fixation by including the anterior and posterior resection plane and a curved part such as angle or symphysis area. the iliac cutting guide is also improved for adaptation and location by including the asis area. without screw fixation, we can achieve exact location of guides without compromising the blood supply of the donor bone marrow. in addition, stereolithographic model of neomandible and pre - bending of the reconstruction plate is done to preserve positions of resected mandible and bone flap during fixation. first, optimal selection of resection plane considering important anatomical structures and the safety margin of the lesion are possible by using virtual simulation surgery of the same size as the patient. second, preoperative contouring of the reconstruction plate to the planned neomandible model could shorten the operation time and flap ischemia time, improving the flap success rate. third, achieving plane - to - plane bone contact could be beneficial to bony union, and the functions of tmjs and the occlusion can be preserved. finally, using this technique helps with explanation to patients of problems and challenges of reconstruction. herein, the authors proposed a protocol for mandible reconstruction based on a case - virtual - surgical - plan classification suggested after satisfactory results acquired with dcia flap using vsp and stereolithographic surgical guides (fig. the protocol consisted in case categorization into four types of vsp, according to the reconstruction approach (immediate or delayed) and condyle preservation described by jewer.. the detailed procedures are chosen by the reconstruction methods such as fff, dcia flap. in type lc) and immediate reconstruction, vsp, mandibular resection guide, fibular or iliac cutting guide, and fixation using miniplate by double plating technique with a pre - bent reconstruction plate on the neomandibular model are needed. in type ii cases, not preserving condyle (jewer classification h) and immediate reconstruction, vsp, mandibular resection guide, fibular cutting guide, and fixation using a pre - bent reconstruction plate on the neomandibular model are needed. in type iii, delayed reconstruction, the condyle is preserved, and the same procedure as other types is performed except for the mandibular resection guide. if the occlusion is to be restored, virtual surgery planning with additional laser cast scan as being applied in orthognathic surgery is used. mandibular reconstruction with dcia flap by vsp and stereolithographic - guided osteotomy offers an easier, faster and more accurate way to obtain excellent functional and aesthetic results. high costs and the difficulty of prompt change of resection margins during malignant tumor surgery are shortcomings. the development of intraoperative model manufacturing for changes of resection plane and the more advanced physical properties of the surgical guides are necessary for better results after surgery. | purpose : the reconstruction of mandibular defects poses many difficulties due to the unique, complex shape of the mandible and the temporomandibular joints. with development of microvascular anastomosis, free tissue transplantation techniques, such as deep circumflex iliac artery (dcia) flap and fibular free flap (fff), were developed. the dcia offers good quality and quantity of bone tissue for mandibular segmental defect and implant for dental rehabilitation. virtual surgical planning (vsp) and stereolithography - guided osteotomy are currently successfully applied in three - dimensional mandibular reconstruction, but most use fff. there are only a few articles on reconstruction with the dcia that assess the postoperative results.methods:three patients admitted during a five month period (april of 2013 to august of 2013) underwent resection of mandible and dcia musculo - osseous reconstruction using a vsp and stereolithographic modeling and assessment of outcomes included technical accuracy, esthetic contour, and functional outcomes.results:this technique yielded iliac bone segment with excellent apposition and duplication of the preoperative plan. flap survival was 100 percent and all patients maintained preoperative occlusion and contour.conclusion:based on our experience, we offer considerations and logically consistent protocols by classification of mandibular defects, and demonstrate the benefits in vsp and stereolithographic modeling of mandibular reconstructive surgery with dcia flap. |
it is currently the mainstay immunotherapy of relapsing - remitting multiple sclerosis (ms). we present a case of an ms patient who developed nephrotic - range proteinuria following ifn- treatment over a timespan of more than 9 years. the histology was suggestive of membrano proliferative glomerulonephritis (mpgn), but the absence of immune - complex deposition suggested a non - immune ifn--related disease. this is a case report of a 37-year - old female who was referred to our renal outpatient clinic with the incidental finding of proteinuria determined by dipstick. she had been on long - term ifn- 44 g three times weekly for most of this time. she had a normal pregnancy 10 years previously (a year prior to her ms diagnosis). she denied haematuria, rash, arthralgia, weight loss, nose bleeds and haemoptysis. at the time of presentation in clinic, she had a urine albumin creatinine ratio (uacr) of 268 mmol / l, equivalent to a protein leak of about 2.5 g/24 h. her serum creatinine (cr) was 60 mol / l, albumin 28 g / l, haemoglobin 11.5 g / dl, adjusted calcium 2.3 mmol / l, c - reactive protein (crp) of 2 mg / l and normal complement levels (c3 0.98 g / l and c4 0.19 an angiotensin - converting enzyme (ace) inhibitor was introduced at this point (figure 1). subsequently, urine culture, anti - glomerular basement membrane antibodies, myeloma screen, hepatitis b and c serology and human immunodeficiency virus were all negative. the ultrasonography showed unequal size kidneys, the right kidney measuring 10 cm and the left 12.5 cm in length., we represent the timing of biochemical parameters : serum creatinine levels, serum albumin levels, albuminuria (expressed as uacr, urine albumin creatinine ratio) and serum inflammatory markers as c - reactive protein (crp). our patient was on long - term ifn- treatment since the diagnosis of multiple sclerosis (ms), 9 years prior to the clinic review. on july 2012, she developed nephroticrange proteinuria with slight deterioration in kidney function and a drop in the serum albumin levels but normal crp. at this point, an ace inhibitor was introduced and the renal biopsy date was arranged. based on the biopsy results, ifn- was switched to glatiramer acetate, with progressive improvement of the proteinuria even after stopping the ace inhibitor. above, we represent the timing of biochemical parameters : serum creatinine levels, serum albumin levels, albuminuria (expressed as uacr, urine albumin creatinine ratio) and serum inflammatory markers as c - reactive protein (crp). our patient was on long - term ifn- treatment since the diagnosis of multiple sclerosis (ms), 9 years prior to the clinic review. on july 2012, she developed nephroticrange proteinuria with slight deterioration in kidney function and a drop in the serum albumin levels but normal crp. at this point, an ace inhibitor was introduced and the renal biopsy date was arranged. based on the biopsy results, ifn- was switched to glatiramer acetate, with progressive improvement of the proteinuria even after stopping the ace inhibitor. the renal biopsy showed 11 glomeruli with a general increase in the cellularity predominantly in the mesangium (figure 2a). the glomerular capillary basement membrane showed foci of basement membrane irregularity and few foci of the electron micrographs confirmed focal mesangial cell interposition (figure 2d), with new basement membrane material deposited on the internal surface. woolly accumulations of slightly electron - dense material, but these did not have the typical morphology of immune complex - type electron deposit (figure 2c). there were mild mesangial cell interposition and basement membrane irregularities, with no more than a hint of glomerular hyper cellularity. the immunofluorescence showed a partly particulate deposition of predominantly c3 within the mesangium with a lesser deposition of igg, m and a. the renal biopsy report concluded that in the absence of immune - complex deposition (or linear dense deposits), these findings justified a search for other possible causes of low - grade endothelial damage, such as the use of interferon therapy. (d) by transmission electron microscopy, photograph showing interposition of mesangial cytoplasm. kidney biopsy specimen. (b) by methenamine silver stain, glomeruli showing reduplication of the glomerular basement. unfortunately, following the renal biopsy, she bled and required embolization. in view of the biopsy report, ifn- was switched to glatiramer acetate (copaxone) 20 mg daily with a resultant progressive improvement of the proteinuria (figure 1). we present the case report of a 37-year - old woman with nephrotic - range proteinuria secondary to mpgn - like glomerulonephritis without associated immune - complex deposition in the context of long - term ifn- therapy. the complete remission of the proteinuria after cessation of ifn- treatment strongly suggests an aetiological role for ifn- in the development of the glomerulonephritis. to our knowledge, this is the second case of mpgn associated with ifn- therapy but the negative immune screen, normal inflammatory markers and the absence of immune complex deposition would imply a different pathway than that previously suggested. its efficacy is thought to be related to its ability in restoring the impaired trafficking of inflammatory cells into the central nervous system and the modification of the pro - inflammatory / anti - inflammatory cytokine homeostasis. the endogenous production of type i ifns (ifn- and ifn-) has a central role in the mediation of antiviral immunity and in many types of kidney diseases. ifn treatment has been linked to different kinds of glomerulonephritis, including minimal change [3, 4 ], focal and segmental glomerulosclerosis (fsgs) [5, 6 ] and collapsing fsgs. glen. reported the largest cohort (n = 11) of collapsing fsgs that developed in patients during treatment with different kinds of ifn. in this patient cohort with collapsing fsgs, three patients were on ifn- for ms. the median and mean duration of ifn therapy at the time of renal biopsy was 4.0 and 12.6 months, respectively, in this cohort. typically, features of mpgn on light microscopy include mesangial hyper cellularity, endocapillary proliferation and capillary wall remodelling with the formation of double contours. recently, it has been divided on the basis of pathophysiology, i.e. based on immune complex - mediation and complement mediation. mpgn without immune - complexes or complement deposition has been described in thrombotic micro - angiopathies, thrombotic thrombocytopenic purpura or haemolytic uraemic syndrome, atypical haemolytic uraemic syndrome associated with complement abnormalities, the anti - phospholipid antibody syndrome, drug - induced thrombotic micro - angiopathies, nephropathy associated with bone marrow transplantation, radiation nephritis, malignant hypertension and connective - tissue disorders resulting from injury to the endothelial cells. recently published a case report of a 40-year - old woman with ms on treatment with ifn- who developed nephrotic syndrome. the kidney biopsy showed tubulo reticular inclusions and positive results for all five major immunofluorescence stains (iga, igg, igm, c1q and c3). on the basis of these biopsy findings, together with other reports of sle induced by ifn [1214 ], suggested that ifn- has the potential to induce and maintain immune complex mediated mpgn. this is in spite of normal complement levels and absence of serological or clinical findings of sle. the pathway of how ifn- therapy triggered an mpgn - like glomerulonephritis without immune complex deposits is uncertain. viral infections activate systemic antiviral immune responses interfering with systemic autologous ifn production and contributing to the triggering of glomerular diseases. it has been reported that, ifn-4, but not ifn-5 or ifn-, was increasingly expressed by intrinsic renal cells during autologous nephrotoxic serum nephritis and that the amount of renal ifn-4 expression correlated with proteinuria and renal excretory function. adenovirus has been hypothesized as the trigger to make this ifn-4 over - expression functionally relevant. although these described glomerulonephritis had immune complex deposits, the hypothesis of a viral infection triggering nephrotic syndrome could not explain its onset after several years of treatment (8 years in our patient, mean 12.6 months in glen. it has been reported that ifn- significantly induced podocyte death and increased the permeability of podocyte monolayers and suppressed renal progenitor differentiation into mature podocytes. on the contrary, there are some reports of amelioration of glomerular injury by recombinant ifn- treatment on animal models [19, 20 ]. therefore, further larger controlled, randomized trials should clarify the effect of ifn- on proteinuria. on the other hand, the absence of immune - complex deposition in our patient 's renal biopsy may explain the complete remission of the proteinuria (in contrast with the partial remission in patients reported by wallbach. and glen.),, we report an ifn- related nephrotic - range proteinuria secondary to mpgn - like glomerulonephritis without immune complex deposits. it is therefore our opinion that the complete remission of proteinuria after stopping ifn- therapy, together with the negative immune screen, normal inflammatory markers and the absence of immune complex deposition, would justify consideration of a different pathway for the development of glomerulonephritis not previously reported. | we present a case report of a 37-year - old woman with multiple sclerosis (ms) who developed nephrotic - range proteinuria secondary to membrano proliferative glomerulonephritis (mpgn)-like disease with mesangial c3 deposition without evidence of immune - complex deposition in the context of long - term interferon- (ifn-) therapy. the complete remission of proteinuria following cessation of ifn-, strongly suggests causality. to our knowledge, this is the second case report of mpgn associated with ifn- use. this being the case, the negative immune screen, normal inflammatory markers and the absence of immune complex deposits would imply a different pathway to that previously suggested. |
kaposiform hemangioendothelioma (khe) is a rare neoplasm of vascular origin typically arising from the skin or soft tissues, and it is more often described in children. the tumor is composed of irregular lobules of small malformed vessels with sheets of spindle cells and infiltrating nodules. previously, khe has been compared to other vascular abnormalities such as juvenile hemangiomas, tufted angiomas, capillary hemangiomas, and kaposi sarcoma. tumor cells typically express endothelial markers, such as cd31 and cd34. in the largest cohort of patients with khe including 107 cases, the disease occurred in infancy in 93% of cases ; only 11% of patients presented with noncutaneous khe, the majority of them in bone, mediastinum, or retroperitoneum. khe can be associated with the development of kasabach - merritt phenomenon (kmp) in up to 71% of the cases. in this life - threatening syndrome, severe thrombocytopenia and hypofibrinogenemia occur as a result of intralesional platelet trapping. despite the locally aggressive behavior and the potential for lethal complications due to consumptive coagulopathy in the setting of kmp, metastatic spread of khe seldom occurs. here, we present the case of a patient with khe with several atypical features, namely onset in adulthood, primary presentation in the spleen, and metastatic spread to the liver and bones, with a partial response to treatment with paclitaxel. a 36-year - old caucasian male presented to the emergency department with acute onset of abdominal pain in the left upper quadrant in november 2012. abdominal tenderness in the left upper quadrant was noteworthy. a computed tomography (ct) scan revealed a 16 11 24 cm splenic mass with signs of splenic rupture. exploratory laparotomy was performed with an intraoperative finding of spontaneous splenic rupture and active subcapsular bleeding. a splenectomy was performed, and the patient was discharged without any additional complications. initial external pathology assessment of the mass indicated a nonspecific vascular proliferation, and the patient started follow - up with his primary care physician. surveillance ct scans performed in january 2013 revealed enlarged cervical and mediastinal lymph nodes, lytic bone lesions in vertebral bodies, a 1.7-cm peritoneal node, and a punctate lesion in the skullcap. short - interval imaging repeated in may 2013 detected additional vertebral lesions and multiple hepatic metastases. the initial recommendation included systemic treatment with gemcitabine and docetaxel, and treatment was started in july 2013, with significant hematological toxicity, leading to treatment discontinuation. a pathology review showed endothelial cells with lobular - pattern proliferation ; immunohistochemical staining revealed positivity for vimentin, cd34, and cd31 and negativity for hhv8, s-100, and smooth muscle actin. ki-67 the decision was made to withhold the systemic treatment and periodically repeat the scans every 3 months, since there was no clear evidence of progression. although the patient remained asymptomatic for 6 months, ct scans showed an increase in the number and volume of hepatic lesions (fig. after 18 weeks, restaging ct scans revealed stability of the vertebral lesions and reduction of the hepatic lesions (table 1 ; fig. 2b), consistent with a 40% reduction based on investigator - assessed recist criteria. paclitaxel was continued until october 2014 (total of 24 weeks), when restaging scans revealed disease progression. subsequent lines of treatment included oral prednisone, doxorubicin, interferon-, gemcitabine, and ifosfamide, with no radiologic response or clinical benefit. although the patient remained with mild to moderate symptoms and adequate performance status, the decision was made to offer best supportive care, and the patient was referred to a palliative care service. after 2 months, the patient had acute onset of thrombocytopenia, hypofibrinogenemia, petechiae, and epistaxis. one week after the presumptive diagnosis of kmp was made, the patient died due to a major episode of gastrointestinal bleeding. atypical presentations of khe have been reported in the bones, breast, thymus, thyroid, ductus choledochus, or retroperitoneum. khe from the spleen has been previously described in a single case by yu and yang, with long - term recurrence - free survival after splenectomy. the case reported here presents several atypical features, namely the age at onset and the pattern of distant spread. in addition, kmp was described in 70% of the cases in the largest published cohort of patients with khe, usually in patients with muscle or fascia involvement, retroperitoneal disease, or mediastinal involvement. described multilevel spinal involvement in a 6-year - old girl with khe that responded to systemic therapy. due to the rarity of this condition, the optimal management of patients with khe remains unclear, and treatment options are supported by small series and single case reports. whenever feasible, complete surgical resection should be recommended. however, complete resection is not always possible due to the infiltrative and locally aggressive nature of khe. medical therapies in the setting of unresectable / metastatic khe aim to decrease the tumor size, revert kmp, and palliate symptoms. corticosteroids are widely used. a consensus meeting suggested oral prednisolone 2 mg / kg / day as the first - line therapy for infant cases with enlarging, unresectable masses requiring intervention without kmp. additional responses were seen with vincristine, interferon-, actinomycin d, cyclophosphamide, and sirolimus. based on the lack of prospective data addressing the optimal treatment for khe, extrapolating the experience with the treatment of other vascular tumors was a strategy for the present case. paclitaxel was recommended in the present case and resulted in partial response after 3 cycles. this semisynthetic taxane showed clinical benefit in a phase ii trial in patients with angiosarcoma. interestingly, very low concentrations of paclitaxel proved to inhibit endothelial cell proliferation in an in vitro environment. had previously described the first evidence of paclitaxel - based chemotherapy in a case of metastatic khe. to the best of our knowledge, our report is the second khe case in the literature documenting an objective response after treatment with paclitaxel. in a retrospective analysis from the memorial sloan kettering cancer center, first - line doxorubicin, liposomal doxorubicin, or paclitaxel produced response rates of 2531%, with equivalent overall survivals in patients with angiosarcoma. molecular therapies may also benefit patients with vascular sarcomas. despite disappointing results in a phase ii study with nonselected patients with sarcomas, sorafenib led to clinically relevant responses in radiation - associated breast angiosarcomas with myc and flt4 amplifications. of note, tyrosine kinase or mtor inhibitors or antiangiogenic agents were not offered to the present patient since these molecules are not available for routine use in our institution for this indication. in summary, this case illustrates an unusual presentation of khe with metastatic spread to the liver and bones, in which the patient experienced a partial response to paclitaxel - based chemotherapy. due to the lack of prospective data, we consider reports such as ours of importance for improving the knowledge about this rare and challenging condition. written informed consent was obtained from the patient for publication of this case report and accompanying images. | backgroundkaposiform hemangioendothelioma (khe) is a rare neoplasm of vascular origin that typically arises from the skin or soft tissues as a solitary tumor. the optimal therapy for this disease is still unknown. we report the case of an adult patient presenting with metastatic khe of the spleen, who had a partial response after treatment with paclitaxel.case presentationa 36-year - old man presented in november 2012 with a nontraumatic rupture of the spleen. a splenectomy was performed, and the pathology was consistent with a nonspecific vascular proliferation. follow - up scans revealed lytic bone lesions and liver metastasis. a biopsy of the liver was performed and confirmed khe. the decision was made to proceed with treatment with gemcitabine and docetaxel, which was discontinued due to myelotoxicity. the patient was then transferred to our institution, and a pathology review supported the diagnosis of metastatic khe. his disease remained stable until february 2014, when he developed progression in the liver. chemotherapy was restarted with paclitaxel, and a partial response was documented after 3 cycles. unfortunately, disease progression occurred after 24 weeks, and subsequent treatments included prednisone, doxorubicin, interferon-, gemcitabine, and ifosfamide, without any response. the patient developed kasabach - merritt phenomenon and passed away 1 week later due to a major gastrointestinal bleeding.conclusionsthis case report suggests that paclitaxel could be considered as a treatment option for advanced khe, a rare condition for which no standard treatment exists. |
historically, research has focused on understanding the mechanisms that give rise to the unwanted choices expressed by chronic cocaine users (ccus) and treatment efforts have been aimed at stopping them altogether. as with many addictions, stimulant addiction is portrayed as the hijacking of natural valuation and learning signals in the brain, such as those in the dopamine - rich striatum. indeed, much scientific evidence supports the hypothesis that the neural systems that balance immediate desires with long - term gains [35 ] function suboptimally in ccus [68 ] and the very mechanisms evolved to protect are compromised in ways that drive addicts to choose cocaine. while this narrative organizes a wealth of experimental data, it also deemphasizes an important observation : not all decisions made by ccus are unhealthy choices for drugs. for example, it has been demonstrated that cocaine users decrease cocaine self - administration when offered merchandise vouchers as an alternative to cocaine, with levels decreasing even further for direct money vouchers. furthermore, when cocaine users are given the choice between a 10 mg unit dose of intranasal cocaine and increasing amounts of money, choices for cocaine decrease as the amount of money offered increases. the preference for money, as opposed to cocaine, in cocaine users has been referred to as a difficult but rational choice in the sense that individuals must forgo their abused drug for the opportunity to obtain other desired commodities. what if cocaine users are choosing money with the intent to obtain cocaine and/or other commodities later ? if so, then these individuals are planning for the future and executing an advantageous preference away from an available drug of abuse. hence, a tension exists between the observations that neural valuation and decision - making systems are compromised in ccus ; yet these individuals execute choice preferences away from cocaine in many domains of their life and for variable periods of time. this supports a complementary approach that seeks to understand the similarities and differences in the expression of sensible choice preferences of ccus and non - cocaine using control participants (controls). this will help inform therapeutic strategies that aim to increase the expression of healthier choices in ccus. as a first step, the current study uses single- and cross - commodity intertemporal choice tasks to examine the times when individuals choose money in the presence of the opportunity to choose cocaine. whereas single - commodity choice tasks (i.e., money now versus money later (mm) and cocaine now versus cocaine later (cc)) have been used extensively, cross - commodity tasks (i.e., money now versus cocaine later (mc) and cocaine now versus money later (cm)) have recently been introduced to examine the real - world trade - offs that ccus experience on a daily basis. in a simple depiction of the claim that reward - guided valuation and decision - making systems are compromised in ccus, choices in favor of money, as opposed to cocaine, are likely to involve neural responses in structures other than the striatum. hence, our working hypothesis was that choosing money instead of cocaine would not correlate with activity in the striatum but rather with other structures related to reward - guided choice furthermore, compensatory mechanisms would differentiate between the preference for money in ccus and that in controls and be observed as functional differences outside of the striatum. fifty right - handed participants were recruited from the virginia tech carilion research center (vtcri ; roanoke, va, usa) or the baylor college of medicine (bcm ; houston, tx, usa). twenty - five individuals were non - treatment seeking chronic cocaine users (ccus) who met dsm - iv criteria for cocaine dependence and 25 individuals were control participants (controls). individuals were recruited via local advertisements and word of mouth and provided informed consent in accordance with the institutional review board at virginia tech or baylor college of medicine. the first laboratory visit was a screening and consenting visit and the second was a scanning visit. at the beginning of each visit urinalysis tests urinalysis kits (quicktox drug screen dip card by branan medical corporation) tested for the use of cocaine (benzoylecgonine ; 300 ng / ml), amphetamine (500 ng / ml), opiates (300 ng / ml), benzodiazepines (300 ng / ml), and marijuana (delta-9-tetrahydrocannabinol ; 50 ng / ml). individuals who tested positive for any drug other than cocaine in individuals considered for the ccus group were excluded from participation. following initial urinalysis screening, individuals answered a series of questions related to demographic variables and were administered the structured clinical interview for the diagnostic and statistical manual of mental disorders (dsm - iv). individuals who presented with a history of substance abuse for substances other than cocaine or nicotine were excluded from the study. in addition, individuals were excluded if they presented with systemic diseases of the central nervous system, head trauma, neurological disorders, or axis - i psychiatric disorders (other than cocaine or nicotine dependence). specifically, individuals were asked to reflect on their cocaine use over the past 12 months and answer a series of 7 questions that addressed tolerance ; withdrawal ; taking more drug than expected ; failed quit attempts ; time spent sequestering or thinking about cocaine ; time spent away from family, hobbies, and friends ; and mental health problems, respectively. the questions read as follows : (1) have you found that you needed to use much more cocaine to get the same effect that you did when you first started taking it ? (2) when you reduced or stopped using cocaine, did you have withdrawal symptoms such as aches, shaking, fever, weakness, diarrhea, nausea, sweating, heart beat irregularities ? (3) have you found that when you used cocaine, you ended up taking more than you thought you would ? " (4) have you tried to reduce or stop taking cocaine but failed ? (5) on the days that you used cocaine, did you spend substantial time (> 2 hours), obtaining, using, or recovering from cocaine, or thinking about using it ? (6) did you spend less time working, enjoying hobbies, or being with family or friends because of your cocaine use ? (7) if cocaine caused you health or mental problems, did you still keep on using it ? whereas individuals who responded positively (e.g., yes) to at least 3 of the 7 questions met criteria for cocaine dependence, individuals who responded positively to 5 or more questions were considered to be addicted to cocaine. individuals who met criteria for cocaine addiction were included as participants in the ccus group. all individuals who passed screening criteria on the first laboratory visit were invited back to the laboratory for the scanning visit. participants in the ccus group were asked to abstain from using cocaine starting at midnight the night before the scheduled scanning visit. on the day of the scanning visit, participants arrived at the laboratory approximately two hours before the fmri scanning session. upon arrival individuals were administered a second urinalysis test. since it is feasible that a positive screen for cocaine could be obtained from individuals remaining abstinent since the previous midnight (e.g., the cocaine metabolite benzoylecgonine can typically be detected in urine up to 3 - 4 days following use in chronic users), participants in the ccus group who tested positive for cocaine use (100% of participants) were asked to provide the exact time since last use. they were also visually monitored for overt symptoms of acute cocaine intoxication (e.g., anxiety and agitation, excessive and/or uncontrolled motor movements, jaw clinching and/or mouth gnawing, erratic behavior, dilated pupils, etc.). participants in the ccus group who reported using cocaine after the previous midnight and/or were judged to be acutely intoxicated by a trained research assistant were excluded from participation (0% of participants). participants in the controls group were required to test negative for all drugs screened by urinalysis. the behavioral task parameters used for the current study have been described in detail elsewhere. participants performed two single - commodity and two cross - commodity discounting tasks in which they chose between hypothetical amounts of money or cocaine available immediately or after some delay (figure 1(a)). single - commodity tasks were tasks where participants chose between money available now or money in the future (mm) and cocaine available now or cocaine in the future (cc). cross - commodity tasks were tasks where participants chose between money available now or cocaine in the future (mc) and cocaine available now or money in the future (cm). prior to the task participants gave an estimate of the number of grams of cocaine that they equated with $ 1000. controls were asked to treat cocaine as a commodity that they had experience with. prior to performing behavioral tasks in the fmri scanner, participants completed two similar single - commodity and two similar cross - commodity adjusting amount discounting tasks outside of the scanner. indifference points, representing the amounts at which individuals are equally likely to choose a presently available commodity versus a future commodity, were calculated from the tasks performed outside the scanner and used to parameterize the scanner tasks. this was done to limit the sampling space while participants were in the scanner, maximizing the likelihood that indifference points could be calculated from data obtained from the scanner task., participants chose between immediate and delayed amounts six times for each of seven delays (1 day, 1 week, 1 month, 6 months, 1 year, 5 years, and 25 years). the sixth choice for each delay was used as the estimated indifference point, or the value at which the participant would be indifferent between immediate and delayed options. specifically, the initial choice at each delay was between the full large delayed amount and 50% of that amount available immediately. when participants chose one of the two options offered, the immediate amount offered in the next trial was first adjusted by 50% of the current offer, with adjustments on subsequent trials being half of the previous adjustment. if the participant chose the immediate amount, the immediate amount decreased for the next trial ; if the participant chose the delayed amount, the immediate offer increased. the indifference values obtained from 1 week, 1 month, 6 months, and 1 year were used to parameterize the offers given in the scanner tasks at 1 week, 4 weeks, 26 weeks, and 52 weeks, respectively. an example trial from the modified behavioral task performed inside the fmri scanner can be seen in figure 1(b). a trial consisted of a viewing period lasting a maximum of 5 seconds. during this period, a question appeared on the top of the screen that asked, would you rather have ? and the immediate and future choice options appeared randomly on the left or right side of the screen below the question. participants selected their preferred option by pressing buttons on boxes positioned in their right and left hands. as soon as a participant selected an option a box appeared around the selected option for approximately 1 second followed by a fixation screen that jittered 1 second around and average presentation period of 5 seconds. at the end of the fixation period for each future time point, the amount of the immediate commodity was varied six times. images were acquired on siemens 3 t allegra scanners either at vtcri or at bcm. structural t1-weighted images were first acquired (0.5 0.5 1 mm) followed by functional images with a 2 s repetition time, 25 ms echo time, and 90 flip angle in 37 interleaved slices (3.4 3.4 4 mm). slices were hyperangulated approximately 30 from the anterior commissure - posterior commissure line in an effort to avoid orbital frontal washout. functional data were adjusted to correct slice timing, realigned, coregistered to t1 anatomical image, normalized to montreal neurological institute (mni) coordinates, resliced (4 4 4 mm), and smoothed using an 8 mm gaussian kernel. these steps were conducted using statistical parametric mapping (spm08, institute of neurology, london, uk). data were also high - pass - filtered at 128 s. comparisons of parametric (i.e., age, education, and monthly income) and categorical (i.e., gender and collection site) demographic variables were conducted with independent samples t - tests and chi - squared tests, respectively, with significant thresholds set to p 0.05. the average (sd) length of education in controls was 14 (6.7) years and did not differ from 14 (6.3) years in ccus, t(48) = 0.678, p > 0.05. the average (sd) number of cigarettes smoked by controls was 2.1 (3.6) per day and did not differ from 4.5 (4.2) cigarettes smoked by ccus, t(48) = 0.18, p > 0.05. the average (sd) monthly income of controls was $ 888.29 (619.26) and did not differ from $ 857.65 (782.37) in ccus, t(48) = 0.127, p > 0.05. the distribution of controls and ccus collected at the two experimental locations did not differ from expected values (bcm : controls = 14, ccus = 20 ; vtcri : controls = 11, ccus = 5), (1, n = 50) = 3.309, p > 0.05. the number of males and females in each experimental group significantly differed from expected values (controls : males = 13, females = 12, ccus : males = 20, females = 5), (1, n = 50) = 4.367, p < 0.05. within- and between - group analyses of both behavioral and brain imaging data, however, yielded no significant differences related to gender. participants in the ccus group reported an average (sd) of 14.58 (4.5) years of experience using cocaine and reported using cocaine for 24.14 (7) days out of the previous month. fourteen participants in the ccus group reported smoking crack cocaine as their primary method of delivery. all participants in the ccus group met criteria for cocaine addiction according to questions derived from the dsm - iv (see materials and methods). ten members of the controls group reported having previous experience with marijuana use, while twenty - five members of the ccus group reported having previous experience with marijuana use. all participants reported having previous experience with alcohol use. at the time of the study, however, all participants tested negative for illicit drug use other than cocaine in ccus. furthermore, participants did not reach criteria for substance dependence, with the exception of cocaine and nicotine dependence in ccus and nicotine dependence in controls. nicotine use (cigarettes smoked per day) was treated as a nuisance variable in all imaging analyses (see section 2). based on choice behaviors on the discounting tasks performed in the fmri scanner individuals were included in two analysis streams (figure 2). for the single - commodity tasks, this resulted in 92% of ccus (n = 23) and 88% of controls (n = 22) included in the mm groups. the cc groups consisted of 84% of ccus (n = 21) and 52% of controls (n = 13). in the cross - commodity tasks, 92% of ccus (n = 23) and 40% of controls the cm groups consisted of 76% of ccus (n = 19) and 24% of controls (n = 06). in the second analysis stream, all individuals (exclusive and nonexclusive responders) were included according to their choices for immediate and delayed commodities. the numbers of participants included in behavioral and corresponding imaging analyses of the second analysis stream can be observed in tables 1 and 2. in the first stream, average indifference magnitudes (sem) revealed the value each group placed on future commodities. these results are shown in figure 3. in the single - commodity tasks a main effect of group was observed when examining indifference magnitudes (f(1, 75) = 39.871, p < 0.001). post hoc analysis demonstrated that the indifference magnitudes (mean sem) in the mm task were significantly less in ccus (837 146), compared to controls (1896 152) (t(43) = 5.170, p < 0.001). similarly, indifference magnitudes in the cc task were significantly less in ccus (612 132), compared to controls (1921 232) (t(32) = 3.783, p = 0.001). within - group analysis of single - commodity tasks revealed that indifference magnitudes did not differ between mm and cc within controls (t(31) = 0.92, n.s). similarly, indifference magnitudes did not differ between mm and cc within ccus (t(31) = 0.92, n.s.). in the cross - commodity tasks main effects were observed for group (f(1, 54) = 5.98, p = 0.018) and task type (f(1, 54) = 20.18, p < 0.001). post hoc comparisons revealed that ccus (457 124) and controls (640 203) did not differ in indifference magnitudes during the mc task (t(31) = 0.791, n.s.). however, during the cm task, indifference magnitudes in ccus (942 129) were significantly less than controls (1860 281) (t(23) = 2.304, p < 0.031). within - group analysis of cross - commodity task types revealed that indifference magnitudes for controls were significantly greater in cm, compared to mc (t(14) = 3.59, p < 0.003). similarly, indifference magnitudes in ccus were significantly greater in cm, compared to mc (t(40) = 2.85, p = 0.007), suggesting that both controls and ccus value future money more than future cocaine. the group percentages (mean sem) for all immediate and delayed choices in each group are displayed in figure 4. during the mm task, the percentage of delayed money choices made by ccus (40 4.9) was significantly less than controls (58 4.7) (t(48) = 2.5, p = 0.016). no difference was observed between groups during the cc task (t(48) = 0.62, n.s.). during the cross - commodity mc task, ccus made significantly more delayed cocaine responses (35 4.3) than controls (17 5.3) (t(48) = 2.7, p = 0.01). during the cross - commodity cm task, ccus made significantly less delayed money responses (50 6.4) than controls (92 3.8) (t(40) = 5.5, p < 0.001). examining the proportion of choices within groups during the cross - commodity tasks revealed that behavioral choices followed the money commodity in both groups. this can be seen as the proportion of delayed responses between mc and cm significantly differed, in favor of the money option, in both controls (t(24) = 9.0, p <.001) and ccus (t(24) = 2.4, p = 0.03). general linear models targeted brain activity while submitting immediate (now) or delayed (later) choices and retrogradely isolated activity while viewing options that became now or later choices. very few differences between groups were observed in the single - commodity tasks (table 1). in the mm task, viewing choice options for what became money now choices resulted in greater precuneus activity in ccus, compared to controls. on the other hand, viewing what became money later choices resulted in greater inferior frontal gyrus and inferior temporal lobe activity in controls, compared to ccus. in the cc task, submitting cocaine now choices produced greater temporal lobe and postcentral gyral activity in controls, compared to ccus. cocaine later choices resulted in greater medial prefrontal activity in controls, compared to ccus. imaging results during cross - commodity tasks are presented in figure 5 and table 2. during cross - commodity tasks, signals in the striatum and left lateral prefrontal cortex differentiated ccus from controls. while viewing options for what became money now choices, ccus had significantly greater activity in bilateral putamen, globus pallidus, and the left caudate (figure 5(a)). activity in the left putamen persisted as ccus executed money now choices (figure 5(b)). groups did not differ while viewing options that became money later choices (figure 5(c)). however, spatially coincident striatal responses, as well as a response in the left dorsal lateral prefrontal cortex, emerged when ccus executed money later choices (figure 5(d)). we used single- and cross - commodity choice tasks and fmri to examine the neurofunctional events that distinguish non - cocaine choice preferences made by chronic cocaine users (ccus) from those of controls (controls). consistent with previous behavioral studies, we observed that ccus devalued the future more than controls, as measured by diminished indifference magnitudes in the single - commodity context. in the cross - commodity context, both ccus and controls executed several choices for money when cocaine was the available alternative. compared to controls, choices for immediate money as opposed to future cocaine were associated with greater activity in the dorsal striatum of ccus. in addition to greater dorsal striatal activity, choices for future money as opposed to immediate cocaine, were associated with greater left dorsal lateral prefrontal cortex (dlpfc) activity in ccus, compared to controls. our behavioral analyses of the single - commodity tasks (i.e., money now versus money later (mm) and cocaine now versus cocaine later (cc)) revealed that ccus devalued future money significantly more than controls. this was observed in the first analysis as significantly smaller indifference magnitudes during the mm task in ccus, compared to controls, and was confirmed in the second analysis where ccus chose future money 18% less often than controls (40% versus 58%). this is consistent with previous single - commodity studies demonstrating that, relative to non - cocaine using individuals, cocaine addicts devalue the future. in the cross - commodity context (i.e., money now versus cocaine later (mc) and cocaine now versus money later (cm)), we found further evidence that ccus devalue future money, relative to controls. ccus had significantly smaller indifference magnitudes during the cm task, compared to controls, and chose future money 42% less often than controls (50% versus 92%). perhaps not surprisingly, when given the choice between money and cocaine, the majority of choices in controls were for money (83% in mc and 92% in cm). interestingly, a large portion of responses in ccus was also for money (65% in mc and 50% in cm). this allowed direct comparisons to be made between groups for money choices in the cross - commodity context. also of note, in the cross - commodity context offering money in the future resulted in a 15% increase in future choices made by ccus. this is a clear demonstration that a desired commodity engenders the ability to shift choices in ccus away from their drug of abuse towards a nondrug alternative for the future and is consistent with previous studies demonstrating the ability of cocaine users to forgo cocaine for money [9, 10 ]. in addition to demonstrating that ccus retain the ability to shift choices away from cocaine, these behavioral results are interesting for another reason. using a single dataset, we present data with seemingly conflicting interpretations. on one hand, we show that ccus devalue the future, a finding so prevalent in various disease states that it is considered a transdisease process. on the other hand, we confirm previous behavioral results that ccus will forgo available drug when an alternative commodity is incentivized appropriately. this highlights the complexity in understanding real - world trade - offs in ccus and should motivate future studies to tease apart such complexities. in the current study, given that both controls and ccus executed choice preferences for money, as opposed to cocaine, we were able to probe the neurofunctional signals that differentiated ccus from controls. imaging analysis of the single - commodity choices revealed that during the mm task, inferior frontal and temporal lobe activity was greater in controls, compared to ccus, while viewing future money choices. on the other hand, ccus exhibited greater precuneus activity while viewing immediate money choices, relative to controls. during the cc task, controls displayed greater temporal lobe and postcentral gyrus activity while choosing immediate cocaine, compared to ccus, and greater medial prefrontal activity (mpfc) for cocaine later choices. these results suggest that controls place more value on future commodities, compared to ccus, regardless of the commodity type and this is associated with increased executive function - related activity in controls, as opposed to ccus. we found that when choosing money as opposed to cocaine, ccus relied on greater activity than controls in areas known to be involved in normal valuation and decision - making [4, 1720 ]. namely, activity in the caudate and putamen of the dorsal striatum was greater as ccus viewed and executed money choices. in addition to the role it plays in the expression of habitual behaviors, the midbrain dopamine system, including the dorsal striatum, has been studied extensively for its specific role in the picoeconomics and neuroeconomics of gambling disorders. furthermore, it is hypothesized that disruptions in the dorsal striatum underlie the cocaine seeking behaviors that accompany chronic cocaine exposure in both rodent [22, 23 ] and primate [24, 25 ] models of addiction. in addition, ccus displayed greater activity in the left dlpfc when choosing future money over immediate cocaine, compared to controls. the left dlpfc is well known for its executive functioning role in on - going decision - making, and our results are consistent with a recent meta - analysis demonstrating that this area is where temporal discounting processes related to the future and working memory processes related to the recent past overlap in the brain. therefore, it is feasible that greater activity in the left dlpfc of ccus is required when executing a difficult choice preference for a future commodity while forgoing immediate cocaine. these results are consistent with a model where ccus exhibit brain function above the levels of controls when executing a choice preference for money as opposed to cocaine. the results from the current study are novel and relevant in light of previous studies that have suggested inhibiting cue - induced striatal dopamine increases as a therapeutic strategy for cocaine addiction. this suggestion logically follows the observation that dopamine levels in the dorsal striatum are positively associated with drug craving and drug craving is known to contribute to relapse. this approach is also supported by data from experiments in primates demonstrating that the transition from acute to chronic cocaine self - administration results in a progressive shift in dopamine receptor expression and glucose utilization from the ventral to the dorsal striatum [24, 25 ]. the shift from acute cocaine effects in the ventral striatum, impairing goal - directed choice, to chronic cocaine effects in the dorsal striatum, impairing habitual choice, has been hypothesized to underlie many addiction - related dysfunctions in cocaine abusing humans (for review see). our results suggest, however, that simply inhibiting activity in the dorsal striatum, while being potentially beneficial for treating some of the negative aspects of addiction, may inadvertently compromise the ability of cocaine addicts to execute choice preferences away from available cocaine. the current data suggests that therapeutic approaches should consider the potential confound of compromising real - world relevant, cross - commodity choices. we contend that successful strategies will benefit from more detailed understanding of the brain activity associated with nondrug choices in ccus. activity in the left dlpfc was greater in ccus, compared to controls, while executing choices for future money as opposed to immediate cocaine. the lateral pfc is known to be active during decision - making and is particularly active when considering the costs and benefits of alternatives. recently, a transcranial magnetic stimulation (tms) study found that disrupting the left, but not the right, dlpfc increased choices of immediate rewards over delayed rewards, providing a causal link between activity in the left dlpfc and self - control mechanisms of future choice. our data are consistent with these results and the competing neurobehavioral decision systems hypothesis, which suggests that the evolutionarily young executive system (including the dlpfc) works in concert with the older limbic system for optimal decision - making [31, 32 ]. for example, hypofrontal activity is also associated with compromised executive abilities in various disease states, including schizophrenia and major depression. consistent with rodent studies demonstrating that repeated self - administration of cocaine decreases basal levels of pfc activity, methamphetamine - abusing populations have been observed to have prefrontal hypoactivity during future choice tasks, compared to controls. our data suggests that, compared to controls, increased dlpfc is needed in conjunction with increased dorsal striatal activity for ccus to execute choices for alternative future commodities when immediate cocaine is also an available commodity. our results suggest that the left dlpfc is a unique therapeutic target for shifting choices in ccus towards healthier alternatives for the future. although we relied on strict methodological standards for screening out current illicit drug use and dependence on substances other than cocaine (our key experimental variable) and nicotine (not different between groups and controlled for in imaging analyses), results may be influenced by extraneous variables not under our explicit experimental control (e.g., gender, age, education, cocaine use patterns, and poly drug, and nontarget drug use). while analysis of these variables, with the exception of gender, revealed no significant difference between groups, it is possible that these variables contributed in some way to the observed results. it should be noted, however, that dividing groups by gender reduced group sample sizes to numbers often deemed underpowered for neuroimaging studies. future studies will explicitly test gender differences associated with nondrug choices in chronic cocaine users. as such, they are less likely to evoke visceral responses understood to influence decision - making processes. it is worth noting, however, that fictive and real money gains and losses have been demonstrated to produce similar behaviors and activate similar brain networks during intertemporal choice tasks, including activity in the striatum and lateral prefrontal cortices. nonetheless, it is plausible that due to their inexperience with cocaine, controls need not exert self - control comparable to that of the ccus when choosing money over cocaine. it is a possibility that ccus, on the other hand, experience willpower or planning proficiency (even in the hypothetical case) not present in controls. to inform this possibility, and in search of additional neural signals associated with choosing money, we performed additional random - effects analyses in each experimental group comparing single- and cross - commodity tasks (i.e., mm versus cm and mm versus mc). this may reflect a lack of statistical power due to the current design and the limited choice behaviors exhibited by participants. finally, in the current study, addiction was operationalized as affirmative responses to five or more (out of seven) dsm - iv criteria used to establish cocaine dependence (which requires a minimum of three of seven affirmative responses ; see materials and methods). therefore, the definition of addiction in the current study is somewhat arbitrary and our results may be generalizable only to a subgroup of cocaine dependent individuals who meet a high number of dependence criteria as established by the dsm - iv. in light of these potential limitations, we encourage readers to treat these data as preliminary. notwithstanding, we hold that the significant results obtained from between - group comparisons of money choices in the cross - commodity context, when cocaine is the nonchosen commodity, are novel, valid, and informative. these data should be considered in future accounts of therapies aimed at shifting choice behaviors in chronic cocaine users. in conclusion, the cross - commodity context can be used as an experimental paradigm to isolate times when ccus forgo their drug of abuse for a preferred alternative. our hypothesis that money choices in ccus would not rely on brain activity in the striatum was rejected. updating this hypothesis, we propose that the difficult choice to forgo cocaine in ccus relies on hyperactivity in the dorsal striatum and dlpfc, known valuation and decision - making brain areas. specifically, hyperactivity in the dorsal striatum is needed for immediate and future choice preferences away from cocaine, and coincident hyperactivity in the left dlpfc is needed for future choice preferences away from cocaine. activity in these areas represents therapeutic targets for shifting choices in ccus away from cocaine options. | addiction is considered a disorder that drives individuals to choose drugs at the expense of healthier alternatives. however, chronic cocaine users (ccus) who meet addiction criteria retain the ability to choose money in the presence of the opportunity to choose cocaine. the neural mechanisms that differentiate ccus from non - cocaine using controls (controls) while executing these preferred choices remain unknown. thus, therapeutic strategies aimed at shifting preferences towards healthier alternatives remain somewhat uninformed. this study used bold neuroimaging to examine brain activity as fifty ccus and controls performed single- and cross - commodity intertemporal choice tasks for money and/or cocaine. behavioral analyses revealed preferences for each commodity type. imaging analyses revealed the brain activity that differentiated ccus from controls while choosing money over cocaine. we observed that ccus devalued future commodities more than controls. choices for money as opposed to cocaine correlated with greater activity in dorsal striatum of ccus, compared to controls. in addition, choices for future money as opposed to immediate cocaine engaged the left dorsolateral prefrontal cortex (dlpfc) of ccus more than controls. these data suggest that the ability of ccus to execute choices away from cocaine relies on activity in the dorsal striatum and left dlpfc. |
fast excitatory synaptic transmission in the mammalian central nervous system (cns) is mediated predominantly by ionotropic glutamate receptors of the ampa- and nmda - type. these receptors are composed of different subunits that determine the biophysical and trafficking properties of the channel. depending on the particular subunit composition of the ionotropic receptors both synaptic transmission and synaptic plasticity can vary considerably. the ampa receptors (ampars) are the workhorses of fast excitatory transmission in the cns. they are tetrameric receptor assemblies of glua14 subunits, which are differently expressed in the brain and are encoded by separate genes (hollmann and heinemann, 1994 ; kato., 2008 ; traynelis., 2010) the subunits that are predominantly expressed in the cns are the glua1 and glua2 subunits and the majority of receptors at the synapse are heteromers incorporating both. the glua2 subunit is critical in determining the properties of ampars ; the presence of an arginine (r) residue in its pore membrane domain, introduced by post - translational editing, renders the receptor impermeable to ca (ci - ampars) and therefore results in a linear current - voltage (i v) curve that reverses at 0 mv. conversely, when the glua2 subunit is absent, the receptors are ca - permeable (cp - ampars) and the i v curve becomes inwardly rectifying because the currents at positive potentials are inhibited by endogenous polyamines (donevan and rogawski, 1995 ; liu and zukin, 2007) and because the unitary current is larger. many reversible post - translational modifications of both glua1 and glua2 subunits regulate ampar expression and function ; among them subunit phosphorylation as well as palmitoylation and ubiquitination have been shown to modulate both biophysical properties and receptor trafficking (lu and roche, 2011). how this post - translational modification affects the trafficking of ampars in a subunit - specific manner, and how this determines synaptic plasticity, remains elusive. tight control of cp - ampar expression ensures that the majority of cells in the brain contain ci - ampars (shin., 2005). although the cp - ampars may mediate excitotoxicity associated with different neurological disorders (liu and zukin, 2007) it has been shown that these receptors also play an important role in synaptic plasticity (plant., 2006 ; adesnik and nicoll, 2007). moreover there are several synapses in which cp - ampars are developmentally expressed, in contrast to the adult where cp - ampars are mainly found on gabaegic interneurons (goldberg., 2003). because of their characteristics, the cp - ampars confer novel properties to the synapses where they are expressed and their specific role in synaptic function as well as synaptic plasticity is beginning to be elucidated. two other nmdar subunits exist : glun2 and glun3 (moriyoshi., 1991 ; traynelis., given that they each can be expressed in distinct subunit isoforms (e.g., glun2a/2b) their combination gives rise to multiple functionally distinct nmdars (paoletti, 2011). it has been shown that nmdar subunit composition is developmentally regulated ; at many synapses glun2b is the predominant isoform during the first 2 postnatal weeks (monyer., 1994). the glun2a isoform gradually increases after birth while the glun2b and glun2d expression progressively decrease. this developmental switch results in a change in the kinetics and subunit - specific modulation of nmdar currents. the glun3 family can be expressed as two isoforms (glun3a and glun3b) that arise from two different genes that have different expression profiles. glun3a is mainly expressed during early postnatal life and decreases in adulthood while glun3b expression is low at early stages and increases in adult animals (henson., 2010). there is accumulating evidence suggesting that nmdars are not a static component of the postsynaptic density but that their function and expression could be regulated by activity - dependent mechanisms (bellone and nicoll, 2007). further investigations are now necessary for understanding the subunit - specific control of receptor activity both during development and in neurological disorders. during brain development, the prenatal and postnatal periods are characterized by rapid changes in neuronal organization, thus providing a critical period during which environmental experiences can lead to long - term changes of both the brain and behavior. at the cellular level changes in synaptic morphology and receptor expression while the regulation of nmdar composition during development has been studied in detail in many brain regions, ampar subunit regulation has been less thoroughly investigated. compelling evidence has shown that glua2 content and consequently the expression of cp - ampars is developmentally regulated in principal neurons in different systems (pellegri., 1998 ; rohrbough and spitzer, 1999 ; lawrence and trussell, 2000 ; pickard., 2000). the principal neurons in the cortex have been extensively studied and it has been shown that the functional properties of ampars on layer 5 pyramidal neurons change with respect to the glua2 subunit during early postnatal development (kumar., 2002). v curve, ca permeability and blockade of the ampar - epsc by either intracellular or extracellular polyamines. it has been shown that the developmental switch of glua2 happens in layer 2/3 pyramidal cells as well as in layer 4 stellate cells. although the change in ampar subunit composition seems to be a general property of principal neocortical neurons, the timing of the switch is different among the layers (brill and huguenard, 2008). the switch occurs between p14p16 in layer 5 pyramidal neurons, but between p12p14 in layer 2/3 pyramidal cells and between p7p8 in layer 4 stellate cells. interestingly the timing of the switch corresponds to their positions within the canonical neocortical circuits rather that the ontogenetic age of the layer (crair and malenka, 1995 ; isaac., 1997). moreover the timing of the glua2 switch in layer 2/3 pyramidal neurons coincides with the critical period for the developmental plasticity in somatosensory cortex (stern., 2001). what still needs to be determined is the role of cp - ampars at developmental synapses. it has been suggested that in the neocortex the early low expression of glua2 subunits could underlie the increased seizure susceptibility of the immature brain (sensi., 1999). a similar developmental expression of cp - ampars has also been observed at mossy fiber - ca3 pyramidal synapses where they dominate the transmission up to p17. here it has been shown that the expression of cp - ampars is regulated by the pdz (postsynaptic density-95/disc large / zona occludens-1) domain - containing protein interacting with c kinase 1 (pick1) since pick1 knock - out mice have a linear i v curves and are insensitive to phylantotoxin 433, a specific glua2-lacking ampar blocker (ho., 2007). interestingly cp - ampars are removed by depolarization - induced long - term depression (diltd), a form of plasticity specifically observed only at young synapses that relies on postsynaptic calcium influx through l - type voltage gated calcium channels and release from intracellular stores (ho., 2007). what was still unknown is the physiological trigger for the developmental loss of cp - ampars. recently we have observed that at excitatory synapses onto dopaminergic (da) neurons in the ventral tegmental area (vta) the postnatal maturation of ampars and nmdars develops in parallel. indeed during the first week the transmission is dominated by cp - ampars and glun2b - containing nmdars. subsequently metabotropic glutamate receptor 1 (mglur1) drives the insertion of ci - ampars and glun2a - containing nmdars through a signaling cascade that involves phospholipase c (plc) (bellone., 2011), indicating that glutamatergic synaptic transmission in the vta relies on the activation of these metabotropic receptors. recently it has been proposed that in the hippocampus the activity - dependent glun2b to glun2a subunit switch during development is mediated by group - i mglurs (matta., 2011). taken together these data suggest a common role for mglurs in the postnatal development of excitatory synapses. future studies will have to address whether at the other synapses the mechanism implicated in the ampar switch during synaptic maturation is an active mechanism similar to the one in the vta or whether it is a passive one in which glua2 expression slowly increases. the switch in the expression of the glua2 subunit has important functional implications since the permeability of ca through the receptors changes during development. interestingly, imaging data in the vta have suggested that synaptic calcium entry was almost exclusively mediated by cp - ampars at neonatal synapses, despite the fact that substantial nmdar - epscs were observed in simultaneous whole - cell recordings (bellone., 2011). one explanation for these data is that the imaging and electrophysiological methods monitored distinct pools of synapses with contrasting properties. in fact, it has been shown that calcium - impermeable glun3a - containing receptors are preferentially expressed during a narrow temporal window in postnatal development at certain synapses (roberts., 2009). many developing synapses have been described as silent, where the transmission is mediated by nmdars, and become functional with the insertion of ampars during the first postnatal week (isaac, 2003). the presence of cp - ampars could represent another form of synaptic maturation and could have strong implications for synaptic transmission and plasticity. calcium influx in response to synaptic activity triggers many intracellular signaling cascades including activity - dependent forms of synaptic plasticity. in order for calcium to enter the neurons through nmdars in other words the activity requirements for synaptic calcium entry change radically once the cell switches from cp - ampars to nmdars. beside principal cells during development, cp - ampars have been found in naive brain mostly in interneurons and it has been shown that they participate in novel forms of long - lasting synaptic plasticity. parallel fiber to stellate cell synapses express glua2-lacking ampars and it has been shown that repetitive activation of these synapses produces a long - lasting switch to glua2-containing ampars. as this subunit is ca impermeable and shows slow channel kinetics, the switch in ampars could affect the stellate cell waveform shape and modify the efficacy of inhibitory synaptic transmission between stellate and purkinje cells (liu and cull - candy, 2000 ; liu and zukin, 2007). the presence of cp - ampars has also been shown in interneurons from the amygdala. at these synapses a novel form of long - term potentiation the induction of this form of plasticity was dependent on postsynaptic calcium and also, while not directly proven, has been suggested to depend on ca entry through cp - ampars (mahanty and sah, 1998). recently kullmann and colleagues described a novel form of plasticity at interneurons in the stratum oriens of hippocampus (lamsa., 2007). this form of ltp does n't follow the hebbian rules since it can be induced by a hyperpolarization of the postsynaptic terminal in concomitance with presynaptic glutamate release. cp - ampars play a major role in this type of plasticity as was demonstrated by showing that blocking these receptors during the induction protocol abolished the ltp. although many forms of learning are nmdar - dependent, certain types of memories and learning processes occur independently of these receptors, yet are clearly experience - dependent. supporting this hypothesis it has been suggested that glua2 deletion in ca1 selectively impairs nmdar - dependent learning and that the nmdar - independent learning requires the activation of cp - ampars (wiltgen., 2010). this finding strongly suggests that cp - ampars play a functional role in nmdar - independent learning. experience - dependent trafficking of both ampars and nmdars in the cns play an important role in many learning processes. it has been suggested that in adult brain the nmdar trafficking occurs at much slower rates compared to ampars. this finding was supported by the relative stability of nmdars at the synapses in contrast to the rapid ampar turnover (bredt and nicoll, 2003). moreover with standard ltp protocols little or no change in the nmdar - mediated synaptic transmission has been observed. in the last few years this idea has been challenged by many lines of evidence suggesting a change in nmdar function as a result of different behavioral experiences. it has been shown that while visual deprivation changes the ratio between glun2a / glun2b leading to an increase in the glun2b expression (he., 2006), sleep deprivation shifts the ratio and increases the glun2a subunit (kopp., 2006). although further studies are needed to investigate the mechanisms controlling the nmdar subunit composition and their role in synaptic plasticity, this dynamic regulation of nmdars in adulthood demonstrates that the adult brain is continuously modified by experiences. there is a wealth of evidence indicating ampar trafficking at glutamatergic synapses but only recently has it been shown that cp - ampars can be trafficked to the synapse after experience - dependent plasticity in different brain regions. one very elegant study has demonstrated that a single whisker experience protocol in vivo can induce a pathway - specific strengthening of neocortical excitatory synapses with a delivery of glu2a - lacking receptors at spared but not deprived inputs. it has been hypothesized that the presence of cp - ampars at layer 4 to layer 2/3 synapses may alter the rules for plasticity facilitating an nmdar - independent form of plasticity (clem and barth, 2006). in the hippocampus the insertion of cp - ampars following an ltp protocol is debated ; it has been observed that pairing - induced ltp resulted in a transient incorporation of synaptic cp - ampars (plant. 2008), whereas two other reports did not see this subunit recomposition using either pairing - induced ltp or hfs - ltp (adesnik and nicoll, 2007 ; gray., 2007). it has been suggested that the ability of hfs to induce ampar subunit reassembly may be dependent on the age of the rats (lu., 2007). what still is not clear is the importance of the transient expression of cp - ampars for the ltp and it has been suggested that these types of receptors, due to their ability to flux ca, may regulate the signaling cascade events important for the maintenance of the plasticity. taken together these findings suggest that experience - dependent insertion of cp - ampars could alter synaptic plasticity and engage different forms of learning that are independent of nmdars (tayler., 2011). in the mesocorticolimbic da system, which originates from the da neurons of vta that project onto the nucleus accumbens (nac) and prefrontal cortex (pfc), many common cellular adaptations have been described following drug exposure (nestler, 2005). in particular addictive drugs, following acute and chronic exposure, are able to leave a trace on transmission at many synapse of the mesocorticolimbic system, a phenomenon termed drug - evoked synaptic plasticity although many different forms of drug - evoked synaptic plasticity have been described in the pfc and nac, for the purpose of this review we will focus on the effects of addictive drugs at the excitatory afferents onto da neurons of the vta. a day after the exposure to a single dose of addictive substance it has been shown that at least two parameters of synaptic transmission are altered at excitatory afferents onto da neurons in the vta. first the ampa / nmda ratio is increased (ungless., 2001). second the current - voltage relationship of ampar - epscs is no longer linear, but is inwardly rectifying (bellone and lscher, 2006). after drug treatment the rectification index (ratio of the chord conductance at negative over positive potential) as well as electron microscopy experiments have suggested the presence of a fraction of ampars that lack the glu2a subunit (bellone and lscher, 2006 ; mameli., 2007). it is still not clear if auxiliary subunits such as the tarps could contribute to the rectification (soto., 2007) or which are the mechanisms that underlie the insertion of glua2-lacking ampars instead of glua2-containing. but, regardless of the exact molecular mechanism, the appearance of rectifying ampar - epscs after drug exposure suggested that in some synapses ampars are redistributed. how can the ampa / nmda ratio increase (particularly when both components are measured at + 40 mv) if the inserted ampars do not conduct current at positive potentials ? initial studies indeed suggested that the nmdar component remained unchanged (ungless., this was based on the observation that the bath application of nmda elicited currents of similar magnitude in rat brain slices regardless of whether they were exposed to cocaine or saline. this approach however has the disadvantage of activating extrasynaptic as well as synaptic receptors. using a two - photon laser to photolyse caged glutamate we compared epsc amplitudes in slices from saline- with cocaine - treated animals (mameli., 2011). this approach not only confirmed the existence of rectifying unitary ampar - epscs but also showed that unitary nmdar - epscs were significantly smaller after the drug treatment. while the molecular mechanisms underlying drug - evoked plasticity of nmdar - mediated transmission have yet to be investigated, preliminary data from our laboratory suggest that the overall reduction of the amplitude is indeed due to a subunit switch such that after the drug treatment the receptors are enhanced in glun2b content (unpublished data). in summary, at excitatory synapses onto da neurons, after a single injection of different addictive drugs, glua2-lacking (and maybe glun2b - containing nmdars) are driven into the synapses and contribute to the transmission up to 5 days after the exposure (bellone and lscher, 2006 ; mameli., 2009 ; brown., 2010) (figure 1). during the first postnatal week (neonatal), transmission is dominated by cp - ampars and glun2b - containing nmdars. subsequently (juvenile) mglur1 receptors play a role in the synaptic insertion of ci - ampars and glun2a - containing nmdar. at juvenile synapses, a single cocaine injection exchanges cp - ampars for ci ones and causes decrease in nmdar function changing the relative contribution of glun2a- and glun2b - containing nmdars. this switch in the glutamatergic transmission may contribute to the re - opening of a critical period seen during the development. we have previously described a form of synaptic plasticity induced by mglur1 that is responsible for reverting drug - evoked synaptic plasticity in the vta (bellone and lscher, 2006). after a single cocaine injection, both pharmacological and electrophysiological activation of mglur1 drive the insertion of glua2-containing and the removal of glua2-lacking ampars (mameli., 2007). interestingly a similar form of plasticity has been described in the nac where activation of mglur1 reversed the accumulation of glua2-lacking ampars after cue - induced cocaine craving (mccutcheon., 2011). moreover mglur1 mediated removal of glua2-lacking ampars is also involved in modulation of fear memory in the amygdala (clem and barth, 2006). an emerging hypothesis in the field is that in different brain regions, mglur1 receptors control the basal level of glua2-containing ampars and that any reduction of their function leads to an up - regulation of glua2-lacking ampars. we do n't know if cocaine induces a change in the number of mglur1 or whether the drug interferes with the intracellular signaling pathway. recently we have demonstrated that in utero cocaine exposure leads to a delay in the normal maturation of both ampars and nmdars (bellone., 2011). since the postnatal synaptic maturation of excitatory transmission onto da neurons of the vta relies on the activation of mglur1, a consequence of in utero cocaine exposure is that a substantial fraction of ampars in adolescent and young adult mice remains calcium permeable while the glun2b subunit still contributes to transmission during the first 2 months of life (figure 2). it will now be necessary to determine the consequences of such a delay in postnatal maturation of excitatory synapses onto da vta neurons for functional plasticity. mglur1 activation drives the postnatal maturation of glutamatergic synapses onto da neurons in the vta. in particular the activation of the metabotropic receptors drives the insertion of glun2a - containing nmdars and glua2-containing ampars from neonatal (p26) to juvenile synapses (p1426). the composition of glutamatergic synapses then remains unaltered from juvenile to adult age (p > 60). in utero cocaine exposure interfere with the mglur1-signaling pathway delaying the postnatal maturation of excitatory transmission at these synapses. over the last few years, the idea has emerged that changes in the excitatory transmission onto da vta neurons are permissive for further changes in the mesolimbic system. while a single injection of cocaine is sufficient to causes a rapid, but transient, potentiation of excitatory inputs in the vta, several injections are required to induce plasticity in the nac (mameli., 2009). moreover in this area, cp - ampars appear after prolonged withdrawal from cocaine and have been implicated in a behavioral model of cue - triggered relapse, termed the incubation of craving (conrad., 2008). although no apparent changes in the nmdar expression have been seen after prolonged withdrawal in the nac, it has been suggested that new glun2b - containing silent synapses are replace by glun2a - containing synapses after that period (ferrario., 2010). further studies are needed to better characterize the nmdar - dependent trafficking in the ventral striatum both during development and after addictive drugs. in conclusion, both in the vta and in the nac, mglur1 activation is sufficient to reverse the cocaine - induced plasticity with similar mechanisms (bellone and lscher, 2006 ; mameli., 2007 ; mccutcheon., 2011). therefore, we can speculate that recruitment of mglur1 could function as a protective mechanism to counteract drug exposure and those individuals with a deficient mglur1-dependent ltd mechanism may be particularly at risk of addiction. many questions remain unsolved. which are the mechanisms that enable the synaptic incorporation and retention of cp - ampars after a single cocaine injection in the vta and after prolonged withdrawal in the nac ? what is the significance of the insertion of cp - ampars and the concomitant decrease in nmdar function at these synapses ? previous findings in hippocampal interneurons expressing cp - ampars show that pairing of a hyperpolarizing current injection with synaptic release can enhance synaptic transmission (lamsa., 2007). in the vta, it has recently been shown that the cocaine exposure enables the induction of a similar form of anti - hebbian plasticity that also relies on calcium entrance from cp - ampars and is independent on nmdars. in contrast, in drug nave or saline - treated mice, where the transmission is mediated by ci - ampars, the ltp induction follows hebbian rules, i.e., ltp is induced by concomitant glutamate release and postsynaptic depolarization. it has therefore been suggested that the exchange of glua2-containing ampars for glu2a - lacking ones and a decrease in nmdar function is sufficient to invert the rules of plasticity induction (mameli., 2011). therefore, the drug - evoked plasticity in the vta is merely permissive for a specific form of activity - dependent synaptic plasticity. future studies will have to address whether anti - hebbian plasticity can occur in vivo, for example during a hyperpolarization of da neurons when upstream gaba neurons are strongly activated. it is important to underline that the drug - related adaptations here described may represent an aberrant form of plasticity that drives long - term adaptations in the downstream reward circuit. conversely, during early development this form of plasticity may be the default and enhance special forms of learning. it is conceivable that during development da neurons do not need to discriminate among the inputs they receive and it is only with the time that they learn to differentiate by strengthening certain connections. the cp - ampars and the plasticity they are associated with could be important for this type of learning. the capacity to discriminate among different stimuli is important in order to select the most valuable reward during the decision - making process. it has been well described in adult brain that da vta neurons respond to neutral, aversive and explicit non - reward predicting stimuli in a similar manner (schultz, 2011). we could speculate that during the development, since the brain has not been yet exposed to different stimuli, the da neurons are prone to generalization and discriminate poorly. subsequently with advanced development various stimuli will acquire a valence and therefore the da vta neurons will be activated only by salient stimuli. drug of abuse enhanced generalized dopamine activation, resulting in decreased reward discrimination. in this context after drug exposure, neutral or aversive stimuli would lead to stronger phasic striatal dopamine changes and would increase dopamine - dependent postsynaptic responsiveness and plasticity. when we give cocaine at an adult age, the drugs re - open a critical period previously seen during development and induce a switch in the ampar subunits composition with an insertion of cp - ampars. we can speculate that this switch could contribute to the re - programming of the da system resulting in reduced reward discrimination. while we are only beginning to understand the repercussions of drug - evoked receptor redistribution, the striking resemblance with the processes occurring during early postnatal development brings a new, exciting perspective that may help us to understand both normal development and the development of addiction. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | as in other parts of the central nervous system (cns) of the mouse, glutamatergic synapses onto dopamine (da) neurons in the ventral tegmental area (vta) mature postnatally. at birth many ampa receptors (ampars) lack glua2r subunit and most nmdars contain the glun2b subunit. within 2 weeks these receptors are replaced with glua2- and glun2a- containing ampars and nmdars, respectively. recent data suggest that a single injection of cocaine (or another drug of addiction) triggers glutamate receptor redistribution with the reappearance of the subunits typically present in immature synapses, as if addictive drugs reopen the developmental critical period. here we review the experimental evidence for this hypothesis and discuss the implications for circuit function. |
diabetes is a common chronic disease that is characterized by impaired insulin secretion and insulin resistance. psychiatric disorders frequently occur in type 2 dm patients and about 15% of the diabetic patients are susceptible to depression. in iranian traditional medicine (itm), the relationship between diabetes and depression is mentioned and special attention to their psychiatric problems is considered in the treatment of diabetes. this study is a descriptive review according to available iranian traditional medicine literature such as canon compared with modern medicine by using pubmed and scopus databases. in itm, diabetes is divided into warm and cold categories where the warm type is more common. emotions such as anger and grief can play an important role in creating the warm or cold diabetes, respectively. in modern medicine, several studies found that the odds of depression in the diabetic group were more than the non - diabetic comparison group. on the other hand, depression may have a role in the pathogenesis of dm with stimulation of the hypothalamic pituitary adrenal axis, which results in increased cortical level and blood glucose, eventually progressing to diabetes. evidence in modern medicine suggests that diabetes and depression can be a risk or exacerbating factor to each other. in itm, the theory of association between depression and diabetes is more highlighted than modern medicine, which is mentioned since more than 1000 years ago. it seems that emphasis on the treatment of depression in diabetic patients may have significant effects in the course of their disease. | background : diabetes is a common chronic disease that is characterized by impaired insulin secretion and insulin resistance. it is considered an urgent public - health issue because of its epidemic perspective. depression is a highly prevalent disease with a lifetime prevalence of 17%. individuals with depression experience reduced functioning and decreased quality of life. psychiatric disorders frequently occur in type 2 dm patients and about 15% of the diabetic patients are susceptible to depression. in iranian traditional medicine (itm), the relationship between diabetes and depression is mentioned and special attention to their psychiatric problems is considered in the treatment of diabetes.methods:this study is a descriptive review according to available iranian traditional medicine literature such as canon compared with modern medicine by using pubmed and scopus databases.results:in itm, diabetes is divided into warm and cold categories where the warm type is more common. emotions such as anger and grief can play an important role in creating the warm or cold diabetes, respectively.in modern medicine, several studies found that the odds of depression in the diabetic group were more than the non - diabetic comparison group. on the other hand, depressed mood was associated with an increase of developing type 2 diabetes. depression may have a role in the pathogenesis of dm with stimulation of the hypothalamic pituitary adrenal axis, which results in increased cortical level and blood glucose, eventually progressing to diabetes.conclusion:evidence in modern medicine suggests that diabetes and depression can be a risk or exacerbating factor to each other. in itm, the theory of association between depression and diabetes is more highlighted than modern medicine, which is mentioned since more than 1000 years ago. it seems that emphasis on the treatment of depression in diabetic patients may have significant effects in the course of their disease. |
hypothyroidism has various cardiovascular manifestations including impaired diastolic function, reduced contractility and infrequently pericardial effusion and heart failure (1 - 3). electrocardiographic (ecg) changes in hypothyroidism were bradycardia, rbbb, flat or inverted t wave, qrs prolongation, qt prolongation and infrequently ventricular arrhythmia, torsades de pointes (1 - 3). the qt dispersion is the interlead variability of qt interval on surface ecg that reflects regional variations in myocardial repolarization. increased the qt dispersion has been linked to the occurrence of malignant ventricular arrhythmias and sudden cardiac death (4 - 6). however, it was reported that the deficiency of thyroid hormone did not affect ecg findings of congenitally hypothyroid neonates (7). therefore, the effects of l - thyroxine treatment on ecg parameters, such as qt dispersion, in patients with primary hypothyroidism were examined. this retrospective study involved 18 patients (3 men, 15 women, age : 4818 yr) with primary hypothyroidism. the patient taking the qt - prolonging drugs or who had histories of cardiac disease were excluded. all patient were checked with serum free t4, thyroid - stimulating hormone (tsh) and standard 12-lead ecgs before and after l - thyroxine treatment. after a rest period, the electrocardiogram had been checked with 12-lead by electrocardiography recorder (25 mm / sec, marquette, fairfield, connecticut, u.s.a.). all measurements were obtained manually by 1 observer unaware of patient identity, diagnosis, and therapy. the qt interval was measured as the distance between onset of the qrs complex and the end of the t wave in all 12-lead. when the u wave interrupted, the end of the t wave was estimated by extrapolation of the descending t - wave lined down to the baseline. the qtd was defined as the difference between the maximum qt and the minimum qt across all 12 leads. the data were analyzed using a statistical package (spss window 10.0 ver). whenever the data could be paired, these were analyzed using paired t - tests. the thyroid function test, before and after l - thyroixine treatment, is shown in table 1. the qt prolongation was detected in 6 patients (33%) at the study baseline. the t - inversion appeared in 3 patients, and after the l - thyroxine treatment, all patients were normalized (fig. the qtc prolongation correlated positively with the baseline tsh level (r=0.484, p=0.042). it was found that after the l - thyroxine treatment, the qt interval (p<0.05), qtc interval (p<0.05), qt dispersion (p=0.008), and the qtc dispersion (p=0.005) significantly decreased. there were no significant changes in the pr interval, rr interval, nor the qrs duration (table 2). the thyroid function test, before and after l - thyroixine treatment, is shown in table 1. the qt prolongation was detected in 6 patients (33%) at the study baseline. the t - inversion appeared in 3 patients, and after the l - thyroxine treatment, all patients were normalized (fig. the qtc prolongation correlated positively with the baseline tsh level (r=0.484, p=0.042). it was found that after the l - thyroxine treatment, the qt interval (p<0.05), qtc interval (p<0.05), qt dispersion (p=0.008), and the qtc dispersion (p=0.005) significantly decreased. there were no significant changes in the pr interval, rr interval, nor the qrs duration (table 2). it is well documented that hypothyroidism affects cardiac functions and have various cardiovascular manifestations including impaired diastolic functions, reduced contractility and infrequently pericardial effusion and heart failure. hypothyroidism classically has been associated with bradycardia, but the degree of slowing heart rate is often modest. other electrocardiographic changes in hypothyroidism were rbbb, flat or inverted t waves, qrs prolongation, qt prolongation and infrequently ventricular arrhythmia, torsades de pointes (1 - 3). the function of the atrial pacemaker is normal and atrial ectopy is rare, but premature ventricular beats and occasionally ventricular tachycardia can occur (8). this is in contrasts to thyrotoxicosis, in which atrial tachyarrhythmias are common and ventricular arrhythmias are rare. the syndrome of torsade de pointes with a long qt interval and ventricular tachycardia can occur with hypothyroidism and be resolved with t4 treatment alone (3, 9). the duration of the action potential may be prolonged, perhaps reflecting a decrease in voltage - gated potassium channels. nathaniel. reported that significant prolongation of the qtc interval occurred in inadequately treated hypothyroidism and the degree of the qtc prolongation was directly related to the severity of hypothyroidism (2). showed that qt prologation and increased qtd were directly related to the tsh levels in hypothyroidism (10). increased qtd has been found to be associated with an increased incidence of malignant ventricular arrhythmias and sudden death (4 - 6). the qtd provides a potentially simple, inexpensive, noninvasive method of measuring underlying dispersion recovery of ventricular excitability, and should be defined in a way that most accurately reflects this state (11). clinical observations showed that ventricular arrhythmias and sudden death are uncommon in hypothyroidism, despite the marked lengthening of the qt interval (3, 12, 13). however, increased qtd in hypothyroidism may facilitate ventricular arrhythmias with hypokalemia, hypomagnesemia, long qt syndrome or drugs. abnormal changes of heart rate variability measurements and its improvement after treatment in hypothyroidism were reported (14). however, the deficiency of the thyroid hormone did not affect ecg findings of congenitally hypothyroid neonate (7). it was necessary to clarify the effects of l - thyroxine treatment on ecg parameters in primary hypothyroidism. in this study, our results showed that the qtc and qt dispersions improved after the l - thyroxine treatment in patients with primary hypothyroidism. this suggests that the thyroid hormone affects ventricular inhomogenicity, and subsequent l - thyroxine replacement therapy may reduce malignant ventricular arrhythmia and sudden cardiac death in primary hypothyroidism. | hypothyroidism has various cardiovascular manifestation and exhibits electrocardiographic change. the qt dispersion on surface ecg reflects regional variations in myocardial repolarization. the effect of l - thyroxine treatment on ecg parameters, such as qt dispersion, in patients with primary hypothyroidism were investigated. this study involved 18 patients (3 men, 15 women, ages : 4818 yr) with primary hypothyroidism. all patients were checked with a standard 12-lead ecg before and after l - thyroxine treatment. various ecg parameters were then measured twice. the mean l - thyroxine treatment duration was 222.7 months. the mean thyroid - stimulating hormone levels of patients before and after therapy were 40.229.8 u / ml, 3.64.6 u / ml (p<0.001) and free - t4 levels were 0.440.38 ng / dl, 1.510.39 ng / dl (p<0.001). after l - thyroxine treatment, qt interval (39542 vs. 38024 msec, p<0.05), qtc interval (43432 vs. 41723 msec, p<0.05), qt dispersion (4523 vs. 3013 msec, p=0.008), qtc dispersion (4923 vs. 3214 msec, p=0.005) significantly decreased. there were no significant changes in the pr and rr intervals, as well as the qrs duration. our findings suggest that the thyroid hormone affects ventricular inhomogenicity, and that l - thyroxine replacement therapy may reduce malignant ventricular arrhythmia and sudden cardiac death in primary hypothyroidism. |
coins are the most commonly ingested foreign bodies (fb), with button batteries, fish bone, marble, stone, and pieces of meat, etc., being other forms of ingested fb. in majority of cases, it is accidental in nature but can be occasionally homicidal, as was probably the case in our patient. we report the case of a female neonate who had a large button battery impacted in the upper one - third of esophagus. a 3.1 kg, 12-day - old newborn girl was referred to the otorhinolaryngology (ent) department with complaints of vomiting, poor feeding, drooling of saliva, and mild cough for the past 10 days. she suddenly developed complaints of vomiting, poor feeding, drooling of saliva, and later cough. she had been kept at home under the care of her grandmother, despite these ailments, for almost a week before medical consultation was sought. she had been shown to a local general practitioner who had treated her for 2 days and then referred her to the pediatric department in our hospital. the pediatrician sent her for an ent consultation in view of the suspicious circumstances associated with the history. on examination, patient 's pulse rate was 142/ min, blood pressure was 75/50 mmhg, and respiratory rate was 40/ min. she was mildly dehydrated, had no stridor but had reduced air entry in the basal region of chest. suspecting an fb, an x - ray neck, lateral and ap view, was done which revealed a rounded fb, 1 1 cm in size, impacted in the upper one - third of the esophagus at the level of t1-t2 [figure 1 ]. preoperative chest x - ray pa view with foreign body in situ anesthesia was induced with sevoflurane in oxygen and fentanyl 6 mcg was given intravenously. rocuronium 3 mg was given intravenously and the airway was secured with a 3 mm i d endotracheal tube. the fb was deeply impacted with intense surrounding edema. repeated attempts at retrieval proved unsuccessful. after about 3 h the procedure was abandoned and the patient was shifted to pediatric intensive care unit (picu). patient was put on synchronized intermittent mandatory ventilation (simv) with pressure support ventilation (psv). she was gradually weaned off and the trachea extubated after 6 h. the patient developed stridor with decrease in oxygen saturation and trachea was reintubated. she was kept on the ventilator, on psv mode, till she was taken up for a repeat surgical procedure. general anesthesia was induced with inhalation of sevoflurane in oxygen and fentanyl 6 mcg was given intravenously. intraoperatively, because of tracheal compression and vagal stimulation by the rigid esophagoscopy, patient had polymorphic electrocardiographic (ecg) and heart rate changes, such as sinus bradycardia and tachycardia, low voltage qrs complexes, st segment depression, and st elevation. other vital parameters including oxygen saturation remained stable. the esophageal mucosa was inflammed and edematous but surprisingly without any charring, blackening, or perforation. the patient was shifted to the picu for observation and 2 days later to the ward after an uneventful recovery. eighty percent of all fb esophagus occur in children, with a peak incidence in the age group of 6 months to 3 years. of the fbs that come to medical attention, 8090% passes through gastrointestinal tract without any difficulty, 1020% requires endoscopic removal, and only about 1% requires surgical intervention. the fb in our case was lodged at the level of upper one - third of the esophagus. x - ray neck, ap and lateral view, is most commonly done for diagnosis. if the incident is not witnessed and the ingested object is radiolucent, the diagnosis of fb ingestion can be very difficult. barium esophagoscopy, computed tomography scans of the neck, ultrasonography, and magnetic resonance imaging may be required for diagnosis. apart from eroding into the trachea, the object can erode into the aorta, leading to exsanguinations and death. fbs should be immediately removed on diagnosis, because they may rapidly cause direct tissue damage (blackening, charring, liquefaction necrosis, and esophageal perforation), by pressure and by chemical and electrical burns. surprisingly, in this case, the button battery had not caused much tissue damage, in spite of lying in situ for almost 10 days. occurrence of fb ingestion in neonates is rare with only a few reported cases in literature. it is seen in circumstances where it has been inserted into the mouth playfully by an elder sibling or homicidal attempts on an unwelcome female child. in the present case, the child was a third female child in a family wherein the only earning member was a poor rickshaw puller. the grandmother had probably put the battery in the child 's mouth and then kept her at home for so many days hoping that she would succumb to it. in such cases, must be maintained when the child presents to a medical facility with symptoms related either to the respiratory or gastrointestinal tract. respiratory distress is the most common manifestation of an fb in esophagus in neonates, and it can lead to misdiagnosis of a respiratory disorder. however, in this case, no history was forthcoming and there were no respiratory symptoms at presentation which is unusual. only high indexes of suspicion lead to the diagnosis. to conclude, we recommend that in case a female neonate, of lower socioeconomic status, presents with sudden onset of respiratory and gastrointestinal symptom a few days after birth, ingestion of a fb, constituting a form of child abuse or neglect with homicidal intentions should be suspected. with this in mind, an early diagnosis can be made and appropriate treatment instituted to avert serious morbidity and even mortality. | foreign body ingestion in neonatal period is an uncommon occurrence, despite foreign ingestion being common among pediatric age group. we report a rare case of foreign body esophagus in a 12-day - old female neonate causing obstructive symptoms after a homicidal attempt. the unusual age and circumstances involving the ingestion of the foreign body prompted us to report this case. |
subarachnoid hemorrhage (sah) cases represent approximately 7% of all types of stroke ], and have a notoriously high mortality rate. despite fluctuations, the overall incidence of sah has been stable at around 10 per 100,000 population per year in most countries for the past few decades. however, in one region of japan, the incidence of sah was reported to be as high as 23 - 25 per 100,000 people per year, one of the highest worldwide. it is possible that this extraordinary incidence might be the consequence of more assiduous diagnosis by early computed tomography (ct). mortality after sah is directly related to the severity of illness, and functional outcomes after sah vary widely. the age, sex, and grade - adjusted overall 30-d fatality rate for sah is about 45 - 50%. critical care requirements could differ depending on a patient 's presentation based on the initial hunt and hess scale, which can be graded by performing a careful clinical assessment of the patient 's neurologic status as follows : when sah is at a low grade (hunt and hess sah grade 1 - 2), perioperative monitoring and management in the intensive care unit (icu) is quite straightforward. it consists of hemodynamic and cerebral assessment at the bedside, routine cardiac monitoring, careful and repetitive clinical examination, and basic blood work. the risk of symptomatic vasospasm is low, and when it does develop, the neurological change is clinically obvious. nonetheless, a low grade of neurological injury may become acutely worse when it is complicated by acute vasospasm that induces symptomatic acute and/or delayed cerebral ischemia. a characteristic example is a patient who initially presents with an alert, oriented neurologic status (low grade hunt and hess status). however, the patient has a diffuse, thick subarachnoid blood collection consistent with classic fisher scale 3, which may induce sudden and rapid deterioration. most of the time, these patients do not require tracheal intubation but must be closely followed with frequent neurological examination by the icu nursing and physician staff. a high grade of sah is evidenced by hunt and hess grade 3 - 5, a large amount of subarachnoid and intraventricular hemorrhage, radiographic evidence of hydrocephalus, and deteriorating mental status. this is a considerably more challenging situation, and the fundamental goal is to maintain adequate cerebral perfusion pressure (cpp). the cpp is the difference between the mean arterial blood pressure (map) and the intracranial pressure (icp) : cpp = map - icp initially, these patients develop very labile intracranial hypertension, a harbinger of potentially impaired cpp. as always, the first step is to ensure adequate resuscitation and control of airway, breathing, and circulation (abc). next, an external ventricular drain (evd) should be placed to ensure continuous icp monitoring. then, to maintain adequate cpp and avoid further injury to the brain tissue, the map must be adjusted relative to the icp. unfortunately, there is little evidence to promote an ideal cpp, which is not only unpredictable, but which, because the disease is so dynamic, may fluctuate widely from day to day. in general, it is preferable to keep the map under tight control while maintaining a cpp of 50 to 70 mmhg. this mandates keeping the icp less than 20 mmhg, with map 70 - 90 mmhg and systolic blood pressure (sbp) preferably constrained to less than 140 mmhg. 1 outlines a reasonable step - by - step management plan for intracranial hypertension in patients with high - grade sah. a recently introduced paradigm shift toward aggressive protocolized management has demonstrated that mortality in patients with hunt and hess grade 4 and 5 sah can be significantly reduced compared with historical controls. the paradigm is initiated by emergent rescue of sah victims with abc support, followed by immediate admission to a highly specialized neurointensive care unit. the fundamental principles of management include early aggressive medical and surgical therapy and the use of advanced technologies to monitor the patient 's injured brain. recommended interventions include immediate placement of an external ventricular drain, timely securing of the ruptured aneurysm, prophylaxis against rebleeding, control of elevated icp, and optimization of cpp. in a sense, the key is to provide a physiologically optimal environment to promote recovery of the injured neurons. the paradigm also encompasses compassionate end - of - life care without premature withdrawal of life support. the most meaningful way to follow a brain - injured patient is to perform serial bedside neurological examinations. unfortunately, with high - grade sah, there is no examination to follow - the patient may remain completely comatose for days or weeks, and the only clinical finding of any portent may be that of intact brainstem reflexes. this frequently leads the clinicians to disclose a dismal prognosis and likelihood of extremely poor outcome to the family, which in turn understandably culminates in withdrawal of medical support.. suggest that clinicians who use initial data to predict outcome may be overly pessimistic, leading to possibly premature withdrawal of support and a " self - fulfilling prophecy ". traditionally, clinicians tend to react to their observations of patients in the icu. for example, if the map decreases, an assessment of intravascular volume status is initiated, quickly followed by a combination of fluid administration and vasopressor therapy with or without inotropic support. if arterial oxygen saturation (sao2) falls, an investigation of possible etiologies begins and a treatment algorithm follows based upon the findings. such " reactive " treatment strategies may be successful for some disease processes, but they do not achieve the best results in advanced - grade sah. the patient 's brain is severely injured, and there is an intense, active inflammatory reaction to the presence of blood in the subarachnoid space. the clinical examination of a stuporous or comatose patient typically remains unchanged for a protracted length of time. when meaningful changes finally occur, it may be too late to mount an effective therapeutic response. the paucity of meaningful information from clinical examination in patients with high - grade sah has driven the development of multi - modal monitoring and goal - directed therapy (table 1). in this approach, the daily neurological examination is augmented by continuous electroencephalography (eeg), real - time measurement of partial pressure of brain oxygen tension (pbto2), and cerebral microdialysis. the clinician utilizes input from multiple variables proactively to adjust patient hemodynamics and achieve the desired level of brain perfusion, which may vary day - to - day or even hour - to - hour. in this way, the entire icu team works toward a clearly defined, common goal. although there is not sufficient data to support the idea that any of these goal - directed therapies might lead to improved outcome, these targets are physiologically reasonable goals until further data is available. for example, the following monitoring goals could be set at any given time : keep icp 60 mmhg, and pbto2 > 10 mmhg. the primary mechanism to achieve these goals is continuous assessment and correction of cardiac output and performance, hemoglobin concentration, and intravascular volume status. successful achievement of these goals implies adequate pulmonary gas exchange, avoidance and prompt treatment of fluid overload (and risk of pneumonia), and optimal cerebral oxygen delivery. this in turn provides a physiologically optimal environment to the injured brain, giving it the best chance for ultimate recovery. intensivists devote a considerable portion of each day to attempting to determine the intravascular volume status of critically ill patients through the use of clinical examination and standard parameters that are often quite misleading. the mainstay of assessment, cvp, is a stagnant pressure measurement that is criticized for its unreliable value as a surrogate for circulating blood volume or preload responsiveness in the operating room or icu. it therefore seems logical to use more than one hemodynamic value to determine the appropriate fluid therapy. this rationale has provided the impetus for the introduction of semi - invasive, pulse contour methods of continuous hemodynamic monitoring, highlighted by doppler ultrasonography. for example, insonation of the inferior vena cava diameter and its collapsibility correlates quite well with other parameters and provides dynamic assessment of intravascular fluid status in critically ill patients. regardless of which variables one follows, it is of paramount importance to ensure a patient condition that is as close to euvolemia as possible. the severe decrease in cerebral blood flow that follows is detrimental to the injured brain, especially during the vasospastic period, and is associated with an unacceptably high risk of cerebral infarction. at the other extreme, volume overload (which includes prophylactic hypervolemic therapy even before a patient suffers any symptomatic vasospasm) may increase urine output and cardiac filing pressures, but may not provide any benefits in cerebral perfusion. when hypervolemia results in pulmonary congestion and edema, arterial hypoxemia further compromises oxygen delivery to the injured brain. the icu team should provide 24 - 7 coverage for sah patients, preferably in a dedicated neurointensive care unit, because these patients require continuous, round - the - clock neurological monitoring. at the first sign of symptomatic cerebral vasospasm current standards of care mandate that this should occur within 2 h. while there is considerable variability among clinicians in therapy for cerebral vasospasm, certain principles are accepted by all. first, it is mandatory to avoid volume depletion and systemic hypotension, which decrease cerebral perfusion and increase the risk of cerebral ischemia. at the onset of symptomatic vasospasm, it is essential to assure euvolemia and preferable to initiate hypertensive therapy. elevation in systemic blood pressure increases perfusion of the vasospastic, ischemic brain. while so - called " triple - h " therapy (hypervolemia, hypertension, and hemodilution) has never been shown to improve long - term outcome in any randomized controlled settings, it is reasonable to initiate interventions that assure euvolemia and mild to moderate systemic hypertension. there is no evidence to support prophylactic hypervolemic therapy or induction of hypertensive therapy prior to the onset of symptomatic vasospasm. a recent survey performed by the neurocritical care society (ncs) revealed that medical centers without a dedicated neurointensive care unit are more likely to use prophylactic hypervolemia. all 375 members of the ncs reported that they agree with the induction of hypertension for severe or symptomatic vasospasm. the options for endovascular and neurosurgical intervention in patients with symptomatic cerebral vasospasm are beyond the scope of this article and will not be discussed further here. acute sah is not uncommonly accompanied by an excess of sympathoadrenal activity that is described by a variety of terms, including paroxysmal sympathetic hyperactivity (psh), central nervous system storming, autonomic storming, and dysautonomic hyperactivity, among others. its exact pathophysiology has not been elucidated, and this syndrome is not confined to sah. it has been observed after a variety of other sources of acute brain injury including traumatic brain injury, intracranial hemorrhage, anoxic brain injury, and encephalitis. rarely, acute parasympathetic reactions have been reported. it is characterized by hypertension, sinus tachycardia, hyperventilation, facial flushing, hyperemia, diaphoresis, hypertonia, hyperreflexia, and fever. they have prolonged icu and hospital stays, increased medical complications, and poor long - term functional outcomes. a rational critical care approach includes prompt diagnosis, the ruling out of other underlying medical etiologies, and the initiation of appropriate medical therapy. a number of different agents have reported to be useful in psh patients, including benzodiazepines, bromocriptine, opioids, beta - blockers, baclofen, alpha-2 agonists, and antiepileptic drugs. it is intuitive that these patients would benefit from deep sedation, intubation, and mechanical ventilation with high - dose iv midazolam or propofol. however, outcomes appear to be enhanced by minimizing sedation so that patients can stay awake, remain extubated, and ultimately move out of the icu. maintaining the balance between adequate pharmacological treatment and avoidance of excessive sedation is challenging but crucial. a variable percentage of patients with high - grade sah may present with acute heart failure, with dyspnea, substernal chest pain, pulmonary congestion and edema, and systolic hypotension. electrocardiographic changes are quite common even without myocardial dysfunction, and include st elevation, inverted t waves, and prolonged qt interval. a wide variety of arrhythmias may occur, especially within the first 48 h. these include sinus bradycardia, supraventricular tachycardia, atrial fibrillation, ventricular ectopy and tachycardia, and in the presence of prolonged qt interval, polymorphic ventricular tachycardia (torsades de pointes). echocardiography reveals impaired myocardial contractility and a substantially decreased ejection fraction, often to less than 25%. this condition may be characterized as an acute neurogenic cardiomyopathy, akin to the stunned myocardium encountered after ischemia - reperfusion injury. a considerable proportion of patients, mostly postmenopausal women, develop a specific variant that causes ballooning of the ventricular apex with sparing of the basal segments. because of the characteristic shape of the ventricle, this syndrome is often called takotsubo cardiomyopathy, after the japanese for an octopus trap, or more formally, transient left ventricular apical ballooning syndrome. this phenomenon, however, has been described in all age groups and both sexes. care for these patients is largely supportive, and the primary goal is to minimize myocardial oxygen consumption and maximize myocardial perfusion. this includes prompt treatment of cardiac arrhythmias, sedation and mechanical ventilation, aspirin, nitrites, statins, beta - blockers, calcium channel blockers, and angiotensin - converting enzyme inhibitors. potent inotropic agents should be avoided if possible, because they increase myocardial oxygen demand. judicious use of a non - catecholamine alpha - adrenergic agent, phenylephrine, is preferred to treat severe systemic hypotension, but in severe cases, intra - aortic balloon counterpulsation may be required to assure adequate myocardial perfusion pressure and decreased myocardial demand. clearly, the use of pure vasopressors without any inotropic properties should also be avoided as this increases the afterload and could potentially make heart failure worse. as with any hypotensive case, the first step would be to assess the volume status rather than jumping to start vasopressors to elevate the blood pressure. typically, the syndrome is reversible after 3 - 5 d as long as no further injury occurs to the myocardium. the perioperative management of patients with sah is challenging, especially when the grade is high. typically, the initial medical battle is to deal with critical intracranial hypertension and cerebral edema (" icp crisis "). this is followed by what may be life - threatening cerebral vasospasm identified by angiography or ultrasonography, preferably before it becomes symptomatic. after a struggle with vasospasm, decisions loom regarding placement of a ventriculo - peritoneal shunt versus weaning the evd. if the patient survives the first 1 or 2 weeks, psh may occur at any time and without any warning. improving the long - term outcome after sah truly requires a well - trained, experienced icu team, preferably in a specialized neurointensive care unit, to provide optimal monitoring and timely and appropriate therapy. a multidisciplinary effort is needed to promptly diagnose and treat the multiple challenges of the dynamic disease that is sah. | despite significant regional and risk factor - related variations, the overall mortality rate in patients suffering from aneurysmal subarachnoid hemorrhage (sah) remains high. compared to ischemic stroke, which is typically irreversible, hemorrhagic stroke tends to carry a higher mortality, but patients who do survive have less disability. technologies to monitor and treat complications of sah have advanced considerably in recent years, but good long - term functional outcome still depends on prompt diagnosis, early aggressive management, and avoidance of premature withdrawal of support. endovascular procedures and open craniotomy to secure a ruptured aneurysm represent some of the numerous critical steps required to achieve the best possible result. in this review, we have attempted to provide a contemporary, evidence - based outline of the perioperative critical care management of patients with sah. this is a challenging and potentially fatal disease with a wide spectrum of severity and complications and an often protracted course. the dynamic nature of this illness, especially in its most severe forms, requires considerable flexibility in clinician management, especially given the panoply of available treatment modalities. judicious hemodynamic monitoring and adaptive therapy are essential to respond to the fluctuating nature of cerebral vasospasm and the varying oxygen demands of the injured brain that may readily induce acute or delayed cerebral ischemia. |
primary retroperitoneal mucinous cystadenocarcinoma is a rare tumor with only about 30 reported cases in the english literature. surgical exploration is needed for the diagnosis and treatment, and many authors recommend extensive excision including total hysterectomy and bilateral salpingo - oophorectomy with enucleation of retroperitoneal tumor1 - 9). adjuvant chemotherapy is sometimes administered following complete surgical excision4, 6, 7, 10, 11). however, the most desirable treatment for this rare tumor is still controversial. we report here on a patient who had tumor excision and adjuvant chemotherapy, and the patient is without evidence of recurrence 42 months after surgery. a 32-year - old married woman was referred to the surgical unit because of a self - palpable mass in her left lower abdomen for over 1 month and the abdominal pain for the recent several days. physical examination revealed a slightly tender, ill - defined mass about 10 cm in size over the left lower abdomen. the laboratory tests, including the complete blood count, the chemistry profile, urinalysis and chest x - ray, were all within normal limits. abdominopelvic computed tomography scanning revealed a huge unilocular cystic mass with an enhancing solid portion, and this was probably located in the retroperitoneal space (figure 1a, 1b). laparotomy revealed a grossly normal uterus, 2 ovaries, 2 fallopian tubes and an appendix. there were no abnormal findings in the liver, stomach, small bowel, large bowel and spleen. a large, encapsulated cystic mass about 10 cm in diameter was found in the retroperitoneum. the descending colon was displaced medially to the mass, and no ascites was found. the inner surface of the cyst was smooth except for three solid mural nodules that were 0.8, 1 and 1.5 cm in diameter, respectively. microscopically, the cyst was lined by atypical columnar cells of mucinous cystadenocarcinoma (figure 2a, 2b). the solid mural nodules showed highly pleomorphic spindle cells and anaplastic giant cells with numerous atypical mitosis, and there were intervening atypical glands of adenocarcinoma (figure 3a). the spindle and giant cells were positive for cytokeratin, vimentin and smooth muscle actin, but negative for s-100 protein and desmin (figure 3b, 3c). the carcinoma cells were positive for cytokeratin and they were negative for vimentin, smooth muscle actin, s-100 protein and desmin. the diagnosis of the mass was primary retroperitoneal mucinous cystadenocarcinoma with mural nodules of sarcomatoid change. the serum tumor markers were not checked before tumor excision. at 10 days after operation, the serum concentration of ca125 was elevated (76.95 iu / ml), which decreased to within normal limits 3 months later, and the serum concentration of cea was normal. adjuvant chemotherapy with six courses of cyclophosphamide 600 mg / m and cisplatin 60 mg / m was administered to the patient. there was no evidence of recurrence on the follow - up abdominopelvic computed tomography 42 months after the operation. the first case was described in 1977 by roth.12), and about 30 cases have been reported in the english medical literature to date. several hypotheses have been suggested to explain the histogenesis of primary retroperitoneal mucinous cystic neoplasm, and they are heterotopic or supernumerary ovary11, 12), retroperitoneal teratoma13), intestinal duplication and coelomic metaplasia. to date, the hypothesis that has gained increasing support is coelomic metaplasia, that is, retroperitoneal mucinous cystadenocarcinomas arise from invaginations of the peritoneal mesothelium, with subsequent mucinous metaplasia1, 3, 4, 5, 12, 14 - 16). the age at diagnosis ranges from 17 to 86 years and only 2 of the reported cases were male15). the patients usually visit the hospital with a complaint of abdominal pain or a palpable mass. radiologic tests including ultrasonography, computed tomography or magnetic resonance imaging are used to localize the tumor and evaluate its nature. the papillary nodules demonstrated within a cyst by ultrasonography or computed tomography have been reported to suggest malignancy16). however, it is often difficult to differentiate a benign from a malignant neoplasm, or even to determine the origin site of the tumor with using the preoperative radiologic images7). motoyama. suggested that the presence of glandular epithelial cells and high levels of cea in the cystic fluid was very useful for making the diagnosis15). the immunohistochemical findings of this neoplasm resemble those of its ovarian counterpart ; thus, a large number of authors recommend management according to the treatment protocol for ovarian neoplasms, including the staging procedure3, 7). although almost all the cases, except the one reported by uematsu16) were diagnosed in the early stage, there have been many relapses cases in the literature6, 10 - 14). roth. reported on a patient who had undergone only tumor excision and he died of spreading disease 6 months postoperatively12). two patients received tumor excision and adjuvant chemotherapy, but one patient had a paraovarian recurrence 21 months after surgery10) and another patient died of disease spread 4 months after surgery11). the patient with histologically borderline malignancy and who was reported on by banerjee underwent tumor excision, left salpingo - oophorectomy and descending colon resection, and mediastinal metastasis developed 4 years later14). mikami. noted a patient who experienced rupture of the tumor capsule during the operation with subsequent peritoneal tumor implantation, and the patient died 18 months after the initial surgery6). for the korean case reported on by song., the tumor was removed laparoscopically after cyst aspiration. five of the reported recurrent cases did n't undergo extensive surgical excision, including hysterectomy and bilateral salpingo - oophorectomy 10 - 14). hysterectomy and bilateral salpingo - oophorectomy were conducted after tumor excision in about half of the reported cases1 - 9), and these case were alive without evidence of recurrence 3 to 36 months postoperatively, except for one case6). so, extensive exploratory laparotomy, including removal of both ovaries and the uterus with extirpation of the retroperitoneal tumor, may improve the prognosis and prevent recurrence1, 2, 5, 11, 17). the resected genital organs in several reports showed no evidence of tumor involvement or infiltration1 - 5, 7 - 9). the only three of all the reported cases showed long - term survival (> 5 years) of 6 years, 5 years and 5 years, respectively, and these cases had not undergone hysterectomy and bilateral salpingo - oophorectomy16, 18, 19). law. recommended only tumor excision for young women who have no evidence of disease dissemination and who hope to stay fertile19). six cases, including the present case, have reported tumor that had one or more mural nodules6, 8, 9, 11, 12), and these nodules satisfied the diagnostic criteria presented by baergen. mikami. reported a patient with primary retroperitoneal mucinous cystadenocarcinoma with a mural nodule, and this nodule was composed of pleomorphic sarcomatoid cells with mitotic figures6). the authors commented that intraoperative rupture of the cyst in this case may have resulted in its peritoneal dissemination and recurrence. two patients with mural nodules died of disease at 4 months and 6 months postoperatively, respectively11, 12). cases with a mural nodule may have an aggressive prognosis like ovarian cancers with a mural nodule ; therefore, all mural nodules warrant careful histological assessment and careful postoperative follow - up6, 8). although our patient was diagnosed as having a neoplasm with mural nodules of sarcomatoid change, she is doing well without evidence of recurrence 42 months postoperatively. cystic tumor rupture during operation may occur, and malignant cells may exist in the spilled fluid ; this results in peritoneal dissemination. among the patients with cystic rupture4, 6), one patient died 18 months postoperatively6). in contrast, another patient reported by tenti. was free of tumor 33 months postoperatively, and that patients underwent adjuvant chemotherapy4). in the literatures, adjuvant chemotherapy was performed in 5 patients after tumor resection4, 6, 7, 10, 11). one of them underwent cytoxan chemotherapy for 21 months, but paraovarian recurrence developed10). 2 other patients died 4 months and 18 months after surgery, respectively, of widespread metastasis6, 11). the advantage of adjuvant chemotherapy has not been proved, and tumor might be hard to cure when it recurs, but patients may have benefit from chemotherapy if the cyst ruptures or if extracystic extension is present16). in the present case, tumor resection and adjuvant chemotherapy were performed. capsular rupture did not occur, but 3 mural nodules of sarcomatoid change were observed. the patient has no evidence of recurrence on follow - up computed tomography and according to the level of tumor marker. we recommend careful histologic examination of the excised specimen and if possible, aggressive management with extensive surgical excision and adjuvant chemotherapy to prevent recurrence, and consideration must be given to the patient 's age, the extent of disease and the histologic findings. primary retroperitoneal mucinous cystadenocarcinoma is a rare tumor and in all reported cases, the duration of follow - up was not more than 5 years. long - term follow - up is essential to evaluate the course and prognosis of this disease. | primary retroperitoneal mucinous cystadenocarcinoma is a rare tumor. only about 30 such cases have been reported in the worldwide literature, and a few korean cases have been reported. the pathogenesis is not clear, and coelomic metaplasia of the retroperitoneal mesothelium has gained wide support. there is no consensus on the appropriate treatment, but surgical exploration is needed for the diagnosis and treatment, and adjuvant chemotherapy may be recommended following complete surgical excision. the long - term prognosis has not been established.we report here on a 32-year - old woman who was diagnosed as having a retroperitoneal mucinous cystadenocarcinoma with mural nodules of sarcomatoid change. tumor excision and adjuvant chemotherapy were done and the patient is doing well without any evidence of recurrence at 42 months postoperatively. |
aortic regurgitation (ar) is characterized by the diastolic reflux of blood flow from the aorta to the left ventricle due to incomplete coaptation of the aortic valve. the overall prevalence of ar was 4.9% in the framingham heart study and 10% in the strong heart study. there are studies that have been performed to visualize the ar on the multi - detector computed tomography (mdct) scans [36 ]. in this study, we intend to mention a part of the aortic valve that becomes incompetent in aortic regurgitation and is visible on the mdct scans. normally, the aortic valve consists of three leaflets and three sinuses. the leaflets are mobile parts of the aortic valve whereas the sinuses are the cavities behind the leaflets. each leaflet is attached to the aortic wall and free margin of each leaflet coaptate with each other along the line of leaflet coaptation during diastole. during systole, the leaflets are open to allow blood flow across the aortic valve from the left ventricle to the aorta. during diastole, these leaflets close to hold the column of blood in the aorta while the left ventricle is being filled during diastole. normally, these valves are competent enough to withhold any blood flow from aorta to the left ventricle during diastole. but in aortic regurgitation, this mechanism fails and blood regurgitates across the aortic valve to the left ventricle. this is visualized as regurgitant jet on the echocardiography and as regurgitant orifice area (roa) on multidetector computed tomography (mdct) [37 ]. in normal tricuspid aortic valves, the leaflets coapt along the line of leaflet coaptation and become visible as hypodense area between aortic leaflets on mdct scans. the central part of this coaptation is due to coaptation of nodules of arantius present in middle of free margin of the aortic leaflets. central aortic valve coaptation area. normally, this area is competent and does not allow any regurgitation. in ar, incomplete coaptation of this area can be visualized on mdct scans as regurgitant orifice area (roa). in this study, we looked at this central area of aortic valve coaptation, performing its planimetric measurements and also determining its hounsfield units to find its tissue properties. retrospective analysis of mdct scans of 400 symptomatic consecutive patients whose scans were performed at our outpatient cardiac computed tomography scanner for the evaluation of coronary artery disease (los angeles biomedical research institute at harbor - ucla medical center) were analyzed. patients with poor image quality (columnation, streak artifacts, motion artifacts, and image noise), bicuspid aortic valves and heavy calcification of aortic valve leaflets that made the aortic valve region non - evaluable were excluded. sixteen mdct scans were excluded due to the above reason and 384 scans were included for evaluation of the aortic valve area. all mdct scans were performed with 64-detector row lightspeed vct scanner (general electric healthcare, milwaukee, wisconsin). individuals presenting with baseline heart rates > 65 beats per minute (bpm) were administered oral beta - blocker therapy as the preferred method for slowing down the heart rate. iv administration was allowed in the protocol, using intravenous metoprolol at 5 mg increments to a total possible dose of 40 mg in order to achieve a resting heart rate 65 beats per minute (bpm) were administered oral beta - blocker therapy as the preferred method for slowing down the heart rate. iv administration was allowed in the protocol, using intravenous metoprolol at 5 mg increments to a total possible dose of 40 mg in order to achieve a resting heart rate < 65 bpm. following a scout radiograph of the chest (anteroposterior and lateral), a timing bolus (using 10 cc of contrast) was performed to detect time to reach optimal contrast opacification in the axial image at a level immediately superior to the ostium of the left main artery. nitroglycerin 0.4 mg sublingual was administered immediately prior to contrast injection. during mdct image acquisition, 60 cc iodinated contrast was injected utilizing a triple - phase contrast protocol : 40 cc iodixanol, followed by 40 cc of a 50:50 mixture of iodixanol and saline, followed by a 50 cc saline flush. the scan parameters were 64 0.625 mm collimation, tube voltage 100120 kvp, and effective current 350780 ma. after scan completion, reconstruction of mdct scans was performed at 595% using 10% increment on retrospective scans and 7080% using 5% interval on prospective scans. for measurement purpose, this particular phase was utilized for measurement because this phase is available on both prospective and retrospective scans and it has been shown to be an optimum phase for coronary artery disease evaluation. central aortic valve coaptation area is identified as circular to triangular shape area present centrally in the aortic valve during diastolic phases. planimetric measurements of this area were performed on cross sectional images of the aortic valve (as shown in fig. 1). this central coaptation area is closed in normal persons, visualized as no contrast present in this area. in patients with aortic regurgitation, this area is incompetent and is visualized as regurgitant orifice area (roa) (as shown in fig. 2). planimetric measurement of the roa when observed was also performed in crosses sectional views of the aortic valve at 75% phase of the r - r interval (fig. 2figure showing regurgitant orifice area (roa) in patient with aortic regurgitation central aortic valve coaptation area as seen on mdct scan figure showing regurgitant orifice area (roa) in patient with aortic regurgitation continuous variables were measured using student t test and categorical variables using chi square test. the data was summarized as mean standard deviation or numbers (percentages). altman correlation was utilized for correlation between central aortic valve coaptation area and central roa. the data was analyzed using commercially available statistical software stata (version 10, college station, tx). mean area of the central aortic valve coaptation area on planimetric measurement was 5.34 5.19 mm. incomplete co - aptation of the aortic valve cusps as roa was noticed on 29 patients. we followed trans - thoracic echocardiography (tte) reports on these 29 patients and all of them were found to have aortic regurgitation of various severities on tte. mean central aortic valve coaptation area (mm) in patients with aortic regurgitation was 10.53 0.26 and in patients without aortic regurgitation were 4.90 0.17. there was correlation found between central aortic valve coaptation area and central roa using bland altman correlation which showed that with increase in roa area, the central aortic valve coaptation area also increases in size (r = 0.80, p = < 0.001) (fig. 3). the average aortic valve calcium score in the whole cohort was 8.49 64.45 au.table 1general characteristics of patient populationpopulation characteristics (no. (%) age (years)60.37 12.86males257 (67.3)hypertension108 (43.03%)diabetes mellitus48 (19.43%)hyperlipidemia88 (35.48%)family history of cad113 (49.78%)central aortic valve coaptation area (mm)5.34 5.19aortic valve calcium score (au)8.49 64.45hounsfield units of aortic pad area (hu)123.69 31.31regurgitant orifice area (roa) mm6.33 8.13fig. 3bland altman correlation between central aortic valve coaptation area and aortic regurgitation orifice area general characteristics of patient population bland altman correlation between central aortic valve coaptation area and aortic regurgitation orifice area figure 1 shows central aortic valve coaptation area on cross - sectional views of the aortic valve as an area present in between the cusps. figure 2 shows a patient with aortic regurgitation showing the regurgitant orifice area on cross - sectional view of the aortic valve. in this study, we looked at an area of the aortic valve present in the central space between the aortic cusps, central aortic valve coaptation area seen during diastole. this is visualized as a circular to triangular area present centrally in between the aortic cusps during diastole. this area is widely open during systole to allow normal flow of blood from the left ventricle to the aorta. during diastole, this area is normally closed and competent enough to prevent any regurgitation of blood flow from the aorta to the ventricle. in ar, we can observe incomplete coaptation of this area in case of central aortic regurgitation jet. eccentric aortic regurgitation jets can be visualized as present in between the aortic cusps on the sides. there was a strong correlation found between increased central aortic valve coaptation area and the presence of increasing roa due to ar. this area may not be well demarcated in patients with bicuspid aortic valves. a significant difference in the hounsfield units was found between the contrast enhanced roa (contrast usually 350500 hu) and the surrounding aortic valve coaptation area (123.69 31.31 hu). this observation will be helpful for further studies looking at the diagnostic accuracy of mdct for ar, especially among scanners using low dose protocols, obtaining images in diastole [8, 9 ]. this area may be difficult to be analyzed if there are motion artifacts, columnation artifacts, and heavy calcifications. the hounsfield measurement in this area may be helpful but it suffers from partial volume effect. it is critical to understand that the normal aortic valve cusps have a small but defined central area visible on current cardiac ct scanners that does not represent aortic regurgitation. it is the increase in this central area along with visible roa that represents aortic regurgitation. | as multiple new procedures now require better visualization of the aortic valve, we sought to better define the central aortic valve coaptation area seen during diastole on multi - detector row cardiac computed tomography (mdct). 64-mdct images of 384 symptomatic consecutive patients referred for coronary artery disease evaluation were included in the study. planimetric measurements of this area were performed on cross - sectional views of the aortic valve at 75% phase of the cardiac cycle. planimetric measurement of central regurgitation orifice area (roa) seen in patients with aortic regurgitation and hounsfield units of the central aortic valve coaptation area were performed. mean area of the central aortic valve coaptation area was 5.34 5.19 mm2 and hounsfield units in this area were 123.69 31.31 hu. the aortic valve coaptation area (mm2) measurement in patients without ar was : 4.90 0.17 and in patients with ar : 10.53 0.26 (p = < 0.05). on bland altman analysis a very good correlation between central aortic valve coaptation area and central roa was found (r = 0.80, p = < 0.001). central aortic valve coaptation area is a central area present at the coaptation of nodules of arantius of aortic cusps during diastole ; it is incompetent and increased in size in patients with aortic regurgitation. |
a significant change from conventional castings is the introduction of direct metal laser sintered (dmls) technology used for metal - ceramic restoration. various in vitro studies on dmls have yielded promising results for its wider clinical usage. yet studies regarding clinical longevity and the survival rates for posterior metal - ceramic fixed partial dentures done with dmls technique is lacking. hence, this study was undertaken to assess the clinical acceptability of posterior metal - ceramic fixed partial denture prosthesis made with dmls technique. forty - five patients with the mean age group of 40 years with missing maxillary or mandibular second premolar or first molar who reported to the department of prosthodontics, thai moogambigai dental college and hospital and were in need of three - unit fixed partial denture formed the study group. the criteria for case selection include the missing teeth that were removed due to irreversible pulpal reasons. only the vital abutment teeth were selected, and they were evaluated for proper positioning in the dental arch without any rotation, tipping, malalignment, and periodontal problems. it was also made sure that abutment teeth were opposed to natural dentition and had no supraeruption. radiographic evaluation was done to rule out any periodontal or periapical pathologies of the abutments. occlusal examination was done to rule out any para functional habits and temporomandibular joint ailments. the preparation design protocols were followed based on the study done by tara. the preparation design had an occlusal reduction of 1.5 mm, which was evaluated using wax check - bite and measured using wax calipers. the preparation had a circumferential chamfer finish line design with a circumferential reduction of 0.8 mm and a total convergence of 6. all internal angles were carefully rounded. impression was made using addition polyvinyl siloxane material (aquasil soft putty / regular set, dentsply de trey gmbh, germany) and poured with type iv die stone (fuji rock, gc). the cast was sent to dent care dental lab (muvattupuzha, kerala, india) for the construction of dmls posterior three - unit metal - ceramic fixed partial denture. provisional restoration was done using poly methyl methacrylate (dental products india [dpi ], rapid repair cold cure, dpi, mumbai). the digital construction of the metal framework was done using computer software, and the laser sintered processing was done by the laser sintering unit (eosint m 270, eos germany) where a high energy focused laser beam directly fuses a localized region of a thin layer of cobalt chromium metal powder to build up the restoration gradually. the thickness of the metal copings was a minimum of 0.35 mm with a connector thickness of 3 mm. the thickness of the veneered ceramic (vita vm 13 ceramic) was 1.15 mm occlusally and 0.8 mm cervically. intraoral evaluation of the restorations were made for marginal integrity, and occlusal contacts were evaluated with articulating film and adjustments were made using porcelain polishing kit to attain contacts in maximum intercuspation, and to eliminate lateral interferences. cementation was done using type i glass ionomer cement (gc corporation tokyo, japan). post - insertion oral hygiene instructions including interdental brushing were explained to the patients and recommended to follow regularly. recall visits were made at 6, 12 months interval and annually thereafter for the next 60 months to follow - up the restorations that were made. though recall visits were done at 6 months interval, only annual evaluation was done to assess the longevity of the restorations. the clinical evaluation was done by qualified prosthodontists by visual and clinical examination using conventional dental diagnostic instruments. to standardize the assessment on the longevity of restorations, the restorations in the study group were evaluated using the recommended clinical indices called the modified ryge clinical criteria [table 1 ]. these criteria through visual and probing examination assesses the fracture resistance of the veneered ceramic, connector failure occurring in the fixed partial denture prosthesis, discoloration at the marginal areas of the veneered ceramic, and marginal integrity of the fixed denture prosthesis. according to the criteria, all categories were given scores namely alpha, bravo, charlie, and delta ratings to determine whether the restorations is in excellent state or failing during the study period. radiographic periapical assessment of the abutments and proximal caries assessments were also done during the annual evaluation. the variables were graded based on the clinical evaluation and the probability distributions of these were calculated. an analysis of survival using the kaplan meier method with approximate 95% confidence intervals was performed for the survival of these posterior fixed partial dentures. in this study group of 45 patients, (24 male and 21 female patients) who received the metal - ceramic posterior fixed partial denture done with dmls technique had been evaluated periodically using modified ryge criteria for the period of 60 months to assess the clinical longevity of these restorations. at the end of evaluation period of 60 months for fracture resistance 39 restorations which had smooth, shiny surface and without any defects were rated alpha [figure 1 ], four restorations with mild chipping of the veneering porcelain and was not impairing the functioning of the metal - ceramic prosthesis was reported bravo [figure 2 ], and two restorations reported charlie had chipping of veneering porcelain impairing esthetics and exposing the framework material. the exposure was reported at the distolingual cusp areas of one case [figure 3 ] and at the area of central fossa in the other case. these two restorations which had bravo rating during the end of the 3 year gradually progressed to the charlie rating at the end of the 5 year of the study. on visual and probing examination for marginal adaptation, 43 restorations reported with alpha rating and one each was reported for bravo [figure 4 ], and charlie and no cases reported delta ratings. for marginal discoloration, only two cases reported bravo rating and all other cases reported alpha rating. the distribution of clinical criteria and its ratings for the study group is shown graphically [figure 5 ]. radiographic assessment for the period of 60 months had 43 cases with no evidence of any periapical changes [figure 6 ]. only one case reported with radiographic evidence of incipient proximal caries requiring restoration of the proximal teeth adjacent to the abutments, and the other with abutment teeth exhibiting periapical changes requiring root canal therapy during the study period. none of the cases had fracture of the framework, and retention was maintained for all the posterior fixed partial denture cases. the survival rate of the laser sintered posterior metal - ceramic fixed partial denture without any major fracture of the ceramic material, or the connector framework was 95.5% with confidence interval of 7896% during the observation period of 60 months [figure 7 ], and mechanical complication [figure 8 ] occurred as a result of major chipping of the veneer porcelain was only 4.5% with confidence interval of 8598%. smooth, shiny surface without defects (alpha rating) mild chipping of the veneering porcelain and not impairing the function (bravo rating) chipping of veneering porcelain impairing esthetics (charlie rating) visible evidence of crevice and penetration of explorer distribution of the 3 clinical criteria and its ratings radiographic assessment of the fixed partial denture survival curve for the direct metal laser sintered posterior metal - ceramic fixed partial denture mechanical complication during the follow - up period taggart was successful in using wax to form the pattern and used pressure to cast the alloys which formed the basis of conventional casting technique. conventional form of casting is still widely practiced and had become the mainstay of casting alloys used in dentistry. due to the use of computer - aided design and computer - aided manufacturing in dentistry, the trend in dental alloy castings has also undergone tremendous change. the laser sintering process was introduced by deckard and beaman, referred to as three dimensional printing because it builds up the framework in a series of successively thin layers in the range of 0.020.06 mm. a laser beam is focused on a bed of powdered metal, and these areas fuse into a thin solid metal layer. after the formation of the first layer, another layer of alloy powder is then laid down, and the next slice of the framework is produced and fused with the first. when all these layers have been built up, the solid copings and bridge framework are taken from the machines which are then sandblasted and ultrasonically cleaned. the other ingredients include tungsten, molybdenum, iron, silicon, cerium, manganese, and carbon. laser sintered crowns and bridges are of a particle size of 314 m and when combined with very fine point laser of 0.1 mm results in a higher density of around 99.9%, resulting in stronger copings with practically no voids. hence, the laser sintered process results in highly accurate and well - detailed restorations. laser sintered metal crowns were compared with conventionally made cast crowns for the internal fit and the results indicate the marginal gap for laser sintered crowns was on an average of 65 m when compared to the conventionally made crowns with a value of 150125 m. the earlier clinical studies conducted with this laser sintered technology were made only for single unit metal - ceramic crowns. based on the promising results obtained from various in vitro studies and an in vivo study for single unit crowns for dmls technique, this study on fixed partial denture made from dmls technique was undertaken since the complex biomechanical functions of posterior metal - ceramic fixed partial dentures which have connectors is entirely different from the function of single unit metal - ceramic crowns. the clinical follow - up of laser sintered posterior metal - ceramic fixed partial denture indicated chipping of the veneering porcelain at 60 months for two cases indicating the need to replace the posterior fixed partial denture due to esthetic concerns over the study period. chipping might be attributed to the anatomical preparation of the abutment teeth in the arch, the homogeneous dimensions of the veneer ceramic, and the ceramic bonding to the metal copings. minor chipping which occurred for the 4 cases was not further progressing during the study period. the failure rate in this study for the direct metal sintered posterior fixed partial denture obtained was 4.5% [figure 7 ] which was in the range comparable to conventionally done cast metal - ceramic restorations which ranged from a value of 2.50% to maximum of 7.60% obtained from other study. the veneer fracture reported with opposing natural dentition denotes the biomechanical differentiation of load applied onto the veneered ceramic of the laser sintered posterior fixed partial denture. marginal discoloration and caries obtained in this study were 4% in comparison to range up to 21.20% obtained for conventional cast metal - ceramic restorations during the same study period denoting the lack in hygienic measures taken by the patient to manage the marginal gingiva and crevices. these patients were instructed, and measures were made to improve the hygienic conditions during the study period. the survival rate of laser sintered metal - ceramic posterior fixed partial denture restorations was 95.5% in comparison to the conventional cast metal - ceramic fixed partial denture of 84.3%. mechanical complication occurring as a result of major chipping of the veneer porcelain was only 4.5% which indicated promising clinical efficacy of the laser sintered metal - ceramic posterior fixed partial dentures. clinical performance of posterior fixed partial dentures done with dmls technique needs more years of clinical research to be comparable with conventional metal - ceramic which has reports of more than 20 years of clinical service. laser sintered metal - ceramic posterior fixed partial dentures have yielded promising results during the observation period of 60 months by proving their clinical survival rate of 95.5% indicating greater clinical acceptability for use in day - to - day clinical practice. | statement of problem : in recent years, direct metal laser sintered (dmls) metal - ceramic - based fixed partial denture prostheses have been used as an alternative to conventional metal - ceramic fixed partial denture prostheses. however, clinical studies for evaluating their long - term clinical survivability and acceptability are limited.aims and objective : the aim of this study was to assess the efficacy of metal - ceramic fixed dental prosthesis fabricated with dmls technique, and its clinical acceptance on long - term clinical use.materials and methods : the study group consisted of 45 patients who were restored with posterior three - unit fixed partial denture prosthesis made using direct laser sintered metal - ceramic restorations. patient recall and clinical examination of the restorations were done after 6months and every 12 months thereafter for the period of 60 months. clinical examination for evaluation of longevity of restorations was done using modified ryge criteria which included chipping of the veneered ceramic, connector failure occurring in the fixed partial denture prosthesis, discoloration at the marginal areas of the veneered ceramic, and marginal adaptation of the metal and ceramic of the fixed denture prosthesis. periapical status was assessed using periodical radiographs during the study period. survival analysis was made using the kaplan meier method.results:none of the patients had failure of the connector of the fixed partial denture prostheses during the study period. two exhibited biological changes which included periapical changes and proximal caries adjacent to the abutments.conclusion:dmls metal - ceramic fixed partial denture prosthesis had a survival rate of 95.5% and yielded promising results during the 5-year clinical study. |
the cytotoxicity of a high purity cornish kaolinite toward mouse peritoneal macrophages in vitro was examined. the material was cytotoxic towards these cells, the activity could be decreased substantially by pretreating the dust with poly(2-vinylpyridine n - oxide). pretreatment of the dusts with poly(acrylic acid) had a small effect on cytotoxicity, but combinations of the polymer treatments virtually abolished the material 's biological activity towards macrophages. these studies indicated that the cytotoxicity of kaolinite is not due to its flakelike morphology.imagesfigure 1. |
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on the day of surgery, all donor tissues are dissected in the operating room prior to the start of the first dsaek surgery. dissection is performed using a microkeratome (altk cbm ; moria japan kk, tokyo, japan) equipped with a 300 m head. after microkeratome - dissection, the donor - endothelial lamella (with the repositioned, dissected cap) is transferred to a punching system, and cut with an 8.0 mm diameter punch (barron donor cornea punch ; katena products inc, denville, nj). the busin glide surface is then wetted with several drops of balanced salt solution (figure 2a). after the composite of donor - endothelial lamella and microkeratome - dissected cap is punched out, several drops of dispersive ophthalmic viscosurgical device are placed onto the endothelial surface (figure 2b). next, hydrodissection of the potential space between the donor - endothelial lamella and the microkeratome - dissected cap is carefully performed, using ophthalmic irrigation solution (bss plus ; alcon, fort worth, tx) to enable smooth detachment of these two lamellae (figure 2d). this technique enables smooth detachment of the composite, without causing the formation of any wrinkles or folds (figure 2e). the donor lamella is then pulled into the busin glide opening, using a 25-gauge anterior - capsular forceps (cat # mf801l ; inami co, ltd, tokyo, japan) (figure 2f). we reported herein a modified surgical technique, prewetting of the glide, and hydrodissection of the donor lamella aimed at optimizing the use of the busin glide for dsaek surgery. hydrodissection of the donor lamella enables the smooth detachment of the composite of donor - endothelial lamella and microkeratome - dissected cap on the busin glide without the formation of any wrinkles or folds. this technique is quite useful when the dsaek is performed using pre - cut tissue, as these tissues are usually distributed with the cap adhered to the dissected stromal bed. in ten out of ten consecutive cases, we saw that simply dragging donor - endothelial lamella directly onto the glide caused macroscopic wrinkling or folding of the donor lamella. it has been shown previously with in vitro vital dye staining9 that folding of the donor endothelial lamella resulting from the forceps manipulation can cause endothelial cell damage. we too, have preliminary vital dye staining data (not shown here) that wrinkling or folding of the donor lamella as it is dragged onto the busin glide causes endothelial cell damage. to date, we have used this modified technique in more than 50 consecutive dsaek cases, and no wrinkles or folds were caused during loading of the donor lamella onto the busin glide. furthermore, prewetting of the glide enabled quite smooth pull - through of the donor lamella in all cases. we strongly believe that this technique eliminates additional endothelial - cell loss caused by wrinkle formation, during donor manipulation. however, care should be exercised in using viscoelastic materials over the graft, especially on the stromal side, because of the increased risk of donor dislocation. currently, these modifications are our preferred endothelial keratoplasty technique during busin glide use. | we describe a modified technique for loading donor corneal endothelial lamella onto a busin glide without causing wrinkles, as part of the procedure of descemet - stripping automated endothelial keratoplasty. briefly, after punching out a composite of the donor - endothelial lamella and a microkeratome - dissected cap, several drops of dispersive ophthalmic viscosurgical device are placed onto the endothelial surface. the busin glide surface is then wetted with several drops of balanced salt solution. after the composite is transferred onto the busin glide, hydrodissection of the potential space between the donor - endothelial lamella and the microkeratome - dissected cap is carefully performed to enable smooth detachment of these two lamellae. whereas simply dragging the donor - endothelial lamella directly onto the glide can cause wrinkling or folding of the donor lamella, this technique enables smooth detachment of the composite without wrinkle or fold formation, and results in less endothelial cell damage. |
hepatitis c virus (hcv) is an enveloped, single - stranded ribonucleic acid (rna) virus with positive polarity. this virus is one of the most important pathogens of the human and is able to cause mild to severe liver diseases. the virus is a member of the hepaciviruses genus in the flaviviridae family (1). hepatitis c virus infection is a major blood - borne infection worldwide, with a silent epidemiology, that it has reached pandemic proportions (2). the chronic infection with hcv, remains a troublesome health problem worldwide, which approximately 3% of the population suffering from it. its prevalence is higher in some countries of asia and africa and approximately 14.5% in egypt (3 - 5). in developing countries, the chronic hepatitis c is the most prominent cause of liver cirrhosis, hepatocellular carcinoma and liver transplantation (6). available estimates indicate that worldwide there were 54,000 deaths and 955,000 disability- adjusted lifeyears associated with acute hcv infection (4). the main routes of transmission in hcv are exposure to infected blood or blood product, intravenous drug use, infected medical equipment, tattooing, needle stick, hemodialysis, sexual activity and organ transplantation (2, 3, 5). the prevalence of anti - hcv from population - based studies is used to compare hcv infection levels globally. hepatitis c virus infected an estimated 185 million people worldwide and is a significant cause of morbidity and mortality. also, in developed countries, hcv predominantly infects people who inject drugs (9). peoples in some closed settings such as prisons, jails, juvenile detention facilities, pretrial detention centers, and extrajudicial detention centers for people who use drug showed higher prevalence of hcv (10). however, it seems that the prevalence of this virus in the general population of iran is less than 1% (11), which is approximately lower than the reports of the neighbor countries. the prevalence of hcv in the general population of different countries of the developed world around iran has been reported to be between 0.9 and 4% (11). there are no previous reports about the prevalence of hcv in rural population of iran and due to the differences in hcv - related risk factors between these two populations, performing of the study on the prevalence of hcv in the rural population is seriously needed. this study was designed to determine the prevalence of anti - hcv antibody in general population of two villages, farmashkan and akbarabad, of the kavar city in fars province, iran, and evaluate the related risk factors in these areas. moreover, the possible associations between all risk factors with anti - hcv antibody prevalence were also evaluated in this population. the present study is a part of kavar cohort study (k.c.s), which is started from 2006 in kavar town with about 71856 populations. this town is located in 35 kilometer southeast of shiraz, the capital of fars province, iran. gastroenterohepatology and endocrine research centers affiliated to shiraz university of medical sciences are implementing k.c.s. from the start of k.c.s till now, all peoples are followed every two years. in the original cohort study, demographic and anthropometric characteristics of participants are documented in the questionnaire. in a cross - sectional study, which approved in ethical committee of shiraz university of medical sciences, by using cochran formula and assuming p = 0.03, q = 0.97, d = 0.01 and = 0.95, the sample size of 1049 was calculated. performing of the study we used census method sampling and therefore, blood samples of all of the iranian peoples aged 7 years old in the two villages, farmashkan and akbarabad of the kavar city in fars province, iran, which referred to the k.c.s center were used. our inclusion criteria were age equal or more than 7 years and using no medication for the hcv treatment. demographic information included age, gender, marital status, occupation, history of blood and its product transfusion, hemodialysis, organ transplantation, dental procedure, accident, war injury, tattooing, injection drug use, addiction, alcohol use, personal or family history of liver disease, imprisonment, extramarital sexual contact, hepatitis b virus (hbv) vaccination, concomitant diseases and its symptoms were extracted from their medical records. the blood samples (10 ml) were cooled on ice and taken to a specialized laboratory affiliated to gastroenterohepatology research center, shiraz university of medical sciences, shiraz, iran. the blood samples were centrifuged at 3500 rpm for 15 min at 4c and the serum was separated and stored at -70c until further analysis. the serum igg antibody against hcv was determined using a third - generation indirect enzyme immunoassay (eia) kit (acon laboratories, inc, usa). this test can detect various subtypes of hcv antibodies using the recombinant antigens specific for core, ns3, ns4 and ns5. the clinical sensitivity and specificity of this kit data were reported as frequency and percentage and analyzed using spss software version 17.0 (washington, usa) for windows. qualitative data were analyzed by chi - square and fisher s exact tests to find any association between risk factors and hcv - antibody positivity. an independent samples t test was used to compare the age variable between the two villages. data were reported as frequency and percentage and analyzed using spss software version 17.0 (washington, usa) for windows. qualitative data were analyzed by chi - square and fisher s exact tests to find any association between risk factors and hcv - antibody positivity. an independent samples t test was used to compare the age variable between the two villages. a p value 0.05). individuals with a history of blood transfusion had11-fold higher risk for anti - hcv antibody positivity than those with negative history of blood transfusion (or : 11.40, 95% ci : 1.02 - 127.21). this study calculated the prevalence of hcv infection in the general population of the two kavar villages, akbarabad and farmashkan, and assessed its association with gender, age, marital status, jobs / position and history of some related risk factors. our participants were rural general populations and most of them were without any known hcv - related risk factors. the general prevalence of hcv in this study was detected as 0.25% and the highest prevalence was seen in age 12 years old (1%). although there was a significant association between blood transfusion and anti - hcv antibody positivity, no significant associations were detected between other related risk factors and anti - hcv positivity. based on a systematic review in 2009, hcv infection prevalence in general population was calculated as 1.6% (3) that is much higher that our results. in another study, which performed in the civilian population of the united states, it had been demonstrated that the prevalence of anti - hcv in the united sates decreased from 1.9% in 2001to 1.3% in 2005, and remained stable up to 2010 (12). the total viremic hcv population of australia in 2012 was estimated at 230000 with a viremic prevalence rate of 1.0%. in addition, the anti - hcv prevalence was estimated at 1.3%, equivalent to 308000 anti - hcv positive individuals (13). ferthermore, several studies were performed about the prevalence of hcv in the european, american, african and asian countries. in a study from austria in 2008, the anti - hcv prevalence rate of 0.5% (0.10.7%) different low (0.12%) and high (1.23%) prevalence rates were reported in two studies performed in 2007 (15) and 2012 (13), respectively. total seroprevalence of 1.38, 0.96, 0.6,0.63, 12.5, 0.4, 0.5, 2.6, 1.6 and 0.95 % in brazil (16), canada (17), czech republic (18), denmark (19), egypt (20), england (21), germany (22),spain (23), switzerland (24) and turkey (25), were also recorded, respectively. our detected hcv prevalence is lower than all previous reports and approximately near to the reports from england and germany. also, our prevalence is similar to a recently report from mashhad, iran. in that study, the prevalence of hepatitis c seropositivity in the general population of mashhad, northeast of iran, was evaluated and its results demonstrated that the overall seroprevalence of hepatitis c was 0.2% using the elisa method (1). reported that the hcv prevalence in iran is 0.5% in individuals with age range of 18 - 65 years and higher and about 1% was seen in male (26). the probable reason for this difference may be the nature of two population, the urban population in merat. (26) and shakeri. (1) studies and the rural population in our study. therefore, the hcv infection related risk factors and consequently anti - hcv antibody prevalence were lower in this study. however, the hcv viral load evaluation using rt - pcr is highly recommended to confirm this difference. although, the prevalence of hcv infection in children ranges from 0.05 to 0.4% (27), the higher prevalence was seen in this study for the persons with age of 12 years old (1%). it is necessary to evaluate the hcv antibody of both children and their mothers to rule out the vertical transmission of hcv. in our study, similar to previous reports from iran (1, 26), the hcv - positive male was dominant in comparison to female (0.3% vs. 0.2%, respectively). however, this difference was not significant and therefore there was no association between gender and anti - hcv antibody positivity. in an iranian study performed by kohan. in 2006, 11.1% of hcv infected persons (23 out of 207) co - infected with hbv (28). in our study, the hbv - hcv co - infection was detected in 13.3% of the persons (two out of 15). also, the high anti - hcv antibody positivity was detected in the student groups (24.8%) based on the job / position categorization. this can be a warning for education and health ministry for learning mostly about viral hepatitis transmission routes and prevention, especially hbv and hcv. transfusion of the infected blood and blood product is one of the major routes of the hcv infection. this is more important in some populations such as thalassemia, hemophilia, and hemodialysis because they are dependent on the blood products during their lifetime. it has been demonstrated that the hcv infection rate in these groups is higher than the normal population (29 - 40) as demonstrated in our study. our study suffered from some limitations including limitations in the univariate analysis and some limitations related to the effects of a long - time of the cross - sectional study. also, lack of confirmation of the positive anti - hcv antibody samples with molecular methods, such as pcr and rt - pcr is another limitation. however, this issue is being set up and performed in our center for larger cohort study. in conclusion, due to this fact that hcv infection is a preventable and curable disease, we recommend more attention for the control of patient - to - patient hcv transmission in hospitals. although prevalence rate in this study was lower than other studies and no statistical significant associations were found with common risk factors except blood transfusion, paying the especial attention to the all risk factors is seriously advised. finally, further studies are required in other rural and urban populations for better evaluation of the hcv - infection prevalence and real source of transmission. | background : hepatitis c virus (hcv) infection is a major blood - borne infection with silent epidemic, major global public health problem and diverse prevalence worldwide.objectives:this study aimed to evaluate the prevalence of hcv infection and related risk factors in the general population of two villages, farmashkan and akbarabad, of the kavar city in fars province, iran.patients and methods : a 34-month cross - sectional study was performed on all people of the villages aged 7 years from july 2007 to april 2010. demographic information and history of hcv - related risk factors were extracted from their medical records. for each participant, the serum anti - hcv igg was assessed by the commercial enzyme - linked immunosorbent assay (elisa) kits.results:a total of 6095 participants (36.4% male and 65.6% female) with the mean age of 92 (7 - 95) and mean sd of 34.6 17.3 years were included in this study. fifteen persons (0.24%) were detected as hcv - positive and the highest prevalence was seen in age 12 years old (1%). a significant association was only detected between blood transfusion and hcv infection ; therefore, those persons with history of blood transfusion had 15-fold higher risk for hcv seropositivity (odds ratio 15.54, 95% ci = 4.89 - 49.41).conclusions : our reported rate of hcv seropositivity is similar to the previous iranian reports. however, future evaluations should be focused on the polymerase chain reaction method for the detection of hcv and determining and evaluating of other related risk factors. moreover, more attention should be paid to blood donors as a reservoir population of hcv. |
immediate / early promoter / enhancer of cytomegalovirus (cmv promoter) is one of the most commonly used promoters for expression of transgenes in mammalian cells for research or therapeutic purposes. although it is often thought of as a constitutive and unregulated pan - specific promoter, it has been shown that its activity is strongly dependent on the host - cell transcriptional environment. many reports demonstrated that it does not direct persistent transgene expression and that its transcriptional activity varies according to cell type and developmental age and can also be upregulated under specific conditions [113 ]. dna methylation of the promoter is the most frequently given explanation for transient gene expression of the transgene constructs containing the cmv promoter, independently of the delivery system used [15 ]. for instance, the cmv promoter was shown to be susceptible to transcriptional inactivation by methylation after transient transfection using different non - viral delivery vectors [2, 4, 5 ]. the same observation was also demonstrated for a stably transfected cell line using lipofection in vitro. inhibition of transgene expression caused by methylation of the cmv promoter was also reported in vivo following adenoviral gene delivery to muscle. on the other hand, some studies demonstrated that methylation is not always responsible for the observed transient expression in vivo. for instance, methylation was investigated as a potential cause of the transient transgene expression from non - viral vectors in the mouse lung, but results suggested that the plasmid dna does not become de novo methylated in the mouse airways. similarly, transcriptional inactivation of cmv after adenovirus gene transfer was demonstrated in the mouse liver although there was no evidence for the methylation of adenovirus dna. in the later study, silenced cmv promoter was reactivated after treatment with lipopolysaccharide (lps). there have also been other reports suggesting that the cmv promoter can be upregulated under specific conditions such as treatment with lps, cyclic amp, phorbol esters, bacterial cpgs [811 ], and by a variety of environmental stresses. recently, chemotherapeutic agents and radiation (ir) were shown to upregulate expression of transgenes driven by the cmv promoter in vitro and in vivo [13, 14 ]. upregulation with chemotherapeutics was reported to occur in multiple cell lines independently of the transfection method used, and radiation was reported to enhance adenoviral gene therapy in pancreatic cancer via activation of the cmv promoter. non - invasive whole body optical methods (luminescence or fluorescence) are very competitive approaches to assess tumor development and responses to drug and gene therapies in vivo and are far more affordable compared to other imaging methods, such as positron emission tomography, x - ray computed tomography, and magnetic resonance imaging [15, 16 ]. recently, they were employed also for following transgene expression and activity of promoters in vivo. however, the in vivo reports on methylation and upregulation of the cmv promoter by conventional treatments, such as ir and cisplatin (cddp), are scarce ; and results are contradictory [1, 6, 7, 14, 17 ]. therefore, the aim of our study was to evaluate the role of methylation and upregulation of the cmv promoter by ir and cddp in vivo using non - invasive fluorescence in vivo imaging, which enables long - term follow - up of reporter gene fluorescence in the animals and consequently, the activity of promoters that control reporter gene expression. different systems in which expression of the reporter gene was controlled either by an inducible promoter or cmv promoter (stable cell lines, stably transfected experimental tumors, and transiently transfected muscles) were set up using the non - viral delivery system of electroporation and the influence of treatment with 5-azacytidine, ir, and cddp on the activity of the cmv promoter in vitro and in vivo was assessed. a plasmid encoding green fluorescence protein (gfp) under the control of the cmv promoter and neomycin resistance gene (pegfp - n1, clontech, basingstoke, uk) and a plasmid encoding gfp under the control of the human p21 promoter and neomycin resistance gene (p21-egfp, kind gift from irena hreljac, national institute of biology, ljubljana, slovenia) were used. human p21 promoter is an inducible mammalian promoter that is induced by dna damage [18, 19 ]. in the study, it was used as a control for the cmv promoter. cell lines murine adenocarcinoma of the mammary glands ts / a and murine fibrosarcoma lpb cell lines were maintained in eagle s minimum essential medium (emem, sigma, taufkirchen, germany), supplemented with 10% fetal calf serum (sigma, taufkirchen, germany), 2 mm l - glutamine, 1 mm sodium pyruvate, and 100 iu / ml penicillin / streptomycin (pliva, zagreb, croatia) in a 5% co2 humidified incubator at 37c. preparation of stable cell lines carrying cmv or p21 promoter - driven reporter gene constructs lpb and ts / a cells were transfected with the pegfp - n1 plasmid and the lpb cell line was transfected with the p21-egfp plasmid. specifically, cells grown as a monolayer were harvested and a 2.5 10 cells / ml cell suspension was prepared in electroporation buffer (125 mm sucrose, 10 mm k2hpo4, 2.5 mm kh2po4, 2 mm mgcl2 6 h2o). a dense cell suspension with a concentration of 1 10 cells and 10 g of pegfp - n1 in 50 l of electroporation buffer was placed between two flat parallel stainless steel electrodes with a 2 mm gap connected to the gt-1 electroporator (university of ljubljana, faculty of electrical engineering, ljubljana, slovenia) and subjected to eight square - wave electric pulses with an amplitude per distance ratio 700 v / cm, 5 ms duration time, and 1 hz repetition frequency. after electroporation cells were incubated for 5 min at room temperature, plated into petri dishes, and then cultured for 2 months under increasing concentrations (5001,000 g / ml for lpb cells and 1,2002,000 g / ml for ts / a cells) of the selection agent geneticin (gibco invitrogen, san diego, ca, usa) to obtain resistant clones. clones with the highest gfp expression were identified by fluorescence microscopy (olympus, hamburg, germany), isolated, propagated, and frozen in liquid nitrogen for subsequent experiments. to determine the number of fluorescent cells, flow cytometry analysis of stable cell lines carrying cmv promoter - driven reporter gene constructs was performed : cells were trypsinized, collected, and 2 10 cells from each stable line were analyzed using flow cytometry (becton dickinson, calibur, franklin lakes, nj, usa). the percentage of cells stably expressing gfp was determined from the histograms. to test the influence of dna methylation and treatment with ir or cddp on cmv activity in in vitro conditions, cells were treated with the dna methylation inhibitor 5-azacytidine (5-aza-2dc, sigma) and exposed to ir and cddp (cis - diamminedichloroplatinum (ii), pharmacia & upjohn s.p.a, milan, italy). for the methylation tests, cells were first passaged for 1 month to allow methylation of dna to occur and then (at the sixth passage) treated with a dna methylation inhibitor, while for the ir and cddp exposure freshly thawed (first passage) cells were used. the treatment protocols were as follows : 5-aza-2dc : cells were plated at a density of 1.5 10 cells / cm and treated with 5-aza-2dc for 3 days. a stock solution of 5-aza-2dc was prepared in phosphate buffered saline (pbs) and was freshly diluted to a working concentration of 1 m in emem each day of the experiment.ir : cells were plated at a density of 1.7 10 cells / cm and irradiated with a dose of 6 gy using an x - ray unit drapac 2000 (gulmay medical ltd, shepperton, uk) operating at 220 kv, 10 ma, and with 0.55 mm cu and 1.8 mm al filtration.cddp : cddp in the form of crystalline powder was dissolved in sterile h2o at a stock concentration of 2 mg / ml. cells were plated at a density of 2.0 10 cells / cm and the stock solution of cddp was added to emem to get a working concentration of 3 g / ml. 5-aza-2dc : cells were plated at a density of 1.5 10 cells / cm and treated with 5-aza-2dc for 3 days. a stock solution of 5-aza-2dc was prepared in phosphate buffered saline (pbs) and was freshly diluted to a working concentration of 1 m in emem each day of the experiment. ir : cells were plated at a density of 1.7 10 cells / cm and irradiated with a dose of 6 gy using an x - ray unit drapac 2000 (gulmay medical ltd, shepperton, uk) operating at 220 kv, 10 ma, and with 0.55 mm cu and 1.8 mm al filtration. cddp : cddp in the form of crystalline powder was dissolved in sterile h2o at a stock concentration of 2 mg / ml. cells were plated at a density of 2.0 10 cells / cm and the stock solution of cddp was added to emem to get a working concentration of 3 g / ml. three days after the treatments, cells were trypsinized, collected, and 2 10 cells were plated in 96-well microplates. expression of the reporter gene was determined using the fluorescence microplate reader infinite 200 (tecan, mnnedorf, switzerland) and fluorescence microscopy (olympus, hamburg, germany) for the in vivo experiments, female balb / c and female c57bl/6 mice obtained from the institute of pathology, faculty of medicine, university of ljubljana, slovenia were used. at the beginning of the experiments, mice were housed and maintained in a specific pathogen - free animal colony at constant room temperature (21c) and 12 h light / dark cycle. animals were subjected to an adaptation period of 710 days before the experiments were carried out. all procedures on animals were performed in accordance with the official guidelines of the ministry of agriculture, forestry, and food of the republic of slovenia (permission no. induction of solid subcutaneous tumors viable ts / a egfp tumor cells (2 10) prepared from cell culture in vitro were injected dorsolaterally in balb / c mice. when the tumors reached approximately 40 mm in volume (710 days), mice were randomly divided into experimental groups and subjected to a specific experimental protocol. preparation of transiently transfected muscle model carrying cmv promoter - driven reporter gene construct c57bl/6 mice were anesthetized with isofluran (torrex chiesi gmbh, wien, austria) using an isoflurane vaporizer (datex ohmeda, helsinki, finland). plasmid pegfp - n1 (20 g in 20 l of water) was injected into both m. tibialis cranialis with a thin (26 g) needle. the hind legs were placed between two flat parallel stainless steel electrodes with rounded corners (dimensions 20 mm 10 mm) with a 6 mm gap between the electrodes connected to the electric pulse generator cliniporator (igea s.r.l., carpi, italy) and subjected to one high - voltage square - wave electric pulse with an amplitude per distance 600 v / cm and 100 s duration and four low - voltage square - wave electric pulses with an amplitude per distance 80 v / cm, 100 ms duration, and 1 hz repetition frequency. good contact between the electrodes and legs was assured by hair removal using hair removal cream (vitaskin, krka, d.d., novo mesto, slovenia) and use of a conductive gel (kameleon d.o.o., when the fluorescence intensity declined to 50% of the highest level (approximately 4 weeks after transfection), mice were randomly divided into experimental groups and subjected to a specific experimental protocol. to test the influence of dna methylation and treatment with ir and cddp on cmv activity in vivo, tumor - bearing balb / c mice and c57bl/6 mice with transiently transfected muscles the treatment protocols were as follows : 5-aza-2dc : mice were treated twice by intraperitoneal injection of 0.2 mg / kg of 5-azacytidine in 300 l of pbs on two consecutive days. a stock solution of 5-aza-2dc was freshly diluted to the working concentration each day of the experiment.ir : tumors and transfected muscles were irradiated with a dose of 6 gy using the same x - ray unit as used for the in vitro experiments. during irradiation, mice were restrained in special lead holders with apertures for irradiation of the tumors / legs, exposing only the tumors / legs and shielding the rest of the body from irradiation.cddp : mice were injected intravenously with 8 mg / kg of cisplatin in 100 l of sterile h2o. for each experiment, a fresh stock solution was prepared from crystalline powder and diluted to the working concentration. 5-aza-2dc : mice were treated twice by intraperitoneal injection of 0.2 mg / kg of 5-azacytidine in 300 l of pbs on two consecutive days. a stock solution of 5-aza-2dc ir : tumors and transfected muscles were irradiated with a dose of 6 gy using the same x - ray unit as used for the in vitro experiments. during irradiation, mice were restrained in special lead holders with apertures for irradiation of the tumors / legs, exposing only the tumors / legs and shielding the rest of the body from irradiation. cddp : mice were injected intravenously with 8 mg / kg of cisplatin in 100 l of sterile h2o. for each experiment, a fresh stock solution was prepared from crystalline powder and diluted to the working concentration. after the treatments, fluorescence intensity of the tumors and muscles expressing gfp was followed transcutaneously using a fluorescence stereo microscope, which enabled non - invasive follow - up of the intensity and duration of gfp expression. for each observation under the microscope, hair over the tumor or muscle was removed using an electric shaver and/or hair removal cream and animals were anesthetized with isoflurane as described above. digital images of fluorescence were recorded everyday post - treatment for 8 days for tumors and every 23 days for 12 days in the case of the muscle with a digital color camera (axiocam mrc5, zeiss, jena, germany) connected to the fluorescence stereo microscope (lumar.v12, zeiss, jena, germany). during capture, tumors or legs were placed in a special holder to minimize the movement of animals caused by breathing and to ensure the same positioning at each observation. images were analyzed using the imagej software tool (national institute of mental health, research services branch, bethesda, ma, usa). 2) taken at different time points were stacked together, and the fluorescence intensity was determined by adjusting the threshold values for each stack. adjusted mean fluorescence intensity of each tumor or muscle picture in the stack was then normalized to the mean fluorescence intensity at day 0. b fluorescence of tumors was separated from background fluorescence using the lower threshold value (blue). the upper threshold value (green) was used to exclude necrotic areas of the tumor.fig. b fluorescence of muscle fibers was separated from the non - transfected region of the leg and background fluorescence by adjusting the lower threshold value (blue). a fluorescent image of the tumor. b fluorescence of tumors was separated from background fluorescence using the lower threshold value (blue). the upper threshold value (green) was used to exclude necrotic areas of the tumor. a fluorescent image of the muscle. b fluorescence of muscle fibers was separated from the non - transfected region of the leg and background fluorescence by adjusting the lower threshold value (blue). a plasmid encoding green fluorescence protein (gfp) under the control of the cmv promoter and neomycin resistance gene (pegfp - n1, clontech, basingstoke, uk) and a plasmid encoding gfp under the control of the human p21 promoter and neomycin resistance gene (p21-egfp, kind gift from irena hreljac, national institute of biology, ljubljana, slovenia) were used. human p21 promoter is an inducible mammalian promoter that is induced by dna damage [18, 19 ]. in the study, it was used as a control for the cmv promoter. cell lines murine adenocarcinoma of the mammary glands ts / a and murine fibrosarcoma lpb cell lines were maintained in eagle s minimum essential medium (emem, sigma, taufkirchen, germany), supplemented with 10% fetal calf serum (sigma, taufkirchen, germany), 2 mm l - glutamine, 1 mm sodium pyruvate, and 100 iu / ml penicillin / streptomycin (pliva, zagreb, croatia) in a 5% co2 humidified incubator at 37c. preparation of stable cell lines carrying cmv or p21 promoter - driven reporter gene constructs lpb and ts / a cells were transfected with the pegfp - n1 plasmid and the lpb cell line was transfected with the p21-egfp plasmid. specifically, cells grown as a monolayer were harvested and a 2.5 10 cells / ml cell suspension was prepared in electroporation buffer (125 mm sucrose, 10 mm k2hpo4, 2.5 mm kh2po4, 2 mm mgcl2 6 h2o). a dense cell suspension with a concentration of 1 10 cells and 10 g of pegfp - n1 in 50 l of electroporation buffer was placed between two flat parallel stainless steel electrodes with a 2 mm gap connected to the gt-1 electroporator (university of ljubljana, faculty of electrical engineering, ljubljana, slovenia) and subjected to eight square - wave electric pulses with an amplitude per distance ratio 700 v / cm, 5 ms duration time, and 1 hz repetition frequency. after electroporation cells were incubated for 5 min at room temperature, plated into petri dishes, and then cultured for 2 months under increasing concentrations (5001,000 g / ml for lpb cells and 1,2002,000 g / ml for ts / a cells) of the selection agent geneticin (gibco invitrogen, san diego, ca, usa) to obtain resistant clones. clones with the highest gfp expression were identified by fluorescence microscopy (olympus, hamburg, germany), isolated, propagated, and frozen in liquid nitrogen for subsequent experiments. to determine the number of fluorescent cells, flow cytometry analysis of stable cell lines carrying cmv promoter - driven reporter gene constructs was performed : cells were trypsinized, collected, and 2 10 cells from each stable line were analyzed using flow cytometry (becton dickinson, calibur, franklin lakes, nj, usa). the percentage of cells stably expressing gfp was determined from the histograms. to test the influence of dna methylation and treatment with ir or cddp on cmv activity in in vitro conditions, cells were treated with the dna methylation inhibitor 5-azacytidine (5-aza-2dc, sigma) and exposed to ir and cddp (cis - diamminedichloroplatinum (ii), pharmacia & upjohn s.p.a, milan, italy). for the methylation tests, cells were first passaged for 1 month to allow methylation of dna to occur and then (at the sixth passage) treated with a dna methylation inhibitor, while for the ir and cddp exposure freshly thawed (first passage) cells were used. the treatment protocols were as follows : 5-aza-2dc : cells were plated at a density of 1.5 10 cells / cm and treated with 5-aza-2dc for 3 days. a stock solution of 5-aza-2dc was prepared in phosphate buffered saline (pbs) and was freshly diluted to a working concentration of 1 m in emem each day of the experiment.ir : cells were plated at a density of 1.7 10 cells / cm and irradiated with a dose of 6 gy using an x - ray unit drapac 2000 (gulmay medical ltd, shepperton, uk) operating at 220 kv, 10 ma, and with 0.55 mm cu and 1.8 mm al filtration.cddp : cddp in the form of crystalline powder was dissolved in sterile h2o at a stock concentration of 2 mg / ml. cells were plated at a density of 2.0 10 cells / cm and the stock solution of cddp was added to emem to get a working concentration of 3 g / ml. 5-aza-2dc : cells were plated at a density of 1.5 10 cells / cm and treated with 5-aza-2dc for 3 days. a stock solution of 5-aza-2dc was prepared in phosphate buffered saline (pbs) and was freshly diluted to a working concentration of 1 m in emem each day of the experiment. ir : cells were plated at a density of 1.7 10 cells / cm and irradiated with a dose of 6 gy using an x - ray unit drapac 2000 (gulmay medical ltd, shepperton, uk) operating at 220 kv, 10 ma, and with 0.55 mm cu and 1.8 mm al filtration. cddp : cddp in the form of crystalline powder was dissolved in sterile h2o at a stock concentration of 2 mg / ml. cells were plated at a density of 2.0 10 cells / cm and the stock solution of cddp was added to emem to get a working concentration of 3 g / ml. three days after the treatments, cells were trypsinized, collected, and 2 10 cells were plated in 96-well microplates. expression of the reporter gene was determined using the fluorescence microplate reader infinite 200 (tecan, mnnedorf, switzerland) and fluorescence microscopy (olympus, hamburg, germany) for the in vivo experiments, female balb / c and female c57bl/6 mice obtained from the institute of pathology, faculty of medicine, university of ljubljana, slovenia were used. at the beginning of the experiments, mice were housed and maintained in a specific pathogen - free animal colony at constant room temperature (21c) and 12 h light / dark cycle. animals were subjected to an adaptation period of 710 days before the experiments were carried out. all procedures on animals were performed in accordance with the official guidelines of the ministry of agriculture, forestry, and food of the republic of slovenia (permission no. induction of solid subcutaneous tumors viable ts / a egfp tumor cells (2 10) prepared from cell culture in vitro were injected dorsolaterally in balb / c mice. when the tumors reached approximately 40 mm in volume (710 days), mice were randomly divided into experimental groups and subjected to a specific experimental protocol. preparation of transiently transfected muscle model carrying cmv promoter - driven reporter gene construct c57bl/6 mice were anesthetized with isofluran (torrex chiesi gmbh, wien, austria) using an isoflurane vaporizer (datex ohmeda, helsinki, finland). plasmid pegfp - n1 (20 g in 20 l of water) was injected into both m. tibialis cranialis with a thin (26 g) needle. the hind legs were placed between two flat parallel stainless steel electrodes with rounded corners (dimensions 20 mm 10 mm) with a 6 mm gap between the electrodes connected to the electric pulse generator cliniporator (igea s.r.l., carpi, italy) and subjected to one high - voltage square - wave electric pulse with an amplitude per distance 600 v / cm and 100 s duration and four low - voltage square - wave electric pulses with an amplitude per distance 80 v / cm, 100 ms duration, and 1 hz repetition frequency. good contact between the electrodes and legs was assured by hair removal using hair removal cream (vitaskin, krka, d.d., novo mesto, slovenia) and use of a conductive gel (kameleon d.o.o., when the fluorescence intensity declined to 50% of the highest level (approximately 4 weeks after transfection), mice were randomly divided into experimental groups and subjected to a specific experimental protocol. to test the influence of dna methylation and treatment with ir and cddp on cmv activity in vivo, tumor - bearing balb / c mice and c57bl/6 mice with transiently transfected muscles were treated with either 5-azacytidine, ir, or cddp. the treatment protocols were as follows : 5-aza-2dc : mice were treated twice by intraperitoneal injection of 0.2 mg / kg of 5-azacytidine in 300 l of pbs on two consecutive days. a stock solution of 5-aza-2dc was freshly diluted to the working concentration each day of the experiment.ir : tumors and transfected muscles were irradiated with a dose of 6 gy using the same x - ray unit as used for the in vitro experiments. during irradiation, mice were restrained in special lead holders with apertures for irradiation of the tumors / legs, exposing only the tumors / legs and shielding the rest of the body from irradiation.cddp : mice were injected intravenously with 8 mg / kg of cisplatin in 100 l of sterile h2o. for each experiment, a fresh stock solution was prepared from crystalline powder and diluted to the working concentration. 5-aza-2dc : mice were treated twice by intraperitoneal injection of 0.2 mg / kg of 5-azacytidine in 300 l of pbs on two consecutive days. a stock solution of 5-aza-2dc ir : tumors and transfected muscles were irradiated with a dose of 6 gy using the same x - ray unit as used for the in vitro experiments. during irradiation, mice were restrained in special lead holders with apertures for irradiation of the tumors / legs, exposing only the tumors / legs and shielding the rest of the body from irradiation. cddp : mice were injected intravenously with 8 mg / kg of cisplatin in 100 l of sterile h2o. for each experiment, a fresh stock solution was prepared from crystalline powder and diluted to the working concentration. after the treatments, fluorescence intensity of the tumors and muscles expressing gfp was followed transcutaneously using a fluorescence stereo microscope, which enabled non - invasive follow - up of the intensity and duration of gfp expression. for each observation under the microscope, hair over the tumor or muscle was removed using an electric shaver and/or hair removal cream and animals were anesthetized with isoflurane as described above. digital images of fluorescence were recorded everyday post - treatment for 8 days for tumors and every 23 days for 12 days in the case of the muscle with a digital color camera (axiocam mrc5, zeiss, jena, germany) connected to the fluorescence stereo microscope (lumar.v12, zeiss, jena, germany). during capture, tumors or legs were placed in a special holder to minimize the movement of animals caused by breathing and to ensure the same positioning at each observation. images were analyzed using the imagej software tool (national institute of mental health, research services branch, bethesda, ma, usa). 2) taken at different time points were stacked together, and the fluorescence intensity was determined by adjusting the threshold values for each stack. adjusted mean fluorescence intensity of each tumor or muscle picture in the stack was then normalized to the mean fluorescence intensity at day 0. b fluorescence of tumors was separated from background fluorescence using the lower threshold value (blue). the upper threshold value (green) was used to exclude necrotic areas of the tumor.fig. b fluorescence of muscle fibers was separated from the non - transfected region of the leg and background fluorescence by adjusting the lower threshold value (blue). a fluorescent image of the tumor. b fluorescence of tumors was separated from background fluorescence using the lower threshold value (blue). the upper threshold value (green) was used to exclude necrotic areas of the tumor. a fluorescent image of the muscle. b fluorescence of muscle fibers was separated from the non - transfected region of the leg and background fluorescence by adjusting the lower threshold value (blue). the data were tested for normality of distribution using the kolmogorov smirnov test. differences between experimental groups were statistically evaluated by the student s t test. statistical analysis was performed using sigmastat software (systat software inc., san jose, ca, usa). lpb and ts / a stable lines expressing the gfp reporter gene under the control of the cmv promoter were prepared and designated as lpb egfp and ts / a egfp, and the lpb cell line expressing gfp under the control of the p21 promoter was prepared and designated as lpb p21-egfp. flow cytometry analysis showed that 70% of cells stably expressed gfp in the ts / a egfp cell line and only 20% in the lpb egfp cell line, whereas in the lpb p21-egfp cell line almost all cells expressed gfp (93% ; fig. 3histograms of gfp fluorescence in a lpb egfp, b lpb p21-egfp, and c ts / a egfp cells. histograms of gfp fluorescence in a lpb egfp, b lpb p21-egfp, and c ts / a egfp cells. cells were first passaged for 1 month (six passages) to allow methylation of dna to occur. during this time, fluorescence intensity decreased by a factor of 2.8 in ts / a egfp and by 1.2 in lpb egfp, but not in lpb p21-egfp cells. after treatment with 5-aza-2dc, a statistically significant increase in fluorescence intensity was obtained in ts / a egfp and lpb egfp cell lines by a factor of 6.9 and 1.4 compared to the control (sixth passage), respectively. the increase in the lpb p21-egfp cell line was by a factor of 1.3 (fig. 4visible and fluorescence images of a ts / a egfp, b lpb egfp, and c lpb p21-egfp cells. results are expressed as the mean se (p < 0.05). visible and fluorescence images of a ts / a egfp, b lpb egfp, and c lpb p21-egfp cells. results are expressed as the mean se (p < 0.05). exposure of cells to ir or cddp resulted in significant upregulation of expression in both cell lines with the cmv promoter, although the effects were more evident in the ts / a egfp cell line. after ir, fluorescence intensity increased 1.82-fold in ts / a egfp and 1.05-fold in lpb egfp cells compared to the control. after treatment with cddp, the increase in fluorescence intensity was comparable to that after ir and was 1.73-fold in the ts / a egfp cell line and 1.03-fold in the lpb egfp cell line. in the control group with the inducible p21 promoter the induction was by a factor of 1.29 after ir and 1.81 after cddp compared to the control (fig. 5visible and fluorescence images of a ts / a egfp, b lpb egfp, and c lpb p21-egfp cells. results are expressed as the mean se (p < 0.05). visible and fluorescence images of a ts / a egfp, b lpb egfp, and c lpb p21-egfp cells. results are expressed as the mean se (p < 0.05). treatment of tumors 710 days post - induction with 5-aza-2dc did not significantly increase fluorescence intensity of ts / a egfp tumors during the observation period. only a trend of increased fluorescence intensity towards the end of the observation period was observed. however, in the transiently transfected muscles, treatment with 5-aza-2dc 4 weeks post - transfection resulted in significantly increased fluorescence intensity towards the end of the observation period on day 12 by a factor of 1.40 (fig. 6fluorescence images of a ts / a egfp tumors, and b transiently transfected muscles after different treatments. influence of treatment with 5-aza-2dc, ir, and cddp on cmv activity in c ts / a - egfp tumor model, and d transiently transfected muscles. results are expressed as the mean se (p < 0.05). fluorescence images of a ts / a egfp tumors, and b transiently transfected muscles after different treatments. influence of treatment with 5-aza-2dc, ir, and cddp on cmv activity in c ts / a - egfp tumor model, and d results are expressed as the mean se (p < 0.05). treatment with ir or cddp resulted in significant upregulation of gfp expression in both in vivo models (p < 0.05, vs. control). after ir, the fluorescence intensity of ts / a egfp tumors was significantly increased by a factor of 1.26 already on the third day after treatment and by a factor of 1.24 on the fourth day. similarly, the increase in fluorescence intensity of transiently transfected muscles following ir was by a factor of 1.17 on the second day and by a factor of 1.25 on the fifth day post - treatment. systemic treatment with cddp upregulated the cmv promoter, resulting in significantly increased fluorescence intensity in both ts / a egfp tumors and transiently transfected muscles. upregulation was the most pronounced on the fourth day post - treatment for tumors and on the fifth day for muscles with a factor of increase 1.35 and 1.18, respectively (fig. lpb and ts / a stable lines expressing the gfp reporter gene under the control of the cmv promoter were prepared and designated as lpb egfp and ts / a egfp, and the lpb cell line expressing gfp under the control of the p21 promoter was prepared and designated as lpb p21-egfp. flow cytometry analysis showed that 70% of cells stably expressed gfp in the ts / a egfp cell line and only 20% in the lpb egfp cell line, whereas in the lpb p21-egfp cell line almost all cells expressed gfp (93% ; fig. 3histograms of gfp fluorescence in a lpb egfp, b lpb p21-egfp, and c ts / a egfp cells. histograms of gfp fluorescence in a lpb egfp, b lpb p21-egfp, and c ts / a egfp cells. cells were first passaged for 1 month (six passages) to allow methylation of dna to occur. during this time, fluorescence intensity decreased by a factor of 2.8 in ts / a egfp and by 1.2 in lpb egfp, but not in lpb p21-egfp cells. after treatment with 5-aza-2dc, a statistically significant increase in fluorescence intensity was obtained in ts / a egfp and lpb egfp cell lines by a factor of 6.9 and 1.4 compared to the control (sixth passage), respectively. the increase in the lpb p21-egfp cell line was by a factor of 1.3 (fig. 4visible and fluorescence images of a ts / a egfp, b lpb egfp, and c lpb p21-egfp cells. results are expressed as the mean se (p < 0.05). visible and fluorescence images of a ts / a egfp, b lpb egfp, and c lpb p21-egfp cells. results are expressed as the mean se (p < 0.05). exposure of cells to ir or cddp resulted in significant upregulation of expression in both cell lines with the cmv promoter, although the effects were more evident in the ts / a egfp cell line. after ir, fluorescence intensity increased 1.82-fold in ts / a egfp and 1.05-fold in lpb egfp cells compared to the control. after treatment with cddp, the increase in fluorescence intensity was comparable to that after ir and was 1.73-fold in the ts / a egfp cell line and 1.03-fold in the lpb egfp cell line. in the control group with the inducible p21 promoter the induction was by a factor of 1.29 after ir and 1.81 after cddp compared to the control (fig. 5visible and fluorescence images of a ts / a egfp, b lpb egfp, and c lpb p21-egfp cells. results are expressed as the mean se (p < 0.05). visible and fluorescence images of a ts / a egfp, b lpb egfp, and c lpb p21-egfp cells. results are expressed as the mean se (p < 0.05). treatment of tumors 710 days post - induction with 5-aza-2dc did not significantly increase fluorescence intensity of ts / a egfp tumors during the observation period. only a trend of increased fluorescence intensity towards the end of the observation period was observed. however, in the transiently transfected muscles, treatment with 5-aza-2dc 4 weeks post - transfection resulted in significantly increased fluorescence intensity towards the end of the observation period on day 12 by a factor of 1.40 (fig. 6fluorescence images of a ts / a egfp tumors, and b transiently transfected muscles after different treatments. influence of treatment with 5-aza-2dc, ir, and cddp on cmv activity in c ts / a - egfp tumor model, and d transiently transfected muscles. results are expressed as the mean se (p < 0.05). fluorescence images of a ts / a egfp tumors, and b transiently transfected muscles after different treatments. influence of treatment with 5-aza-2dc, ir, and cddp on cmv activity in c ts / a - egfp tumor model, and d transiently transfected muscles. results are expressed as the mean se (p < 0.05). treatment with ir or cddp resulted in significant upregulation of gfp expression in both in vivo models (p < 0.05, vs. control). after ir, the fluorescence intensity of ts / a egfp tumors was significantly increased by a factor of 1.26 already on the third day after treatment and by a factor of 1.24 on the fourth day. similarly, the increase in fluorescence intensity of transiently transfected muscles following ir was by a factor of 1.17 on the second day and by a factor of 1.25 on the fifth day post - treatment. systemic treatment with cddp upregulated the cmv promoter, resulting in significantly increased fluorescence intensity in both ts / a egfp tumors and transiently transfected muscles. upregulation was the most pronounced on the fourth day post - treatment for tumors and on the fifth day for muscles with a factor of increase 1.35 and 1.18, respectively (fig. the results of our study demonstrate that the cmv promoter can not be considered a constitutive promoter. we show that it is silenced with time and that it can be (re)activated after treatment with a demethylating agent. we also showed that the cmv promoter can be induced by treatment with ir and cddp in stably transformed cell lines in vitro and in vivo and after transient delivery to muscle. furthermore, we demonstrated that non - invasive fluorescence imaging is an appropriate and convenient method to monitor the activity of the promoter in vivo. sustained and well - defined or controllable expression levels of the transgene since expression levels of transgenes are dependent on the promoter, proper choice of promoter linked to the gene of interest is essential for the success of these vectors, especially if they are going to be used in human gene therapy. the cmv promoter is one of the most commonly used promoters for expression of transgenes in mammalian cells. despite the fact that many reports demonstrated that its transcriptional activity can be changed under specific conditions, it is still used as a constitutive promoter in many in vitro and in vivo studies and even in clinical gene therapy trials. the basis of our experiments was construction of different systems (stably transfected cell lines, stably transfected tumors, and transiently transfected muscles) in which expression of the gfp reporter gene was regulated either by the inducible p21 or cmv promoter. these systems allowed us to follow promoter activity simply by quantifying fluorescence intensities either by fluorescence microscopy and fluorometer in vitro or by non - invasive in vivo imaging using a fluorescence stereomicroscope, assuming that fluorescence of the cells is proportional to promoter - driven expression. for construction of stably transfected tumor cell lines, two histologically different murine cell lines were used : mammary adenocarcinoma and fibrosarcoma. at the end of the culturing period in the selection agent namely, studies demonstrated that the cmv promoter exhibits cellular specificity and that it is active only in cell types which are naturally infected by the virus [11, 25, 26 ]. these studies were done on transgenic mice expressing a reporter gene under the control of cmv, thus, in non - transformed non - cancer cells. in our case, ts / a, which is murine adenocarcinoma of the mammary glands, and lpb, which is murine fibrosarcoma, cell lines were used. although the cell lines in our study were transformed cancer cell lines, they are derived from two different cell types. therefore, it can be speculated that differences in the percentage of stably transfected cells were due to the inherent differences in these cell lines with regard to susceptibility to infection with cmv. another observation during the preparation of stable cell lines was that initial high fluorescence intensity decreased after every round of replication in cell lines with the cmv promoter, but not in the cell line with the p21 promoter. several mechanisms for the transient expression of the delivered transgene have been described in the literature, such as loss of vector dna from the transduced cells and transcriptional inactivation of the promoter [27, 28 ]. in our case, the loss of vector dna could not be a major limiting factor for sustained transgene expression, since stably transfected cells were resistant to increasing concentrations of geneticin, meaning that they expressed the selective marker encoded on the plasmid, which demonstrates that the plasmid dna is present in the cells. in addition, results of our previous study, showing that long - term transgene expression can be maintained even after transient transfection in muscle, support the argument that the major cause for the observed loss of transgene expression was promoter inactivation rather than the loss of plasmid. dna methylation of the promoter is the most frequent justification for inactivation of the promoter [1, 2, 4, 5, 29 ]. the process of promoter methylation is mediated by a class of enzymes that covalently link a methyl group to the promoter region of the gene leading to modification of accessibility of transcriptional factors to the promoter. to test if the observed decrease in reporter gene expression in our study was due to methylation of the cmv promoter, freshly thawed cells were first passaged for 1 month to allow methylation of dna to take place and then treated with the demethylation agent 5-aza-2dc, which is one of the best characterized drugs used for reactivation of methylated promoters [2, 3032 ]. the fluorescence intensity of the first passage of freshly thawed cells was taken as the reference for the unmethylated state. after the treatment, an obvious increase in fluorescence was obtained, which was more pronounced in the ts / a egfp cell line (6.9) than in the lpb egfp cell line (1.4). similar to our results, reactivation of cmv - driven reporter gene expression after treatment with 5-aza-2dc was reported in a study using lipofection to stably transfect a human glioblastoma cell line. interestingly, in our study, the increase in fluorescence after (re)activation was higher than would be expected from the reference (fluorescence intensity of the cells from the first passage), especially in the ts / a cell line, indicating that some other mechanisms were involved in the observed (re)activation. namely, in addition to its demethylating function, 5-aza-2-dc has also been shown to possess significant cytotoxic and anticancer activity. in fact, it was originally developed as an anticancer agent and was as such used in many preclinical and clinical trials. there are two lines of evidence supporting the involvement of stress in the observed upregulation in our study. the first is that some upregulation occurred also after treatment with 5-aza-2dc in the control group with the p21 promoter. these results are in accordance with other studies [3438 ], where cytotoxicity of 5-aza-2dc was linked to its ability to induce dna damage, leading to activation of the p53 and consequently p21 pathways. the next line of evidence supporting the involvement of stress is upregulation of the cmv promoter which was obtained after exposure of cells to ir and treatment with cddp. therefore, the increase in fluorescence intensity above the reference value in the two stably transfected cell lines with the cmv promoter is most probably the sum of demethylation of the cmv promoter and upregulation of the cmv promoter due to the cytotoxic action of 5-aza-2dc. however, to fully validate the involvement of dna methylation in the (re)activation of the cmv promoter, further experiments, such as methylation - sensitive restriction enzyme analysis, should be performed. to test whether inactivation of the cmv promoter also occurs in in vivo conditions, in vitro results we only used the ts / a egfp tumor model since the reporter gene expression in the lpb egfp cell line was too low and could not be detected transcutaneously due to technical limitations of the fluorescence stereomicroscope. in the ts / a egfp tumors, the effects of treatment with 5-aza-2dc were not as obvious as in vitro ; only the trend of increased fluorescence intensity was indicated. the reason for the observed difference between the in vitro and in vivo results is most probably due to the fast growth of tumors which limit the duration of the experiment (10 days compared to 1 month used in in vitro experiments) and consequently the time needed for methylation to occur. this observation is also supported by the fact that the trend was more pronounced toward the end of the experiment. to our knowledge, our study is the first study dealing with methylation of the cmv promoter in tumors. other studies dealing with methylation of cmv were done in normal tissues and their results are inconsistent. association of transcriptional silencing with methylation of the cmv promoter was demonstrated in rat muscles using adenoviral gene delivery. on the other hand, the results of other studies showed that methylation is not responsible for transient transgene expression in vivo after adenovirus gene transfer in the mouse liver and after non - viral vectors delivery in the mouse lung. in these reports, promoter attenuation due to inflammatory processes was proposed as an alternative mechanism for the decrease in transgene expression. to confirm that methylation of the cmv promoter is associated with transcriptional silencing in muscle also after non - viral gene delivery methods, as was demonstrated for viral delivery, additional experiments were performed in transiently transfected muscles carrying cmv promoter - driven gfp. treatment with 5-aza-2dc was performed 1 month after transfection, when the fluorescence intensity of gfp reached 50% of the highest value, and we could anticipate that there was enough time for methylation to occur. therefore, our results indicate that methylation could be responsible for the observed increase in fluorescence intensity after transient transfection in muscle and support the results obtained in rat muscle after adenoviral - mediated transfection. the next part of our study dealt with the stress - induced upregulation of the cmv promoter. we used the same systems as for the methylation tests, but instead of treatment with 5-aza-2dc, we utilized two standard anticancer treatments : local ir and systemic treatment with the chemotherapeutic agent cddp that cause dna damage. to avoid the effect of methylation the exposure of cells to ir and cddp induced a significant increase in fluorescence intensity compared to untreated controls. however, the magnitude of this increase varied among the cell lines, being high in ts / a egfp cells and low in lpb egfp cells. furthermore, the magnitude of induction was similar to that obtained with the inducible p21 promoter. therefore, in our study in lpb cells, the cmv promoter was as inducible as the known inducible p21 promoter [18, 19 ]. similar observations have been previously demonstrated for different cell lines using different reporter genes and different delivery systems [13, 14, 39 ]. for example, the chemotherapeutic agent doxorubicin was reported to upregulate cmv promoter - driven reporter gene (luciferase) expression in stably transfected cell lines [14, 39 ] and after transient transfection using lipofectamine and an adenoviral vector. ir (10 gy) was also reported to upregulate a cmv promoter - driven reporter gene in cells transfected using electroporation. in our study, significant upregulation of the cmv promoter with ir and cddp was also demonstrated in vivo in tumors as well as in transiently transfected muscles. similar to our study, luminescence - based whole body imaging showed upregulation of the cmv promoter between 4 and 10 days after treatment of animals with doxorubicin. this upregulation of the cmv promoter is of special importance when gene therapy is intended to be combined with other standard cancer treatments, such as radiotherapy and chemotherapy. increased expression of the therapeutic protein and consequently an increased therapeutic effect can occur after treatment of patients with standard therapy. non - invasive imaging methods should be taken into consideration when gene therapy is combined with treatments that upregulate the promoter used in the gene therapy vector, as non - invasive imaging can give the exact time dependence of the activity of the promoter and therefore optimal planning for the combination of therapies can be achieved. fluorescence imaging has in this case, an advantage over luminescence imaging, since no additional substrate is required for visualization of transgene expression and can be safely used over a prolonged period of time, although with bioluminescence imaging lower levels of the reporter gene can be detected. our study demonstrated that the cmv promoter can be upregulated by different treatments. observed alterations in the activity of the cmv promoter limit the usefulness of this widely used promoter as a constitutive promoter and highlight the importance of proper choice of promoter linked to the gene of interest for success of gene therapy as well as basic research. on the other hand, inducibility of cmv promoters can be beneficially used in gene therapy when combined with standard cancer treatment, such as radiotherapy and chemotherapy. non - invasive imaging methods have great potential for development of these treatment combinations that will have an enhanced antitumor effect at the same level of normal tissue damage. | purposethe cytomegalovirus (cmv) promoter is one of the most commonly used promoters for expression of transgenes in mammalian cells. the aim of our study was to evaluate the role of methylation and upregulation of the cmv promoter by irradiation and the chemotherapeutic agent cisplatin in vivo using non - invasive fluorescence in vivo imaging.proceduresmurine fibrosarcoma lpb and mammary carcinoma ts / a cells were stably transfected with plasmids encoding cmv and p21 promoter - driven green fluorescent protein (gfp) gene. solid ts / a tumors were induced by subcutaneous injection of fluorescent tumor cells, while leg muscles were transiently transfected with plasmid encoding gfp under the control of the cmv promoter. cells, tumors, and legs were treated either by dna methylation inhibitor 5-azacytidine, irradiation, or cisplatin. gfp expression was determined using a fluorescence microplate reader in vitro and by non - invasive fluorescence imaging in vivo.resultstreatment of cells, tumors, and legs with 5-azacytidine (re)activated the cmv promoter. furthermore, treatment with irradiation or cisplatin resulted in significant upregulation of gfp expression both in vitro and in vivo.conclusionsobserved alterations in the activity of the cmv promoter limit the usefulness of this widely used promoter as a constitutive promoter. on the other hand, inducibility of cmv promoters can be beneficially used in gene therapy when combined with standard cancer treatment, such as radiotherapy and chemotherapy. |
female stress urinary incontinence is an ancient problem. following the " hammock hypothesis " by delancey, which describes the pathophysiology of female stress incontinence (1), the tension - free vaginal tape (tvt) procedure for treatment of stress urinary incontinence in women was introduced in 1996 (2). the tvt procedure is a minimally invasive surgical method for treating female urinary incontinence that has gained widespread popularity. the procedure requires positioning of a vaginal tape precisely underneath the mid - urethra with no tension, to restore urethral support. with techniques used previously, most studies report a high cure rate and long - term durability (3). surgeons who consider adopting the tvt procedure for their patients need to give attention not only to the efficacy of the procedure but also to the potential complications and untoward effects which may impact upon patient satisfaction and quality of life. voiding dysfunction is a well recognized complication when using suburethral sling procedures (4, 5). it has been reported that subjective and objective voiding patterns could change after the tvt procedure (6). voiding dysfunction has been estimated to occur at a rate of 1.5 - 20% after the tvt procedure (7, 8). therefore, it is of interest to understand the reason why significant postoperative voiding dysfunctions follow the procedure and to determine whether those symptoms are predictable preoperatively. in this study, we evaluated comprehensive risk factors that may be predictive of postoperative voiding dysfunction, and factors having impact on patient satisfaction after the tvt procedure. between march 1999 and may 2002, 437 women with stress urinary incontinence underwent the tvt procedure in the urology department at our institution. we excluded 90 patients with a maximum follow up of less than six months and/or incomplete postoperative data and a further 62 patients without preoperative data to comprehensively analyze risk factors for postoperative voiding dysfunction. we retrospectively analyzed the remaining 285 patients by chart - reviews or telephone survey (by one person). the evaluation of surgical results including satisfaction, sense of urine loss, pad test, and others was performed six months after the surgery. average age, mean symptom period, and mean parity were 51 yr, 6.9 yr, and 2.8 times, respectively. preoperative symptom grade was i (loss of urine only with coughing, sneezing or lifting heavy objects) in 128 patients, ii (loss of urine with minimal activity such as walking or arising from the sitting position) in 153 and iii (totally incontinent while upright) in four patients (9). the study population included 10 patients who had previously undergone anti - incontinence surgeries, such as the raz procedure, fascia lata sling, burch colposuspension and anterior vaginal wall sling. six patients had a history of vaginal hysterectomy and 45 patients underwent transabdominal hysterectomy in the past. the reasons for hysterectomy were uterine prolapse (n=2), cervical cancer (n=3), and abnormal uterine bleeding or uterine myoma (n=46). concurrent hysterectomy, correction of cystocele, stone removal from the ureter or bladder, urethral caruncle excision and posterior vaginal repair were performed in seven, six, two, one and 20 patients, respectively. most of the procedures (n=249) were performed under local and intravenous anesthesia (midazolam 1.5 mg and propofol 1.5 mg / kg) except for 36 women who needed general or spinal anesthesia for their concurrent procedures (table 1). preoperative evaluation included medical history, physical examination, urinalysis, urine culture, a 3 days voiding diary, 1-hr pad test proposed by the international continence society (10), postvoid residual urine volume measurement, uroflowmetry, and urodynamic study including measurement of valsalva leak point pressure (vlpp), maximal urethral closing pressure (mucp), and pressure flow study. all surgeries were performed by one urologist with even tension adjustment using long mayo scissors as a spacer. the bladder was emptied and catheter was not indwelled at the end of the procedure. if an adequate voiding was noted, determined as two consecutive voidings with a residual urine volume less than 100 ml, the patients were discharged within 24 hr. if a patient failed to void or if the bladder was injured during the procedure, a urethral catheter was placed and then removed the next day. for patients who failed to void or who were found to have a postvoid residual urine volume greater than 100 ml on two consecutive voidings, intermittent self - catheterization was recommended. voiding dysfunction was defined as having a peak flow rate less than 10 ml / sec (straining voiding) or a residual urine volume greater than 30% of the maximum cystometric capacity (poor emptying) on two or more readings (11). an ultrasound postvoid residual was measured. between patients with (n=29) and without (n=256) voiding dysfunction, multiple factors related to patient characteristics (age, parity, body mass index, history of hysterectomy or anti - incontinence surgery), symptoms and physical examination (symptom grade, 1-hr pad test, presence of concomitant urge incontinence, and cystocele), preoperative urodynamic study (peak urinary flow, volume of residual urine, mucp, vlpp, maximal bladder capacity, maximal detrusor pressure, and detrusor pressure at peak flow rate), combined surgeries (simultaneous hysterectomy, anterior and posterior colporrhaphy, or correction of cystocele), and bladder perforation by a needle instrument were compared respectively. uroflowmetry pattern had a normal configuration (uninterrupted) and bell - shaped form in most subjects and was therefore not analyzed. we determined the subjective and objective cure rate by a questionnaire and clinical examination, respectively. if patients had no urine loss after the procedure or social continence, a term coined in a previous report meaning that any perceived wetting by the patient was controlled by tissues or a small minipad, we considered them to be subjectively cured. we determined the objective cure rate by a 1-hr pad test and a stress test. patients with pads containing less than 2 g of urine and a negative stress test were considered cured. all patients were requested to answer a global satisfaction question in one of four ways, such as " very satisfied ", " satisfied ", " so - so ", and " dissatisfied ", and an intention to recommend the surgery to others in two ways, such as " yes " or " no ", at six months after their surgery. we correlated patients ' satisfaction with postoperative voiding status and investigated which factor is related to the patients ' global satisfaction. univariate analyses were performed using the student 's t - test, with p<0.05 considered statistically significant. between march 1999 and may 2002, 437 women with stress urinary incontinence underwent the tvt procedure in the urology department at our institution. we excluded 90 patients with a maximum follow up of less than six months and/or incomplete postoperative data and a further 62 patients without preoperative data to comprehensively analyze risk factors for postoperative voiding dysfunction. we retrospectively analyzed the remaining 285 patients by chart - reviews or telephone survey (by one person). the evaluation of surgical results including satisfaction, sense of urine loss, pad test, and others was performed six months after the surgery. average age, mean symptom period, and mean parity were 51 yr, 6.9 yr, and 2.8 times, respectively. preoperative symptom grade was i (loss of urine only with coughing, sneezing or lifting heavy objects) in 128 patients, ii (loss of urine with minimal activity such as walking or arising from the sitting position) in 153 and iii (totally incontinent while upright) in four patients (9). the study population included 10 patients who had previously undergone anti - incontinence surgeries, such as the raz procedure, fascia lata sling, burch colposuspension and anterior vaginal wall sling. six patients had a history of vaginal hysterectomy and 45 patients underwent transabdominal hysterectomy in the past. the reasons for hysterectomy were uterine prolapse (n=2), cervical cancer (n=3), and abnormal uterine bleeding or uterine myoma (n=46). concurrent hysterectomy, correction of cystocele, stone removal from the ureter or bladder, urethral caruncle excision and posterior vaginal repair were performed in seven, six, two, one and 20 patients, respectively. most of the procedures (n=249) were performed under local and intravenous anesthesia (midazolam 1.5 mg and propofol 1.5 mg / kg) except for 36 women who needed general or spinal anesthesia for their concurrent procedures (table 1). preoperative evaluation included medical history, physical examination, urinalysis, urine culture, a 3 days voiding diary, 1-hr pad test proposed by the international continence society (10), postvoid residual urine volume measurement, uroflowmetry, and urodynamic study including measurement of valsalva leak point pressure (vlpp), maximal urethral closing pressure (mucp), and pressure flow study. all surgeries were performed by one urologist with even tension adjustment using long mayo scissors as a spacer. the bladder was emptied and catheter was not indwelled at the end of the procedure. if an adequate voiding was noted, determined as two consecutive voidings with a residual urine volume less than 100 ml, the patients were discharged within 24 hr. if a patient failed to void or if the bladder was injured during the procedure, a urethral catheter was placed and then removed the next day. for patients who failed to void or who were found to have a postvoid residual urine volume greater than 100 ml on two consecutive voidings, intermittent self - catheterization was recommended. voiding dysfunction was defined as having a peak flow rate less than 10 ml / sec (straining voiding) or a residual urine volume greater than 30% of the maximum cystometric capacity (poor emptying) on two or more readings (11). between patients with (n=29) and without (n=256) voiding dysfunction, multiple factors related to patient characteristics (age, parity, body mass index, history of hysterectomy or anti - incontinence surgery), symptoms and physical examination (symptom grade, 1-hr pad test, presence of concomitant urge incontinence, and cystocele), preoperative urodynamic study (peak urinary flow, volume of residual urine, mucp, vlpp, maximal bladder capacity, maximal detrusor pressure, and detrusor pressure at peak flow rate), combined surgeries (simultaneous hysterectomy, anterior and posterior colporrhaphy, or correction of cystocele), and bladder perforation by a needle instrument were compared respectively. uroflowmetry pattern had a normal configuration (uninterrupted) and bell - shaped form in most subjects and was therefore not analyzed. we determined the subjective and objective cure rate by a questionnaire and clinical examination, respectively. if patients had no urine loss after the procedure or social continence, a term coined in a previous report meaning that any perceived wetting by the patient was controlled by tissues or a small minipad, we considered them to be subjectively cured. we determined the objective cure rate by a 1-hr pad test and a stress test. patients with pads containing less than 2 g of urine and a negative stress test were considered cured. all patients were requested to answer a global satisfaction question in one of four ways, such as " very satisfied ", " satisfied ", " so - so ", and " dissatisfied ", and an intention to recommend the surgery to others in two ways, such as " yes " or " no ", at six months after their surgery. we correlated patients ' satisfaction with postoperative voiding status and investigated which factor is related to the patients ' global satisfaction. univariate analyses were performed using the student 's t - test, with p<0.05 considered statistically significant. mean operative time and the length of hospital stay were 28.4 min and 1.4 days, respectively. there was a significant decrease in peak flow rate by uroflowmetry preoperatively vs. postoperatively (30.1 vs. 23.5 ml / sec, p=0.0001) with a mean decline of 6.6 ml / sec. however, change in volume of residual urine was not significant (16.7 vs. 20.5 ml, p=0.271). two hundred and sixty - one patients (91.6%) favored the procedure in answers to a global satisfaction questionnaire. there were 268 patients (94.0%) with an intention to recommend the surgery to others. patient 's peak flow rate only showed significant differences between the normal voiding group and voiding dysfunction group (preoperative peak urinary flow rate : 30.8 vs. 23.7 ml / sec, p=0.004, table 2). when we generated an roc curve for peak urinary flow rate, the area under the curve was 0.684 ; sensitivity and specificity were 22.3% and 55.2% at 20.9 ml / sec, and 33.6% and 34.5% at 23.9 ml / s, respectively (fig. when we compared the difference in patients ' satisfaction rate between patients with preoperative peak flow rate lower and higher than 20 ml / sec according to this result, there was no statistical difference between them (data not shown). also, there was no statistically significant difference between those with and without voiding dysfunction in terms of cure rates, patients ' satisfaction and intention to recommend the surgery to others (table 3). postoperative peak urinary flow rate was less than 12 ml / sec in 23 patients, less than 10 ml / sec in 17 patients, and less than 8 ml / sec in six patients. the mean decline in peak urinary flow rate from their preoperative value was 13.4 ml / sec for patients with less than 12 ml / sec, 12.7 ml / sec for patients with less than 10 ml / sec, and 12.9 ml / sec for patients with less than 8 ml / sec. when we stratified all patients by these 3 postoperative peak flow rate values, significantly lower level of global satisfaction was noted from patients with a peak urinary flow rate less than 8 - 10 ml / sec, though the statistics were not significant for patients passing 12 ml / sec (table 4). the mean preoperative peak urinary flow rates were 21.6 and 20.3 ml / sec in patients with less than 10 and 8 ml / sec postoperatively. in patients with a preoperative peak urinary flow rate less than 20 ml / sec, of less than 10 ml / sec than more than 20 ml / sec (table 5). it offers advantages over conventional repairs : it is quick and can be done under local anesthesia. though the procedure is well known as an effective approach with a high long - term cure rate, boustead (12) indicated that 1.5 - 20% of patients can develop postoperative voiding dysfunction. the question is whether these postoperative voiding dysfunctions are predictable preoperatively, whether patients ' satisfaction is related with voiding dysfunction, and whether there are pre- or postoperative risk factors predictive of patients ' satisfaction. we previously reported that peak urinary flow rate is an independent prognostic factor for immediate postoperative urinary retention after the tvt procedure and its significant influence on patient satisfaction (13). prevalence and predictors of voiding dysfunction after various sling procedures have been identified but unfortunately vary among different institutions. the most likely reason for this difference is the lack of standard definitions of voiding dysfunction. for instance, groutz. (14) defined it as a maximum flow rate less than 12 ml / sec, or residual urine volume greater than 100 ml. (15) defined voiding difficulty as a peak flow rate less than 15 ml / sec (if at least 200 ml had been voided), or greater than 200 ml of residual urine on two or more readings. lose. (9) defined impaired bladder emptying as a peak flow rate less than 15 ml / sec for patients younger than 60 yr and less than 10 ml / sec for patients 60 yr or older (at a voided volume greater than 150 ml). in terms of the tvt procedure, (16) defined normal voiding as a postvoid residual urine < 100 ml, frequency of six of fewer voids per day and two or fewer per night, and a urinary stream considered normal by the patient. the definition of voiding dysfunction in the current study is a peak urinary flow rate less than 10 ml / sec (voided volume greater than 100 ml) as straining voiding, or residual urine volume greater than 30% of the maximum bladder capacity as poor emptying on two or more readings. based on our definition, 29 (10.2%) patients were included in the voiding dysfunction group. among the clinical factors, preoperative peak flow rate only showed significant differences between the normal voiding and voiding dysfunction groups (table 2). when we generated an roc curve for peak urinary flow rate, the area under the curve was 0.684 ; sensitivity and specificity were 22.3% and 55.2% at 20.9 ml / sec, and 33.6% and 34.5% at 23.9 ml / sec, respectively (fig. 1). this means peak urinary flow rate can effectively estimate the risk of voiding dysfunction and predict postoperative urinary retention. anti - incontinence surgeries can not avoid inducing some degree of obstruction to the lower urinary tract. the tvt procedure is not much different in this regard, although this procedure is completely tension free. sander. (6) reported the peak urinary flow rate decreased from a preoperative 29.4 ml / sec to postoperative 24.5 ml / sec (p=0.036). there was a significant decrease in the peak urinary flow rate preoperatively versus postoperatively (30.1 ml / sec ; 23.5 ml / sec, respectively, p<0.001) with an average decline of 6.6 ml / sec in our study. from our results, the risk of developing voiding dysfunction can be effectively stratified by the average decline in peak urinary flow rates. the intriguing point is that voiding dysfunction itself did not have a significant impact on patient satisfaction in most patients. this means most patients are still satisfied with gaining continence at the expense of a decrease in flow rate and a 6.6 ml / sec decline in peak urinary flow rate is modest enough to endure for most of the patients. likewise, the preoperative peak flow rate predictive of voiding dysfunction, 20 ml / sec in the current study, could not effectively predict patients ' satisfaction after the surgery. furthermore, we could not find any relevance between patients ' satisfaction and preor postoperative risk factors in patients with normal voiding and even in patients with voiding dysfunction. however, when we stratified all patients into subgroups according to specific postoperative peak flow rates, if a patient already had a low peak urinary flow rate that was not able to buffer the inevitable decline, or the decline in peak urinary flow rate was severe enough to be less than 10 ml / sec, global satisfaction with the procedure was significantly compromised. this result signifies patients ' satisfaction depends much more on postoperative peak flow rate itself than on voiding dysfunction. though we could not find any significant correlation between preoperative peak flow rate and patients'satisfaction in this study, the descent of peak flow rate was more significant in patients with less than 10 and 8 ml / sec postoperatively and preoperative peak flow rate was already lower in them (about 20 ml / sec) comparing to the mean preoperative peak flow in all patients (about 30 ml / sec). in this context, this ostensibly " tension - free " procedure may actually only be " tension - tolerable " in a sense. in any situation where the tension is intolerable, patients ' satisfaction on the gaining of continence can be overcome by the disappointing urinary flow rate as shown in this study. therefore, all efforts decreasing tension should be taken in applying vaginal tape and particular attention should be given to patients with already low preoperative peak flow rate in terms of placement of vaginal tape and preoperative warning of potential voiding risk after the procedure. patient satisfaction on the highly curative tvt procedure is not significantly influenced by postoperative voiding dysfunction based on the current definition. however, the inevitable decline in peak urinary flow rates may compromise patient satisfaction with the procedure when their postoperative flow rate is less than 10 ml / sec. | this study was undertaken to identify risk factors for postoperative voiding dysfunction and factors having impact on patient global satisfaction after a tension - free vaginal tape (tvt) procedure. two hundred and eighty - five women who underwent the tvt procedure for stress urinary incontinence were analyzed to identify risk factors predictive of voiding dysfunction. postoperative voiding dysfunction was defined as a peak urinary flow rate (pfr) 30% of bladder capacity (incomplete emptying, n=13). the global satisfaction rate was 91.6%. voiding dysfunction developed in 29 (10.2%) patients. among the factors, pfr was only factor of significance for voiding dysfunction. there was no significant difference between patients with and without voiding dysfunction in terms of their satisfaction. but postoperative pfr < 10 ml / sec significantly compromised global satisfaction after the surgery. in those patients with a preoperative pfr < 20 ml / sec, there were more patients with postoperative pfr < 10 ml / sec. peak urinary flow rate is an important factor for the postoperative voiding dysfunction. the inevitable decline in pfr can compromise patients ' satisfaction with the procedure, when their postoperative pfr was < 10 ml / sec. |
excess adiposity increases the risk of developing a variety of pathological conditions, including type 2 diabetes, cardiovascular disease, steatohepatitis, and several types of cancer [1 - 4 ]. in the past two decades, advances in obesity research have led to the recognition that adipose tissue is an endocrine organ that secretes hormones or cytokines termed adipokines. adipokines play crucial roles in the regulation of appetite and satiety control, energy expenditure, insulin sensitivity and insulin secretion, endothelial function, and blood pressure. the adipokines leptin and adiponectin, which are primarily secreted by adipocytes, play a major role in the pathogenesis of obesity and its comorbidities, including type 2 diabetes and cardiovascular disease. on the other hand, recently, areas of active investigation focus on the molecular bases of metabolic inflammation and potential pathogenic roles in diabetes and cardiovascular disease. this inflammation develops in response to an excess of nutrient flux into metabolic tissues including adipose tissue, liver, and skeletal muscle and is currently recognized as an important link between obesity and insulin resistance. obesity - associated systemic inflammation is characterized by increased circulating concentrations of proinflammatory cytokines and chemokines, and activation of several kinases that regulate inflammation, including c - jun nh2 terminal kinase, ib - kinase /nuclear factor b and mammalian target of rapamycin / s6 kinase that interfere with insulin action in adipocytes and hepatocytes. increasing evidence supports that obesity - induced inflammation is mediated primarily by immune cells such as the macrophages and t lymphocytes in metabolic tissues. in particular, a significant advance in our understanding of obesity - associated inflammation and insulin resistance has been recognition of the critical role of adipose tissue macrophages (atms). atms are a prominent source of proinflammatory cytokines, such as tumor necrosis factor (tnf)- and interleukin (il)-6, that can block insulin action in adipocytes via autocrine / paracrine signaling and cause systemic insulin resistance via endocrine signaling, providing a potential link between inflammation and insulin resistance [10 - 12 ]. in both humans and rodents, atms accumulate in adipose tissue with increasing body weight and their content correlates positively with insulin resistance [13 - 15 ]. importantly, tissue macrophages are phenotypically heterogeneous and have been characterized according to their activation / polarization state as m1 (or " classically activated " proinflammatory macrophages) or m2 (or " alternatively activated " noninflammatory macrophages [16 - 18 ]). m2 atms predominate in lean mice, whereas obesity induces the accumulation of m1 atms with high expression of tnf-, il-6 and inducible nitric oxide synthase, leading to a proinflammatory environment in white adipose tissue (wat). thus, both recruitment and proinflammatory activation of atms is required for the development of insulin resistance in obese mice. chemokines, a family of cytokines that induce leukocyte chemotaxis, are deeply involved in the development of allergic diseases and autoimmune diseases. more than 50 chemokines which exhibit various physiological and pathological properties have been discovered to date. recent studies have shown that preadipocytes and adipocytes express chemokines, and have demonstrated the infiltration of bone marrow - derived macrophages into obese adipose tissue, which is involved in the development of insulin resistance. this review summarizes various roles of chemokine and chemokine receptor (chemokine system) in inflammation, and also focuses on some of the latest findings on the chemokine systems that link adipose tissue inflammation with the pathology of insulin resistance. chemokines attract leukocytes to areas of inflammation and lesions and play a key role in leukocyte activation. ever since, many studies have been conducted to identify the roles of chemokines in acute, neutrophil - predominant inflammation and chronic, monocyte- and lymphocyte - predominant inflammation. to date, more than 50 chemokines have been identified in humans (table 1). chemokines have four conserved cysteine residues. based on the motif patterns involving two n - terminal cysteine residues, chemokines can be classified into the following four subfamilies : cxc, cc, c, and cx3c (where x is any amino acid residue) (table 1). the cxc chemokines are mainly chemotactic for neutrophils and are known for their involvement in acute inflammation whereas most of cc chemokines act on monocytes, t cells, eosinophils, and basophils, which mediate chronic inflammation and allergy. all chemokines signal via seven - transmembrane g - protein - coupled receptors (or chemokine receptors). to date, 18 chemokine receptors have been identified, including 11 cc chemokine receptors, 6 cxc chemokine receptors, and one for each c (xcr1) and cx3c chemokine receptor (table 1). most chemokines bind to several chemokine receptors and chemokine receptors have overlapping ligand specificities. although some chemokines have a one - to - one specificity (specific receptors), most chemokines bind to the same receptor (shared receptors) (table 1). for example, four chemokine ligands, including monocyte chemoattractant protein (mcp)-1, mcp-2, mcp-3, and mcp-4 bind to c - c motif chemokine receptor 2 (ccr2) receptor. even when multiple ligands interact with a single receptor, diverse effects are produced by different ligands because the binding affinity and the resulting effect differ across ligands. furthermore, as chemokines are differently expressed, distributed, and regulated in cells and tissue, they may play different roles in physiological conditions or diseases. although chemokines appear to exhibit a high degree of functional redundancy, the functions of the individual chemokines can be classified into two types : ' inducible ' or ' inflammatory ' and ' constitutive ' or ' homeostatic '. inflammatory cxc and cc chemokines promptly recruit neutrophils, monocytes and eosinophils to the site of injury or inflammation, cluster on chromosomes 4 and 17, and share the same receptor. furthermore, a number of inflammatory chemokines act together as a potent inducer of cell migration (quantitative regulation). since the late 1990s, it has been clearly demonstrated that several chemokines exhibit different properties from those of inflammatory chemokines. these homeostatic chemokines act specifically and constantly on lymphocytes and dendritic cells, and are characterized by relatively specific ligand - receptor pairs. it is believed that homeostatic chemokines modulate the physiological homing of naive lymphocytes and dendritic cells to secondary lymphoid tissue, and that they are involved in highly specific migration control (qualitative control). there have been great advances in chemokine research through the elucidation of the potential pathogenic roles of inflammatory chemokines in allergic diseases and autoimmune diseases, including bronchial asthma, rheumatoid arthritis, and inflammatory bowel disease. in such diseases, chemokines and their receptors promote the onset and modify the severity of each of these diseases through the selective accumulation of leukocytes in the site of inflammation. mcp-1, a prototype of the chemokine, as well as mip-1 and mip-1 are the cc chemokine subfamily that are isolated from monocyte and macrophages, and have been shown to be closely linked with the development of many inflammatory diseases. the signals of cc chemokine receptors ccr1, ccr2, and ccr5 are also involved in the pathogenesis of multiple sclerosis, characterized by chronic inflammation induced by monocytes, macrophages, and t cells (table 1). in acute respiratory distress syndrome, on the other hand, cxc chemokines act on neutrophils, causing a large number of neutrophils to infiltrate into the lung, thus triggering a severe acute inflammatory reaction. furthermore, in recent years, increasing evidence has shown that chemokines and their receptors play pathogenic roles on cancer, cardiovascular disease, neurodegenerative disorder, and metabolic diseases which have led many researchers to explore the role of chemokines on various other diseases associated with chronic inflammation. it has become increasingly evident that chemokine system also involves chronic subacute inflammation that is the common underlying condition of obesity, insulin resistance, and type 2 diabetes. the interaction of mcp-1 with its receptor ccr2 is considered pivotal in obesity - induced insulin resistance. several groups have reported that mice with targeted deletions in the genes for mcp-1/ccl2 and its receptor ccr2 have decreased atm content, decreased inflammation in fat, and protection from high - fat (hf) diet - induced insulin resistance. conversely, mice overexpressing mcp-1 in adipose tissues have increased numbers of atms along with insulin resistance. therefore, the mcp-1-ccr2 axis is of central importance for promoting atm recruitment and insulin resistance in mice. more recent studies, however, have produced conflicting results and indicated greater complexity than suggested by earlier reports. loss of mcp-1 neither attenuates obesity - associated macrophage recruitment to wat nor improves metabolic function, suggesting that mcp-1 is not critical for obesity - induced atm recruitment and systemic insulin resistance. furthermore, although ccr2 mice fed a hf diet have fewer macrophages in wat compared with wt mice ccr2 deficiency does not normalize atm content and insulin resistance to the levels in lean animals, indicating that atm recruitment and subsequent insulin resistance are also regulated by mcp-1-ccr2 independent signals. in fact, other chemokine systems have also been implicated in atm infiltration in obese mice [30 - 32 ]. indeed, serum levels of cxcl5 produced by atm are increased in obese mice and humans, and mice lacking cxcr2, the receptor of cxcl5, show resistance to the onset of obesity - induced disorders of glucose metabolism. moreover, previous research has suggested that the concentration of keratinocyte, which is homologous to human il-8, increases in the adipose tissue and plasma in an obese model, and that bone marrow chimera mice with bone marrow cells from cxcr2 deficient mice show decreased obesity - induced inflammation and insulin resistance compared to controls. however, additional unidentified chemokine / chemokine rereceptor pathways that may play significant roles in atm recruitment and insulin sensitivity remain to be fully identified. we recently identified and characterized a critical role for ccr5, a different cc chemokine receptor, in the regulation of the adipose tissue inflammatory response to obesity and the development of insulin resistance (fig. first, expression of ccr5 and its ligands is significantly increased and is equal to that of ccr2 and its ligands in the wat of obese mice, particularly in the macrophage fraction. second, fluorescence - activated cell sorter analysis clearly demonstrates a robust increase in ccr5 atms in response to a hf diet even after normalizing for stromal vascular cell number and fat weight. third, and most important, ccr5 mice are protected from insulin resistance, hepatic steatosis, and diabetes induced by hf feeding. it is noteworthy that two distinct models, both ccr5 mice and chimeric mice lacking ccr5 only in myeloid cells, are protected from hf diet - induced hyperinsulinemia and glucose intolerance through a reduction in atm accumulation. finally, it is interesting that an m2-dominant shift in atm is induced in obese ccr5 mice. therefore, we conclude that deficiency of ccr5 causes an m2-dominant phenotypic shift in atms, which contributes to the attenuation of obesity - induced insulin resistance. our study provides new information about the role of ccr5, a new chemokine system, in obesity - induced insulin resistance in an animal model. it is important that the effects of ccr5 do not appear to result from global alterations in adipocyte biology. thus, decreased atm recruitment does not appear to be secondary to changes in adiposity because the adipocyte size of obese ccr5 mice and age - matched controls is similar. moreover, expression of adipocyte - derived factors such as leptin and adiponectin in wat and plasma levels are similar between genotypes. additionally, a bone marrow transplantation study revealed that lack of ccr5 expression in macrophages alone was sufficient to protect mice from the hf diet - induced insulin resistance ; this was associated with a marked reduction in atm infiltration. these data support the conclusion that ccr5 atms are important in the development and maintenance of obesity - induced adipose tissue inflammation and insulin resistance in mice. recent human studies have also shown upregulation of the expression of not only mcp-1-ccr2 but also other cc chemokines (ccl5, ccl7, ccl8, ccl11) and their receptors (ccr1, ccr3, ccr5) in the visceral fat of morbidly obese individuals in whom macrophage infiltration has been confirmed. taken together, ccr5-mediated signals in the adipose tissue may be involved, in some way, in the induction and maintenance of obesity - induced inflammation and in the development of insulin resistance in both rodents and humans. do the two cc chemokine receptors, ccr2 and ccr5, play common or unique roles in obesity - induced adipose tissue inflammation and insulin resistance ? importantly, no significant compensatory increase in the expression for ccr2, or vice versa, has been found. therefore, ccr5, independently from and/or cooperatively with ccr2, plays a role in the maintenance of atm dysfunction and insulin resistance once obesity and its metabolic consequences have been established (fig. moreover, similar to the case in ccr5 mice, hf diet - induced increased fat mass are minimally affected by ccr2 deficiency, and obese ccr2 mice matched for adiposity with controls showed reduced atm recruitment and improved systemic insulin sensitivity. therefore, the effects of either ccr5 or ccr2 do not appear to result from global alterations in adipocyte biology. however, hf feeding promotes accumulation of m1 macrophages in wat of wt mice, whereas increase in m1 atms are markedly suppressed in ccr5 mice. in contrast, m2 expression within atms is increased in ccr5 mice on a hf diet, suggesting that deficiency of ccr5 causes an m2-dominant phenotypic shift in atms, which contributes to the attenuation of obesity - induced insulin resistance. recent studies have demonstrated that obesity is associated with increased accumulation of not only macrophages but also t cells in adipose tissue. wu., showed that rantes / ccl5 mrna levels are highly correlated with the t cell marker cd3 in human visceral adipose tissue. however, the numbers of cd3 t cells, cd4 t cells and cd8 t cells did not differ in wat of hf diet - fed wt and ccr5 mice, suggesting that ccr5 deficiency affects atm recruitment more prominently. one important question concerns whether the loss of ccr5 affects the m1/m2 status in the bone marrow or peripheral blood (fig. the former, called proinflammatory / classical monocytes, preferentially accumulate in atherosclerotic plaques and exhibit a strong inflammatory response to lipopolysaccharide. in contrast, the latter, known as resident / remodeling / patrolling monocytes, participate in the resolution of inflammation. both ly6c and ly6c monocytes are recruited to sites of inflammation or injury (fig. although the relationship between the monocyte subtypes and their fate as m1/m2 macrophages remains unknown, that loss of ccr5 could cause alteration of ly6c and ly6c monocytes subsets at the level of either bone marrow or peripheral blood, and that this contributes to the m2-dominant shift of atm in obese ccr5 mice. our study provides new information regarding the role of ccr5 as a novel link among obesity, adipose tissue inflammation, and insulin resistance (fig. 1). however, many questions have yet to be answered, including how ccr5 and its ligands are induced in response to either a hf diet or obesity ; how ccr5 regulates m2 macrophages ; which metabolic tissue / organ is responsible for enhanced insulin sensitivity in ccr5 ; and of the 50 chemokines in metabolic diseases, what distinct roles are played by ccr5 ? as cytokines and chemokines work in networks, the effect of individual molecules on metabolic function depends on their place in the hierarchy of the network. several recent reports suggest that obesity increases macrophage infiltration to insulin target organs other than adipose tissue. for example, mcp-1-mediated infiltration of ccr2 bone marrow cells was observed in the liver of the ob / ob mice, and adenovirus - induced overexpression of mcp-1 in the liver of wild - type mice treated with hf diet was found to increase lipogenesis and to promote fatty liver disease. chemokine - mediated macrophage infiltration into the liver might be therefore related with the pathology of not only insulin resistance but also steatohepatitis. in light of these new data, however, ccr5 may be a promising therapeutic target for insulin resistance, metabolic syndrome, and type 2 diabetes. further work is required to gain a systematic understanding of how ccr5 and mcp-1-ccr2 as well as other chemokine systems, connect obesity, inflammation, and insulin resistance. vigorous research has been conducted to explore the physiological functions of chemokines in controlling inflammatory and immune response, as well as to elucidate their pathophysiological roles in the development of autoimmune diseases and allergic diseases. it has also become increasingly evident that chemokines play an important role in obesity - induced insulin resistance and its comorbidities, including type 2 diabetes and cardiovascular disease. mcp-1-ccr2 as well as other chemokine systems may be deeply involved not only in tissue- and organ - level inflammation caused by interaction between adipose tissue and macrophages, but also in the onset of localized inflammation caused by cross - talk between adipose tissue and other organs and the subsequent development of systemic insulin resistance. there are many questions, however, that have yet to be answered, including how chemokine expression is regulated in obesity, how chemokines regulate macrophage polarization, and what the different roles of over 50 chemokines are in metabolic diseases. further research on chemokines in relation to the molecular bases of metabolic inflammation and pathogenic roles in insulin resistance is expected to contribute to the development of new drugs that could control inflammation- or immune - mediated disorders such as metabolic syndrome and type 2 diabetes. | obesity is a state of chronic low - grade systemic inflammation. this chronic inflammation is deeply involved in insulin resistance, which is the underlying condition of type 2 diabetes and metabolic syndrome. a significant advance in our understanding of obesity - associated inflammation and insulin resistance has been recognition of the critical role of adipose tissue macrophages (atms). chemokines are small proteins that direct the trafficking of immune cells to sites of inflammation. in addition, chemokines activate the production and secretion of inflammatory cytokines through specific g protein - coupled receptors. atm accumulation through c - c motif chemokine receptor 2 and its ligand monocyte chemoattractant protein-1 is considered pivotal in the development of insulin resistance. however, chemokine systems appear to exhibit a high degree of functional redundancy. currently, more than 50 chemokines and 18 chemokine receptors exhibiting various physiological and pathological properties have been discovered. therefore, additional, unidentified chemokine / chemokine receptor pathways that may play significant roles in atm recruitment and insulin sensitivity remain to be fully identified. this review focuses on some of the latest findings on chemokine systems linking obesity to inflammation and subsequent development of insulin resistance. |
the aging, demographics, and memory study (adams) currently estimates that 14 percent of people aged 71 and older in the united states have dementia. the number of people aged 65 and older living with alzheimer 's disease is expected to nearly triple by 2050. however, research estimates that about one - third of general practitioners find the clinical management of dementia difficult. yet many medical students find the complexities of older people 's medical problems, including dementia, overwhelming [4, 5 ]. medical trainees struggle to see persons with adrd in a positive light [39 ]. point to how students ' aging - related attitudes negatively impact career and treatment decisions thereby contributing to the deficit of practitioners available to care for people with adrd. experiential learning models, designed to stretch thinking modalities, without increasing time requirements, may sufficiently alter medical trainees attitudes, so they feel more comfortable communicating with and caring for people with adrd [3, 6, 9, 10 ]. studies support that artistic interventions affect the attitudes of people caring for those with adrd [7, 9, 1113 ]. a study examining the impact of a poetry intervention at one care home and one day center found that poetry helped caregivers recognize the humanness of the adrd residents. in turn, this recognition motivated the caregivers to develop positive attitudes toward people with adrd and a desire to provide more humanistic care. at a nursing home, an artistic intervention exposing medical students to the joy dementia patients derived from the activity time slips, a creative story telling program including persons with dementia, expanded participants ' ability to see sufferers of adrd in a more positive light. at columbia school of medicine, nineteen medical students attended a single 90-minute museum - based art - centered program that engages individuals with adrd and caregivers. after the artistic intervention at columbia, the medical students indicated significant increases in comfort levels with adrd patients. research supports both how attitudinal shifts impact caregiving and the use of nonclinical learning interventions for helping medical trainees see people with adrd in a positive light. us news and world report ranked rowan university school of osteopathic medicine, a large medical school in the suburban philadelphia region of southern new jersey, as a 2016 top program in geriatric medical education. second - year medical students take a month - long required geriatrics course which covers the physiology of aging and public health topics. the third - year medical students complete a required four - week clinical rotation in geriatric medicine at local facilities. rowan university school of osteopathic medicine medical students at all levels of training received an invitation to participate in the workshop. researchers selected study participants by sending an email out to the first fifteen students who signed up to attend the workshop. this email informed the fifteen students about the research study and offered them an opportunity to participate in the study. within the week prior to the workshop, researchers sent out a second email confirming that the student would participate in the workshop and the research study. the students participating in the study reviewed and signed the consent form prior to attending the workshop. fourteen out of the fifteen workshop participants met the inclusion criteria of currently attending rowan university school of osteopathic medicine. the present pilot study only includes research data for the eleven medical students who completed all three study components : preintervention and postintervention das and half - hour postintervention semistructured interviews. eight first - year medical students, two third - year medical students, and one second - year student completed the preintervention and postintervention das and half - hour postintervention semistructured interviews. of these students, seven identified themselves as asian (asian = indian, pakistani, filipino, and chinese) (63%), one participant as black (9%), and three as white (27%). eight out of eleven student participants were male (57%). four out of the eleven study participants knew family members with adrd and five out of eleven expressed an interest in geriatrics prior to attending the workshop. researchers compared the present pilot study 's demographics to that of the student body which consisted of 47.3% males, 45.4% white, 39.6% asian, 8.3% black, and 3.5% hispanic out of a total of 647 students. medical students and assisted living facility residents with adrd gathered at a local continuing care community, located in new jersey, to work with gary glazner, the founder and executive director of the alzheimer 's poetry project, who ran the poetry workshop. residents with adrd, admitted to the dementia unit where the intervention took place, scored less than a twenty - four on the mini mental state examination (mmse is the accepted state - wide assessment as a part of the diagnostic protocol for dementia. a person that scores < 24 on they lacked the ability to independently perform adls (adl : feeding oneself, bathing, dressing, grooming, work, homemaking, and leisure) and iadls (iadl : ability to use telephone, shopping, food preparation, laundry, mode of transportation, responsibility for own medications, housekeeping, and ability to hand finances). a board certified geriatrician evaluated and deemed each person on the unit appropriate for admission based on a physical exam, mmse, adl, and iadls. requiring a short time commitment on the part of participants defines this intervention as a low - intensity learning intervention. prior to the poetry with adrd participants, the rowan students attended a one - hour overview lecture of the alzheimer 's poetry project by glazner at rowan university. at the dementia unit, glazner gave a brief, fifteen - minute introduction of his work to those in attendance. after the lecture, students and adrd residents, led by glazner, constructed a group poem about love. after the session, students conversed with the facility 's residents. in total, the intervention lasted three and a half hours. researchers measured preintervention attitudes with the das within the week of the intervention. one week following the intervention, researchers administered the postintervention das and conducted the postintervention, half - hour, semistructured qualitative interviews. das data exists for multiple studies evaluating medical students ' attitudes after participation in artistic interventions and would allow researchers to compare cross - study outcomes [7, 8 ]. being a statistically valid, 20-item scale, each item of the das falls on a 7-point likert scale ranging from 1 (strongly agree) to 7 (strongly disagree). the das holds convergent validity with the kogan attitudes toward older people scale, fraboni scale of ageism, attitudes towards disabled persons scale, and the interaction with disabled person scale. researchers completed statistical analysis of the das using spss (version 20.0, ibm corp. in addition to das data collected in this study, researchers performed ipa of the postintervention qualitative interviews, analyzing verbatim transcripts. the postintervention qualitative interview questions sought to discover students ' perceived knowledge of dementia, general outlook of dementia as a diagnosis, and viewpoints on caring for dementia patients. conducting half an hour postworkshop semistructured interview sessions with each participant, researchers asked participants the following questions : did you attend gary glazner 's poetry workshop ? why did you involve yourself in this activity ? have you had high contact, low contact, or no contact with dementia patients ? the two researchers recorded the postintervention qualitative interviews with a digital recorder with dragon naturally speaking software. did you attend gary glazner 's poetry workshop ? why did you involve yourself in this activity ? have you had high contact, low contact, or no contact with dementia patients ? the software electronically transcribed each interview and the two researchers reviewed transcription and made certain the transcripts held a verbatim accounting of students ' postintervention interviews. after data collection and transcription of the interview audio, noting changes in tempo, flow of speech, intonation, and volume, a researcher trained in medical anthropological ethnographic techniques performed first - pass qualitative coding of the transcript, identifying initial themes. the researcher then went on to conduct second - pass coding critically assessing the first - pass themes and collating the coded transcripts into general theme sets. after this aggregation of second - pass themes, the researcher structured the themes into primary and secondary data sets supported with verbatim quotations. another researcher working on the pilot study juxtaposed verbatim quotations to the primary and secondary themes ensuring accurate reporting. the workshop gave me more confidence i guess in not being timid or one foot back in terms of approaching people with dementia (4.3234) (4 = transcript number / student number, 3234 = line number where statement is located on transcript ; researchers used transcript and line number for the multiple review of themes derived from verbatim quotes) matched peers statements. for instance, student 3 's comments echoed those of student 10 's statement : this experience has made me more comfortable with speaking and conversing and interaction with people that are older ipa supports the grouping of comments like those from student 3 and student 10 into a singular theme. the secondary theme 3 (the workshop increased students ' confidence and comfort with approaching those with adrd) formed from this type of analytical deconstruction of the interview transcripts. during the postintervention qualitative interview sessions, participants also filled out the postintervention das questionnaire. previous researchers of medical students ' attitudes of geriatrics found that ipa delved deeper into the complexities of students ' behaviors. when soliciting medical students ' perspectives on geriatric education using ipa at the miller school of medicine, researchers uncovered themes often neglected by attitude scales. public health researchers also use ipa to overcome the limitation of quantitative approaches, the failure to expand upon the lived experience of people affected by changes in healthcare policy. many researchers accept that ipa offers necessary insights for making effective educational policies with the potential to impact healthcare outcomes. researchers assigned both the questionnaires and recordings a study i d number linking to the participant and left no identifiers on either the questionnaire or the audio recording. researchers deleted the associated key with study ids and associated participants after the labeling of the questionnaires and audio recordings with an i d study number. analysis of the postintervention semistructured interview transcripts occurred without access to the names of the participants. the students participated in the workshop freely, the students did not receive monetary incentives, and the intervention did not represent a course requirement although completion of the workshop would contribute to consideration for the new jersey institute of successful aging distinguished student award. the protocol for this research was reviewed and approved by the institutional review board of rowan university school of osteopathic medicine. this arts - based, poetry workshop significantly improved medical students ' attitudes towards individuals with adrd. the das data (n = 11) showed statistically significant results and the ipa (n = 11) offered insight into the positive impact of the poetry intervention. when administering the das, the lowest score is 20, neutral score is 80, and the highest score is 140. eleven students completed both pre- and postintervention tests with a preintervention mean of 107.09 11.85 (t(10) = 8.77, p < 0.001). after the postintervention administration, the overall das score changed positively and significantly to a mean of 121.82 10.38 (t(10) = 8.77, p < 0.001). o'connor and mcfadden when developing the das found that individuals with adrd family members may score higher at preintervention level. delving deeper into the mean das score data set, the present pilot study also examined whether or not das responses of rowan medical students with family members affected by adrd (4 out of 11 participants) showed significant variance from the students without family members affected by adrd (7 out of 12 participants). researchers conducted an independent samples test comparing overall das scores between participants with adrd family members to those without adrd relatives. the independent samples test shows that the p values for the overall preintervention das score p = 0.251 and overall postintervention das p = 0.707 lacked a significant difference (t(9) = 1.227, p = 0.636). these results support an equal variance between the two groups and suggest that the group with adrd family members did not score higher than the group without adrd family. table 4 summarizes the results of the paired samples t - tests which compared overall, subdomain, and item level das scores before and after the poetry workshop. ten out of twenty items showed statistically significant differences (table 4). based on the paired t - test analysis, researchers found that the das items 1, 3, 4, 8, 1216, and 19 show score improvements between the preintervention and postintervention tests using the das (table 3). on the das, these items are grouped under the subdomains of comfort and knowledge. representing items from the das knowledge subdomain items, 3, 12, 14, 15, and 19 displayed statistical significance. researchers calculated the greatest positive preintervention to postintervention change on items 14 and 19. on item 14 (people with adrd can enjoy life), nine out of eleven participants had a positive change between preintervention and postintervention das, all participants showing a 1 point increase. on item 19 (we can do a lot now to improve the lives of people with adrd), six out of eleven participants improved their scores between the preintervention and postintervention, all participants showing a 1 point increase. the das items which fall under the umbrella of the comfort subdomain, 1, 4, 8, 13, and 16, displayed statistical significance. item 16 (i feel frustrated because i do not know how to help people with adrd) stood out as a comfort subdomain das item whose scores increased at postintervention in comparison to preintervention. item 8 (i 'm not very familiar with adrd) showed a greater than one point increase in eight out of eleven participants ' scores. this section includes a brief discussion of several of the primary themes (tables 1 and 2), an example highlighting a correlate between das data and ipa, and the voices of student responses to the intervention. the ipa of verbatim semistructured interview transcripts (n = 14) yielded five primary themes (table 1) and five secondary themes (table 2). for instance, in the case of das item 4 (i feel confident around people with adrd) with significance (p < 0.019), the ipa secondary theme 3 (workshop increased confidence in communicating with adrd patients) directly supported the quantitative gains reported by das. going beyond the das data, after the intervention, the medical students underwent a positive shift in the way they described persons with adrd. students demonstrated this shift as they started articulating that those living with adrd could enjoy themselves and express themselves creatively : student 5 : i guess what i viewed as what someone with severe dementia would be like was nothing i saw at the workshop. i think that one thing that the workshop definitely showed me though was that these people can be very creative, they seemed to be enjoying themselves i think to the point that i did n't even realize a lot of them had dementia (a lot of them, in reference to the adrd of the facility 's dementia unit). it did influence me positively more so in the sense that they are still capable of a lot. (5.2529) student 6 : honestly, when they were going around and sitting in front of him and i was waiting for someone to not be receptive. i think it helped me see firsthand that they can have a lot of fun and enjoy themselves and almost forget like they have dementia. so you know i might have thought a certain way about them but when i was actually there interacting with them, you know everything went out the window and it 's like forming a new experience and a new perspective of how they are (14.40 - 8) watching glazner 's work and participating through poetry encouraged students to also consider poetry as a tool for treating those with adrd, stating the following : student 9 : i think the workshop helped me determine that there are other resources available to people with ad that are not just medications [] with the poetry and with the book that we got of poetry i think that i would be able to do some of that in the office if patients were willing to do it with me. so i have tools to use both in the office and with patients in between visits as well. (9.1317) student 15 : i did n't know that poetry could do such a difference [] today just increased my own knowledge that you know there 's no barrier to helping people you can use a lot of tools to make a difference. (15.1216) student 8 : maybe hopefully even in the future it [referring to poetry ] could even be part of, ah, treatment, ah, that physicians would assign to patient 's family or even nursing homes. (8.33 - 5) the poetry intervention left students expressing that they now feel more comfortable interacting with people with adrd. student 10 : i think i may have patients from the elderly that come to my practice and this experience has made me more comfortable with speaking and conversing with people that are older. i like do n't have to stick with people my age to go out and have a good time. i feel like i could go and hang out with people with dementia, i dunno i just feel more comfortable. (10.2226) overall, the ipa and das analysis demonstrated significant and positive results. student 5 : i guess what i viewed as what someone with severe dementia would be like was nothing i saw at the workshop. i think that one thing that the workshop definitely showed me though was that these people can be very creative, they seemed to be enjoying themselves i think to the point that i did n't even realize a lot of them had dementia (a lot of them, in reference to the adrd of the facility 's dementia unit). it did influence me positively more so in the sense that they are still capable of a lot. (5.2529) student 6 : honestly, when they were going around and sitting in front of him and i was waiting for someone to not be receptive. i think it helped me see firsthand that they can have a lot of fun and enjoy themselves and almost forget like they have dementia. so you know i might have thought a certain way about them but when i was actually there interacting with them, you know everything went out the window and it 's like forming a new experience and a new perspective of how they are (14.40 - 8) student 9 : i think the workshop helped me determine that there are other resources available to people with ad that are not just medications [] with the poetry and with the book that we got of poetry i think that i would be able to do some of that in the office if patients were willing to do it with me. so i have tools to use both in the office and with patients in between visits as well. (9.1317) student 15 : i did n't know that poetry could do such a difference [] today just increased my own knowledge that you know there 's no barrier to helping people you can use a lot of tools to make a difference. (15.1216) student 8 : maybe hopefully even in the future it [referring to poetry ] could even be part of, ah, treatment, ah, that physicians would assign to patient 's family or even nursing homes. (8.33 - 5) student 10 : i think i may have patients from the elderly that come to my practice and this experience has made me more comfortable with speaking and conversing with people that are older. i like do n't have to stick with people my age to go out and have a good time. i feel like i could go and hang out with people with dementia, i dunno i just feel more comfortable. the present pilot study adds depth to the previous das studies which lacked the added insights of ipa. this study required a short time commitment of about 3 hours on the part of the participants but still managed to affect students ' attitudes. finding an artistic intervention with a short time commitment on the part of participants fits the hectic pace of medical students ' schedules. conducting a longitudinal study of similar interventions would reveal whether or not students ' attitudes remain altered by this type of low - intensity intervention. the structure of the poetry workshop in and of itself may have influenced study outcomes. rowan university used a small grant from the arnold p. gold foundation to pay for glazner to come to the school and run the workshop. he instructed and demonstrated for participants how to employ poetry to augment the quality of life of individuals with dementia. glazner performed call and response during the construction of the love poem ; this encouraged participation from both the medical students and people with dementia. other poetry preceptors might not carry out the workshop as effectively and might not produce the same effects in students ' perceptions of people with adrd [17, 18 ]. the rowan participants trained in gary 's methods could theoretically carry out sessions with new sets of students and people with adrd. using the small group model to hold multiple workshops throughout the year, a greater number of medical students could benefit from this type of learning intervention. however, the changes attributed to the workshop might not have been as significant from students just attending the workshop. the postinterview semistructured interviews and das administration themselves may have also served as a therapeutic tool (self - reflection). a student remarked on the experience of filling out the das, stating the survey forced him to face what you are scared to think about, it helped me think about myself and what i thought of working with people with dementia (2.6769). the previous study with columbia medical students that attended the museum intervention which spurred a positive change in their attitudes towards people with adrd also filled out a preintervention and postintervention das. future researchers could examine whether or not an additional reflection component beyond the das would produce an even greater effect on students ' attitudes. although researchers employed randomization techniques for selecting the study sample, only eleven rowan medical students completed the study. these students ' exposure to geriatrics in medical school may have also augmented the measured positive effects of the poetry workshop. student 14 reinforces this point when stating : i have participated in many, um, nursing home projects and geriatrics related seminars and i just feel a connection and i 'm really interested (14.7, 8). educators working to run the poetry intervention workshops may face the difficulty of soliciting student participation. many students view adrd individuals and elderly in a negative light which may represent a challenge to recruitment especially in institutions without geriatric focused curriculums [12, 13, 17 ]. introducing this workshop in the preclinical years may prove useful in giving medical students the tools and confidence to work with adrd individuals by the time they reach their clinical years. in our sample, ideally, medical students would get the opportunity to participate in this type of workshop each year of training. the use of ipa and the short time frame between preintervention and postintervention administration perhaps mitigated some of the weakness associated with not using a comparison group. studies with a larger sample size and conducted at a variety of medical institutions with various curriculums would shed even more light on the key motivating factors behind students ' decisions and attitudes towards people with adrd. both the ipa and das data offered insights into students ' motivational factors for attending the workshop. although student 6 's motivation to attend the workshop was as follows, my grandma has parkinson 's in the late stages, and i was just looking to get exposure to other dementia patients (6.4 - 5), the independent samples tests performed on das data demonstrated that familiar connection did not influence statistical outcomes. the positive effect of the workshop superseded familiar relationships although a larger sample may change this outcome. researchers of the present pilot study support the use of ipa for the clarification of students ' motivating factors as measured by the das as supported by student quotations and the primary and secondary themes (tables 2 and 3). the results of the present pilot study support the need for workshops like the poetry intervention to help medical students to see people with adrd outside the confines of biomedical definitions of adrd and social media outlets ' depictions of adrd as hopeless and negative. student 10 highlights this point, when stating : i see someone like meredith gray 's mother on gray 's anatomy, the stereotype where the person is in their own mindset at some time in their life, so they are too caught up in their own world to go and pay attention to what is going on around them really. the patients i saw do not look like they are out of touch with reality. (10.5356) (meredith gray 's mother, a fictional tv character on gray 's anatomy, has dementia)the poetry workshop 's positive influence on the students ' descriptors of people with adrd, like those of student 10 and the overall positive change in mean das scores, supports the use of a poetry workshop as means to improve medical students ' attitudes toward people with adrd. i see someone like meredith gray 's mother on gray 's anatomy, the stereotype where the person is in their own mindset at some time in their life, so they are too caught up in their own world to go and pay attention to what is going on around them really. the patients i saw do not look like they are out of touch with reality. (10.5356) (meredith gray 's mother, a fictional tv character on gray 's anatomy, has dementia) | purpose. researchers assessed whether medical students ' participation in a poetry workshop with people with alzheimer 's disease and related dementias (adrd) affected their attitudes towards persons with adrd. objective. to add to the growing body of research summarizing the impact of nonclinical interventions on medical students ' perspectives about people with adrd. design. researchers used dementia attitudes scale (das) and interpretive phenomenological analysis (ipa) to analyze participants ' attitudes. setting. osteopathic medical school and dementia care unit in the state of new jersey. participants. eleven out of fourteen medical students completed the study. measurements. emerging themes were classified from the postintervention semistructured interviews and descriptive statistics were used to compare the preintervention to postintervention das. results. researchers found statistically significant differences between preintervention and postintervention das scores. study participants scored a preintervention das mean, 107.09 (sd = 11.85), that changed positively and significantly to the postintervention das mean, 121.82 (sd = 10.38). das subdomains, comfort (p = 0.002) and knowledge (p = 0.01), and eleven of the twenty das items underwent a positive and statistically significant shift from preintervention to postintervention. ipa of the interviews yielded five primary and five secondary themes, supporting the measured statistical outcomes. conclusion. medical students ' participation in a poetry workshop, with people with adrd, positively impacts their attitudes. |
premature ejaculation (pe) is defined as ejaculation that occurs with minimal stimulation, and which happens before or shortly after penetration, resulting in harassment and anxiety. pe is the most common sexual dysfunction in men younger than 40 years old, affecting approximately 30% of men. however, pe is more common in blacks, hispanic males and muslims (2, 3). there are two types of premature ejaculation, lifelong or primary, and acquired or secondary (4). primary, or lifelong, premature ejaculation is defined as pe that has been a problem since initially beginning coitus. in secondary, or acquired, premature ejaculation the patient has previously had desirable intercourse with subsequent development of pe. measuring the time of ejaculation, usually defined as intravaginal ejaculation latency time (ielt) is very important (5). self - estimated and stopwatch - measured ielt accurately determine pe status with 80% sensitivity and 80% specificity and are interchangeable (6). in addition, the primary method of evaluation of men with pe is physical examination to diagnose the underlying medical problems, which can be related to pe or other sexual dysfunctions such as chronic disease, endocrinopathy, autonomic neuropathy, peyronie s disease, urethritis and prostatitis. without specific findings from a history or physical examination, conducting laboratory tests or physiological testing are not commonly recommended (7). sometimes the most effective treatment method is medication combined with non - medication treatment (8). there are several behavior therapies, such as the squeezing and start - stop methods, but many couples find these cumbersome. pharmacologic therapy may include selective serotonin reuptake inhibitor (ssri) therapy (citalopram, sertraline, fluoxetine, dapoxetine or paroxetine), phosphodiesterase type 5 (pde5) inhibitor therapy (tadalafil or sildenafil), topical desensitizing agents (prilocaine or lidocaine) and other agents (tramadol or pindolol). nowadays, the first choice of treatment for pe is ssris (9). it is worth mentioning that an increase of ielt may begin a few days after initiation of daily ssri intake, and the maximum delay is not observed until after a 1 - 2-week course (10). the therapeutic effect of daily ssris on pe is supported by many double - blind, placebo - controlled and well - designed trials (11, 12). fluoxetine is inferior to sertraline, while the effect of clomipramine does not significantly differ from fluoxetine and sertraline. doses of paroxetine, sertraline, fluoxetine and clomipramine were 20 - 40 mg, 25 - 200 mg, 10 - 60 mg and 25 - 50 mg, respectively. there is little evidence to show that citalopram is less effective than other ssris and that fluvoxamine is not effective (13, 14). a few days after drug intake, ejaculation delay may occur, but 1 - 2 weeks later, the effect is more obvious because the receptor desensitization takes time (15). diarrhea, fatigue, yawning, perspiration, nausea, dry mouth, drowsiness and vomiting are the usual side effects of ssris. at first, these side effects are usually mild, and they gradually decline after two to three weeks (15). the medication enhances the increase of ielt 6 - 20 times more than before treatment. although the males are satisfied with the treatment, many of them interrupt it within a year (16). the aim of this study was to compare the efficacy of paroxetine alone and paroxetine plus tadalafil in patients complaining of premature ejaculation. in 2014, this quasi - experimental study was performed on 100 consecutive 17 to 49-year - old potent men with premature ejaculation and without any clear organic disease. all married patients with lifelong pe and ielt shorter than 1.5 minutes were included in the study. additional exclusion criteria included patients with erectile dysfunction (ed), prostatitis, severe drug complications and poor control over ejaculation. the study participants were equally classified into two groups, a and b, using a computer - generated random tabulation list. this research was approved by the ethical committee of babol university of medical sciences and informed consent was obtained from all patients. the assessment before treatment contained a history, physical examination, u / c and stamey test to exclude genital tract infection. all patients self - completed the international index of erectile function (iief) questionnaire. group a included 50 patients who received 10 mg daily doses of paroxetine four hours before planned sexual activity for 30 days, and if it was needed the dose was increased to 20 mg. group b included 50 patients who received daily doses of 10 mg tadalafil one hour before intercourse, plus 10 mg paroxetine four hours before planned sexual activity for 30 days. the primary outcome was ielt and secondary outcomes were intercourse satisfaction in men and drug side effects. other variables, such as age and number of intercourses per week, were recorded for all patients. patients were followed - up with at three and six months after beginning therapy to assess primary and secondary outcomes. the data were analyzed using spss version 15 statistical software, and the independent t - test with intention to treat method was used. a value of p 0.05). the side effects in group a included gastrointestinal upset and/or nausea in 5 (10%) patients, headache in 4 (8%) patients, decreased libido in 2 (4%) patients and delayed ejaculation in 1 (2%) patient. the side effects in group b included headache in 11 (22%) patients, flushing in 8 (16%) patients and gastrointestinal upset and/or nausea in 4 (8%) patients. flushing episodes were more significant in group b (p = 0.000) than in group a. other side effects did not show a significant difference between the two groups. recently, some studies have investigated the therapeutic role of phosphodiesterase 5 (pde5) inhibitors in pe. pde5s may decrease performance anxiety because of improved erections and may downregulate the erectile limen to a lower state of arousal, so that greater arousal is necessary to reach the ejaculation limen ; however, many of the mechanisms involved are speculative (17 - 19). therefore, the aim of this study was to compare the efficacy of paroxetine alone and paroxetine combined with tadalafil in patients complaining of premature ejaculation. the results of the present study show that ielt at the 3-month and 6-month follow up, in the group with combination therapy of paroxetine and tadalafil, was higher than that of the paroxetine group, but this difference was not significant. previously, sildenafil was compared with placebo in a well - designed, randomized, double - blind, placebo - controlled study (20). in this study, sildenafil enhanced the perception of ejaculatory control, overall sexual satisfaction and confidence, reduced anxiety and the demand time to reach a second erection after ejaculation, though ielt was not significantly increased. in another randomized, double - blind, placebo - controlled study, the combination of sildenafil (50 mg before intercourse) with lidocaine or prilocaine had the same effect, but showed no superiority to placebo (ielt was not considered) (21). one double - blind randomized study showed that sildenafil improved ielt and satisfaction and reduced anxiety in comparison with several ssris and the pause - squeeze technique. sildenafil, clomipramine, sertraline, paroxetine and the pause - squeeze technique improved the baseline ielt from 1 minute to 15 minutes, 4 minutes, 3 minutes, 4 minutes and 3 minutes, respectively (22). various open - label studies have indicated that sildenafil combined with an ssri is superior to ssri monotherapy. in contrast to paroxetine alone, sildenafil plus paroxetine improved ielt and satisfaction (23). sildenafil plus sertraline, in comparison to sertraline alone, significantly improved ielt and satisfaction (24). the combination of sildenafil with paroxetine and psychological and behavioral counseling in patients who had failed with other treatments considerably improved ielt and satisfaction (25). finally, behavioral therapy alone compared to sildenafil plus behavioral therapy was significantly inferior in the improvement of ielt and satisfaction (26). other pde5 inhibitors (tadalafil and vardenafil) have limited data on their effect in pe (19, 27). in the current study, pe was treated by paroxetine alone and in combination with tadalafil and showed that ielt increases with combination therapy. in summary, the role of pde5 inhibitors is not established in pe patients without ed, and there are few double - blind placebo controlled studies. paroxetine is more effective than sertraline, clomipramine and fluoxetine (13, 14). the current study is limited because it was not blind and this could affect the result. nevertheless, the results show that paroxetine plus tadalafil provides better results in terms of ielt and intercourse satisfaction, versus paroxetine alone in potent patients with premature ejaculation ; however, combined treatment mildly increases drug side effects. | background : several recent studies have investigated the therapeutic role of phosphodiesterase type 5 (pde5) inhibitors in premature ejaculation (pe) used in the treatment of erectile dysfunction.objectives:in the present research, the efficacy of paroxetine alone and paroxetine plus tadalafil was compared in patients referred because of premature ejaculation.patients and methods : this quasi - experimental study was performed on 100 consecutive 17 to 49-year - old potent men with premature ejaculation and without any clear organic disease. all patients had lifelong pe with an intravaginal ejaculation latency time (ielt) shorter than 1.5 minutes. informed consent was obtained from all patients who were randomly divided into two groups using a computer - generated random tabulation list. in group a, patients received 10 mg paroxetine daily, in addition to four hours before planned sexual activity. in group b, 10 mg paroxetine was taken daily, plus 10 mg tadalafil one hour before planned sexual activity. the duration of the intervention was six months and patients were evaluated for ielt three and six months after the beginning of therapy.results:the mean age of patients in groups a and b were 33 9.6 and 31.2 9.3 years, respectively (p = 0.368). the mean number of intercourses were 1.08 0.6 and 1.12 0.6 per week in groups a and b, respectively (p = 0.791). mean ielt at the 3-month follow up in groups a and b was 4.5 1.5 and 5 2.4 minutes, respectively (p = 0.285) and at the 6-month follow up was 4.8 1 and 5.3 2 minutes, respectively (p = 0.278).conclusions : the results of the study show that tadalafil can increase the mean ielt and can be used for treatment of premature ejaculation in combination with paroxetine. |
celiac disease (cd) is a disorder of the small intestine known by mucosal inflammation, villous atrophy, and crypt hyperplasia, which appear upon exposure to dietary gluten and improve after withdrawal of gluten from the diet. however, the serologic tests for celiac disease and the common use of upper endoscopy have complicated the definition. these tests have identified patients who may be involved in the disease but have variable degrees of histop - athologic symptoms and changes. no definite single test can lead to a definite diagnosis of celiac disease for every individual. so, the most important step in diagnosis as a general rule is using serologic tests. the specific tests like iga anti - tissue transglutaminase and iga endomysial antibody can be recom - mended [2, 3 ]. patients with a positive iga endomysial or transglutaminase antibody test should undergo a small bowel biopsy. the exact minimal number is uncertain ; however, some experts believe that at least four should be obtained. the duodenal mucosa may be atrophic with loss of folds, contain visible fissures, have nodular appearance or scalloped folds, but such findings are not universally acceptable and may be seen with some other disorders. the diagnosis is usually established if there is a correlation between the serologic results and the biopsy findings. tissue transglutaminase is a ubiquitous intracellular enzyme released by inflammatory and endothelial cells and fibroblasts in response to mechanical irritation or inflammation. once it has been secreted, it cross - links glutamine - rich proteins like gluten proteins from wheat. however, it can also deamidate glutamine residues in gluten to glutamic acid. deamidation causes a negative charge in gluten peptides, which increases their binding to hla - dq2 and dq8, which potentiates their capacity to stimulate t - cells [6, 7 ]. in addition to activation of pathogenic t cells, innate responses to gliadin are also involved in the immune response, and perhaps even necessary to trigger the gliadin - specific (adaptive) t - cell response in genetically potential individuals. pattern recognition to provide an early response to stimuli such as rna, dna, lipopolysaccharide, or viral proteins, in contrast to the adaptive immune system, which depends on hla - presentation and t - cell recognition and expansion. in celiac disease, certain cereal peptides can apparently initiate innate immune responses in macrophages, monocytes, dendritic cells, and intestinal epithelia via yet unknown receptors and mechanisms [8, 9 ] in the immune system. when t - cells are stimulated, two types of t - cells are produced. one type is t - helper and the other one is memory t - cell. therefore, every time gluten enters the body of celiac patients, memory t - cells will be activated and trigger innate immune response. this study tries to answer this question whether the immune response starts if gluten enters the body of celiac patients through skin and whether gluten skin patch test can be used for celiac disease diagnosis. a patch test is based on the principle of a type iv (delayed) hypersensitivity reaction. this is where a substance is recognized by immune cells in the skin known as antigen - presenting cells (apcs). the apc moves down the lymphatic system to a lymph node where it presents antigen to t - cells. if the t - cell identifies the substance as a threat, it sends out all types of immune cells including more of its own type to where the antigen has entered the skin. patch test is the gold standard of food allergy diagnosis, and celiac disease is one of the non - ige mediated types of food allergy. when gluten comes into contact with the skin of a celiac patient (through patch test), tissue transglutaminase in skin cells can transmute gluten to glutamic acid, and apcs move down to the lymphatic node (where there are gluten - specific memory t - cells) and present gluten as an antigen to t - cells, the immune system is thus activated and produces hypersensitivity reaction type iv. therefore, it can be stated that skin patch test can be considered as a method for diagnosis of celiac disease. in this study, we try to determine the effectiveness of patch test in diagnosis of celiac disease compared to that of biopsy. this study was carried out in al - zahra university hospital affiliated with isfahan university of medical sciences, isfahan, iran. we collected comprehensive clinical and histological data of all patients suspected of celiac disease undergoing endoscopy from november 2005 until june 2009. the patients were divided into two groups, the case group and the control group. in the case group, there were 30 patients with typical celiac disease criteria ; they had symptoms of celiac disease, positive serologic test results, villous atrophy in biopsy, and their symptom resolved subsequently on a gluten - free diet. in the control group, we investigated whether these patients suffered from dyspepsia or had intestinal diseases other than celiac disease, such as gerd, h. pylori infection, or gastroenteritis. all patients in the control group had normal villous architecture. in this study, we used viaskin, which is a type of gluten patch test produced by dbv technology, france. viaskin has a polymer plate containing gluten, with positive and negative poles (the negative pole is placed on the skin of patient. when viaskin is placed on the skin, the gluten in the polymer plate is solved by natural moisture of the skin and absorbed, as a result of which the immune system is activated. in this study, we placed both gluten patch test and placebo patch test on the skin between scapulas of the patient. after 48 hours, we removed both patch tests and reported the responses twice ; after 48 hours and 96 hours. the result of patch tests had five grades : grade 0 meant no reaction, grade i : mild erythema, grade ii : severe erythema, grade iii : erythema and eventually papules or only papules, and grade iv meant erythema, vesicles, and pustules. four patients in the case group and eight patients in the control group who showed irritation reactions were withdrawn from this study. in order to determine the diagnostic value of gluten patch test, once we chose grade 0 as negative response and once grade 0 and i as negative response. we determined sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) of gluten patch test both after 48 and 96 hours. statistical differences between the study groups were evaluated using spearman 's test, mann whitney test, or chi square test, as appropriate. sensitivity and specificity, positive predictive value (ppv), and negative predictive value (npv) of gluten patch test in detecting celiac disease were determined. median age of control group was 9.08 (range : 316) and in control group was 11.27 (range : 431). twelve patients (40) in case group and 15 patients (55.6%) in control group were females. the values for gluten patch test after 48 hours and 96 hours are shown in table 2. the best results were obtained when we reported the responses of gluten patch test after 96 hours, in which sensitivity 95%, specificity 8%, ppv 67%, and npv 43% were reported. therefore, gluten patch test is not efficient in screening celiac disease. primary reason for endoscopy and small intestine biopsy in study and control groups in this study, we compared responses to gluten patch test with severity of villous atrophy in patients with celiac disease (partial atrophy of villus was considered as mild and marsh iiic was of the greatest severity), spearman 's test showed that there is a direct relationship between gpt response after 48 h and severity of villous atrophy (r=0.26 p=0.04) ; for example, we can say patients with marsh iiic will show a higher grade of gpt after 48 h than patients with partial atrophy, but there is no relationship between response to gpt after 96 and severity of villous atrophy. there is an inverse relationship between patients age and response to gpt after 96 h (confirmed by spearman 's test results : r=0.25, p=0.04). this means that after 96 hours, older patients will show a lower grade of gpt response than younger patients. but there is no relationship between age and response to gpt after 48 h. the value for gluten patch test in diagnosis of celiac disease in comparison with small intestine biopsy gpt : gluten patch test ; npv : negative predictive value ; ppv : positive predictive value in 30 patients with celiac disease, six patients discontinued gluten - free diet. there was no relationship between response to gpt and continuing gluten - free diet (confirmed by chi - square test results, p=0.16). in this study, there was a direct relationship between serology test results (anti t - tg igg, anti gliadin iga) of cd patients and responses to gpt after 96 hours (by mann we can therefore say there is a relationship between negative anti gliadin iga and negative anti t - tg igg on the one hand and grade 0 in gluten patch test after 96 h on the other hand. but there is no relationship between results of these serology tests with results of gpt after 48 hours, and there is no relationship between the results of other serology tests (anti endomysial ab iga, igg, anti gliadin ab igg, anti t - tg iga) and responses to gpt. for the first time, the diagnostic value of gluten patch test for celiac disease was investigated in this study. different studies have shown that skin patch test is a reliable test for diagnosis of food allergy and delayed hypersensitivity reaction [13, 14 ] and useful to diagnose both mediated and non - mediated ige reactions. several studies have shown that skin patch test is sensitive in diagnosis of food allergies associated with atopic dermatitis, particularly in young children [16, 17 ]. in a study, specificity of skin patch test in diagnosis of cow milk allergy was determined to be 95% and for hen eggs to be100%. in 46 patients with allergic eosinophilic esophagitis, the sensitivity of both skin patch test and skin prick test were 97% and specificity of both was 5% in diagnosis of foods that cause aee. in another study on 19 patients with food protein - induced entrocolitis syndrome (fpies), sensitivity of skin patch test was shown to be 10% and its specificity to be 71% in diagnosis of foods associated with fpies. all the studies showed that skin patch test is useful for food allergy diagnosis and because celiac disease is a food allergy and is a type of non - ige mediated reaction, it was expected that gluten patch test could be an effective test in diagnosis of celiac disease. however, this study showed that gluten patch test can not be such an efficient test in screening of celiac disease. nevertheless, the gluten patch test can be useful in diagnosis of cd when employed with clinical symptoms, serology test, and biopsy of small intestine. we should doubt our diagnosis if a patient with cd shows positive response to gpt after 96 h. regarding the value of this study, we should doubt the statement that cd is a kind of food allergy ; perhaps it is better to present celiac disease as an autoimmune disease. this hypothesis is reinforced by association of celiac disease with other autoimmune diseases such as diabetes type i [21, 22 ], selective iga deficiency [23, 24 ], autoimmune thyroid disease [25, 26 ], and autoimmune myocarditis [27, 28 ]. in this hypothesis, in cd patients, gluten is probably similar to epithelium of small intestine and when patients use gluten, the autoimmune system will be activated. this study showed that there is a direct relationship between severity of villous atrophy in cd patients and the response to gluten patch test after 48 h. is the grade of villous atrophy predictable by the grade of gluten patch test result after 48 hours ? there is a direct relationship between gluten patch test results after 96 h and anti gliadin iga and anti t - tg igg results. will gluten patch test after 96 h be able to be replaced with anti gliadin iga and anti t - tg igg ? | objectiveceliac disease is an intestinal disorder identified by mucus inflammation, villous atrophy and crypt hyperplasia. this disorder can be controlled by elimination of gluten from daily diet. patients with celiac disease are at greater risk of gastrointestinal malignancy and non - hodgkin lymphoma than are the general population. this study tries to present the value of gluten patch test for diagnosis of celiac disease.methodsin this investigation, the study population was divided into case and control groups. the case group consisted of patients with celiac disease. the control group were patients involved in celiac disease but suffering from other gastrointestinal disorders. both gluten patch and placebo patch were attached to the skin between the scapulas. the results were read twice : 48 hours and 96 hours after the patch was applied. patients who showed irritation reactions were withdrawn from this study. the results were analysed by spss software, spearman 's test, chi square, and mann whitney tests.findingsthe value obtained from the gluten patch test after 96 hours are as follows : specification at 95%, sensitivity at 8%, positive prediction value at 67%, and negative prediction value at 43%.conclusionit can be concluded that the gluten patch test is not an efficient test for screening of celiac disease, however, it can be useful for diagnosis of celiac disease if employed and studied with clinical symptoms and serologic and biopsy tests. furthermore, we should doubt our judgment if the result of gluten patch test for the patient with celiac disease is positive. |
leclercia adecarboxylata, an ubiquitous gram - negative bacillus of the enterobacteriaceae family, has been rarely isolated from environmental and clinical specimens. l. adecarboxylata shares characteristics of enterobacteriaceae ; they are gram - negative, facultatively - anaerobic, oxidase - negative, mesophilic, peritrichate - flagellated bacilli. in clinical specimens, l. adecarboxylata has been commonly isolated from polymicrobial wound infections. the majority of infections are seen in immunocompromised patients but there are reports of l. adecarboxylata infections in immunocompetent patients. we present in this paper 3 patients infected with this bacillus one of whom succumbed. a 41-year - old otherwise healthy male presented to casualty with severe head injury following a road traffic accident. vital signs on admission were unremarkable but on physical examination, decorticate posturing was noted. while his chest x - ray was normal, computed tomography (ct) scan of head without contrast revealed right fronto - temporo - parietal acute subdural hemorrhage. the patient was intubated, resuscitated and underwent right craniotomy and evacuation. on the third post - operative day he developed fever and tachypnea. culture revealed pure growth of large grey non - hemolytic colonies on blood agar and pale non - lactose - fermenting colonies on macconkey agar after 24 hours of incubation. the strain was motile, produced indole, fermented mannitol and did not produce hydrogen sulfide and oxidase. the organism was identified as leclercia adecarboxylata by vitek 2 system (biomrieux, marcy - letoile, france) and was found to be resistant to ampicillin (mic 32 g / ml) but sensitive to amoxicillin - clavulanic acid (mic 2 g / ml), ciprofloxacin (mic 0.25 g / ml), cotrimoxazole (mic 20 g / ml), amikacin (mic 2 g / ml), gentamicin (mic 1 g / ml), piperacillin - tazobactum (mic 4 g / ml), ceftriaxone (mic 1 g / ml), cefuroxime (mic 8 g / ml), cefepime (mic 1 g / ml), cefoperazone - sulbactum (mic 8 g / ml), tigecycline (mic 0.5 g / ml), meropenem (mic 0.25 g / ml) and imipenem (mic 0.25 g / ml). a 32-year - old otherwise healthy female patient presented to casualty with history of headache, vomiting and loss of consciousness. vital signs on admission were notable for a blood pressure of 170/80 mm hg but were otherwise unremarkable. seven weeks into her hospital stay after multiple surgical procedures and prolonged ventilation, she developed fever of 102f and her leukocyte count was 12,300/l. tracheal aspirate sent during this febrile episode revealed polymorphonuclear leukocytes and few gram - negative bacilli. culture yielded pure growth of l. adecarboxylata identified by vitek 2 system and sensitive to ampicillin (mic 1 g / ml), amoxicillin clavulanic acid (mic 0.5 g / ml), ciprofloxacin (mic 0.5 g / ml), cotrimoxazole (mic 20 g / ml), amikacin (mic 2 g / ml), gentamicin (mic 1 g / ml), piperacillin - tazobactum (mic 1 g / ml), ceftriaxone (mic 1 g / ml), cefuroxime (mic 2 g / ml), cefepime (mic 1 g / ml), cefoperazone - sulbactum (mic 2 g / ml), tigecycline (mic 0.25 g / ml), meropenem (mic 0.5 g / ml) and imipenem (mic 0.5 g / ml). blood and urine culture demonstrated no growth. the patient received imipenem 1 g intravenous every 6 hourly and amikacin 80 mg intravenous daily for 1 week. a 32-year - old male presented to the out - patient department with history of upper back pain for 1 month and lower limb weakness for 6 days. magnetic resonance imaging revealed multiple vertebral body signal changes with complete destruction of the c6 vertebral body and a paravertebral collection. with the provisional diagnosis of vertebral tuberculosis, he underwent laminectomy and c6-c7 decompression. on the fifth post - operative day, he developed fever of 101 f. leukocyte count was 9200/l. tracheal culture demonstrated polymorphonuclear leukocytes and gram - negative bacilli and yielded pure growth of l. adecarboxylata, identified by vitek 2 system. it was found to be sensitive to ampicillin (mic 2 g / ml), amoxicillin - clavulanic acid (mic 1 g / ml), ciprofloxacin (mic 0.5 g / ml), cotrimoxazole (mic 20 g / ml), amikacin (mic 1 g / ml), gentamicin (mic 1 g / ml), piperacillin - tazobactum (mic 2 g / ml), ceftriaxone (mic 2 g / ml), cefuroxime (mic 2 g / ml), cefepime (mic 1 g / ml), cefoperazone - sulbactum (mic < = 2 g / ml), tigecycline (mic 0.5 g / ml), meropenem (mic 0.25 g / ml) and imipenem (mic 0.5 g / ml). a 41-year - old otherwise healthy male presented to casualty with severe head injury following a road traffic accident. vital signs on admission were unremarkable but on physical examination, decorticate posturing was noted. while his chest x - ray was normal, computed tomography (ct) scan of head without contrast revealed right fronto - temporo - parietal acute subdural hemorrhage. the patient was intubated, resuscitated and underwent right craniotomy and evacuation. on the third post - operative day he developed fever and tachypnea. culture revealed pure growth of large grey non - hemolytic colonies on blood agar and pale non - lactose - fermenting colonies on macconkey agar after 24 hours of incubation. the strain was motile, produced indole, fermented mannitol and did not produce hydrogen sulfide and oxidase. the organism was identified as leclercia adecarboxylata by vitek 2 system (biomrieux, marcy - letoile, france) and was found to be resistant to ampicillin (mic 32 g / ml) but sensitive to amoxicillin - clavulanic acid (mic 2 g / ml), ciprofloxacin (mic 0.25 g / ml), cotrimoxazole (mic 20 g / ml), amikacin (mic 2 g / ml), gentamicin (mic 1 g / ml), piperacillin - tazobactum (mic 4 g / ml), ceftriaxone (mic 1 g / ml), cefuroxime (mic 8 g / ml), cefepime (mic 1 g / ml), cefoperazone - sulbactum (mic 8 g / ml), tigecycline (mic 0.5 g / ml), meropenem (mic 0.25 g / ml) and imipenem (mic 0.25 g / ml). a 32-year - old otherwise healthy female patient presented to casualty with history of headache, vomiting and loss of consciousness. vital signs on admission were notable for a blood pressure of 170/80 mm hg but were otherwise unremarkable. seven weeks into her hospital stay after multiple surgical procedures and prolonged ventilation, she developed fever of 102f and her leukocyte count was 12,300/l. tracheal aspirate sent during this febrile episode revealed polymorphonuclear leukocytes and few gram - negative bacilli. culture yielded pure growth of l. adecarboxylata identified by vitek 2 system and sensitive to ampicillin (mic 1 g / ml), amoxicillin clavulanic acid (mic 0.5 g / ml), ciprofloxacin (mic 0.5 g / ml), cotrimoxazole (mic 20 g / ml), amikacin (mic 2 g / ml), gentamicin (mic 1 g / ml), piperacillin - tazobactum (mic 1 g / ml), ceftriaxone (mic 1 g / ml), cefuroxime (mic 2 g / ml), cefepime (mic 1 g / ml), cefoperazone - sulbactum (mic 2 g / ml), tigecycline (mic 0.25 g / ml), meropenem (mic 0.5 g / ml) and imipenem (mic 0.5 g / ml). blood and urine culture demonstrated no growth. the patient received imipenem 1 g intravenous every 6 hourly and amikacin 80 mg intravenous daily for 1 week. a 32-year - old male presented to the out - patient department with history of upper back pain for 1 month and lower limb weakness for 6 days. magnetic resonance imaging revealed multiple vertebral body signal changes with complete destruction of the c6 vertebral body and a paravertebral collection. with the provisional diagnosis of vertebral tuberculosis, he underwent laminectomy and c6-c7 decompression. on the fifth post - operative day, he developed fever of 101 f. leukocyte count was 9200/l. tracheal culture demonstrated polymorphonuclear leukocytes and gram - negative bacilli and yielded pure growth of l. adecarboxylata, identified by vitek 2 system. it was found to be sensitive to ampicillin (mic 2 g / ml), amoxicillin - clavulanic acid (mic 1 g / ml), ciprofloxacin (mic 0.5 g / ml), cotrimoxazole (mic 20 g / ml), amikacin (mic 1 g / ml), gentamicin (mic 1 g / ml), piperacillin - tazobactum (mic 2 g / ml), ceftriaxone (mic 2 g / ml), cefuroxime (mic 2 g / ml), cefepime (mic 1 g / ml), cefoperazone - sulbactum (mic < = 2 g / ml), tigecycline (mic 0.5 g / ml), meropenem (mic 0.25 g / ml) and imipenem (mic 0.5 g / ml). l. adecarboxylata is a member of the enterobacteriaceae family, the members of which are regarded as normal flora in the gut of animals. the case series presented includes both immunocompetent and immunocompromised hosts. as per the definition by centre for disease control and prevention (cdc), pneumonia contracted by a patient in a hospital about 48 - 72 hours after admission is considered as nosocomial pneumonia. the patients had been mechanically ventilated and the organisms were isolated from samples collected several days after admission, suggesting that these were nosocomial infections. the presence of polymorphonuclear leukocytes in the gram stain of the aspirate along with the pure growth of the isolate in culture and associated leukocytosis may help to some extent in indicating the clinical significance of this organism that was isolated in the above three cases. the isolates reported here demonstrated sensitivity to most antibiotics ; repeat cultures in patients # 1 and # 2 did not grow the organism. however, the hospital infection control team was notified and the hospital staff was instructed about hospital infection control measures. although nucleic acid analyses like 16s rrna sequencing are considered gold - standard methods for bacterial identification, we could not perform molecular tests as they were not available in our facility. however, in the three cases mentioned above, l adecarboxylata was recovered in pure culture without any co - existing pathogen. similar findings were reported by hess., wherein l. adecarboxylata was isolated from a wound infection as a single pathogen in an immunocompetent patient. l. adecarboxylata has been rarely isolated from cases of pneumonia. in a study done by temesgen., l. adecarboxylata was isolated from sputum in a patient with pneumonia due to multiple bacteria. another case of pneumonia due to multi - drug resistant l. adecarboxylata was described by eiland. in our case series, we have described cases of pneumonia due to a single pathogen which was found to be sensitive to most of the antibiotics tested. forrester. have described isolation of l. adecarboxylata from blood and central venous catheter of a trauma patient. two of the three cases described by us are from trauma cases. due to significant morbidity and mortality associated with nosocomial infections, microbiologists and clinicians though most l. adecarboxylata isolates are sensitive to many of the antibiotics, their co - existence with multi - drug resistant organisms could result in the transmission of resistance elements. in view of prolonged hospital stay in one of the three cases, transfer of drug resistance is a matter of great concern. | leclercia adecarboxylata, a gram - negative bacillus of the enterobacteriaceae family, is an uncommonly identified human pathogen. the organism has been reported worldwide and isolated from various environmental sources. most human infections are polymicrobial and commonly occur in immunocompromised hosts, although nosocomial infections in immunocompetent hosts have been documented. we describe three case reports of l. adecarboxylata isolation from cases of hospital acquired pneumonia admitted to a tertiary care center for neurosurgical care. |
the most common and generally accepted ' typical ' us features of hepatic hemangiomas are their small size, uniform hyperechogenicity, well - defined margin, and posterior echo enhancement (1 - 3) (fig. 1). in addition, follow - up scanning only rarely shows a change in size, appearance, or detectability (4). they are usually composed of large blood - filled cavernous spaces, lined by a single layer of flat endothelial cells and separated by fibrous septa ; multiple interfaces between the walls of the sinuses and the blood within them account for the typical hyperechogenicity seen at us (4). the enhanced through - transmission observed in many cases reflects the low acoustic impedance of blood - filled spaces and tends to occur in hemangiomas larger than 25 mm (2). because hemangiomas may undergo various changes including internal hemorrhage with necrosis, thrombosis, myxomatous change, fibrosis, and- rarely- calcification as they become larger (1, 4), ' atypical ' us features are frequently seen in larger ones. unlike a typical hemangioma, one that is atypical has an internal echo pattern at least partially hypoechoic ; a hyperechoic mass with a hypoechoic central portion or decreased echogenicity can be seen throughout the entire lesion (figs. the most suggestive us feature of this type of hemangioma is an echogenic border, seen as a thick echogenic rind or thin rim around a tumor (4) (figs. 2 and 6). although the prevalence of atypical hemangioma has not been precisely determined, it seems that approximately 20 - 40% of hemangiomas are of this kind (4, 5). the echogenicity of hepatic parenchyma influences the us appearance of a hemangioma. because of the increased echogenicity of attenuating fatty liver parenchyma, diffuse fatty infiltration may lead to an atypical echo - poor appearance (6) (fig. 8). if a ' typical ' hemangioma is present in a liver in which fatty infiltration has occurred, its us appearance will be altered. the lesion will initially appear less hyperechoic than isoechoic, and finally hypoechoic relative to infiltrated liver. fatty infiltration of the liver may also cause obscuration of the echogenic border around the tumor (5). it has recently been shown that the speed of contrast enhancement occurring during incremental dynamic ct or multiphase dynamic mr imaging provides a basis for predicting the echo pattern at us, and vice versa. on dynamic ct or mr images, sonographically hypoechoic hemangiomas tend to show rapid enhancement, but if hyperechoic, they tend to enhance slowly (7, 8) (figs. 9 and 10). as techniques for evaluating the vascularity of various organs and diseases, color and power doppler us have recently shown promise. since power doppler us is superior to color doppler us in detecting slow flow, it has been used in atempts to diagnose hepatic hemangiomas specifically on the basis of their typical hemodynamic features. though it was initially believed that power doppler is able to depict slow flow within hemangiomas (10), it has been suggested that homogeneous noise from highly reflective interfaces can be misinterpreted as true flow (11). to avoid these false signals, a recent study in which a doppler phantom / flow control system and a 2 - 4 mhz transducer were used found that for determining the vascularity of hyperechoic tissue, a pulse repetition frequency of 1,000 hz and a medium wall filter were adequate. using these optimized parameters, the majority of hepatic hemangiomas produced either no power doppler signal or one that was minimal (12) (fig. the majority of hepatic hemangiomas show a typical enhancement pattern : peripheral nodular or globular enhancement during the bolus dynamic phase and centripetal fill - in and persistent enhancement during the delayed phase, and for this reason, dynamic contrast - enhanced ct or mr imaging has been widely used for diagnosis. since microbubble contrast agents for us have become available, the efficacy of these for the characterization of focal hepatic lesions has been the subject of numerous investigations (9, 13 - 15). in hepatocellular carcinoma, the use of contrast agents greatly increases intratumoral doppler signals (13, 14), but in hemangiomas, the use of power doppler us with microbubble agents reveals either no internal vascularity or sparse peripheral flow (13, 15) (fig. 11). due to its lack of sensitivity in detecting slow flow in hemangiomas, power doppler us can not therefore, be used for the specific diagnosis of hepatic hemangioma, even with the use of microbubble agents (15). pulse - inversion harmonic us is a newly introduced technique for displaying the amplitude of second harmonic signals resulting from non - linear echoes. in contrast to power doppler us, pulse - inversion harmonic us with interval delay scanning can provide strong gray - scale enhancement by microbubble contrast agents and detect signals from microbubbles in very slow flow without doppler - related artifacts. it has recently been shown that this technique can depict the typical enhancement patterns of three different types of liver lesions : hemangiomas, metastases, and hepatocellular carcinomas. while most hemangiomas (95%) show peripheral globular or rim - like enhancement with progressive centripetal fill - in, malignant tumors do not show this pattern (9) (fig. pulse - inversion harmonic us with interval delay scanning using a contrast agent is therefore believed to be potentially useful for the specific diagnosis of hepatic hemangiomas by demonstrating their characteristic enhancement features. in the diagnosis of hepatic hemangiomas, the most important aspect is non - invasive differentiation from other tumors. since us plays a key role in the initial evaluation of these hemangiomas, knowledge of the spectrum of those that are atypical is important and can help avoid most diagnostic errors. at the same time, however, in most cases of hemangioma with atypical sonographic features a specific diagnosis can be established by means of dynamic contrast - enhanced studies such as ct or mr imaging. because pulse - inversion harmonic us with interval delay scanning using a contrast agent is also believed to be potentially useful for the specific diagnosis of hepatic hemangiomas by demonstrating their characteristic enhancement features, simple and immediate characterization of a focal hepatic lesion newly detected by initial us examination is possible. | because us plays a key role in the initial evaluation of hepatic hemangiomas, knowledge of the entire spectrum of us appearances of these tumors is important. most hemangiomas have a distinctive us appearance, and even with those with atypical appearances on conventional gray - scale us, specific diagnoses can be made using pulse - inversion harmonic us with contrast agents. in this essay, we review the spectrum of us appearances of hepatic hemangiomas on conventional gray - scale, power doppler, and pulse - inversion harmonic us with contrast agents. |
persistent cloaca is defined as a defect in which rectum ; vagina and urinary tract meet and fuse into a single common channel. this is a rare anomaly seen exclusively in girls with an incidence of 1 in 50,000 live births and accounts for 10% of all anorectal malformations in females. it is a complex malformation with a wide spectrum of severity and remains a difficult reconstructive challenge. achieving bowel and urinary control, as well as normal sexual function are the main management issues, whereas malignancy is rarely of concern in this group of patients. although cloacogenic tumors of anorectum, bladder, and vulva are reported, which are mainly adenocarcinomas ; development of squamous cell carcinoma of bladder in patients with cloacal malformation is unknown. a 36-year - old married female having two children, both delivered by caesarian section, presented to us with complaints of urinary frequency, urgency, and urge incontinence associated with low - grade fever. she had history of recurrent urinary tract infections (uti) since childhood and had received multiple courses of antibiotics. she had been diagnosed as having a persistent cloaca and had undergone multiple cutback procedures at birth. on pelvic examination, ultrasound abdomen showed a mass lesion in urinary bladder [figure 1 ]. computed tomography (ct) abdomen and pelvis with three dimensional reconstruction confirmed the presence of persistent cloaca, a large mass in the bladder with infiltration into the pubic bones [figure 2 ] with uterus didelphys and rectum crossing between the two uteri and opening into the bladder base [figure 3 ]. cystoscopy revealed a large solid, sessile growth arising from dome and anterior wall of urinary bladder, rectum opening just distal to bladder neck and two cervical openings posteriorly. cold cup biopsy from growth revealed a moderately differentiated squamous cell carcinoma (scc). ultrasonogram abdomen showed a mass lesion in urinary bladder ct scan with 3d reconstruction shows the presence of persistent cloaca contrast enhanced ct scan abdomen and pelvis showing uterus didelphys and rectum crossing between the two uteri and opening into the bladder base with a diagnosis of squamous tumor with persistent cloaca, radical cystectomy and urinary diversion were planned. intraoperatively, we found a 15 cm pouch colon along with a large tumor involving anterior wall of bladder, infiltrating the pubic bones. pelvic exenteration, standard bilateral pelvic lymph node dissection, pubectomy, and wet colostomy were performed. operative time was 6 hours with 1200 cc blood loss and the patient received 2 units of packed cell transfusion. postoperative course was uneventful except for a small incision disruption which was managed with secondary suturing. she was started on adjuvant methotrexate and platin - based combination chemotherapy but finally lost to follow up. cloacogenic tumors i.e., tumors arising from remnants of cloacal membrane have been reported in the anorectum, vulva, bladder, and vagina. most of these are adenocarcinomas and less commonly of transitional cell origin. squamous cell carcinoma arising from cloaca to best of our knowledge, this is probably the first case of squamous cell carcinoma of urinary bladder occurring in a patient with persistent cloacal anomaly. the reason for recurrent uti in our patient may be fecal contamination of urinary tract due to persistent cloaca. studies from john hopkins institute have shown that history of uti is an important risk factor (risk ratio = 1.6, confidence interval (ci) = 1.4 - 1.8) for development of scc. these are : i) production of excess nitrites by bacteria, ii) conversion of nitrites into nitrosamines, and iii) increased absorption of carcinogens through bladder mucosa because of inflammation. development of malignancy in patients with cloacal malformations is exceedingly rare but a possibility should be kept in mind, particularly while treating recurrent and unexplained utis in a patient with persistent cloaca. taking all treatment perspectives together, the most serious problem is the lack of any histological or clinical data allowing a reliable prognosis of future bladder growth and long - term storage and voiding function after birth. | a rare case of squamous cell carcinoma of bladder occurring in a 36-year - old female with persistent cloacal anomaly who presented with frequency, urgency, dysuria, and recurrent urinary tract infection is reported. contrast enhanced computed tomography with three dimensional reconstruction showed presence of bladder tumor and persistent cloaca. she underwent pelvic exenteration and wet colostomy. histopathologic findings revealed locally advanced moderately differentiated squamous cell carcinoma. |
the normal prostate luminal cells, which are the histological origin of most prostate malignancies, are physically separated from the stroma by the basal cells and basement membrane (bm). basal cells are joined by intercellular junctions and adhesion molecules, constituting a continuous sheet encircling luminal cells 1 - 2. the bm is composed of type iv collagen, laminins, and other molecules, forming a continuous lining surrounding and attaching to the basal cell layer 3 - 4. the epithelium is normally devoid of blood vessels and lymphatic ducts, and totally relies on the stroma for its metabolic and even survival needs. due to these structural relationships, the disruption of both the basal cell layer and the bm is a pre - requisite for prostate tumor invasion. it is a commonly held belief that human prostate tumor invasion is a multistage process, progressing sequentially from normal to hyperplasia, to prostatic intraepithelial neoplasia (pin), and to invasive or metastatic stages 5 - 8. progression from pin to invasion is believed to be triggered by cancer cells that increasingly produce proteolytic enzymes with tumor progression, which cause degradation of the bm 9 - 10. these theories are consistent with results of studies in tissue cultures and animal models, whereas are hard to interpret the following critical facts : (1) our previous studies revealed that some healthy men between 19 and 29 years old had a spectrum of proliferative lesions, including hyperplasia, pin, and incipient adenocarcinoma 11 - 13, (2) recent studies detected a dna phenotype that is identical to that of invasive prostate cancer in some healthy men, and in morphologically normal prostate tissues adjacent to prostate cancer 14 - 17, (3) a vast majority of pin express high levels of proteolytic enzymes, while only 10 - 30% of untreated pin progress to invasive lesions during patients ' lifetime 18 - 21. unfortunately, none of the current approaches could predict which pin lesions will progress 22 - 25. the only established approach to monitor pin progression is repeat biopsy 22 - 25, which is costly and painful, and (4) results from all at clinical trials of prostate cancer treatment or prevention with corresponding proteolytic enzyme inhibitors have been very disappointing 26 - 28. together, these facts argue that alternative pathways of prostate tumor progression and invasion may exist or even play more direct roles. since over 90% of prostate cancer related mortality result from invasion - related illness, and the incidence of pin could be up to 16.5%-25% prostate biopsies 24 - 28, there is an urgent need to uncover the intrinsic mechanism of tumor invasion. promoted by the fact that the basal cell layer is the sole source of tumor suppressor p63 and maspin 29 - 32, and that degradation of basal cell layers is a pre - requisite for tumor invasion, our recent studies have attempted to identify early signs of basal cell degradation. our initial study examined the physical integrity of basal cell layers in 50 patients with co - existing pre - invasive and invasive prostate tumors. of 2,047 ducts and acini examined, 197 were found to harbor focal disruptions (the absence of basal cells resulting in a gap greater than the combined size of at least 3 basal cells) in their basal cell layers. the frequency of focal basal cell layer disruptions (fbcld) varied from none in 22 cases to over 1/3 of the ducts or acini with fbcld in 17 cases 33. compared to their non - disrupted counterparts, focally disrupted basal cell layers showed a significantly lower frequency of tumor suppressor expression and proliferation, but a significantly higher rate of degeneration and leukocyte infiltration 33. in contrast, epithelial cells overlying focally disrupted basal cell layers had a significantly higher rate of proliferation and expression of tumor invasion related genes 33 - 34. based on these and other findings, we have proposed that prostate tumor invasion or progression is triggered by fbcld induced auto - immunoreactions, which facilitate formation of more aggressive cell clones or monoclonal proliferation of tumor stem cells overlying focally disrupted basal cell layers. our hypothesis and supporting data have been recently published in multiple peer - reviewed journals 33 - 36. as the basement membrane surrounds and attaches to the basal cell layer, our current study attempted to assess whether fbcld would impact the physical integrity of the associated basement membrane. formalin - fixed and paraffin embedded tissue blocks from 25-human prostate tumors with both pre - invasive and invasive components were selected from our previous studies 33 - 36. consecutive sections at 4 m thickness were prepared and placed on positively charged slides. the first and last sections from each case were stained with hematoxylin and eosin (h&e) for morphological classification, based on our published criteria 11. to identify focal basal cell layer disruptions (fbcld), two sections from each case were subjected to double immunohistochemistry with basal cell phenotypic markers p63 (clone : 4a4 ; cell marque, foster city, ca) at a 1:50 dilution and cytokeratin (ck) 34e12 (clone : m0630 ; dako, carpinteria, ca) at a 1:50 dilution according to the manufacturers ' protocols. a fbcld was defined as the focal absence of basal cells resulting in a gap larger than the combined size of at least three basal cells in at least two immediate adjacent sections. to identify the potential impact of fbcld on the physical integrity of the associated basement membrane, two sections immediate adjacent to double immunostained ones that harbored fbcld from each case were subjected to double immunohistochemistry to simultaneously elucidate the basal cell layer and the basement membrane using a previously published protocol. briefly, deparaffinized sections were incubated in 1x antigen retrieval solution (cat # : rv1000 m ; biocare medical, concord, ca) overnight (?) at 70 in a regular oven. after incubation, the sections were washed in tap water and pbs (ph 7.4), each for 5 - 10 minutes, and then, incubated with antibodies to p63 (at a 1:50 dilution) and ck 34e12 (at a 1:50 dilution) for 2 - 3 hours at room temperature. after the incubation, the sections were washed in three changes of pbs, each for 2 - 3 minutes, and then, incubated with the corresponding secondary antibody. the antigen and antibody complex was detected with an abc detection kit and a dab chromogen kit (vector laboratories, burlingame, ca), according to the instructions provided by the manufacturer. after chromogen reaction, the sections were washed in tap water and pbs, each for 5 - 10 minutes. then, the sections were incubated with proteinase k ready - to - use solution (cat # : s3020 ; dako, carpinteria, ca) at room temperature for 3 - 5 minutes. after the proteinase k digestion, sections were incubated with mouse monoclonal antibodies to collagen iv (clone : civ 22 ; dako ; carpinteria, ca) at a 1:50 dilution or laminin (clone : vp - l551 ; vector laboratories, burlingame, ca) at a 1:25 dilution at room temperature for 2 - 3 hours. after the incubation, the sections were washed in three changes of pbs, each for 2 - 3 minutes, and then, incubated with the corresponding secondary antibody. the antigen and antibody complex was detected with an abc detection kit and an ap red - chromogen kit (cat # : 00 - 2203 ; zymad, south san francisco, ca) according to the instructions provided by the manufacturers. to assess the specificity of the immunostaining first, different negative controls were used, which included (1) the substitution of the primary antibody with normal serum, (2) the omission of the secondary antibody from the immunostaining sequence, (3) serial dilutions of the primary antibody, and (4) the inclusion of sections from normal lymph - nodes in the normal immunostaining process. second, the same immunostaining protocol was used on the same cases, but substituting with different detection system and substrates. third, the immunostaining procedure was repeated at least twice using the same protocol and under the same condition and immunostained sections were independently evaluated by at least two investigators. using p63 and cke12 double immunostained sections as references, the corresponding sites of fbcld in sections double immunostained for both basal cell phenotypic and basement membrane markers were photographed, and large prints were made and examined, to determine whether alterations in the basal cell layer and the basement membrane are correlated events. correlated alterations of the basal cell layer and basement membrane were defined as a simultaneous focal loss of both structures on the same sites. distinct immunoreactivities to collagen or laminin were preferentially seen in the basement membrane, but were also seen in the stroma and blood vessels, which contain abundant collagen and laminin as structural elements. all negative controls completely lacked distinct immunoreactivities to any of the markers used. in sections double immunostained for basal cell phenotypic and basement membrane markers, both p63 and cke12 could elucidate the basal cell layer, while p63 appeared to be able to better differentiate between the basal cell layer from the basement membrane (fig 1). a vast majority of the ducts and acini with normal morphology or with hyperplastic or pin lesions contained a non - disrupted basal cell layer and a continuous basement membrane, whereas their adjacent invasive lesions laced both (fig 1). the physical integrity of the basal cell layer and basement membrane appeared to be largely independent of the ductal or acinar lumen or tumor size (fig 1e-1h). of a total of 89 fbcld encountered, 76 (85 %) showed focal disruption or fragmentations in the overlying basement membrane (table 1), whereas none showed the integrity of basement membrane is impaired while the basal cell layer is normal. over 60% of the focal disruptions in both the basal cell layer and the overlying basement membrane were seen in pin (fig 2), while about 30% of these focal disruptions were seen in ducts or acini with benign morphology (fig 3). the size of these focal disruptions varied substantially, from a few cells (fig 2a-2d) to more than a half of the entire basal cell layer and the basement membrane (fig 2e-2h). the size of these focal disruptions in normal or hyperplastic lesions was generally small and varied in numbers (fig 3). the basement membrane overlying the remaining 13 (15%) fbcld showed significant attenuation or reduction of the immunostaining intensity, compared to its adjacent counterpart overlying the non - disrupted basal cell layer (fig 4). the basement membrane in all or nearly all ducts or acini with p63 positive basal cells was substantially thicker and more uniform than that in ducts or acini without p63 positive basal cells (fig 4a-4b), and also, a vast majority of the focal disruptions occurred near basal cells that lack p63 expression (not shown). the pattern and frequency of fbcld seen in our current study are in total agreement with those of our previous studies, and also those from other groups. our findings of the loss or fragmentations of the basement membrane are also in line with previous reports. to our best knowledge, our finding of correlated alterations in the basal cell layer and the underlying basement membrane, and malignancy - associated morphologic alterations (focal disruptions in both the basal cell layer and basement membrane) in morphologically normal or hyperplastic duct or acinar clusters, however, have not been previously reported. since the epithelium is normally devoid of both blood vessels and lymphatic ducts, and the basal cell layer is the sole source of several tumor suppressors 29 - 32, a focal disruption in the basal cell layer and its underlying basement membrane could potentially have a number of consequences, including : (1) a loss or reduction of tumor suppressors and the paracrine inhibitory functions, which allow the luminal cells to undergo elevated proliferation 37 - 41, (2) alterations in the permeability for oxygen or growth factors, which selectively triggers the exit of stem or progenitor cells from quiescence, and favor proliferation of cells overlying fbcld 42 - 44, (3) the exposure of luminal cells to different cytokines, which facilitates vasculogenic mimicry and tumor angiogenesis 45 - 46, (4) the physical contact between luminal and stromal cells, which augments the expression of stromal mmp and facilitates epithelial - mesenchymal transition and cell motility 47 - 49, and (5) the physical contact between luminal and immunoreactive cells, which directly causes genomic or cellular damages that trigger a cascade reaction of malignant transformation 50 - 55. these alterations could individually or collectively trigger elevated proliferation in luminal cells near fbcld, which leads to the enlargement of fbcld and stretching - out of the residual basal cell layer and basement membrane. eventually, the entire basal cell layer and basement membrane becomes dissociated or degenerated (as those shown in fig 2e-2h), which facilitates invasion or progression of the overlying tumor cells. thus, ducts or acini with focal disruptions in both the basal cell layer and the underlying basement membrane are very likely at greater risk to develop invasive prostate lesions. consequently, the development of more practical and quantitative methods to assess the physical and functional integrity of the basal cell layer and basement membrane may lead to the development of a more effective alternative for repeat biopsy to monitor tumor progression and invasion. more importantly, our findings suggest that in addition to the multistage model, in which prostate carcinogenesis is believed to be sequentially progressing from normal, to hyperplasia, to high grade pin, and to invasive lesions, prostate tumor invasion could potentially take place at any stage, if a focal disruption of the basal cell layer and the basement membrane happens to occur near a tumor progenitor 34 - 36. the underling mechanism for the correlated alterations in the basal cell layer and the underlying basement membrane is unknown, but is likely to result from focal degeneration of aged or injured basal cells and resultant auto - immunoreactions. the basal cell belongs to a self - renewal population that has to consistently undergo proliferation and differentiation to replace aged or injured cells. a number of external or internal insults, such as radiation, carcinogens, localized trauma, inflammation, or other factors, could cause the inactivation of, or defects, in basal cell renewal - related genes, which impair the basal cell replenishment process to replace the aged or injured basal cells, resulting in a senesced basal cell population. these senesced basal cells may have significantly reduced functions to produce the major building blocks of the basement membrane or may have significantly reduced affinity in their surface to attract the deposition of collagen, laminin, and other building elements of the basement membrane. consistent with this possibility is the fact that the basement membrane in all or nearly all ducts or acini with p63 positive basal cells was substantially thicker and more uniform than that in ducts or acini without p63 positive basal cells (fig 4a-4b), and also, a vast majority of the focal disruptions occurred near basal cells that lack p63 expression. in summary, our current study reveals for the first time that the basement membrane underlying all focally disrupted basal cell layers encountered showed either correlated focal disruptions (85%) or substantial attenuation (15%), suggesting that the functional or physical status of the basal cells significantly impact the physical integrity of the associated basement membrane. as the degradation of both the basal cell layer and the basement membrane is a pre - requisite for prostate tumor invasion or progression, ducts or acini with focally disrupted basal cell layer and basement membrane are likely at greater risk to develop invasive lesions. thus, further elucidation of the specific molecules and mechanism associated with these events may lead to the development of a more effective alternative for repeat biopsy to monitor tumor progression and invasion. | our recent studies revealed that focal basal cell layer disruption (fbcld) induced auto - immunoreactions represented a contributing factor for human prostate tumor progression and invasion. as the basement membrane surrounds and attaches to the basal cell layer, our current study assessed whether fbcld would impact the physical integrity of the associated basement membrane. paraffin sections from 25-human prostate tumors were subjected to double immunohistochemistry to simultaneously elucidate the basal cell layer and the basement membrane with corresponding biomarkers. the physical integrity of the basement membrane overlying fbcld was examined to determine the extent of correlated alterations. of a total of 89 fbcld encountered, 76 (85 %) showed correlated alterations in the overlying basement membrane, which included distinct focal disruptions or fragmentations. in the remaining 13 (15%) fbcld, the overlying basement membrane showed significant attenuation or reduction of the immunostaining intensity. the basement membrane in all or nearly all ducts or acini with p63 positive basal cells was substantially thicker and more uniform than that in ducts or acini without p63 positive basal cells, and also, a vast majority of the focal disruptions occurred near basal cells that lack p63 expression. these findings suggest that focal disruptions in the basal cell layer and alterations in the basement membrane are correlated events and that the physical and functional status of the basal cells could significantly impact the physical integrity of the overlying basement membrane. as the degradation of both the basal cell layer and the basement membrane is a pre - requisite for prostate tumor invasion or progression, ducts or acini with focally disrupted basal cell layer and basement membrane are likely at greater risk to develop invasive lesions. thus, further elucidation of the specific molecules and mechanism associated with these events may lead to the development of a more effective alternative for repeat biopsy to monitor tumor progression and invasion. |
the reaction of a manganese(v)oxo porphyrinoid complex with the lewis acid b(c6f5)3 leads to reversible stabilization of the valence tautomer mniv(o)(-radical cation). the latter complex, in combination with b(c6f5)3, reacts with aro h substrates via formal hydrogen - atom transfer and exhibits dramatically increased reaction rates over the mnv(o) starting material. |
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erectile dysfunction (ed) remains a common cause of significant postoperative morbidity in patients undergoing radical therapies for prostate cancers or other pelvic malignancies, because the cavernous nerve can be inadvertently axotomized, lacerated, or stretched at the time of surgery. although techniques such as nerve - sparing radical prostatectomy, nerve grafting, and robotic - assisted radical prostatectomy have been developed in which the cavernous nerve is largely preserved, postoperatively, patients still face a high incidence of ed. in recent years, stem cell therapy has evolved as a potential therapy in the prevention of ed following cavernous nerve injury. the multilineage differentiation potential of stem cells can contribute to regenerate the damaged cavernous nerve, thereby providing an autologous and curative therapeutic option. although bone marrow cells have the ability to differentiate into neurons, limitations hinder the widespread application of these cells. among the various stem cells, human muscle - derived stem cells (hmdscs) and human - adipose tissue derived stem cells (hadscs) have great potential in stem cell therapy because of their abundance, ease of isolation, and low immunogenicity. in addition, hmdscs and hadscs have a considerable capacity for self - renewal and can differentiate into neural lineages [5 - 9 ]. although many studies on the neurogenic trans - differentiation of hmdscs and hadscs have been conducted, no in vitro studies are available for comparison of the neurogenic capacity of these cells. in this study, therefore, we compared the neuronal differentiation capacity of hmdscs with that of hadscs in vitro. human adipose tissues were obtained by simple liposuction from the abdominal subcutaneous fat of donors. subcutaneous adipose tissues were washed with phosphate- buffered saline (pbs) and digested with 1 mg / ml collagenase under gentle agitation for 60 minutes at 37. next, the digested tissues were filtered through a 100 m nylon mesh in order to remove cellular debris and centrifuged at 1,500 rpm for 5 min to obtain a pellet. the pellet was re - suspended in rcme (rnl bio, seoul, korea) or dmem containing 10% fetal bovine serum (fbs). the cell fraction was cultured overnight at 37 in 5% co2 in rcme or dmem. adhesion of cells was checked under a microscope the next day. the procedure for preparation of hadscs using scalpels, hmdscs isolated from the rectus muscles of humans were removed and minced into a coarse slurry. louis, mo, usa), dispase (invitrogen, carlsbad, ca, usa), and trypsin - edta (invitrogen). after physical and enzymatic dissociations, muscle - derived cells were centrifuged and re - suspended in proliferation medium (containing dmem [gibco, grand island, ny, usa ], 10% horse serum, 10% fbs, 1% chick embryo extract, and 1% penicillin - streptomycin). for isolation of smnps, cells were plated in collagen - coated flasks (pp1). after 2 hours, cells non - adherent in pp1 were transferred into the next flasks (pp2) and incubated for 16 hours. no adherent cells were observed in pp3 ; therefore, the serial transfer of non - adherent cells was terminated at this point. during cultivation, two morphologically pp2 and pp3 flasks contained approximately 30%, 10%, and < 1% of fibroblast - like cells, respectively. further purification of weakly adherent cells (primarily round - shaped cells) was performed on cells adherent in pp2 and pp3 flasks. cells were trypsinized for 3 min, after which suspended cells from pp2 and pp3 flasks were combined and re - plated in a fresh flask. as these cells proliferated in proliferation medium for 3 to 4 days, were grown as adherent cultures in dmem supplemented with 10% fbs and 1% penicillin - streptomycin at 37 in 5% co2. after 24 hours, cells were washed with pbs and plated in neurobasal media, 1% penicillin, and 50 m / ml of forskolin and 1 g / ml laminin, 20 ng / ml basic fibroblast growth factor (bfgf), and 20 ng / ml epidermal growth factor (egf) with 2% fbs. both hmdscs and hadscs were placed and grown on poly - l - lysine - coated cover slips for 5 days. after 5 days, these cells were washed with pbs, fixed with 4% paraformaldehyde for 10 minutes at room temperature, washed with pbs again, and then permeabilized with 0.1% triton - x 100. after washing twice with pbs, cells were blocked by incubation with 10% normal goat serum for 1 hour at room temperature. nestin (1:100 ; abcam, combride, uk), -tubulin iii (tuj1, 1:500 ; abcam, combride, uk), and gfap (1:100 ; abcam, combride, uk) were then added. primary antibodies were incubated overnight at 4. after washing, the cells were treated with alexa 488-conjugated goat anti - mouse antibody as a secondary antibody, followed by incubation at room temperature for 1 hour. nuclei were stained with dapi for cell counting, and the cells were observed under a fluorescence microscope. rna was extracted from hmdscs, differentiated hmdscs (d - hmdscs), hadscs, and differentiated hadscs (dhadscs). first - strand cdna was synthesized by using oligo dt primer and superscript ii reverse transcriptase according to the manufacturer 's instructions. real - time polymerase chain reaction analysis (pcr) was performed by using rt sybr green / rox qpcr master mix using the real - time thermal cycler (50/2 min, 95/10 min, [95/15 s, 60/30 s, 72/30 s - 40 cycles ], 95/15 s, 60/1 min, 95/15 s). protein was homogenized in ice - cold lysis buffer containing 20 mm tris - cl, ph 8.0, 150 mm nacl, 1 mm edta, 1% triton x-100, 10 m leupeptin, 20 g / ml chymostatin, and 2 mm pmsf. following centrifugation at 12,000 xg for 30 minutes at 4, the supernatant was extracted and quantified by use of the bicinchoninic acid (bca) protein assay kit (pierce, rockford, il, usa) and then electrophoresed on an sds - page gel. after transfer to a nitrocellulose membrane, the membrane was blocked with 5% skim milk and then incubated with antibody against nestin (1:1,000 ; abcam), -tubulin iii (1:1,000 ; abcam), gfap (1:1,000;abcam), or -actin, followed by incubation with the corresponding secondary antibody conjugated to horseradish peroxidase. the ecl method (amersham, arlington heights, il, usa) was used for development of protein bands. statistical analyses were performed by using the two - tailed student 's t - test. human adipose tissues were obtained by simple liposuction from the abdominal subcutaneous fat of donors. subcutaneous adipose tissues were washed with phosphate- buffered saline (pbs) and digested with 1 mg / ml collagenase under gentle agitation for 60 minutes at 37. next, the digested tissues were filtered through a 100 m nylon mesh in order to remove cellular debris and centrifuged at 1,500 rpm for 5 min to obtain a pellet. the pellet was re - suspended in rcme (rnl bio, seoul, korea) or dmem containing 10% fetal bovine serum (fbs). the cell fraction was cultured overnight at 37 in 5% co2 in rcme or dmem. adhesion of cells was checked under a microscope the next day. the procedure for preparation of hadscs using scalpels, hmdscs isolated from the rectus muscles of humans were removed and minced into a coarse slurry. louis, mo, usa), dispase (invitrogen, carlsbad, ca, usa), and trypsin - edta (invitrogen). after physical and enzymatic dissociations, muscle - derived cells were centrifuged and re - suspended in proliferation medium (containing dmem [gibco, grand island, ny, usa ], 10% horse serum, 10% fbs, 1% chick embryo extract, and 1% penicillin - streptomycin). for isolation of smnps, cells were plated in collagen - coated flasks (pp1). after 2 hours, cells non - adherent in pp1 were transferred into the next flasks (pp2) and incubated for 16 hours. no adherent cells were observed in pp3 ; therefore, the serial transfer of non - adherent cells was terminated at this point. during cultivation, two morphologically pp2 and pp3 flasks contained approximately 30%, 10%, and < 1% of fibroblast - like cells, respectively. further purification of weakly adherent cells (primarily round - shaped cells) was performed on cells adherent in pp2 and pp3 flasks. cells were trypsinized for 3 min, after which suspended cells from pp2 and pp3 flasks were combined and re - plated in a fresh flask. as these cells proliferated in proliferation medium for 3 to 4 days, the cells formed additional colonies. human adipose tissues were obtained by simple liposuction from the abdominal subcutaneous fat of donors. subcutaneous adipose tissues were washed with phosphate- buffered saline (pbs) and digested with 1 mg / ml collagenase under gentle agitation for 60 minutes at 37. next, the digested tissues were filtered through a 100 m nylon mesh in order to remove cellular debris and centrifuged at 1,500 rpm for 5 min to obtain a pellet. the pellet was re - suspended in rcme (rnl bio, seoul, korea) or dmem containing 10% fetal bovine serum (fbs). the cell fraction was cultured overnight at 37 in 5% co2 in rcme or dmem. adhesion of cells was checked under a microscope the next day. the procedure for preparation of hadscs using scalpels, hmdscs isolated from the rectus muscles of humans were removed and minced into a coarse slurry. louis, mo, usa), dispase (invitrogen, carlsbad, ca, usa), and trypsin - edta (invitrogen). after physical and enzymatic dissociations, muscle - derived cells were centrifuged and re - suspended in proliferation medium (containing dmem [gibco, grand island, ny, usa ], 10% horse serum, 10% fbs, 1% chick embryo extract, and 1% penicillin - streptomycin). for isolation of smnps after 2 hours, cells non - adherent in pp1 were transferred into the next flasks (pp2) and incubated for 16 hours. no adherent cells were observed in pp3 ; therefore, the serial transfer of non - adherent cells was terminated at this point. during cultivation, two morphologically pp2 and pp3 flasks contained approximately 30%, 10%, and < 1% of fibroblast - like cells, respectively. further purification of weakly adherent cells (primarily round - shaped cells) was performed on cells adherent in pp2 and pp3 flasks. cells were trypsinized for 3 min, after which suspended cells from pp2 and pp3 flasks were combined and re - plated in a fresh flask. as these cells proliferated in proliferation medium for 3 to 4 days, the cells formed additional colonies. both hmdscs and hadscs (1x10cells) were grown as adherent cultures in dmem supplemented with 10% fbs and 1% penicillin - streptomycin at 37 in 5% co2. after 24 hours, cells were washed with pbs and plated in neurobasal media, 1% penicillin, and 50 m / ml of forskolin and 1 g / ml laminin, 20 ng / ml basic fibroblast growth factor (bfgf), and 20 ng / ml epidermal growth factor (egf) with 2% fbs. both hmdscs and hadscs were placed and grown on poly - l - lysine - coated cover slips for 5 days. after 5 days, these cells were washed with pbs, fixed with 4% paraformaldehyde for 10 minutes at room temperature, washed with pbs again, and then permeabilized with 0.1% triton - x 100. after washing twice with pbs, cells were blocked by incubation with 10% normal goat serum for 1 hour at room temperature. nestin (1:100 ; abcam, combride, uk), -tubulin iii (tuj1, 1:500 ; abcam, combride, uk), and gfap (1:100 ; abcam, combride, uk) were then added. primary antibodies were incubated overnight at 4. after washing, the cells were treated with alexa 488-conjugated goat anti - mouse antibody as a secondary antibody, followed by incubation at room temperature for 1 hour. nuclei were stained with dapi for cell counting, and the cells were observed under a fluorescence microscope. rna was extracted from hmdscs, differentiated hmdscs (d - hmdscs), hadscs, and differentiated hadscs (dhadscs). first - strand cdna was synthesized by using oligo dt primer and superscript ii reverse transcriptase according to the manufacturer 's instructions. real - time polymerase chain reaction analysis (pcr) was performed by using rt sybr green / rox qpcr master mix using the real - time thermal cycler (50/2 min, 95/10 min, [95/15 s, 60/30 s, 72/30 s - 40 cycles ], 95/15 s, 60/1 min, 95/15 s). protein was homogenized in ice - cold lysis buffer containing 20 mm tris - cl, ph 8.0, 150 mm nacl, 1 mm edta, 1% triton x-100, 10 m leupeptin, 20 g / ml chymostatin, and 2 mm pmsf. following centrifugation at 12,000 xg for 30 minutes at 4, the supernatant was extracted and quantified by use of the bicinchoninic acid (bca) protein assay kit (pierce, rockford, il, usa) and then electrophoresed on an sds - page gel. after transfer to a nitrocellulose membrane, the membrane was blocked with 5% skim milk and then incubated with antibody against nestin (1:1,000 ; abcam), -tubulin iii (1:1,000 ; abcam), gfap (1:1,000;abcam), or -actin, followed by incubation with the corresponding secondary antibody conjugated to horseradish peroxidase. the ecl method (amersham, arlington heights, il, usa) statistical analyses were performed by using the two - tailed student 's t - test. 1a and i). in immunohistochemical staining, hmdscs and hmdscs were strongly positive for neural stem cell marker (nestin), but weakly positive for neuronal marker (-tubulin iii) and glial marker (gfap) (fig. following 5 days of neural induction, hmdscs and hadscs displayed a differentiated phenotype in morphology (fig. immunohistochemistry showed that nestin expression was decreased, and -tubulin iii and gfap expression were increased (fig. there was no significant difference in nestin expression between d - hmdscs and d - hadscs ; however, -tubulin iii and gfap expression in d - hadscs was increased compared with that in d - hmdscs. according to cell morphology and staining, hadscs had a higher phenotype and neurogenic potential than did hmdscs. to dissect the neurogenic potential of hmdscs and hadscs at the molecular level, the mrna levels of nestin, -tubulin iii, and gfap were detected by real - time pcr, normalized to the housekeeping gapdh. nestin mrna levels in d - hmdscs and d - hadscs were approximately 50% lower than the expression in hmdscs and hadscs, respectively. there was no significant difference in nestin mrna expression between d - hmdscs and d - hadscs (fig. however, -tubulin iii and gfap mrna expression in d - hadscs was significantly higher than that in d - hmdscs (fig. 2b and 2c). we also investigated the protein expression of nestin, -tubulin iii, and gfap in differentiated hmdscs and hadscs by western blot analysis (fig. the protein expression of nestin, -tubulin iii, and gfap before and after differentiation showed almost the same tendency with mrna expression. protein expression of nestin in the hmdscs and hadscs was significantly decreased after neuronal induction (fig. 1a and i). in immunohistochemical staining, hmdscs and hmdscs were strongly positive for neural stem cell marker (nestin), but weakly positive for neuronal marker (-tubulin iii) and glial marker (gfap) (fig. following 5 days of neural induction, hmdscs and hadscs displayed a differentiated phenotype in morphology (fig. immunohistochemistry showed that nestin expression was decreased, and -tubulin iii and gfap expression were increased (fig. there was no significant difference in nestin expression between d - hmdscs and d - hadscs ; however, -tubulin iii and gfap expression in d - hadscs was increased compared with that in d - hmdscs. according to cell morphology and staining, hadscs had a higher phenotype and neurogenic potential than did hmdscs. to dissect the neurogenic potential of hmdscs and hadscs at the molecular level, the mrna levels of nestin, -tubulin iii, and gfap were detected by real - time pcr, normalized to the housekeeping gapdh. nestin mrna levels in d - hmdscs and d - hadscs were approximately 50% lower than the expression in hmdscs and hadscs, respectively. there was no significant difference in nestin mrna expression between d - hmdscs and d - hadscs (fig. however, -tubulin iii and gfap mrna expression in d - hadscs was significantly higher than that in d - hmdscs (fig. 2b and 2c). we also investigated the protein expression of nestin, -tubulin iii, and gfap in differentiated hmdscs and hadscs by western blot analysis (fig. the protein expression of nestin, -tubulin iii, and gfap before and after differentiation showed almost the same tendency with mrna expression. protein expression of nestin in the hmdscs and hadscs was significantly decreased after neuronal induction (fig. -tubulin iii and gfap protein expression in d - hadscs was significantly higher than that in d - hmdscs (fig. this study compared the neurogenic potential of hmdscs and hadscs by detecting several neuron cell markers. our data suggest that differentiated hadscs have higher neuronal / glial marker expression than do hmdscs and thereby are more suitable for nerve regeneration. recently, stem cell treatment has been regarded as a potential treatment for ed. in recent years, tissue engineering strategies have combined living cells and scaffold materials for the development of biological substitutes that can restore tissue functions. both natural and synthetic materials have been fabricated for transplantation of stem cells and their specific differentiation into muscles, bones, cartilage, and neuron - like cells. studies have shown that hmdscs and hadscs can be induced into adipocytes, osteoblasts, chondrocytes, myocytes, hepatocytes, endotheliocytes [5 - 9 ], and neuron - like cells [2,10 - 13 ]. the surface phenotype of hmdscs and hadscs is similar to that of bone marrow - derived stromal cells. previous reports have indicated that neural differentiation of hmdscs and hadscs is achieved with different experimental protocols, including the use of chemical agents, such as forskolin [11,14 - 17 ], laminin, egf, and bfgf. bfgf is known to generate neural precursor cells with a greater capacity for neuronal differentiation. forskolin is a commonly used agent to increase the intracellular levels of cyclic adenosine monophosphate (camp) by activating the enzyme adenylyl cyclase. laminin - stimulated levels of tyrosine hydroxylase of chromaffin cells were associated with their conversion to adrenergic sympathetic cells. laminin has differential action in the proliferation, survival, and differentiation of cultured neuronal cells. although forskolin, laminin, egf, and bfgf enhanced neuron - like cell production, the results were greatly improved when the combination was added to the medium. we succeeded in inducing the neural differentiation of hmdscs and hadscs by treatment with forskolin, laminin, b - fgf, and egf. in the present study, the neuronal phenotype at 5 days following neural induction was observed by staining for nestin - negative and -tubulin iii- and gfap - positive. we found that d - hmdscs and d - hadscs displayed similar immunocytochemical changes following neural induction. hmdscs and hadscs grown under control conditions expressed low -tubulin iii and gfap but detectable levels of nestin. after 5 days of neural induction, we detected strong staining for -tubulin iii as well as gfap in d - hadscs. real - time pcr and western blotting confirmed that the number of undifferentiated hmdscs and hadscs expressing cell type - specific markers was very low. neurons were observed in cultures when compared with that of nestin - negative and -tubulin iii-, gfap - positive. our results showed that differentiated muscle- or adipose - derived stem cells have morphological and phenotypic characteristics that are similar to those of neural cells. in this study, we confirmed that the ability of hadscs to differentiate into neural cells was better than that of hmdscs. in summary, we have shown that both hmdscs and hadscs have morphological and phenotypic characteristics similar to those of neural cells. therefore, hadscs seem to be an ideal alternative source of stem cells for cavernous nerve regeneration. | purposeerectile dysfunction (ed) remains a major complication from cavernous nerve injury during radical prostatectomy. recently, stem cell treatment for ed has been widely reported. this study was conducted to investigate the availability, differentiation into functional cells, and potential of human muscle - derived stem cells (hmdscs) and human adipose - derived stem cells (hadscs) for ed treatment.materials and methodswe compared the neural differentiation of hmdscs and hadscs. human muscle and adipose tissues were digested with collagenase, followed by filtering and centrifugation. for neural induction, isolated hmdscs and hadscs were incubated in neurobasal media containing forskolin, laminin, basic - fibroblast growth factor, and epidermal growth factor for 5 days. following neural induction, hmdscs and hadscs were differentiated into neural cells, including neurons and glia, in vitro.resultsin neural differentiated hmdscs (d - hmdscs) and differentiated hadscs (d - hadscs), neural stem cell marker (nestin) showed a significant decrease by immunocytochemistry, and neuronal marker (-tubulin iii) and glial marker (gfap) showed a significant increase, compared with primary hmdscs and hadscs. real - time chain reaction analysis and western blotting demonstrated significantly elevated levels of mrna and protein of -tubulin iii and gfap in d - hadscs compared with d-hmdscs.conclusionswe demonstrated that hmdscs and hadscs can be induced to undergo phenotypic and molecular changes consistent with neurons. the neural differentiation capacity of hadscs was better than that of hmdscs. |
arachidonic acid (aa) within the cell membrane is metabolized by the enzyme 5-lipoxygenase (5-lo) to produce leukotrienes.1,2 the 5-lo activating protein (flap) binds to 5-lo in this process, enabling transfer of aa to 5-lo. aa metabolism produces leukotriene a4 (lta4), which is subsequently converted to either ltb4 or the cysteinyl leukotrienes (cyslts) ltc4, ltd4, and lte4. cyslts bind to cyslt1 and cyslt2 receptors, causing bronchoconstriction and eosinophilic inflammation, while ltb4 promotes the chemotaxis and activation of immune cells including neutrophils and lymphocytes through blt1 and blt2 receptors. aa metabolism by 5-lo also produces 5-hydroxyeicosatetraenoic acid (5-hete), which is further metabolized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo - ete) ; this activates neutrophils and eosinophils.3 cyslts levels are elevated in the lungs of patients with asthma.4,5 cyslt receptor antagonists are used for the treatment of asthma,1 but do not inhibit ltb4 or 5-hete activity. the 5-lo inhibitor zileuton is approved for the treatment of asthma, but the doses used in clinical practice only partially inhibit leukotriene production, and the therapeutic index is limited by side effects.1 there is no available drug that completely inhibits the actions of all of the mediators produced by the 5-lo pathway. gsk2190915 is a novel potent flap inhibitor currently in development for the treatment of asthma (the flap pathway is detailed in figure 1).6 it inhibits pulmonary cyslts and ltb4 production in animal models,8 and inhibits ltb4 production by whole blood stimulated ex vivo and urine lte4 excretion in healthy subjects.9 the inhaled allergen challenge model is widely used to characterize potential new treatments for asthma ; inhibition of the early asthmatic response (ear) demonstrates the ability to prevent acute allergic bronchoconstriction, whereas inhibition of the late asthmatic response (lar) suggests effective anti - inflammatory properties.7,1015 we have recently shown that gsk2190915 100 mg daily inhibits both the ear and lar in subjects with mild asthma.7 other flap inhibitors also attenuate both the ear and lar.12,13 the therapeutic dose of a new treatment for asthma is usually established by assessing its effect on pulmonary function tests and symptoms. such studies require large numbers of subjects to discriminate between doses.16,17 we have employed an alternative approach ; we used the ear to study the dose response effects of gsk2190915 in subjects with asthma. we have previously reported that gsk2190915 100 mg inhibits the ear,7 but have not reported the effects of lower doses. we knew that gsk2190915 50 mg almost completely suppresses urinary lte4 levels in healthy subjects, whereas 10 mg causes incomplete suppression, ranging from 40% to 60%.9 therefore, we chose to assess the effect of gsk2190915 10 mg and 50 mg on the ear in subjects with mild asthma, and to compare the results with those of the effect of gsk2190915 100 mg daily on the ear from our previously reported study.7 nineteen nonsmoking subjects, aged 18 to 55 years, with mild asthma were recruited. males and females of non - childbearing potential were included ; females of childbearing potential were not included as the appropriate reproductive toxicology studies had not been conducted at the time of the study. other inclusion criteria were : body mass index within the range 18.535.0 kg / m ; forced expiratory volume in 1 second (fev1) > 70% predicted ; current nonsmoker with a pack history of 10 pack years ; current asthma treatment with an inhaled short - acting beta agonist only ; no use of inhaled corticosteroids for at least 1 month before screening ; and asthma diagnosis confirmed by provocative methacholine or histamine concentration causing a 20% fall in fev1 (provocative concentration 20 [pc20 ]) 10% from the pre - saline measurement. subjects then proceeded to inhale methacholine (provocholine ; methapharm europe, geneva, switzerland), starting at a concentration of 0.0625 mg / ml, and increasing two - fold until the pc20 was reached. the concentration of methacholine did not exceed 8 mg / ml. subjects were monitored until lung function returned to within 10% of resting fev1. a 3 ml blood sample was taken on day 3 at 2 hours after dosing, and plasma was separated by centrifugation (4c, 1,500 g, 15 minutes), then stored (20c) for subsequent analysis of gsk2190915 (lower limit of qualification of 5 ng / ml) by a validated method based on protein precipitation, followed by high - performance liquid chromatography tandem mass spectrometry analysis, as described previously.9 adverse events (aes), clinical laboratory tests, physical examination, vital signs, 12-lead electrocardiogram, and pulmonary function tests, were recorded throughout the study. study completion by at least 16 subjects provided at least 90% power to detect a 40% attenuation of the placebo response in the minimum fev1 absolute change from baseline within the 2-hour period following allergen challenge on day 3 ; using a two - sided 5% significance level, assuming a within - subject standard deviation of 0.27 l and a mean placebo response of 0.87 l. the primary endpoint was the minimum fev1 absolute change from baseline within the 2-hour period after allergen challenge on day 3 of each treatment. the change from allergen challenge baseline fev1 over time on day 3 was analyzed using a repeated - measures model (including time point, period, treatment, treatment - by - time). the minimum fev1 absolute change from baseline within the 2-hour period following allergen challenge on day 3 was analyzed using a mixed - effects model (including period, treatment, and covariates for pre - dose fev1 on day 1). a similar analysis was performed for the weighted mean fev1 within the 2-hour period after allergen challenge on day 3. nineteen nonsmoking subjects, aged 18 to 55 years, with mild asthma were recruited. males and females of non - childbearing potential were included ; females of childbearing potential were not included as the appropriate reproductive toxicology studies had not been conducted at the time of the study. other inclusion criteria were : body mass index within the range 18.535.0 kg / m ; forced expiratory volume in 1 second (fev1) > 70% predicted ; current nonsmoker with a pack history of 10 pack years ; current asthma treatment with an inhaled short - acting beta agonist only ; no use of inhaled corticosteroids for at least 1 month before screening ; and asthma diagnosis confirmed by provocative methacholine or histamine concentration causing a 20% fall in fev1 (provocative concentration 20 [pc20 ]) 10% from the pre - saline measurement. subjects then proceeded to inhale methacholine (provocholine ; methapharm europe, geneva, switzerland), starting at a concentration of 0.0625 mg / ml, and increasing two - fold until the pc20 was reached. the concentration of methacholine did not exceed 8 mg / ml. subjects were monitored until lung function returned to within 10% of resting fev1. a 3 ml blood sample was taken on day 3 at 2 hours after dosing, and plasma was separated by centrifugation (4c, 1,500 g, 15 minutes), then stored (20c) for subsequent analysis of gsk2190915 (lower limit of qualification of 5 ng / ml) by a validated method based on protein precipitation, followed by high - performance liquid chromatography tandem mass spectrometry analysis, as described previously.9 adverse events (aes), clinical laboratory tests, physical examination, vital signs, 12-lead electrocardiogram, and pulmonary function tests, were recorded throughout the study. study completion by at least 16 subjects provided at least 90% power to detect a 40% attenuation of the placebo response in the minimum fev1 absolute change from baseline within the 2-hour period following allergen challenge on day 3 ; using a two - sided 5% significance level, assuming a within - subject standard deviation of 0.27 l and a mean placebo response of 0.87 l. the primary endpoint was the minimum fev1 absolute change from baseline within the 2-hour period after allergen challenge on day 3 of each treatment. the change from allergen challenge baseline fev1 over time on day 3 was analyzed using a repeated - measures model (including time point, period, treatment, treatment - by - time). the minimum fev1 absolute change from baseline within the 2-hour period following allergen challenge on day 3 was analyzed using a mixed - effects model (including period, treatment, and covariates for pre - dose fev1 on day 1). a similar analysis was performed for the weighted mean fev1 within the 2-hour period after allergen challenge on day 3. nineteen subjects (18 males and 1 female, mean age 35 years, mean fev1 3.80 l, mean fev1 predicted 92.8%) were enrolled in the study (patient demographics and characteristics are shown in table 1). the mean fev1 percentage fall from baseline during the screening allergen challenge was 33.4% (range 20.8% to 56.5%). all available data from subjects who received at least one dose of study medication were included in the analysis of efficacy, pharmacokinetics, and safety. figure 2 shows the time profile of the ear after treatment with gsk2190915 and placebo on day 3 following allergen challenge ; gsk2190915 10 mg and 50 mg attenuated the placebo response in a dose - dependent manner. the primary endpoint analysis of minimum fev1 absolute change from baseline showed statistically significant attenuation of the fall in fev1 for gsk2190915 10 mg and 50 mg compared with placebo (figure 3) ; the mean treatment differences were 0.21 l (95% confidence interval [ci ] 0.04 l, 0.38 l) and 0.41 l (95% ci 0.24 l, 0.58 l), respectively, corresponding to a mean attenuation of 18.6% and 36.0% of the placebo response to allergen challenge, respectively. there was a significant difference between gsk2190915 50 mg and 10 mg in the minimum fev1 absolute change from baseline ; the mean treatment difference was 0.20 l (95% ci 0.03 l, 0.36 l). compared with placebo, gsk2190915 50 mg, but not gsk2190915 10 mg, significantly attenuated the weighted mean fev1 absolute change from baseline (figure 3). the mean treatment differences were 0.12 l (95% ci 0.01 l, 0.24 l) and 0.30 l (95% ci 0.18 l, 0.43 l) for gsk2190915 10 mg and 50 mg compared with placebo, respectively, corresponding to mean attenuation of 21.5% and 56.2% of the placebo response, respectively. on day 3, at 2 hours after dosing with gsk2190915 10 mg and 50 mg, the mean (95% ci) plasma concentrations were 83.4 ng / ml (62.3, 104.5) and 418.9 ng / ml (311.0, 526.7), respectively. there were 19 mild - to - moderate aes (table 2), all of which resolved spontaneously. eight were deemed at least possibly related to treatment ; of those eight, four occurred on gsk2190915 10 mg, one on gsk2190915 50 mg, and three on placebo. one subject had high fasting blood glucose before dosing on day 1 of the first treatment period, which was not noted until after dosing and could not have been due to treatment. a second subject had a flu - like illness during the third treatment period (placebo), and was withdrawn as it was not considered safe to perform the allergen challenge. a third subject received gsk2190915 50 mg during the first treatment period and subsequently completed a 25-day washout period. alanine aminotransferase (alt) was increased in his pre - dose sample from the second treatment period. he received a single gsk2190915 10 mg dose before being withdrawn from the study ; subsequent monitoring showed a decline in alt. nineteen subjects (18 males and 1 female, mean age 35 years, mean fev1 3.80 l, mean fev1 predicted 92.8%) were enrolled in the study (patient demographics and characteristics are shown in table 1). the mean fev1 percentage fall from baseline during the screening allergen challenge was 33.4% (range 20.8% to 56.5%). all available data from subjects who received at least one dose of study medication were included in the analysis of efficacy, pharmacokinetics, and safety. figure 2 shows the time profile of the ear after treatment with gsk2190915 and placebo on day 3 following allergen challenge ; gsk2190915 10 mg and 50 mg attenuated the placebo response in a dose - dependent manner. the primary endpoint analysis of minimum fev1 absolute change from baseline showed statistically significant attenuation of the fall in fev1 for gsk2190915 10 mg and 50 mg compared with placebo (figure 3) ; the mean treatment differences were 0.21 l (95% confidence interval [ci ] 0.04 l, 0.38 l) and 0.41 l (95% ci 0.24 l, 0.58 l), respectively, corresponding to a mean attenuation of 18.6% and 36.0% of the placebo response to allergen challenge, respectively. there was a significant difference between gsk2190915 50 mg and 10 mg in the minimum fev1 absolute change from baseline ; the mean treatment difference was 0.20 l (95% ci 0.03 l, 0.36 l). compared with placebo, gsk2190915 50 mg, but not gsk2190915 10 mg, significantly attenuated the weighted mean fev1 absolute change from baseline (figure 3). the mean treatment differences were 0.12 l (95% ci 0.01 l, 0.24 l) and 0.30 l (95% ci 0.18 l, 0.43 l) for gsk2190915 10 mg and 50 mg compared with placebo, respectively, corresponding to mean attenuation of 21.5% and 56.2% of the placebo response, respectively. on day 3, at 2 hours after dosing with gsk2190915 10 mg and 50 mg, the mean (95% ci) plasma concentrations were 83.4 ng / ml (62.3, 104.5) and 418.9 ng / ml (311.0, 526.7), respectively. there were 19 mild - to - moderate aes (table 2), all of which resolved spontaneously. eight were deemed at least possibly related to treatment ; of those eight, four occurred on gsk2190915 10 mg, one on gsk2190915 50 mg, and three on placebo. one subject had high fasting blood glucose before dosing on day 1 of the first treatment period, which was not noted until after dosing and could not have been due to treatment. a second subject had a flu - like illness during the third treatment period (placebo), and was withdrawn as it was not considered safe to perform the allergen challenge. a third subject received gsk2190915 50 mg during the first treatment period and subsequently completed a 25-day washout period. alanine aminotransferase (alt) was increased in his pre - dose sample from the second treatment period. he received a single gsk2190915 10 mg dose before being withdrawn from the study ; subsequent monitoring showed a decline in alt. we have shown that 50 mg administered once - daily had a significantly greater effect than 10 mg once - daily. gsk2190915 50 mg once - daily for 3 days caused 36% and 56% mean attenuation of the minimum and weighted mean fev1 change from baseline, respectively, in subjects with mild asthma ; both changes were statistically significant. in our previously reported study,7 gsk2190915 100 mg once - daily caused 33% and 63% mean attenuation of the minimum and weighted mean fev1 change from baseline compared with placebo, respectively. thus, 50 and 100 mg had very similar effects, suggesting both of the doses were at the top of the dose response curve for ear. as the 50 mg dose was shown to have a similar effect on ear to that reported with a 100 mg dose,7 it would be of interest to assess whether the lower doses of gsk2190915 used in this study also protect against lar. assessment of other efficacy outcomes, such as airway hyperresponsiveness to methacholine, and airway or blood markers of inflammation, would also have been useful. however, such assessments would have required a more complex study design ; as the priority of the study was to measure the dose response effect on the ear, additional assessments were not included. the effect of gsk2190915 50 mg and 100 mg once - daily on the ear is similar to that previously reported for the clinical dose of the leukotriene antagonist montelukast.10,11 furthermore, this effect is also similar to that observed in studies using flap inhibitors.12,13 these studies confirm the role of cyslts in acute allergic bronchoconstriction ; however, it would be useful to compare the magnitude of the effect of the gsk2190915 50 mg dose with other classes of drugs, although such studies have not been performed to date. compared with placebo, gsk2190915 10 mg once - daily for 3 days caused 18% and 21% mean attenuation of the minimum and weighted mean fev1 change from baseline, respectively ; only the minimum mean change was significantly different from placebo. the effects of gsk2190915 10 mg and 50 mg doses on the ear correlate well with those of the same doses on urinary lte4 suppression in healthy subjects;9 10 mg suppressed lte4 by 40%, and 50 mg suppressed lte4 almost completely. we did not include urine lte4 in this study ; however, the plasma concentrations were similar to those in healthy volunteers.9 the data suggest that the use of urine lte4 measurements to assess the effects of drugs on leukotriene production could be a good predictor of the likely effects on allergic bronchoconstriction. in our previously reported study on asthma, the geometric mean peak concentration (cmax) for 100 mg of gsk2190915 was 671 (95% ci : 494, 910) ng / ml,7 compared with the observed cmax values for 10 mg (83.4 [95% ci : 62.3, 104.5 ] ng / ml) and 50 mg (418.9 [95% ci : 311.0, 526.7 ] ng / ml) doses in this study. these dose - related increases in gsk2190915 pharmacokinetics in patients with asthma have also been observed in healthy subjects using gsk2190915 doses up to 1,000 mg. ideally, we could have studied more doses of gsk2190915 in the current study, but there is a limit to the number of allergen challenges that subjects with asthma are willing to tolerate. we therefore chose not to include a dose of 100 mg, but to use results from our previously reported study7 for comparison. although the two studies used different subjects, they all had mild asthma, the procedure for allergen challenge was identical, the clinical sites were the same, the two studies were conducted at the same time, and the inclusion criteria were the same, except that subjects in our previously reported study were also required to have a lar in retrospect, a dose between 10 mg and 50 mg would have yielded useful information about the therapeutic dose. nevertheless, the ear data from our previous7 and current studies provide a guide for doses that can be included in larger and longer dose - ranging studies on clinical endpoints such as lung function and asthma control. while allergen challenge studies of more than one dose have been conducted with other drugs such as inhaled corticosteroids, these studies have included both the ear and lar. we are unaware of any other studies that have used the ear alone to study the dose response effects of novel drugs. we have demonstrated that the ear is a model that can be used in relatively small numbers of subjects to define the dose response effects. this approach is faster and more efficient than alternatives such as longer studies of fev1. in general, gsk2190915 was well - tolerated in this short study. of the three subjects who were withdrawn from the study because of aes, two had abnormal blood results in pre - dose blood samples and the other was withdrawn during placebo treatment. studies with longer duration of dosing and larger numbers of subjects are required to define the potential adverse effects of gsk2190915. in conclusion, gsk2190915 caused dose - dependent attenuation of the ear response to inhaled allergen. gsk2190915 50 mg attenuated the ear similarly to gsk2190915 100 mg in our previous study,7 suggesting that 50 mg is at the top of the dose response curve. this study shows how the ear can be used to assess the therapeutic dose of a new treatment for allergic asthma. | backgroundgsk2190915, a 5-lipoxygenase activating protein inhibitor, inhibits the production of cysteinyl leukotrienes and leukotriene b4 and 5-oxo-6,8,11,14-eicosatetraenoic acid. we have previously reported that gsk2190915 100 mg daily inhibits early and late asthmatic responses to inhaled allergen ; the effects of lower doses have not been reported. this study assessed the dose response effects of gsk2190915 10 mg and 50 mg on the early asthmatic response (ear) to inhaled allergen.methodsnineteen subjects with mild asthma and an ear were enrolled in a randomized, double - blind, three - way crossover study of gsk2190915 10 mg, 50 mg, and placebo orally once - daily for 3 days. allergen challenge was performed 2 hours after the third dose.resultscompared with placebo, gsk2190915 10 mg and 50 mg caused significant, dose - dependent attenuation of the minimum forced expiratory volume at 1 second (fev1) absolute change from baseline ; mean treatment differences were 0.21 l (95% confidence interval [ci ] 0.04 l, 0.38 l) and 0.41 l (95% ci 0.24 l, 0.58 l), respectively. gsk2190915 50 mg was more effective than 10 mg ; mean difference between treatments was 0.20 l, (95% ci 0.03 l, 0.36 l). compared with placebo, gsk2190915 50 mg, but not 10 mg, significantly inhibited the weighted mean fev1 absolute change from baseline.conclusiongsk2190915 50 mg attenuated the ear similarly to gsk2190915 100 mg in our previous study, suggesting 50 mg is at the top of the dose response curve. gsk2190915 10 mg is a suboptimal dose. the ear can be used to assess the therapeutic dose of a new treatment for asthma. |
one possible reason for persistent endodontic infection may be retention of microorganisms in the dentine tissue of the root canal walls. an infection of the pulp can result in microbial colonization of the entire root canal system, together with the dentinal tubules adjacent to the canal. selective pressures related to oxidation reduction potential, nutrient supply, and microbial interactions are related to the maintenance of endodontic infections. it is the dominant microorganism in root - filled teeth presenting post - treatment apical periodontitis and can be isolated from the root canal in pure culture. e. faecalis has been the focus of attention as a recognized pathogen, isolated both in mixed microbiota and in monocultures. several virulence factors (aggregation substance, enterococcal surface proteins (esp), gelatinase, cytolysin toxin, extracellular superoxide production, capsular polysaccharides, antibiotic resistance determinant) can facilitate tissue invasion, immunomodulation, and cause toxin - mediated damage. sodium hypochlorite presents antimicrobial activity with action on bacterial essential enzymatic sites promoting irreversible inactivation originated by hydroxyl ions and chloramination action. dissolution of organic tissue can be verified in the saponification reaction when sodium hypochlorite destroys fatty acids and lipids resulting in soap and glycerol. chlorhexidine gluconate solutions of varying concentrations have recently been recommended as endodontic irrigants and dressings.[810 ] chlorhexidine is a cationic agent (biguanide group ; 4-chlorophenyl radical), which exhibits antibacterial activity. the cationic nature of the compound promotes connection with anionic compound at the bacterial surface (phosphate groups from teicoic acid at gram - positive and lipopolysaccharide at gram - negative bacteria) capable of altering its integrity. the potassium ion, being a small entity, is the first substance to appear when the cytoplasmic membrane is damaged. various research results have been inconclusive when comparing the antimicrobial effect of these solutions.[1113 ] disadvantages of conventional root canal treatments include their skill - dependent nature, long treatment time, possible weakening of teeth due to widening of the root canal, and use of medicaments such as sodium hypochlorite. the current problem of bacterial drug resistance perhaps best illustrates the continuing requirement both for new agents and new approaches to eliminate infection from root canal system. the commercial phenothiazine dye, toluidine blue (tb), is an effective photosensitizing agent for the inactivation of pathogenic organisms, including viruses, bacteria, and yeast. in recent years, tb has been reported as an antifungal and antibacterial drug for inactivation of yeast and some gram - positive and gram - negative bacteria. photodynamic therapy (pdt) matured as feasible medical technology in 1980s at several institutions throughout the world to eradicate premalignant and early - stage cancer and reduce the tumor size in end - stage cancers involving 3 components : 1. tissue oxygen. the word photodynamics means the study of activating effects of light on living organisms. employing the same principle, pdt can be described as a medical treatment that utilizes light to activate a photosensitizing agent in presence of oxygen. pdt or photoactivated disinfection uses light of a specific wavelength to activate a nontoxic photoactive dye (photosensitizer) in the presence of oxygen. the photoactivated sensitizer can interact with the biological substrate, leading to the production of highly reactive oxygen species, such as singlet oxygen and free radicals, which can kill microorganisms by damaging essential cellular molecules, including proteins, membrane lipids, and nucleic acid. the aim of this study was to compare the effectiveness of 2% chlorhexidine, 2.5% naocl, pdt, and combination of two factors (pdt plus 2.5% naocl) as intracanal disinfectants in extracted teeth against e. faecalis. ninety freshly extracted, intact, adult, human, single - rooted, mature teeth with a single canal were collected and stored in sterile saline. calculus and stains were removed from the root surface using an ultrasonic scaler (cavitron, dentsply ltd, weybridge, uk). after preparation of coronal two - thirds of all canals using gates glidden files number 4, 3 and 2, they were sequentially prepared within 1 mm apical end of the canal, using hedstrm files (antaeos, munich, germany) up to size 40. the canal was irrigated with physiologic saline after the use of each size file. to remove the smear layer that had developed on the canal wall as a result of the instrumentation, each canal the assembled tooth, lid, and bottle were covered with aluminium foil and autoclaved at 121c, for 15 min. the bottle was then aseptically filled with sterile brain heart infusion broth (bhi) (merk, poole, uk) so that the root was covered. a clinical strain of e. faecalis strain atcc 29212, isolated from a treated root canal at the department of endodontics of shahid beheshti university of medical sciences was used in this experiment. bacterial suspension was prepared by a pure culture of this e. faecalis strain, grown in 1 ml of sterile bhi broth to obtain an optical turbidity of 0.5 mcfarland standard, and incubated for 1 hour at 37c in the incubator. the inoculums were injected into the prepared root canal using a sterile syringe and incubated for 48 h at 37c. teeth were randomly divided into five experimental groups (each composed of 15 teeth) according to the post - instrumentation procedures. groups were as follow : the canals were filled with naocl (2.5% v / v) for 5 min, removed with sterile paper points, and irrigated with normal saline solution (.85% v / v). the canals were filled with naocl (2.5% v / v) for 5 min, removed with sterile paper points and then was irradiated with diode laser. the irradiation source was a diode laser (fotoson cms dental, denmark) with a total power of 200 mw / cm and 625 nm of wavelength. total irradiation time was 60 s. root canal infected with e. faecalis was subjected to lethal photosensitization with toluidine blue with concentration 15 g / ml and diode laser for 60 s. the irradiation source was a diode laser (fotoson cms dental, denmark) with a total power of 200 mw / cm and 625 nm of wavelength. the canals were filled with naocl (2.5% v / v) for 5 min, removed with sterile paper points, and then subjected to pdt as described above. root canals were injected with chlorhexidine (2% v / v) (altrincham, cheshire, wa14 5dh, uk) for 5 min, removed with sterile paper points, and irrigated with normal saline solution (.85% v / v). controls consisted of no treatment (positive control) and without inoculation of bacterium (negative control) for five experimental groups. following all treatments, the liquid content of the root canal canals were filled with sterile 0.85% normal saline solution, and sample was taken by the sequential use of three paper points placed to the working length. paper points were transferred to tubes containing 1 ml of 0.85% normal saline solution and agitated for 1 minute. after 10-fold serial dilutions in saline, aliquots of 0.1 ml were plated onto blood agar plates and incubated at 37c for 48 hours. the colony forming units (cfus) grown were counted and then transformed into actual counts based on the known dilution factors. ninety freshly extracted, intact, adult, human, single - rooted, mature teeth with a single canal were collected and stored in sterile saline. calculus and stains were removed from the root surface using an ultrasonic scaler (cavitron, dentsply ltd, weybridge, uk). after preparation of coronal two - thirds of all canals using gates glidden files number 4, 3 and 2, they were sequentially prepared within 1 mm apical end of the canal, using hedstrm files (antaeos, munich, germany) up to size 40. the canal was irrigated with physiologic saline after the use of each size file. to remove the smear layer that had developed on the canal wall as a result of the instrumentation, each canal the assembled tooth, lid, and bottle were covered with aluminium foil and autoclaved at 121c, for 15 min. the bottle was then aseptically filled with sterile brain heart infusion broth (bhi) (merk, poole, uk) so that the root was covered. a clinical strain of e. faecalis strain atcc 29212, isolated from a treated root canal at the department of endodontics of shahid beheshti university of medical sciences was used in this experiment. bacterial suspension was prepared by a pure culture of this e. faecalis strain, grown in 1 ml of sterile bhi broth to obtain an optical turbidity of 0.5 mcfarland standard, and incubated for 1 hour at 37c in the incubator. the inoculums were injected into the prepared root canal using a sterile syringe and incubated for 48 h at 37c. teeth were randomly divided into five experimental groups (each composed of 15 teeth) according to the post - instrumentation procedures. groups were as follow : the canals were filled with naocl (2.5% v / v) for 5 min, removed with sterile paper points, and irrigated with normal saline solution (.85% v / v). the canals were filled with naocl (2.5% v / v) for 5 min, removed with sterile paper points and then was irradiated with diode laser. the irradiation source was a diode laser (fotoson cms dental, denmark) with a total power of 200 mw / cm and 625 nm of wavelength. total irradiation time was 60 s. root canal infected with e. faecalis was subjected to lethal photosensitization with toluidine blue with concentration 15 g / ml and diode laser for 60 s. the irradiation source was a diode laser (fotoson cms dental, denmark) with a total power of 200 mw / cm and 625 nm of wavelength. the canals were filled with naocl (2.5% v / v) for 5 min, removed with sterile paper points, and then subjected to pdt as described above. root canals were injected with chlorhexidine (2% v / v) (altrincham, cheshire, wa14 5dh, uk) for 5 min, removed with sterile paper points, and irrigated with normal saline solution (.85% v / v). controls consisted of no treatment (positive control) and without inoculation of bacterium (negative control) for five experimental groups. following all treatments, the liquid content of the root canal was carefully absorbed with paper points. canals were filled with sterile 0.85% normal saline solution, and sample was taken by the sequential use of three paper points placed to the working length. paper points were transferred to tubes containing 1 ml of 0.85% normal saline solution and agitated for 1 minute. after 10-fold serial dilutions in saline, aliquots of 0.1 ml were plated onto blood agar plates and incubated at 37c for 48 hours. the colony forming units (cfus) grown were counted and then transformed into actual counts based on the known dilution factors. the canals were filled with naocl (2.5% v / v) for 5 min, removed with sterile paper points, and irrigated with normal saline solution (.85% v / v). the canals were filled with naocl (2.5% v / v) for 5 min, removed with sterile paper points and then was irradiated with diode laser. the irradiation source was a diode laser (fotoson cms dental, denmark) with a total power of 200 mw / cm and 625 nm of wavelength. root canal infected with e. faecalis was subjected to lethal photosensitization with toluidine blue with concentration 15 g / ml and diode laser for 60 s. the irradiation source was a diode laser (fotoson cms dental, denmark) with a total power of 200 mw / cm and 625 nm of wavelength. the canals were filled with naocl (2.5% v / v) for 5 min, removed with sterile paper points, and then subjected to pdt as described above. root canals were injected with chlorhexidine (2% v / v) (altrincham, cheshire, wa14 5dh, uk) for 5 min, removed with sterile paper points, and irrigated with normal saline solution (.85% v / v). controls consisted of no treatment (positive control) and without inoculation of bacterium (negative control) for five experimental groups. following all treatments, the liquid content of the root canal was carefully absorbed with paper points. canals were filled with sterile 0.85% normal saline solution, and sample was taken by the sequential use of three paper points placed to the working length. paper points were transferred to tubes containing 1 ml of 0.85% normal saline solution and agitated for 1 minute. after 10-fold serial dilutions in saline, aliquots of 0.1 ml were plated onto blood agar plates and incubated at 37c for 48 hours. the colony forming units (cfus) grown were counted and then transformed into actual counts based on the known dilution factors. table 1 depicts the mean, median, range, and percent reduction of cfus observed for all groups. counting of all cfu of e. faecalis remaining in the control (no treatment) and groups was done to determine effectiveness of treatments. there was a significant reduction in the cfu counts (p < 0.05) compared with the initial numbers recorded in positive control, photodynamic therapy alone, and chlorohexidine therapy ; both had mild antibacterial effects. a maximum of viable bacterial reduction was observed with a combination of pdt plus 2.5% naocl that resulted in 100% bacterial kill [figure 1 ]. the group analyses were performed to investigate the ability of each procedure for reducing the bacterial counts when compared with the previous conditions. a reduction in the number of cfus was statistically significant for all groups (p < 0.05 for all groups). the reduction factors were significantly higher in group 2 (naocl plus diode laser) compared to group 3 (pdt) and group 5 (chlrohexidine) (p <.05). the mann - whitney u test did not show significant differences when comparing samples from group 2 and group 1 (p = 0.09). however, group 4 (pdt plus naocl) was significantly more effective in eliminating bacteria from the root canals when compared to all [figure 1 ] (p < 0.05). highest cfu of viable bacteria was from positive control group, while the negative control group was free of microorganisms under the experimental conditions [table 1 ]. effect of antibacterial agents against e. faecalis counts of enterococcus faecalis colony - forming units after effect of antibacterial agents although this species has been recently questioned as to its importance in the etiology of treatment failures, such a role can not be surely disregarded in the light of current evidence. moreover e. faecalis has been widely used as a valuable microbiological marker for in vitro studies because it has been shown to successfully colonize the root canal in a biofilm - like style, invade dentinal tubules, and resist to some endodontic treatment procedures. in present study, e. faecalis was chosen as the test microorganism because it is one of the most resistant microorganism found in infected root canals, and it has been reported that cases with refractory endodontic treatment were associated with this bacterial strain.[2426 ] from the results obtained with the control teeth, it is clear that e. faecalis was capable of migrating up to 400 m and deeper into the dentinal tubules. in the present study, 2% chlorhexidine solution was used because previous results have shown that 2% solution was more efficient in the shortest period of time than all other concentrations. previous reports have shown that chlorhexidine has a marked effect against e. faecalis[2730 ] and could be effective even at low concentrations against the microorganisms most frequently present in infected root canals, anaerobic bacteria, and candida albicans. the 2.5% naocl (5 min) alone exhibited reduction of e. faecalis in the root canal system of extracted human teeth. however when combined with diode laser, 2.5% naocl was more effective on e. faecalis killing. the effect of naocl plus pdt was the most effective therapy and achieved 100% kill of e. faecalis after 2-min contact time. in this study 2.5% naocl was used because it was shown to denature bacterial toxins and dissolve organic tissues. studies have shown that the magnitude of the antimicrobial efficacy of a 2.5% naocl and chlorhexidine can be influenced by the methodology, microbial characteristics in the biofilm, exposure time, and concentration of the substance tested. the antimicrobial effect of naocl by direct contact on e. faecalis occurred after 2 min. our results demonstrate that 2% chlorhexidine and 2.5% naocl had a significant effect on the viability of e. faecalis. thus, our result confirms those of previous studies on oral microorganisms. in this study, in previous studies of bactericidal activity against e. faecalis biofilm extracted from human teeth, it was reported that the total energy output of a diode laser was 36 j and total energy level was 76 j and combined with a photosensitizer was adequate for a high level of sterilization. nagayoshi., found that irradiation at 5 w for 60 seconds in the presence of a photosensitizer decreased lesion defect area. in the present study, irradiation with laser, tbo and irrigation with naocl 2.5% (5 min) with an absorption maxima (625 nm) had an excellent efficacy against e. faecalis, because in the total of 15 canals incubated with this bacterial strain and then treated with pdt / laser / naocl, no bacteria could be detected in 100% cases. seal and coworkers (2002) reported effect of 100% killing teeth infected with s. intermedius biofilm treated with 3% naocl for 10 min. previous results have shown that singlet oxygen production was not the most relevant parameter to consider when evaluating the antimicrobial activity of pdt against s. mutans. a major advantage of pdt in treatment of root canal infections is the absence of thermal side - effects in the tissues surrounding the roots, as associated with the use of high power lasers. the anticipated benefits of access of laser light and photosensitizer were more limited than hypothesized. refinement of the laser delivery system by introduction of the laser beam into the root canal and/or increased energy delivery may be needed to achieve a better antimicrobial effect. overall pdt of single species grown in a tooth model was interestingly effective considering the light dose at the access cavity. but ultimately, combination of it with 2.5% naocl and pdt is the best option to maximize root canal disinfection and can predictably disinfect root canals in clinical settings. there are many variables to be taken into account when developing a pdt protocol, including light parameters, photosensitizers, and light delivery techniques. the peak of absorption of a photosensitizer should match the wavelength of the light used for irradiation in order to promote formation of singlet oxygen, a highly reactive oxygen molecule responsible for pdt - mediated bacterial killing. both methylene blue and toluidine blue, are phenothiazine dyes that have a maximum wavelength absorption of 656 nm and 625 nm, respectively. for endodontic disinfection, photosensitizers have been tested at concentrations ranging from 6.25 mg / ml to 25 mg/ ml for methylene blue and from 10 mg / ml to 100 mg/ ml for toluidine blue. souza and workers used tbo with concentration of 15 g / ml against e. faecalis. in this study, we used concentration of 15 g / ml. souza reported that pdt with either methylene blue or toluidine blue did not have a significant additional effect to the chemo - mechanical preparation using 2.5% naocl as an irrigant in the reduction of e. faecalis populations. the additional antibacterial effect of pdt with either photosensitizer (methylene blue and toluidine blue) was reported, but it was statistically significant only for toluidine blue. the results of our study showed that reduction in the number of cfus was statistically significant for all groups : chlorhexidine, naocl, laser plus tbo (pdt), laser / naocl, and pdt / naocl against e. faecalis. one of the aims of root canal treatment is to eliminate the bacteria, their products, and the substrate from the root canal system. the result of this research suggests that effect of pdt plus 2.5% naocl simultaneously is effective in the elimination of e. faecalis from dentinal tubules under the conditions of this study. | background : enterococcus faecalis has been widely used as a valuable microbiological pathogen for in vitro studies due to its ability to successfully colonize the root canal in a biofilm - like style, invade dentinal tubules, and resist endodontic treatment procedures.the aim of this study was to compare the bactericidal efficacy of photodynamic therapy (pdt), 2% chlorhexidine, 2.5% naocl, and combination of pdt and 2.5% naocl against e. faecalis.materials and methods : sixty single - rooted teeth had their canals contaminated with e. faecalis in brain heart infusion broth and were incubated for 48 hours.the canals were then subjected to 2% chlorhexidine, 2.5% naocl, pdt (red light emitting diode 625 nm+ toludine blue) and pdt + 2.5% naocl. controls consisted of no treatment (positive control) and without inoculation of bacterium (negative control). following treatment, the canal contents were sampled with sterile paper points.the samples were dispersed in transport medium, serially diluted, and cultured on blood agar to determine the number of colony forming units (cfu). data were analyzed by mann - whitney u test at 5% significance level. the significance level for all analyses was set at p <.05.results : combination of pdt and 2.5% naocl achieved maximum reduction in recovered viable bacteria, no viable bacteria was observed after treatment of pdt + 2.5% naocl.conclusion:combination of pdt and 2.5% naocl simultaneously is effective in the elimination of e. faecalis from dentinal tubules under the conditions of this study. |
acute interstitial nephritis (ain) defines a pattern of renal injury usually associated with an abrupt deterioration in the renal function characterized histopathologically by inflammation and edema of the renal interstitium. ain is an important cause of acute kidney injury caused by drug hypersensitivity reactions, especially in intensive care units, where patients are usually receiving multiple drugs including antibiotics. recently, linezolid, a member of oxazolidinone antibiotic class, has been found to be associated with ain and drug rash with eosinophilia and systemic symptoms (dress) syndrome. a 54-year - old male case of alcoholic cirrhosis (child a) and diabetes mellitus without any evidence of nephropathy was admitted at our center with 2-day history of altered sensorium and fever. there was no prior history of headache, vomiting, seizures, or decreasing urine output. on physical examination, patient was febrile, disoriented, pulse rate was 100/min, blood pressure was 150/70 mmhg, mild icterus, and pedal edema was present. lab investigations revealed hb 9.6 gm / dl, total leukocyte count (tlc) 16 800/ mm with normal eosinophil counts and platelet count 1.1lac / mm, with normal coagulation profile with inr of 1.3. serum bilirubin was 3.0 mg / dl with ast / alt of 87/96 iu / l. viral markers were positive for igm anti - hev ; however, hbsag, anti - hcv, igm anti - hav, and hiv were negative. ascitic fluid analysis revealed high ascites with high serum - ascites albumin gradient normal cell counts. his ultrasonography abdomen was suggestive of chronic liver disease with coarse echotexture and normal bilateral kidneys. renal functions were normal with 1 + proteinuria with full field leucocytes and 8 - 10 rbc / high - power field on urine analysis. patient remained in altered sensorium, and was diagnosed as a case of hepatic encephalopathy and uro - sepsis on the basis of positive urine culture. linezolid 600 mg twice a day was started in view of positive urine culture for enterococcus. on day 3 of linezolid therapy, he developed pruritus, erythematous macular rash involving all the extremities and trunk. peripheral blood smear showed eosinophilia with an absolute eosinophil count of 2125 cells / mm. repeat urine examination revealed 2 + proteinuria with 15 - 20 leukocytes with wbc casts and rbc cast, with no evidence of eosinophils. renal functions were deranged with serum creatinine rising upto 5.2 mg / dl with decreasing urine output requiring dialytic support. dermatological opinion was taken and a diagnosis of dress syndrome was made. keeping the clinical possibility of drug - induced ain as a cause of renal dysfunction, his drugs interstitium showed edema with moderate inflammatory infiltrate comprising predominantly of mononuclear cells with few eosinophil and proximal tubular dilatation with patchy necrosis [figure 1 ]. birefringent oxalate crystals were also present in tubules and interstitium with foreign body giant cell reaction [figure 2 ]. thus, the renal biopsy established the diagnosis of acute tubulointerstitial nephritis with patchy tubular necrosis. patchy tubular necrosis and foreign body giant cell reaction to oxalate crystals in the interstitium oxalate crystals in tubules and mononuclear cell infiltrate in the interstitium patient was managed with short course of prednisolone for 2 weeks. renal functions gradually improved with serum creatinine level decreasing to 1.4 mg / dl, which corresponds to an estimated creatinine clearance of 56 ml / min. patient is being regularly followed - up and has not shown any deterioration in renal functions. the incidence of ain in patients biopsied for acute renal failure has been reported to be approximately 5 - 15%. the common underlying etiologies include drug - induced, infection - related, autoimmune, and idiopathic forms. the most common etiology of ain is drug - induced disease, which is thought to underlie 60 - 70% of cases. backer. reviewed about 128 patients of ain and found drugs to be responsible for 71% cases with antibiotics responsible for around 1/3 of all. linezolid, potent antibiotic with excellent activity against multidrug - resistant gram - positive bacteria and good safety profile, has recently been found to be associated with dress syndrome and ain presenting as acute kidney injury.[46 ] our patient developed nonoliguric renal failure, macular rash, pruritus, and eosinophilia 3 days after linezolid exposure. all other drugs known to cause ain were excluded one by one ; however, renal dysfunction improved only after withdrawal of linezolid and corticosteroid therapy. this is a strong supporting evidence of linezolid associated dress syndrome with accompanying ain confirmed on renal biopsy. the dress syndrome is a hypersensitivity reaction, characterized by a widespread and long - lasting papulopustular or erythematous skin eruption often progressing to exfoliative dermatitis with fever, lymphadenopathy, and visceral involvement (hepatitis, pneumonitis, myocarditis, pericarditis, and nephritis) and accompanied by blood alterations like eosinophilia in about 90% and mononucleosis in about 40% of cases. anticonvulsants, sulfonamides, dapsone, allopurinol, minocycline, and gold salts are among the most frequent culprit drugs. linezolid - associated ain with or without dress syndrome has been described in only three patients till now. report by savard. described an 88-year - old patient developing dress syndrome with ain and mild hepatitis following linezolid, almost 7 days after initiation of therapy. as in our case, the renal biopsy of this patient also showed deposition of oxalate crystals, without any evidence of secondary hyperoxaluria. in another report, hammer. reported linezolid - induced ain in a patient with tibial osteomyelitis. withdrawal of linezolid, short - term hemodialysis, and corticosteroid therapy led to resolution of symptoms and eventual recovery of the renal function. residual renal insufficiency was not a feature of ain in these patients with native kidneys in contrast to our case. esposito. reported a renal transplant patient developing ain without systemic symptoms on exposure to linezolid for management of an enterococcus faecium abscess of a huge liver cyst. biopsy is usually done when either the diagnosis is not clear or patient does not show improvement in the renal function, despite discontinuation of suspected drug. adverse prognostic factors in ain recovery include diffuse versus patchy inflammation on biopsy, excess number of neutrophils (1% to 6%) and extent or severity of interstitial fibrosis, which correlates with the final glomerular filtration rate. noninvasive tests such as ultrasonography, gallium scintigraphy, and eosinophiluria have limited diagnostic utility. the mainstay of therapy for drug - induced ain is early discontinuation of the suspected drug. there are no randomized trials to support the use of corticosteroids in treatment of ain ; however, the decision to use steroids depends upon the clinical course following withdrawal of offending drug. in general, the prognosis for drug - induced ain is good, and at least partial recovery of the kidney function is normally observed. early recognition is crucial because patients can ultimately develop chronic kidney disease. in conclusion, early detection of this rare adverse reaction and prompt discontinuation of the offending agent may potentially prevent acute kidney injury. this case indicates that linezolid can cause ain, and we recommend close monitoring of renal function in patients who are prescribed this drug. | linezolid, a member of oxazolidinone antibiotic class, is a relatively well - tolerated drug with few side effects. it is active against gram - positive cocci, including multidrug resistant staphylococci and enterococci. we report a case of a 54-year - old diabetic male with alcoholic cirrhosis admitted in intensive care unit with altered sensorium. he was diagnosed as a case of hepatic failure secondary to hepatitis e infection and enterococcal urosepsis. he was started on linezolid based on the urine culture sensitivity report. on day three of linezolid treatment, he developed severe pruritus, macular rash, eosinophilia, and renal dysfunction. renal biopsy showed acute tubulointerstitial nephritis. renal functions improved on discontinuation of linezolid and short course of steroid therapy. |
in the past decade, we have seen a great acceleration in the development of new quantum chemical methods, resulting in dozens of new computational techniques that potentially improve the accuracy of results and/or computational efficiency. among these methods, many contain at least one empirical parameter fitted to reference data. as the value of all scientific models must ultimately be determined by comparison with experimental observations, it would be ideal if the empirical models were based on experimental data. unfortunately, in many cases, the computed quantity can not be isolated in an experiment, and direct comparison is thus not possible.(4) in such cases, it is necessary to establish a set of very accurate, well - characterized computational data that can be used to parametrize and validate empirical models. these benchmark data also serve as a valuable tool for the assessment of the accuracy of nonempirical, but more approximate, methods. one area of intensive development of computational methods in the past decade is in the proper treatment of noncovalent interactions. as these types of interactions occur throughout nature, designing accurate, computationally efficient, quantum chemical techniques that give accurate interaction energies and geometries for intermolecular interactions is critical in the treatment of a vast number of systems relevant in areas of chemistry, biodisciplines, and material science. as it has become increasingly clear in the past several years that there are many possibilities for developing new computational methods that give improved accuracy at lower computational costs, it has also become clear that there should exist standard databases of high - quality data against which new methods can be parametrized and validated. many such databases have recently been developed for several different molecular properties, including heats of formation, ionization potentials, electron affinities, and intermolecular interaction energies. the existence of reliable data for the latter of these properties has historically been very limited because of the large computational expense associated with the calculation of accurate interaction energies for all but the smallest molecular complexes. only recently has it become possible to compute interaction energies for medium - sized complexes (up to approximately 40 atoms) with accuracy that is sufficient for benchmark data. there are two main reasons that it has now become possible to get these types of data. the first of these is the development of new computer hardware (including computer parallelization) that allows for much more efficient computations on molecules and molecular clusters. the second reason is the development of a hybrid estimated ccsd(t)/cbs method, which requires only computation of the extrapolated mp2/cbs result as well as the ccsd(t) interaction energy with a small / medium basis set. this method is the most computationally inexpensive technique that has been shown to give accurate interaction energies for many different types of molecular complexes. our main goal in this work is to create a well - balanced database of benchmark interaction energies for noncovalent interactions relevant to biological chemistry. noncovalent interactions are extremely important in biomolecules, as they play large roles in determining their structure and dynamics, and are also responsible for recognition processes in biological systems. thus, the development of new computational tools that accurately describe noncovalent interactions within biomolecules in an efficient way is crucial if significant advances are to be made in computational biophysical chemistry. the development of such methods, which generally contain several empirical parameters, critically depends on the availability of high - quality reference data. there are several interaction energy databases that have been developed in the past 510 years, each with distinct strengths and weaknesses. the most important of these databases are the ones that use the estimated ccsd(t)/cbs method, or other similar (ccsd(t)-based) methods for the reference values. the s22 reference set,(9) which was developed in this laboratory in 2006, has become the most popular interaction energy database and has been used extensively in the parametrization and validation of many different computational techniques (more in discussion below). at the time of its creation, this database represented the state - of - the - art in terms of the level of accuracy that could be attained for relatively large complexes (the largest complex contains 30 atoms). the interaction energies in this test set were computed using the estimated ccsd(t)/cbs procedure, which had recently been developed. despite the fact that s22 is extremely useful and has served as a model in the development of newer databases, there are some problems associated with this data set. although the interaction energies published in the original s22 paper were very accurate relative to the standard methods of the time, it has been shown that, using more modern computers, it is possible to improve the accuracies of these values by using larger basis sets. thus, s22 interaction energies were recalculated by sherrill and co - workers(17) and by szalewicz and co - workers(18) using larger basis sets consistently for all of the complexes. another potential problem with the s22 set is that it is heavily weighted toward nucleic acid - like structures, containing many base pair - like (cyclic) hydrogen bonds and many examples of stacked aromatic (especially heterocyclic aromatic) species. there are several interaction motifs that are strongly under - represented, such as single hydrogen bonds and aromatic aliphatic dispersion interactions, or practically missing, such as aliphatic several years after the development of s22, dissociation curves of the 22 complexes were calculated by merz and co - workers(19) and in this laboratory.(10) in our approach, the resulting data set (named s22 5) contains five examples of each of the s22 complexes, with relative displacements of 0.9, 1.0 (ie the equilibrium geometry), 1.2, 1.5, and 2.0. in this study, dft - sapt analyses were employed, principally to determine the relative contributions of electrostatic and dispersion terms to the total interaction energy of each complex. recently, grimme and co - workers published the gmtkn30 (general main group thermochemistry, kinetics, and noncovalent interactions) data set, which might actually be classified as a superdatabase containing 30 distinct data sets collected from the literature. as indicated by the title, the gmtkn30 set contains data sets for several different molecular properties, including barrier heights, reaction energies, and properties for noncovalent interactions. among the 30 data sets, there are 10 that deal explicitly with noncovalent interactions, with five interaction energy databases (containing a total of 89 complexes) and five databases of relative energies for molecules containing intramolecular noncovalent interactions (58 molecules). it should be noted that s22 is one of the five interaction energy data sets contained within the gmtkn30 database. zhao and truhlar have also developed a superdatabase of atomic and molecular properties that is divided into six categories : thermochemistry, barrier heights, electronic spectroscopy, transition metal reaction energies, structural data, and noncovalent interactions.(3) the noncovalent interaction category (ncie53) contains eight separate subsets with a total of 53 complexes. as in the case of the gmtkn30 database, the s22 set is contained within the ncie53 set. the additional subsets contain hydrogen bonds, charge - transfer complexes, dipole interactions, weak (dispersion dominated) interactions, and stacking complexes. the ncie53 database can be said to be better balanced than the s22 set, mainly because of its inclusion of single hydrogen - bonding complexes, dipole interactions, and charge transfer complexes. in terms of dispersion - dominated interactions, this data set, like the s22, this is true because the weak interactions subset contains mainly noble gas dimers, which have extremely low interaction energies (often less than 0.1 kcal / mol) ; thus, the s22 dispersion complexes dominate this category. in an ambitious project, friesner and co - workers constructed an extremely large database of interaction energies containing 2027 complexes.(15) this set was constructed by collecting almost all of the interaction energy data that had been produced at (at least) the estimated ccsd(t)/cbs level at the time (december 2010). also included within this test set this collection is, of course, very valuable, as it represents the largest single repository of interaction energy data. there are, however, several reasons that this database is not well suited to certain applications. because of the data set s enormous size, it is not practical to routinely use it for the parametrization of new methods ; this is especially true when the method is computationally demanding. another issue is that the database is not very well balanced in terms of inclusion of different interaction motifs. for example, among the 2027 complexes contained in the set, 1892 of them include at least one aromatic molecule (93.3%, 59.8% contain benzene), while there are only 66 examples of aliphatic finally, the data collected from various sources were calculated using different setups ; this may have a non - negligible impact on the quality of the data found in the set. most importantly, deficiencies in the size of the basis set might lead to inconsistencies of the order of magnitude of the accuracy of the methods parametrized on these data. here, we present a database of accurate interaction energies for 66 molecular complexes, which we refer to as the s66 database, computed at the estimated ccsd(t)/cbs level of theory. the complexes contained within the database represent a wide distribution of interaction motifs, including electrostatic dominated (hydrogen bonding), dispersion dominated, as well as mixed (electrostatic / dispersion) interactions. several variations of each interaction type are also taken into account ; for example, both single and double (cyclic) hydrogen bonds are included. among the dispersion dominated interactions, examples of aromatic aromatic (stacking), aromatic we include molecules containing only carbon, oxygen, nitrogen, and hydrogen, as these are the most commonly encountered elements in biochemistry. one valuable property of this database is that it is easily expandable and the addition of complexes containing additional elements should be straightforward. we include not only accurate interaction energies at the potential energy minima, but also a set of 8 points along the dissociation curve, referred to as the s66 8 data set. the accurate description of the entire potential energy surface is of great importance for any method that is applied to calculations on nonequilibrium geometries, or that is used for geometry minimizations, vibration analyses, or molecular dynamics simulations. the former is especially important in the case of large systems where a given moiety may interact with a great number of other chemical groups, with the number of interactions quickly increasing as a function of distance. our goal is to design a new, larger, data set that covers noncovalent interactions in bioorganic molecules in a balanced way. the data set consists of 66 complexes formed by combining 14 monomers in various configurations. the monomers were chosen so that they represent the motifs and functional groups most commonly found in biomolecules (see table 1). the smaller molecules considered are generally carriers of the functional group of interest (i.e., methanol, methylamine, etc.), while the larger ones are actual biomolecular building blocks (uracil, n - methylacetamide as a peptide bond model). a more detailed list of the interactions obtained by combining these is given below ; the complexes are depicted in figure s1 in the supporting information. the size of the data set was chosen so that various types of interactions are well represented, yet it is small enough to make more demanding calculations on it practical. only complexes with interactions stronger than approximately 1.5 kcal / mol were included in the set to minimize the number of systems that contribute negligibly to the statistical analysis of the errors (when an absolute error measure is used). also, duplicate entries for the same interaction (i.e., hydrogen - bond donor / acceptor group combination) were removed from the set, usually keeping the smaller complex. in this work, we do not consider charged species, for which interaction energies calculated in small gas - phase models are not directly applicable to real system in the condensed phase. also, such interactions are an order of magnitude stronger than the ones in neutral complexes, which would distort the even distribution of interaction energies desired in the data set one of the main goals of this work is to produce a interaction energy data set that is very well balanced. toward that end, it is our goal to include roughly equal amounts of electrostatic - rich, dispersion - rich, and mixed (electrostatic / dispersion) interactions in the set. the s66 set is divided into three categories : hydrogen bonding (23 complexes), dispersion - dominated (23), and other (20). in addition, we provide interaction energy decompositions from dft - sapt calculations that allow quantifying the ratio between electrostatics and dispersion when more accurate characterization of an interaction is desired. each interaction is assigned a category, sapt - electrostatic (23 complexes), sapt - dispersion (27), or sapt - mixed (16), based on the relative contributions of electrostatic and dispersion forces. the heuristic categorization and the actual calculations are in good agreement. in the first group, there are 23 hydrogen - bonded complexes. the single hydrogen bonds cover all possible combinations of donors and acceptors in the water molecule, hydroxyl group, amine group, and carbonyl group, plus some other hydrogen bonds possible within our set of monomers. our selection of complexes therefore allows detailed examination of how a given method performs for different types of hydrogen bonds. five cyclic hydrogen bonds, represented by both small models (acetic acid and acetamide) and the uracil dimer, are included to cover hydrogen bonding in nucleic acid base pairs. ; y = o, n) hydrogen bonds ; there are several electrostatic interactions that can also be classified as hydrogen bonds, such as in ethynewater, included in the others category. the group of dispersion - dominated complexes (23 systems) is built from two types of monomers with different properties : planar, often aromatic molecules and aliphatic hydrocarbons, which results in three possible interaction classes : stacking (10 systems), aliphatic these interactions are often described differently by approximate computational methods ; it is therefore very important to include all of them in the data set. the aliphatic hydrocarbons are represented by three different isomers of pentane to cover linear, branched, and cyclic hydrocarbon chains. the last group, named others, contains 20 complexes that do not fit to the two categories above. (x = c, o, n) interactions, t - shaped aromatic ring complexes, nonspecific interactions of polar molecules, and others. in a well - designed data set, the interaction energy should be equally distributed among all of the systems. the histogram of interaction energies in the s66 data set is shown in figure 1. the majority of complexes have interaction energies that are around 4 kcal / mol, approximately following a normal distribution. the only outlying points are the double hydrogen bonds with interaction energies between 15 and 20 kcal / mol ; these can not be eliminated because we want to include this type of interaction in the set. all of the groups of complexes should ideally have equal interaction energy sums if the data set is used to parametrize or test methods that should describe all types of interaction equally. this is hard to achieve in a set of this size when we also would like to include all of the most important interactions. in s66, the sum of interaction energies in hydrogen - bonded complexes (205 kcal / mol) is more than twice as large as that in dispersion - dominated (80 kcal / mol) or other (70 kcal / mol) complexes. although the hydrogen bonds dominate in such a summation, there are other arguments to consider : in the group other, dispersion still makes a very important contribution, and it can not be neglected even in the h - bonded complexes. overall, the dft - sapt decomposition shows that the dispersion to electrostatics ratio for the entire set is 0.86:1, which is not far from being an even representation of both interaction types. therefore, we consider the s66 to be well balanced for general use. in cases where more control over the separate components is needed, the desired weighting can be applied. in addition to equilibrium geometries, we also provide data for eight points along the dissociation curve of each complex. the displaced complexes are created by scaling the intermolecular distance in the optimized structure ; details are given in the methods. using eight points for each complex enables an accurate reconstruction of the dissociation curve by interpolation. the sampling of the region around equilibrium was improved to allow accurate determination of the minimum of the dissociation curve. this information is very important for parametrization of new methods where fitting to the extended set should lead to better reproduction of the geometries of noncovalent complexes. the possibility to interpolate the optimal distance accurately allows for assessment of the performance of a method based on comparison of equilibrium intermolecular distances with benchmark data, the minimum at the ccsd(t)/cbs level obtained from the s66 8 set. the interaction energies in this set are all calculated at the ccsd(t)/cbs level. the s66 and s66 8 data sets do not overlap exactly, but the s66 geometries are always close to one of the s66 8 points. therefore, they should be used separately, s66 8 to explore the entire dissociation curve and s66 when a more accurate description of the minima is needed. in the past 6 years, the s22 database(9) developed in our laboratory has been widely adopted as a standard data set used to test and develop methods focused on noncovalent interactions. other databases of benchmark data covering interaction energies often include the s22 set, as described above. therefore, a detailed comparison of the s22 and s66 data sets clearly highlights all of the issues s66 attempts to correct, and this comparison also partially applies to other data sets based on s22. this becomes important when one focuses on some particular type of interaction, for example, hydrogen bonds. in such a case, the s22 set does not contain enough complexes of a given type for reliable statistical processing of the data. the s22 set is focused mainly on interactions of nucleic acid bases and does not include other types of interactions with comparable weights. regarding hydrogen bonds, most of the complexes, and an even larger fraction of the total h - bonding energy, feature double hydrogen bonds that are stronger than the single ones. this has been improved by extending the set by four more complexes, forming the s26 set.(23) in dispersion - dominated complexes, the s22 set contains only stacked aromatic molecules, with the only exception being the methane dimer, for which the magnitude of the interaction energy is very small, making its contribution to the entire set negligible. this is, in our opinion, the most important drawback of the s22 set, because many methods parametrized on s22, or performing well on it, fail to describe dispersion interactions between aliphatic hydrocarbon groups (see the discussion of the results below). this has been noted previously but has not been solved systematically.(24) in contrast to s22, the same basis sets are used for benchmark calculations on all of the complexes in the s66 data set, regardless of the size of the system. although more accurate calculations are possible for the smaller complexes, our approach eliminates possible method - dependent errors. the same applies to the geometries ; all complexes in the s66 data set were optimized using the same protocol. with the intermolecular distance interpolated from ccsd(t)/cbs calculations, the geometries of the larger complexes should be more accurate than the ones used previously. the dft - sapt interaction energy decomposition provided for the s66 data unbiased categorization of the nature of the interactions and more detailed analysis of the results, that is, correlating the errors in a tested method with a numerical descriptor of the nature of the interaction. this decomposition is available for the s22 set, but it has been published only very recently.(25) the s66 8 data set provides a better description of the dissociation curve than our extension of the s22 data set, the s22 5 set.(10) better sampling around the minimum allows accurate interpolation of the potential energy surface. despite the size of the data set, we do not consider it complete. now, it covers the most common interactions in biomolecules containing only h, c, n, and o elements. we are working on extension of the data set to other elements and functional groups often present in bioorganic systems. the same methodology is being applied so that the new data will be perfectly compatible with the s66 set. these extensions are another important reason for our choice of a reference method that can routinely be applied to a large number of systems. another extension we are working on is a better coverage of the potential energy surface of the s66 complexes. in addition to the dissociation curves presented in the s66 data set, we will also sample other intermolecular degrees of freedom. interaction energies for all of the complexes considered here were computed using fixed monomer geometries, meaning that deformation energies of monomers are not included. the structures of the monomers were taken directly from the dimer optimizations (described below), and no further geometry optimizations were performed on them. the counterpoise correction was employed in all interaction energy calculations to minimize the effects of the basis set superposition error (bsse). dunning s correlation - consistent series of basis sets(26) with diffuse functions(27) are used throughout this study. the use of diffuse functions is crucial for the accurate description of noncovalent interactions. in the following text, we use an abbreviated form axz (x = d, t, q) instead of the full names (aug - cc - pvxz). the cc - pvtz basis set (abbreviated tz) is used in specific cases discussed below. to calculate interaction energies in the s66 and s66 8 data sets (66 and 528 entries, respectively), we had to choose a method that balances accuracy and available computational resources. we use the method of estimating the ccsd(t)/cbs limit described in refs (58). mp2 terms are calculated separately in suitable basis sets, and the total energy is composed as follows : the hf energy converges with the basis set faster than the correlation energy, and one calculation using a large basis set is adequate.(28) here, we use the aqz basis set. the mp2 correlation energy is extrapolated to the cbs limit using helgaker s formula(29) from the atz and aqz basis sets. we have tested other extrapolation schemes, but none produced smaller errors when interaction energies were compared to accurate cbs limit estimates in a set of small complexes. therefore, we conservatively choose the helgaker scheme, which is robust and free of empirical parameters. the most important part of the ccsd(t)/cbs scheme is the choice of basis set for the ccsd(t) calculation, for which we are much more limited by the steep scaling of the calculation with system size. in contrast to some other works that use customized basis sets, we wanted to use a standard basis so that our protocol can be easily reproduced. in the series of correlation - consistent basis sets, the largest basis sets suitable for these calculations are tz and adz ; the latter has a lower maximum quantum number, but includes diffuse functions. we tested both basis sets against more accurate calculations on a set of small complexes, and adz performed noticeably better. it has been previously noted that adz is the smallest basis set that can be used for the ccsd(t) correction that gives errors of less than 0.1 kcal / mol. for these reasons, we have followed a multistep protocol to prepare high - quality geometries for complexes of this size. complex preparation : for complexes for which the minimum geometry was not known beforehand, we performed a search along the most important degrees of freedom at the scc - dftb - d(34) level to identify possible conformations. if multiple minima close in energy were found, we applied the following steps to all of them until we were able to select the one with the lowest energy using a method with accuracy better than the energy difference between them. preliminary optimization of the geometry has been performed using density functional theory with an empirical dispersion correction(35) (dft - d), using the tpss functional(36) and the tzvp basis set(37) along with a dispersion correction optimized for this combination of functional and basis set. no symmetry was assumed in any calculation, and the starting structures were perturbed randomly to remove any possible symmetry. this procedure is used to avoid possible optimization to a saddle point instead of a minimum. final optimization of the complexes was carried out at the counterpoise - corrected mp2/tz level. the resolution of the identity (ri - mp2 method) was used to accelerate the calculations. this setup has been shown to yield geometries close to those obtained at the coupled clusters level. tight optimization limits (energy change 3 10 kcal / mol (5 10 au), max. component 0.06 kcal / mol / (5 10 au), root - mean - square(rms) gradient 0.03 kcal / mol / (2.5 10 au)) were used to ensure good convergence, even in the intermolecular degrees of freedom. such an optimization requires well converged energy calculations, so an scf convergence threshold of 10 au was used. from these geometries, the s66 8 set was prepared by scaling the closest intermolecular distance in the complex along an intermolecular axis. the definition of the axis is different for different types of complexes. for hydrogen bonds, it is defined by the hydrogen and the acceptor atom. for cyclic hydrogen bonds, the centers of mass of the monomers are used, with some exceptions in, for example, t - shaped complexes, where only some atoms have been arbitrarily chosen as the centers to conserve the original arrangement in the displaced geometries. one of the monomers is moved along the axis so that the minimum distance between them is 0.9, 0.95, 1.05, 1.1, 1.25, 1.5, and 2.0 times the equilibrium value. these seven extensions, along with the mp2 equilibrium geometry, form the s66 8 data set. once the ccsd(t)/cbs interaction energies for the s66 8 set were calculated, we used the first five points of each dissociation curve (factors of 0.91.1 multiplying the equilibrium distance) to obtain a new minimum in the distance coordinate at the coupled clusters level. the selected points were interpolated with a fourth - order polynomial, and the position of the minimum of this function was used to construct a new geometry. the dft - sapt interaction energy decomposition has been performed with the adz basis set to make calculations on larger complexes practical. we have shown that this can be addressed by scaling of the dispersion component;(43) in this work, we apply the scaling factor of 1.193 recommended for this basis set (all dft - sapt dispersion energies listed in this article are already scaled). such a scaling has been shown to improve the results consistently ; the accuracy that can be reached is sufficient for our analysis, which accompanies more accurate ccsd(t)/cbs calculations. the pbe0ac functional recommended by the authors of the method was used for the dft - sapt calculations;(44) the calculations of the monomers have been performed in the basis set of the dimer. the shift needed to correct the asymptotic behavior of the functional was calculated as the difference between the homo energy of each monomer and the true ionization potential obtained from the calculation of its neutral and ionized form, using the same functional and basis set. density fitting was used to speed up these calculations.(45) the interactions of each of the complexes in the s66 set were characterized as being electrostatics dominated, dispersion dominated, or mixed (electrostatic / dispersion). the complexes with dispersion / electrostatics ratios lower than 0.59 are categorized as electrostatic, those with d / e ratios higher than 1.7 (1/0.59) are categorized as dispersion bound, and complexes with d / e ratios between 0.59 and 1.7 are categorized as mixed. the threshold of 0.59 generates groups that agree well with empirical categorization. we have used the newly obtained benchmark data to analyze the performance of several advanced wave function methods that are supposed to closely reproduce ccsd(t)/cbs data. the complete basis set limit for these methods has been calculated analogously as in the ccsd(t)/cbs scheme by combining the mp2/cbs term extrapolated from atz and aqz basis set and the higher order correction calculated in adz basis set. in addition to plain mp2, mp3, and ccsd calculations, we have focused on correlated wft methods that use empirical parameters, mp2.5(46) scs - mp2,(47) scs - mi - mp2,(48) scs - ccsd,(49) scs - mi - ccsd,(50) dispersion - weighted mp2(51) (dw - mp2), and nonempirically corrected mp2c. all of the spin - component - scaled results have been derived from the calculations already performed to obtain the benchmark data. mp2 with a time - dependent dft based dispersion correction (mp2c) represents another approach to improve the performance of the relatively efficient mp2 method. in the spin component scaled methods, we used the following scaling coefficients for the other - spin (singlet) and same - spin (triplet) terms : the scs - mp2 method uses cos = 6/5 and css = 1/3 regardless of the basis set used. the scs - mi - mp2 was optimized to reproduce interaction energies in the s22 set. we use coefficients published for extrapolation from the tz to qz basis sets(48) (cos = 0.4, css = 1.29). we extrapolate from the equivalent, but augmented, basis sets ; the difference in the cbs value is negligible. the scs - ccsd method (cos = 1.27, css = 1.13) was developed using the qz basis set, while we use extrapolation to the cbs limit ; we expect the parameters to be transferable because the quadruple- basis should not be far from the cbs limit. the scs - mi - ccsd coefficients (cos = 1.11, css = 1.28) were optimized on the s22 data set, which uses different basis sets for complexes of varying size ; here, the transferability of the parameters is an open question. the dw - mp2 method was originally based on explicitly correlated mp2 (mp2-f12) calculations ; this approach improves the convergence of the mp2 energy with basis set size and allows the use of a smaller basis set (adz). in this work, we have replaced it with the extrapolated mp2 results as another means to approach the complete basis set limit. therefore, minor differences in our dw - mp2 results can be expected as compared to ref (51). all dft and ri - mp2 optimizations have been carried out in turbomole 6.2.(54) interaction energies at the mp2, mp3, mp2c, ccsd, and ccsd(t) levels and dft - sapt interaction energy decompositions have been calculated using the molpro program(55) in versions 2009 and 2010. a threshold for scf convergence of at least 10 au was used for all of the calculations. density fitting was used for the mp2 calculations used to obtain the mp2/cbs correlation energy term. for the scc - dftb - d calculations, the dftb+ program(56) the performance of the studied methods, with respect to the benchmark calculations, can be described by multiple statistical tools. for the s66 data set, we provide multiple error measures that often carry different information. we consider the root - mean - square error (rmse) as the most important one, because it reflects the overall quality of the tested method well and is widely used in the field. it is also the variable optimized in parametrization of a method using the least - squares algorithm. additionally, we list the mean unsigned error, the average (signed) error, which indicates the systematic component of the error, and the largest (maximum unsigned) error, expressed as a percentage, representing the worst case scenario. we also provide the error separately for different groups of complexes of the data set. because the average interaction energy in these groups differs, an error in the units of energy does not allow comparison between these groups. therefore, we use relative errors, calculated as an rmse divided by the average interaction energy in the group, expressed as percentages. construction of the s66 8 us to interpolate the ccsd(t)/cbs energies around the mp2 minimum to obtain an accurate estimate of the equilibrium distance at the ccsd(t)/cbs level (see methods for description of the procedure). comparing the energy predicted by the interpolation with the actual calculation on the new geometry even the largest difference in the set is only 0.002 kcal / mol (0.05% of the interaction energy), which indicates that our polynomial fit accurately represents the potential energy surface and that the obtained geometry can be safely considered a minimum in this coordinate. comparison of the mp2 and ccsd(t) minima, in terms of distance and ccsd(t)/cbs interaction energies, allows us to measure the quality of the (counterpoise corrected) mp2/tz geometries. the relative difference in geometries, measured as the change of the closest distance, is on average 0.03. in the worst case, for the stacked benzene dimer of course, the changes in geometry affect the interaction energies. because the intermolecular potentials are rather flat, such small changes in geometry translate into comparably small improvements in the interaction energies. over the entire set, we measured an rmse of 0.020, and a maximum of 0.086 kcal / mol (3% of eint). this is comparable to the estimated accuracy of the method used for the interaction energy calculation ; therefore, the mp2 optimization can be safely used in cases when we are interested only in the interaction energy at higher levels. these results confirm previous studies that recommend the tz basis set for mp2 optimizations.(38) the interaction remains attractive even in the geometries with shortest distance (displacement factor 0.9). in all but the stacked systems, the interaction remains very strong ; on average, it amounts to 83% of the interaction energy in equilibrium. in the stacked complexes, the repulsion is steeper due to the large contact area and short equilibrium distance, and the interaction energy becomes as low as 10% of the equilibrium value. to estimate the accuracy of our benchmark data, we compare the scheme used in the s66 data set with more accurate estimates of ccsd(t)/cbs. this has been done on a set of ten small complexes introduced in our previous work.(43) for these, we extrapolated the ccsd(t) correlation energy to the complete basis set limit from calculations using the atz and aqz basis sets. table s4 in the supporting information gives the results of the comparison of the current benchmark technique with the extrapolated ccsd(t)/cbs(atzaqz) method. for this small test set, the s66 benchmark method gives an average error value of 1.2% with the largest error being 2.5%. these results indicate that the errors associated with the current benchmark method should be reasonably small ; for the s66 test set, we expect that the errors should generally be below 3%. we are aware of the fact that the errors present in our benchmark data are close to the errors of some of the studied methods to which they are compared. however, in the comparison of similar methods, using the same extrapolation scheme and basis sets, the major part of the error coming from the contributions shared by both methods is canceled. for example, the ccsd(t)/cbs approach is consistent with all of the ccsd - based methods where the mp2 and ccsd terms are the same and the protocol differs only in omitting the triples. table 2 gives the dft - sapt dispersion / electrostatic ratios and the sapt - electrostatic / dispersion / mixed (sapt - e / d / m) category for all of the complexes in the s66 set. it can be seen in this table that among the interactions in the hydrogen - bonding category, 20 are clasified as sapt - electrostatic, while three are classified as sapt - mixed. the complexes containing cyclic hydrogen bonds or water exhibit the smallest d / e ratios. the range of d / e ratios for single h - bonds involving nh2 or nh donors is 0.330.79, while the corresponding ratios for single h - bonds involving oh donors are 0.290.42. the three sapt - mixed complexes in this group, menh2meoh, menh2menh2, and menh2peptide, represent three of the four complexes that have menh2 as the hydrogen - bond donor. here, the hydrogen bond itself is weaker, while the secondary (dispersion) interactions in the systems are not negligible. the d / e values for the interactions in the dispersion category are in the range from 1.33 to 5.42. within this group, there are 20 sapt - dispersion interactions and three sapt - mixed interactions, which are for the uracilethyne, uraciluracil, and pyridineuracil complexes. generally the interactions that are most electrostatic in nature are interactions involving the two heterocyclic aromatic molecules (uracil and pyridine). this trend stands to reason as the uracil and pyridine heteroatoms give these molecules large charge separations. as would be expected, the aliphatic there are three sapt - electrostatic, 10 sapt - mixed, and seven sapt - dispersion complexes within the mixed category. here, it can be seen that interactions involving ethyne are generally electrostatic in nature, with three of these, ethynewater, ethyneacoh, and pyridineethyne, being categorized as sapt - electrostatic. selected error measures of the tested methods for the s66 test set are listed in table 3 and plotted in figure 2. relative errors for different interaction categories are plotted in figure 3 ; the dispersion category is further divided to, aliphatic aliphatic, and aliphatic interactions, for which the relative errors are plotted in figure 4. the full listing of the errors for these groups is provided in table s1 in the supporting information. the errors are reported as rmse, mean unsigned error (mue), average signed error (avg), and largest error in the set relative to the interaction energy (max). this method is generally regarded as yielding qualitatively, or semiquantitatively, accurate results (when used with the counterpoise correction), with the quality of its interaction energy values depending strongly on the basis set with which it is used. in a previous study in this laboratory, we have shown that mp2 generally yields its best results when it is paired with either the adz or the tz basis.(57) here, we investigate the performance of this method with the adz and tz bases, as well as at the cbs limit. it can be seen in figure 2 that similar errors are produced by the mp2/adz (0.79 kcal / mol), mp2/cbs (0.69 kcal / mol), and mp2/tz (0.70 kcal / mol) methods. these errors are rather high, as compared to those of many of the other methods considered here. however, it should be kept in mind that mp2/adz and mp2/tz are the least computationally expensive methods included in the study and have much better scaling properties than any method that includes any higher order terms (especially when density fitting is used). inspection of figure 3 reveals that, despite the fact that mp2 produces similar overall errors with tz, adz, and at the cbs limit, mp2/tz gives the most balanced description of the various interaction categories, giving its largest relative error for the dispersion category (14%). mp2/cbs gives extremely accurate results for hydrogen bonding complexes (2%), while producing very large errors for interactions in the dispersion category (29%). the scs - mp2 method makes use of a separate scaling of the singlet and triplet mp2 correlation and was originally developed for reaction energies. the scs - mi - mp2 method uses the same scaling scheme, but was parametrized for improved performance in the description of noncovalent interactions (using the s22 data set). results for scs - mp2/cbs and scs - mi - mp2/cbs are depicted in figure 2. not surprisingly, scs - mp2/cbs gives errors that are relatively high (rmse 0.87 kcal / mol), as the method is not parametrized for intermolecular interactions. scs - mi - mp2/cbs, on the other hand, yields an rmse value of 0.38 kcal / mol, which represents a significant improvement over the unscaled mp2 method (for any of the basis sets tested here). comparing scs - mi - mp2/cbs to mp2/tz (the best mp2 performer), it can be seen in figure 3 that the scaled mp2 method gives improved results for hydrogen bonds and interactions in the other category, with especially large improvements for the hydrogen bonds (11% vs 3%). the most computationally expensive part of the mp2.5 method (as utilized here) is the mp3/adz calculation, meaning that this technique is only more computationally intensive than the mp2-based methods. mp3 is known to strongly underestimate dispersion interactions, as opposed to mp2/cbs, which is known to overbind dispersion bound complexes. the basis for the mp2.5 method, which is constructed as an average of mp2 and mp3, is a mutual cancellation of these errors. as might be expected, mp3/cbs yields relatively high rmses (0.62 kcal / mol). on the other hand, mp2.5/cbs yields surprisingly accurate results for this data set, considering its relatively low cost, and the fact that it does not (technically) contain any empirical parameters. kcal / mol) is scs - mi - ccsd / cbs (0.08 kcal / mol). considering the data presented in figure 3, it can be seen that mp2.5 gives a well - balanced description of the three interaction categories, producing relative errors of no more than 7% for any particular interaction type. the lowest errors occur for hydrogen bonds (1%), while the largest errors occur for the dispersion complexes (7%). mp2.5 gives a very well - balanced description of the dispersion - bound complexes, with errors between 6% and 7% for all three dispersion subcategories. like scs - mp2, scs - ccsd was parametrized to improve ccsd s description of reaction energies. it has also been noted that this method gives improved results for noncovalent interactions. as in the case of scs - mp2, scs - ccsd has also been parametrized (against the s22 set) to give improved results for molecular complexes ; with the new parameters the method is designated scs - mi - ccsd. this reparameterization leads to significant improvement of the accuracy from rmse of 0.25 kcal / mol in scs - ccsd / cbs to 0.08 kcal / mol in scs - mi - ccsd / cbs. it can be seen in figure 2 that the rmses produced by both scs - ccsd / cbs and scs - mi - ccsd / cbs are much lower than that of ccsd / cbs (0.70 kcal / mol). to highlight the accuracy that can be obtained with each of these techniques, it will be noted that the maximum relative error produced by each of these methods is 6%. figure 3 shows that both scs - ccsd / cbs and scs - mi - ccsd / cbs produce very small errors for all interaction categories. the scs - mi - ccsd technique gives particularly low errors for all interaction categories, producing its largest relative rmse for the dispersion category (3%). both scs - ccsd and scs - mi - ccsd produce small relative errors for the dispersion subcategories, with scs - ccsd giving its largest error for the and aliphatic aliphatic categories (4%) and scs - mi - ccsd giving its largest error for the aliphatic aliphatic category (6%). the scs - mi - ccsd / cbs is the most accurate method from the studied set. the error with which it reproduces the ccsd(t)/cbs benchmark is smaller than the estimated accuracy of the benchmark calculations. the method is also very robust, as indicated by the narrow range between maximum and minimum error. therefore, it can be recommended as an alternative to ccsd(t) calculations for larger systems or for any purpose where the ultimate accuracy is not required. the dw - mp2 method, which utilizes a (system dependent) weighted average of mp2/cbs and scs - mp2/cbs results to compute interaction energies, yields results that are improved with respect to its parent methods, both in terms of overall errors (rmse 0.40 kcal / mol) and in terms of errors for the three interaction categories. this is somewhat surprising ; given the relatively poor performance of scs - mp2 for dispersion bound complexes, it would be expected that dw - mp2 should give low errors for hydrogen - bonding complexes (as does mp2/cbs) and higher errors for dispersion bound complexes (as does scs - mp2/cbs). however, it should be noted that the signs of the errors given by mp2/cbs and scs - mp2 for dispersion bound complexes are generally opposite in sign ; that is, mp2/cbs tends to overbind, while scs - mp2/cbs tends to underbind. thus, any interaction energy constructed as a linear combination of results from these two methods will exhibit some inherent error cancellation, which is likely responsible for this method s relatively low errors. the mp2c method, which incorporates a td - dft description of dispersion, yields an rmse (0.71 kcal / mol) that is comparable to those of mp2/cbs (0.69 kcal / mol) and mp2/tz (0.70 kcal / mol). this technique gives very low errors for hydrogen - bonding complexes (2%) but rather high errors for both the dispersion (24%) and the other this is somewhat surprising because, from the theoretical point of view, the mp2c offers a well justified improvement over mp2 itself. on the other hand, it is still only a second - order perturbation treatment, which can not describe higher order contributions. the rmse (kcal / mol) with respect to the ccsd(t)/cbs benchmark. the symbol next to the bar is the sign of the average error. plus indicates that the method underestimates the strength of the binding over the whole data set ; minus indicates systematic overbinding. relative errors (%) for the three groups of complexes : hydrogen bonds, dispersion - dominated, and others. the error is calculated as a rmse relative to average interaction energy in the group so that the errors can be compared between the groups. relative errors (%) for the three types of dispersion - dominated complexes :, aliphatic the error is calculated as a rmse relative to average interaction energy in the group so that the errors can be compared between the groups. the availability of dft - sapt electrostatic and dispersion data for all of the s66 complexes allows for the assessment of various methods in terms of the errors they produce for varying dispersion / electrostatic ratios. as an example, we have prepared a plot depicting the (percentagewise) errors as a function of the dispersion / electrostatic ratio for the mp2/cbs and mp2/tz methods (figure 5). the results shown here are in good agreement with the known properties of these two methods. mp2/cbs gives a very good description of electrostatically driven interactions while overbinding substantially for dispersion - bound complexes. mp2/tz, on the other hand, underestimates electrostatically driven interactions and generally gives a better overall description of dispersion dominated and mixed interactions (although the errors are still significant), yielding errors that indicate no specific tendency to overbind or underbind. the type of plot shown for mp2/cbs and mp2/tz can be used for the assessment and development of new methods and as a tool for better determining their strengths and weaknesses. errors of selected methods plotted against the ratio of dispersion to electrostatic term from the dft - sapt decomposition. figure 6 shows the rmses produced by the tested methods for the s66 8 data set. the mp2c method was not applied to these sets because the improvement as compared to mp2 itself is expected to be small, while the computational demands are much larger. it will be noted here that errors for the s66 8 data set are generally smaller than those for the s66 set, which is attributable to the fact that interaction energies for structures far from their equilibrium geometries are generally very small, and the corresponding errors for these structures will also tend to be small. it can be seen in figures 2 and 6 that, in terms of the relative performance of each of the tested methods, the same general trends are followed for the s66 and s66 8 data sets. table s2 (supporting information) gives the s66 8 rmses for each of the tested methods along with a description of the errors that occur at long intermolecular separations (displacement factors 1.12.0) and short intermolecular separations (factor of 0.9). here, it can be seen that the relative errors at the shorter intermolecular distances are always about 23 times larger than those at the longer separations. this is not a surprising result, as it would be expected that larger errors occur in regions where the potential energy curves are the steepest. the rmse (kcal / mol) in the s66 8 (dissociation curves of the 66 complexes). here, we have presented a database of interaction energies for 66 intermolecular complexes, each in 9 distinct geometric configurations. the data set was constructed to include a balanced set of commonly encountered interaction motifs involved in biomolecular structures containing c, o, n, and h. this data set was designed to be expandable, meaning that complexes containing additional binding motifs, or additional elements, can easily be added. reference data were obtained at a high level of theory using the ccsd(t)/cbs scheme consistently for all 594 points. importantly, the reference method and basis sets used for each of the complexes are identical to avoid the introduction of additional random error. geometries of the complexes in s66 and s66 8 data sets, the benchmark ccsd(t)/cbs interaction energies, and results of all of the methods tested here are available through the begdb web site(58) (www.begdb.com) for download and interactive browsing. the geometries of the complexes in the s66 data set have been carefully optimized, and the intermolecular distance is the minimum at the ccsd(t)/cbs level. this is an important advantage over previous data sets where geometries of all but very small complexes had been optimized at only the mp2 level. accurate equilibrium geometries are a valuable tool for parametrization of new methods that should yield not only accurate energies, but also equilibrium geometries, and potential energy surfaces in general. the s66 8 data set contains eight points on the dissociation curves of each of the s66 systems (528 in total). for a given complex, the region around the equilibrium distance is sampled preferentially, which allows for accurate interpolation of the true minimum of the curve. the s66 data set contains 66 ccsd(t)/cbs interaction energies obtained using atz and aqz basis sets for the extrapolation of mp2 correlation energy and adz for the ccsd(t) term. these values have been calculated on refined geometries where the intermolecular distance is a true minimum at this computational level. for the 528 nonequilibrium geometries contained in these sets, we provide ccsd(t)/cbs interaction energies obtained using the same setup. the enormous computational expense associated with the ccsd(t) method, even with a relatively small basis set such as adz, makes it necessary to seek less costly methods that can produce comparable results. of the 12 wave function - based methods tested in this work, there are three that stand out, in terms of their performance on the s66 and s66 8 sets, as producing particularly low errors at a given computational cost point ; these are scs - mi - ccsd / cbs, mp2.5/cbs, and scs - mi - mp2/cbs. both of the scs - mi- (mp2 and ccsd) methods yield errors that are substantially lower than their parent methods, with scs - mi - ccsd / cbs giving errors that are extremely low for both data sets (rmse 0.08 kcal / mol in the s66 set). mp2.5/cbs produces errors that are only slightly higher than those of scs - mi - ccsd / cbs (0.16 kcal / mol), at a much lower computational cost. | with numerous new quantum chemistry methods being developed in recent years and the promise of even more new methods to be developed in the near future, it is clearly critical that highly accurate, well - balanced, reference data for many different atomic and molecular properties be available for the parametrization and validation of these methods. one area of research that is of particular importance in many areas of chemistry, biology, and material science is the study of noncovalent interactions. because these interactions are often strongly influenced by correlation effects, it is necessary to use computationally expensive high - order wave function methods to describe them accurately. here, we present a large new database of interaction energies calculated using an accurate ccsd(t)/cbs scheme. data are presented for 66 molecular complexes, at their reference equilibrium geometries and at 8 points systematically exploring their dissociation curves ; in total, the database contains 594 points : 66 at equilibrium geometries, and 528 in dissociation curves. the data set is designed to cover the most common types of noncovalent interactions in biomolecules, while keeping a balanced representation of dispersion and electrostatic contributions. the data set is therefore well suited for testing and development of methods applicable to bioorganic systems. in addition to the benchmark ccsd(t) results, we also provide decompositions of the interaction energies by means of dft - sapt calculations. the data set was used to test several correlated qm methods, including those parametrized specifically for noncovalent interactions. among these, the scs - mi - ccsd method outperforms all other tested methods, with a root - mean - square error of 0.08 kcal / mol for the s66 data set. |
blood hypereosinophilia (he) remains a frequent finding encountered in a daily clinical practice of different fields of medicine. under various conditions, eosinophils may produce and release a variety of biologically active substances which may invade target organ and lead to its dysfunction and/or damage. the harmful role of eosinophils results from their inflammatory, fibrotic and thrombotic properties. the production and development of eosinophils are regulated by several cytokines, but the role of interleukin (il)-5 was found to be essential. it is secreted by activated t cells and to a lesser extent by mast cells and eosinophils themselves. the underlying causes of he are diverse and can be broadly divided into primary (clonal), secondary (reactive), hereditary (familial) and idiopathic. the term he should be used when blood eosinophilia is greater than 1.5 10/l on two occasions with a minimum of 1-month time interval, and/or tissue he is documented. the contemporary definition of hypereosinophilic syndrome (he s) requires the presence of blood he and an end - organ damage that was proved to be eosinophilia related. as the characteristics of different he s variants are well - known, the outcome of patients with asymptomatic, unexplained and persistent he remains unclear. herein, we present the clinical and laboratory characteristics of 40 consecutive patients with idiopathic blood he. all patients included in the study met the criteria for blood he, and they were recruited from several hematologic centers in poland between 1994 and 2013. the reasons why the patients visited the primary care physician and performed full blood test were following : random blood investigation (n = 30), abdominal pain (n = 2), loss of weight (n = 2), facial swelling (n = 1), joint pain (n = 1), bone pain (n = 1), night sweats (n = 1), diarrhea (n = 1) and dyspnoea (n = 1). all patients underwent the basic evaluations at the primary care level including all necessary tests in order to rule out the most common causes of blood he, but no abnormalities were detected. originally reported complaints resolved spontaneously after 23 days without treatment, and they were found not to be he related. therefore, more detailed imaging and endoscopic studies were not recommended by a treating physician. moreover, these symptoms did not re - appear in the long - term observation. all patients were free of any symptoms. on admission a complete blood test with differential the presence of infections caused by human immunodeficiency virus, cytomegalovirus, epstein - barr virus, hepatitis b virus, hepatitis c virus and toxocara was excluded using serological tests. anti - nuclear antibodies as well as myeloperoxidase and proteinase 3 anti - neutrophil cytoplasmic antibodies were ruled out. peripheral blood t - lymphocytes were determined by flow cytometry technique on epics - xl - mcl (beckman - coulter, usa) using monoclonal antibodies directed against t - cell surface antigens : cd2, cd3, cd4, cd5, cd7, cd8, t - cell receptor (tcr) and tcr, and no aberrant population (including the most common cd3-cd4 +) has been detected. polymerase chain reaction (pcr) was negative for jak2v617f, bcr - abl and fip1l1-pdgfra molecular abnormalities. blood tests with differential, biochemistry, electrocardiogram, echocardiography, chest x - ray and abdominal ultrasound were repeated every 3 months. stool examination for ova and larvae was systematically repeated throughout the study period, and it remained negative. the study was approved by local ethics committee by silesian medical university, katowice, poland. twelve patients with at least moderate blood eosinophilia (defined as greater than 3.0 10/l) for more than 1-year duration were treated with corticosteroids to avoid end - organ damage. the starting cs dose varied between patients, and it was left to physician s discretion. a complete response (cr) was defined as a return of absolute eosinophil count (aec) to normal ranges (10 (10/l)52 % hemoglobin (g / dl)13.0 (8.719.1)hemoglobin 3 10/l42 % eosinophils in bone marrow (%) 30.5 (1178.2)serum ige (iu / ml) 528 (11.94,089)serum ige > n 67 % serum b12 vitamin (pg / ml) 333 (1491,431)serum b12 > n 2 % wbc white blood cell, aec absolute eosinophil count, normal ranges (n) : ige 7 pg / ml) and 6.6 (sensitivity > 1.08 pg / ml), respectively. taken together, the diagnosis of lymphocytic - hes was established. despite the fact that blood aec was greater than 3.0 10/l in more than 40 % of study patients and serum ige levels exceeded normal ranges in almost 70 % of them, the organ involvements were not detected in subsequent imaging studies. however, the median duration of follow - up for our patient population is less than 5 years. older reports demonstrated that he s may develop after 89 years of sustained blood he. currently, it is difficult to support the thesis that a benign idiopathic blood he is a smoldering form of he s. to confirm this hypothesis, the markers of eosinophil activity were measured in a patient with a long - standing blood he. the test showed an impaired function of eosinophils, but it did not explain why and when they become deleterious to the organs. there is no evidence that the introduction of cs, despite the lack of symptoms, will influence the natural history of these patients and prevent the he s development. it should be highlighted that the treatment with cs for heus remains controversial, and currently, there is no data supporting this approach. in a daily clinical practice, the decision who and when should receive the treatment is left to the treating physician, e.g., mayo group preference is to start the treatment when aec is considered too high, but this threshold is not established. some authors recommend an aec greater than 2.0 10/l as an indication for treatment. one should keep in mind that a sustained high blood he remains frustrating both for clinician and for a patient, but it should not justify a durable cs treatment. it was due to a high number of circulating blood eosinophils and the risk of organ damage. all treated patients responded to cs, and the dose was promptly reduced to maintain remission. five patients were left on higher cs maintenance doses (1020 mg daily), and it was due to their very high blood he at diagnosis (from 7.9 10 to 55.0 10/l). in fact, the exposure to the higher cs dose was short, and therefore, the observed side effects were irrelevant. the median duration of cs treatment is 8 weeks (range 224 weeks). a female patient who developed he s was free of cs until the cardiac failure occurred. she responded to the treatment and remains in remission with normal cardiac function while still on cs. every patient with heus requires the comprehensive evaluation to exclude various reactive and neoplastic eosinophilia - related conditions according to a contemporary consensus of multidisciplinary experts in the field of eosinophilia. an international prospective study with a larger patient population and a longer follow - up the use of cs for heus patients should be individualized paying special attention to adverse drug reactions. it seems reasonable to plan a randomized study in order to assess the benefit of cs in heus. | the term hypereosinophilia of undetermined significance (heus) previously known as idiopathic, benign eosinophilia relates to patients who have a long - lasting, unexplained and asymptomatic blood he. these patients have not been studied so far in terms of demographic characteristics and clinical outcome. the aim of this study was to present the clinical characteristics and outcome of heus patients. this is a retrospective, single - center study of 40 patients with heus. all patients underwent the basic and specialized evaluations in order to rule out the most common causes of blood he, but no abnormalities were detected. twelve patients with at least moderate blood hypereosinophilia (defined as greater than 3.0 109/l) for more than 1-year duration were treated with corticosteroids (cs) to avoid end - organ damage. twenty - one patients (52 %) had an increased leukocyte count at diagnosis. median blood eosinophilia was 4.2 109/l (range 1.555.4). he > 3.0 109/l was demonstrated in 17 patients. 65 % of studied population had an increased serum ige levels, whereas only 2 % demonstrated an increased serum vitamin b12 levels. a median bone marrow infiltration by eosinophils was 30.5 % (range 1178.2). all treated patients responded promptly to cs and remained in complete remission while receiving low doses of cs (20 mg / day to 5 mg every 2-day). one patient developed hypereosinophilic syndrome (he s) after 11 years of follow - up. further studies are needed to define risk factors of he s development. the use of cs for heus patients is controversial and should be individualized. |
the effects of small molecules on organisms have long been the focus of biochemistry and pharmacology. over the last years there has been a considerable increase in the number of high - throughput screens that have been performed using chemical libraries (13). at the same time, the molecular targets of individual chemicals are being studied in ever greater detail (4,5). there also is a great interest in chemical biology approaches, using small molecules to perturb cellular functions (6). for the design and interpretation of these studies, the context of the chemicals and proteins needs to be considered. for example, in the case of high - content screening for specific cellular effects, it is important to know whether the active chemicals already have known activities that can explain the observed effects, or whether novel mechanisms of actions might be present. therefore, we have developed a search tool for interactions of chemicals (stitch) both as a large - scale, downloadable database of interaction data and as an interactive web tool for the exploration of interaction networks (figure 1). since its first release (7), stitch is being accessed by over one hundred scientists each week and has been used as a source of protein chemical associations e.g. by prathipati. (8), who used the stitch network to automatically extract the targets of anti - tuberculosis compounds in mycobacterium tuberculosis. human proteins predicted to interact with aspirin according to different sources of evidence are shown. edges are colored according to the source of evidence (magenta : experimental information, cyan : manually curated databases, yellow : text - mining). human proteins predicted to interact with aspirin according to different sources of evidence are shown. edges are colored according to the source of evidence (magenta : experimental information, cyan : manually curated databases, yellow : text - mining). aspirin will display a pop - up showing the structure and description. here, we present the second version of stitch. in addition to the sources of protein chemical interactions included in the previous version pdsp ki database (9), protein data bank (pdb) (10), kegg (11), reactome (12), nci - nature pathway interaction database (http://pid.nci.nih.gov), drugbank (13) and matador (14)we now further include interactions imported from glida (15), pharmgkb (16,17), comparative toxicogenomics database (ctd) (18) and bindingdb (19). these added databases mainly provide information on interactions between human proteins and drugs or drug - like molecules. the imported sources of information are scored separately and then combined with information from text - mining (7). databases which contain manually annotated interactions receive high scores, while interactions based on experimental information are scored by the confidence or relevance of the reported interaction. the number of high - confidence (score 0.7) human chemical for these high - confidence interactions, the number of interacting human proteins increases from 5300 to 7400 (as stitch is locus - based, only one gene product is counted per gene). the stitch network is created by mapping interactions from the sources mentioned above and from text - mining onto a consolidated set of chemicals that has been derived from pubchem, assigning a confidence score for each interaction (7). the newly - derived protein chemical and chemical chemical associations are then complemented with protein protein interactions from the string database (20). in the previous version of stitch (7) these actions specify the nature of the interaction independent of the source of interaction information. we have greatly extended the set of available actions by further importing action types from glida (15), pharmgkb (16,17), ctd (18), bindingdb (19) and a manually annotated set of interactions. this set of interactions has been curated from drugbank (13) records, results from nlp analysis of pubmed abstracts, medical subject headings (mesh) pharmacological actions, anatomical therapeutic chemical classification (atc) entries and a review paper on drugs and their targets (21). the number of available edges with a high - confidence action annotation increased from 44 000 to 65 000 human chemical protein interactions. as described previously (7), stitch creates a consolidated set of chemicals from pubchem (22) by merging stereo isomers and salt forms of the same molecule into one compound. this is done to ensure that all information about the same biologically active entity is merged. while this works very well for drugs that can be supplied in different formulations (e.g. different salt forms) it is our long - term goal to associate interactions both with the individual isomer and the merged structure. for now, we have taken the step to explicitly display all the different compounds that have been deemed biologically equivalent (figure 2). for the drug aspirin, a link to pubchem and a short description is shown. different salts of aspirin that will have the same bioactivity have been consolidated and merged with the main, uncharged form. below each chemical structure, the first part of the inchikey is shown, corresponding to an encoded (hashed) description of the structure. this short string can be used to search for more information about the compound on the internet. different structural scaffolds corresponding to aspirin. for the drug aspirin, a link to pubchem and a short description is shown. different salts of aspirin that will have the same bioactivity have been consolidated and merged with the main, uncharged form. below each chemical structure, the first part of the inchikey is shown, corresponding to an encoded (hashed) description of the structure. this short string can be used to search for more information about the compound on the internet. recently, the international union of pure and applied chemistry (iupac) has standardized an open format for chemical structures, namely the iupac international chemical identifier (inchi). in addition to the existing capability to search chemical structures using smiles string, we have now also implemented a search for inchis. we use the tool open babel to convert inchis to smiles strings, which are in turn searched against our chemical database by using hashed fingerprints as implemented in the open - source chemical development kit (23). furthermore, we have implemented a search for inchikeys, which are short strings that represent an encoded (hashed) form of the chemical structure. inchikeys consist of two parts, the first of which is based on the chemical connectivity, whereas the second part contains information about stereochemistry, tautomers and other structural variations. as stitch currently considers structures with the same connectivity to be equivalent (thus merging stereo isomers), only the first part of the inchikey is queried against our chemical database. we also use this part of the inchikey to provide links to google and chemspider. many proteins, especially drug targets, have a large number of high - scoring interactions with small molecules in the stitch network. in this case, a network centered on such a protein will only show chemicals unless very many interaction partners are requested to be shown (figure 3a). in order to allow the user to see more of the context of the query protein, we now offer the option to show a network in which proteins and chemicals each make up more than a third of the nodes (figure 3b). when this option is selected, only a limited number of the highest - scoring chemicals are displayed. further chemicals are omitted in favor of proteins (or vice versa) and their number is shown to the user. if the network consists of only chemicals, but no proteins are available at the current settings (e.g. due to a minimum score limit), then the option to show more context is not shown. (a) the highest - scoring interaction partners of ptgs1 are non - steroidal anti - inflammatory drugs (nsaids). as the confidence scores for these interactions are very high, no interacting proteins are shown. (b) the user may ask stitch to display more of the interaction context and to let at least one - third of the interaction partners be proteins. in this case, stitch is skipping 19 high - scoring chemicals in order to include four interacting proteins. in both networks, the color of the edge corresponds to the type of connected nodes (e.g. green : chemical protein interaction) and the width of the edge correlates with the confidence score. (a) the highest - scoring interaction partners of ptgs1 are non - steroidal anti - inflammatory drugs (nsaids). as the confidence scores for these interactions are very high, no interacting proteins are shown. (b) the user may ask stitch to display more of the interaction context and to let at least one - third of the interaction partners be proteins. in this case, stitch is skipping 19 high - scoring chemicals in order to include four interacting proteins. in both networks, the color of the edge corresponds to the type of connected nodes (e.g. green : chemical protein interaction) and the width of the edge correlates with the confidence score. previously, stitch required the user to select an organism when searching for interactions with a chemical. now, this is not required anymore. when no organism is selected, the organism with the highest - scoring interaction partners is selected. in case of multiple organisms with equal scores, human and several model organisms (human is one of the highest - ranking species for 60% of the chemicals with protein chemical interactions.) for example, the binding between the antipsychotic agent fluspiperone and the 5hydroxytryptamine (serotonin) receptor 7 has only been studied in mouse and rat. it is also possible to restrict the search to different levels of the ncbi taxonomy (24), e.g. bacteria, fungi or rodents. while central repositories of gene annotations exist, no such information is available in a centralized manner for chemicals. to be able to display text annotation for chemicals, we have imported information from the following databases : drugbank (13), national cancer institute (nci) thesaurus (25), mesh descriptors and qualifiers. using stitch s dictionary of chemical synonyms we mapped compounds from these databases to stitch identifiers. in case where descriptions are available for different forms of the same compound (e.g. different salt forms, which have been merged in stitch), we have assigned the text annotation from only one source, prioritizing sources as follows : nci (descriptions), drugbank (descriptions), drugbank (pharmacology), drugbank (drug category), mesh (pharmacological action), nci (tags) and mesh (scope note). descriptions are available for 33 352 chemicals, covering 33% of the chemicals with interactions. the stitch homepage offers several short tutorials to introduce the different query options (e.g. searching for a single identifier or multiple chemical structures). a search for aspirin on the homepage will lead to the interaction network shown in figure 1. here, the main interactors of the drug are shown in human (which is selected automatically as described above). the known main targets, ptgs1 and ptgs2, are connected by very high scores. while most interaction partners are backed up by evidence from manually curated databases and are therefore very reliable, one interaction is derived only from text - mining : cox1 is actually a false positive arising from an ambiguous synonym. taken together, stitch 2 offers an enlarged set of protein chemical interactions, extended inter - database operability, increased query options and an improved user interface. stitch can be accessed at http://stitch.embl.de/. users can explore the interaction network interactively or download the complete set of interactions. in addition, we provide an application programming interface (api) to let scripts resolve identifiers and retrieve interaction networks either as an image or in standard network formats (20). novo nordisk foundation center for protein research (partial). funding for open access charge : european molecular biology laboratory. conflict of interest statement. | over the last years, the publicly available knowledge on interactions between small molecules and proteins has been steadily increasing. to create a network of interactions, stitch aims to integrate the data dispersed over the literature and various databases of biological pathways, drug target relationships and binding affinities. in stitch 2, the number of relevant interactions is increased by incorporation of bindingdb, pharmgkb and the comparative toxicogenomics database. the resulting network can be explored interactively or used as the basis for large - scale analyses. to facilitate links to other chemical databases, we adopt inchikeys that allow identification of chemicals with a short, checksum - like string. stitch 2.0 connects proteins from 630 organisms to over 74 000 different chemicals, including 2200 drugs. stitch can be accessed at http://stitch.embl.de/. |
hip dislocations are mostly due to low velocity injury in children younger than 3 years. a 25 month old child presented to our casualty following a fall from a slide 3 hours prior to presentation. an x - ray of the pelvis was taken which showed a posterior hip dislocation on the right side. the child had an emergency closed reduction under general anaesthesia followed by a broom stick plaster cast with hips in 30 abduction. the child was reviewed at 6 and 18 months with mri scans at 6 and 18 months. paediatric hip dislocation (less than 3 years of age) is a very rare entity whose incidence is on the rise due to the increase in road traffic accidents. the key stone in proper management is clinical suspicion, early recognition and promptreduction (within 6 hours). also gentle manipulation during reduction has a definite role in preventing iatrogenic chondrolysis and osteonecrosis of femoral head. the periosteum is thick in children, the bones are soft and elastic and can absorb quite some force because of the soft spongy nature. hence dislocations are rare in paediatric age group. however with the increasing number of motor vehicles plying on our roads and the ever present risk of road traffic accidents, there is a recent change in the above trend. most of the paediatric injuries are the result of low velocity accidents. in case of high velocity injuries there has been case reports of traumatic hip dislocations in childhood mostly in the western world. most of the published articles in literature include the adolescent population (12 - 17 years). there are very few case reports of such dislocations in children < 3 years. here we present a case report of a child (age 25 months) with traumatic posterior dislocation of the hip following low velocity injury. a 25 month old male child presented to our casualty following a slip and fall from a slide while playing in a park. he had severe pain in the right hip and limb was adducted and internally rotated. an emergency radiograph of the pelvis taken in emergency showed a posterior dislocation of his right hip[fig.1 ]. gentle traction with minimal rotation was sufficient to achieve reduction, the congruency of which was checked under image intensifier. once congruency was confirmed, an above knee broom stick plaster was applied to both lower limbs with hips in 30 abduction. check radiographs[fig.2 ] was taken along with a mri[fig.3 ] of the hip to check if there was any traumatic injury to the physes. he was kept on the plaster for 6 weeks following which plaster cast was removed and child mobilized with as tolerated weight bearing. follow - up x - ray was taken at 6 weeks[fig.4 ] and serial mri scans taken at 6 and 18 months [fig.5 & fig.6 ] showed no chondrolysis or osteonecrosis. he had restriction of flexion of knee beyond 120 at 6 weeks which improved to 140 by 8 weeks. x - ray following closed reduction and plaster application showing concentric reduction. mri taken immediately following closed reduction showing minimal marrow edema in head and neck area. x - ray taken at 6 weeks following removal of plaster cast showing stable hip with no re - dislocation. m r i at 6 months showing normal physeal plate with no avn m r i at 18 months showing normal femoral head anatomy and physes however with the everpresent danger of motor vehicle accidents in our crowded and chaotic roads, this rarity may become more common. in most children however, hip dislocations are mostly due to low velocity accidents which occur during playing. there are a few reports in western literature of hip dislocation in peadiatric population, most of them are case reports. there has been only two case reports of children less than 3 years with hip dislocation. also most of the reports emphasizes the need to do urgent reduction within the golden period (i.e 6 hours). the risk of osteonecrosis in one study was 5.7% in acute cases vs 47.8% in old neglected cases undergoing reduction procedures. the severity of the injury is also an important predictor for the development of osteonecrosis. also important is that during closed reduction, care is to be taken not to perform forceful manipulation as this greatly increases the risk of iatrogenic physeal separation. as is with our case gentle traction with minimal rotation is enough to reduce the joint. the method of immobilization varies with the authors from skin traction to plaster cast application but we found that results with plaster cast are more predictable with better patient cooperation. child abuse is also a possible etiology of hip dislocation in children when a child presents with a doubtful history and injuries in various stages of healing. in our patient, the immediate post - reduction mri showed a small linear posterior labral tear which had healed in subsequent mri scans taken at 6 months in literature there are case reports of traumatic hip dislocations in childhood (< 3 years) in the western archives. here we present a case report of a traumatic hip dislocations in a child < 3 years in indian population. paediatrichip dislocations are a medical emergency which requires a great deal of suspicion to detect early and treat appropriately. our patient had excellent recovery and functional outcome because of early reduction within the golden period (6 hours). although he had a labral tear, the soft tissues healed with immobilisation without any further intervention with no permanent disability. we therefore conclude that traumatic hip dislocations in children < 3 years is an uncommon injury but a keen lookout is a must for early diagnosis and treatment. however a high degree of suspicion combined with early and prompt reduction is the keystone in management. this is to ensure a good functional outcome and prevention of complications like osteonecrosis and chondrolysis. | introduction : hip dislocation in a child less than 3 years is a very rare event. only a few case reports have been documented in western literature. it is rarely reported from indian population. hip dislocations are mostly due to low velocity injury in children younger than 3 years. we report such a case of hip dislocation in a 2 year old child.case report : a 25 month old child presented to our casualty following a fall from a slide 3 hours prior to presentation. his right lower limb was adducted and internally rotated. there was severe pain on attempting movements. an x - ray of the pelvis was taken which showed a posterior hip dislocation on the right side. the child had an emergency closed reduction under general anaesthesia followed by a broom stick plaster cast with hips in 30 abduction. congruency of reduction was checked with image intensifier before plaster application. the plaster was removed at 6 weeks and gradual weight bearing started. the child was reviewed at 6 and 18 months with mri scans at 6 and 18 months. there were no signs of avascular necrosis or chondrolysis.conclusion:paediatric hip dislocation (less than 3 years of age) is a very rare entity whose incidence is on the rise due to the increase in road traffic accidents. the key stone in proper management is clinical suspicion, early recognition and promptreduction (within 6 hours). also gentle manipulation during reduction has a definite role in preventing iatrogenic chondrolysis and osteonecrosis of femoral head. |
a 30-year - old female patient admitted to our hospital with sudden hearing loss and tinnitus in the right ear. her medical history also included lumbar discopathy. she d been using beta interferon at a dose of 0.3 mg on alternate days for the treatment of ms for the last 3.5 years. physical examination of the patient demonstrated intact bilateral tympanic membranes, weber test was lateralized to the left ear, rinne test was positive (+ /+) in the left, and false negative (+ /- pure tone audiogram was performed on the patient with the initial diagnosis of sudden hearing loss (figure 1). the final diagnosis was total hearing loss in the right ear (air conduction / bone conduction : 113/67 db). the hearing acuity of the left ear was determined to be in normal hearing range (air conduction / bone conduction : 18/12 db). after the patient was admitted to our clinic, routine laboratory tests were run. in the laboratory tests ; there were no abnormal findings except wbc ; 12.710/mm, triglycerides ; 34 mg / dl, ck - mb ; 25 u / l, uibc ; 366 ug / dl. contrast - enhanced cranial magnetic resonance imaging (mri) and neurology consultation were ordered for the patient. as a result of the consultation accordingly, a new ms attack was not anticipated. to investigate vascular, connective tissue pathologies or infectious etiologies, the results of new laboratory tests were as follows : rheumatoid factor < 20, anti - nuclear antibody ; negative (), anti - phospholipid ig g ; negative (), anti - phospholipid igm ; negative (). levels of protein c, protein s, von willebrant factor antigen were in their normal ranges. in elisa tests, levels of anti - toxoplasma ig m, anti - cmv ig m, ebv vca ig m, anti - hsv-1 ig m, anti - hsv-2 ig m, vdrl - rpr, anti - ds dna were within their normal ranges. in the light of these findings, classical parenteral treatment protocol of sudden sensorineural hearing loss was prescribed as prednisolone kg/1 mg / day with tapering dose, ranitidine 50 mg / day, pentoxifylline 900 mg / day, low - molecular - weight heparin 0.6 ml / day, acyclovir 2 g / day. also hyperbaric oxygen therapy was administrated to the patient for 10 days at doses of 2.5 ata / day. pure tone audiogram was performed totally 3 times during hospitalization period. in spite of all our medical treatments the patient was discharged with oral treatment and followed up in our outpatient clinic. at the end of our treatment, audiologic examinations were performed. in november, one month after the onset of the disease, there was not any hearing improvement as detected on her pure tone audiogram (figure 2). three months later spontaneous recovery was observed on her pure tone audiogram air / bone conduction 13/7 db, 30/27 db were reported for the left, and the right ear, respectively (figure 3). depending on these findings, the patient s sudden hearing loss of the right ear might be related to ms disease. sudden hearing loss (shl) is a hearing loss of 30 db or more, over at least three contiguous audiometric frequencies, that develops over a period of a few hours to 3 days [4, 5, 6, 7, 8, 9 ]. the etiology of the disease is not certain, but autoimmune vascular malformations, thrombotic, and central nervous system diseases are among the main reasons [4, 5 ]. ms is a demyelinating disease of the central nervous system that involves the white substance. rarely cerebellum, brain stem and spinal cord can be effected by ms. as a result of autoimmunity, intravascular t cells attack the myelin sheath, and nerve fibers, then it starts as an inflammatory process. on the other hand, neural regeneration begins in order to stop the damage at a minimum level. in 4 - 10 % of ms patients, sensorineural hearing loss occurs during periods of relapse or remission [5, 6 ]. detection of brain lesions of ms in mri, can provide evidence concerning the dissemination of ms lesions in space and time. therefore a diagnosis of ms should be guided by the defined criteria of mri for this abnormality. hearing loss in ms disease may be due to plaques which are placed on brain stem, any area of entrance of cochlear nerve into the brain stem, and auditory cortex [6, 7 ]. in this situation, auditory brainstem response (abr) test is the most appropriate test for the diagnosis of sensorineural hearing loss in ms patients. also, elongations in interpeak latencies of i - iii, iii - v waves or changes in the morphology and amplitudes of iii and v wave s may be observed [7, 8 ]. in the right ear abr of our patient, we observed elongation in absolute latencies of waves iii and v, as well as iii - v waves interpeak latencies. if sudden hearing loss is related with ms, it usually recovers without sequellae. in a retrospective study of hellman and his colleagues, 253 ms patients with sensorineural hearing loss were scanned, and sudden hearing loss was diagnosed in 11 of them. in our patient, sudden hearing loss recovered with a mild sensorineural hearing loss, and no recurrence was observed in one year follow - up. in conclusion, whenever sudden hearing loss is diagnosed in a patient, physicians should request cranial imaging to differentiate for cranial pathologies such as ms. sudden hearing loss can be the first symptom of ms or may indicate relapse of the disease. | multiple sclerosis (ms) is the most common demyelinating disease of the central nervous system. ms involves different regions of the central nervous system in different periods, and causes demyelination. ms is a neuromotor disorder which progresses with remissions and relapses. symptoms of ms may regress completely or heal after the relapses leaving sequelae. sudden sensorinerural hearing loss (sshl) is hearing loss of 30 db or more over at least three contiguous audiometric frequencies that develops over a period of a few hours to 3 days. in 4 - 10 % of the ms patients, sensorineural hearing loss occurs between relapses or remissions. in this case, audiotory brainstem response (abr) test is the most appropriate test for the diagnosis of sensorineural hearing loss in ms patients. in this article, we will discuss a patient diagnosed as ms who presented with sudden sensorineural hearing loss during the remission of the disease. |
methods and any associated references are available in the online version of the paper at http://www.nature.com / naturemedicine/. note : supplementary information is available on the nature medicine website. | cancer stem cells (cscs) or tumor progenitor cells are involved in tumor progression and metastasis1. micrornas (mirnas) regulate both normal stem cells and cscs25 and mirna dysregulation has been implicated in tumorigenesis6. cscs in many tumors, including cancers of the breast7, pancreas8, head and neck9, colon10,11, small intestine12, liver13, stomach14, bladder15, and ovary16 have been identified using adhesion molecule cd44, either individually or in combination with other marker(s). prostate cancer (pca) stem / progenitor cells with enhanced clonogenic17 and tumor - initiating and metastatic18,19 capacities are also enriched in the cd44 + cell population, but whether mirnas regulate the cd44 + pca cells and pca metastasis remains unclear. here we show, through expression analysis, that mir-34a, a p53 target2024, was under - expressed in cd44 + pca cells purified from xenograft and primary tumors. enforced expression of mir-34a in bulk pca cells inhibited clonogenic expansion and tumor development. mir-34a re - expression in cd44 + pca cells blocked whereas mir-34a antagomirs in cd44 pca cells promoted tumor regeneration and metastasis. systemically delivered mir-34a inhibited pca metastasis and extended animal survival. of significance, cd44 was identified and validated as a direct and functional target of mir-34a and cd44 knockdown phenocopied mir-34a over - expression in inhibiting pca regeneration and metastasis. our study reveals mir-34a as a critical negative regulator of cd44 + pca cells and establishes a strong rationale for developing mir-34a as a novel therapeutic against prostate cscs. |
amyotrophic lateral sclerosis (als) is a progressive neurodegenerative disorder characterized by relentless loss of motor neuron function. the selective degeneration of upper and lower motor neurons results in progressive weakness of the limb, bulbar, abdominal, and thoracic muscles. the reported annual prevalence and incidence rates of als were only 9.9 and 2.3 per 100,000 people per year respectively in asian country, japan. however, the importance of als should not be underestimated since there are more than one in 500 people who will die of the disease in the industrialized world. without mechanical ventilation, most patients die within 2 to 5 years after symptom onset because of respiratory failure, although 5% to 10% of patients may live more than 10 years. up to now, riluzole was the only drug approved by the food and drug administration as neuroprotective treatment / disease - modifying treatment for als that has been shown to slightly slow the course of als. oral administration of riluzole 100 mg daily for als patients is reasonably safe and prolongs a limited lengthening of median survival by 2 to 3 months after 18 months treatment. to our knowledge, other than riluzole, there are no other new treatment that can halt or reverse the progressive loss of neurons, leading to an increase of the als life expectancy. given the fatalness of the illness and lack of effective neuroprotective treatment / disease - modifying agents, complementary and/or alternative medicine (cam) is thus increasingly sought to treat als worldwide. traditional chinese medicine (tcm) is a main form of cam originating in ancient china and has been practiced for a history of 3000 years. accordingly, chinese herbal medicines (chms) are frequently used in the treatment of als. studies in vivo and in vitro showed that chms have a great potential for treatment of als, with neuroprotective function against excitatory amino acid toxicity, oxidative stress, calcium cytotoxicity, and neuroinflammation. during song dynasty (9601279 ad), the tcm prescription book shengji zonglu (complete record of sacred benevolence) compiled by zhao ji who was the eighth emperor of song dynasty of china in 1111 to 1117 ad. this book lists 20,000 tcm prescriptions and describes the causes, symptoms, and cures for different diseases. dihuang yinzi (dhyz), a classical tcm prescription for neurological disorders yinfei syndrome that centered on the symptoms of the speech and language disorders such as aphasis and logopathy (yin syndrome) and disorders of motility such as motor paralysis and difficulty in walking (fei syndrome), was first recorded in this book with the name dihuang yin. it is composed of 12 kinds of chms : (a) radix rehmanniae preparata 15 g ; prepared rehmannia root (shudihuang), the dried roots of radix rehmanniae recens ; (b) fructus corni 15 g ; asiatic cornelian cherry fruit (shanzhuyu), the dried ripe pulp of cornus officinalis sieb. (c) herba cistanches 15 g ; desertliving cistanche (rousongrong), the dried roots of cistanche deserticola y. c. ma ; (d) radix morindae officinalis 15 g ; morinda root (bajitian), the dried roots of morinda officinalis how. ; (e) radix aconiti lateralis preparata 15 g ; prepared common monkshood branched root (fuzi), the processed root of aconitum carmichaeli debx. ; (f) cortex cinnamomi 15 g ; cassia bark (rougui), the dried bark of cinnamomum cassia presl ; (g) herba dendrobii 15 g ; dendrobium (shihu), the dried roots of dendrobium loddigesli rolfe. or dendrobium fimbriatum hook. var. oculatum hook. or dendrobium chrysanthum wall. or dendrobium officinale kimra et migo or dendrobium nobile lindl. ; (h) radix ophiopogonis 15 g ; dwarf lilyturf tuber (maidong), the dried roots of ophiopogon japonicus (thunb.) ker - gawl. ; (i) fructus schisandrae chinensis 15 g ; chinese magnoliavine fruit (wuweizi), the dried ripe fruit of s chinensis (turcz.) baill. ; (j) rhizoma acori tatarinowii 15 g ; grassleaf sweetflag rhizome (shichangpu), the dried roots of acorus tatarinowii schott. ; (k) radix polygalae 15 g ; milkwort root (yuanzhi), the dried roots of polygala tenuifolia willd. or polygala sibirica l. ; (l) poria ; indian bread (fuling) 15 g, the dried sclerotia of poria cocos (schw.) wolf. (table 1). in modern times, dhyz is still used continuously and widely for treatment of neurological disorders such as stroke, parkinson disease dementia, and spinal cord injury. here, we reported a case of als patient using modified dhyz who has survived 12 years with significant improvement in bulbar paralysis. on 31 july 2004, a 41-year - old chinese han nationality woman was admitted to the hospital with complaints of weakened bilateral grip, slurred speech, stumbling, and muscular twitchings for 3 years. the initial symptom, the intermittent weakness of right upper limb without numbness and pain, appeared in november 2001. after 6 months, uncontrollable twitching was observed in the right upper limb and the patient 's speech became slurred. on 5 march 2002, the electromyography (emg) done at the huashan hospital affiliated to fudan university showed neurogenic injury in bilateral upper limbs and tongue (table 2). based on the revised el escorial criteria, clinically als was diagnosed. the patient was orally administrated with riluzole (50 mg, twice a day) for 10 months. she stopped the riluzole by herself because symptoms continuously deteriorated and could not bear the economic burden. on 31 july 2004, she was admitted to the hospital with weakness of limbs, dysarthria, dysphagia, and the clumsiness in daily activities. on examination, the four main vital signs, temperature, heart rate, blood pressure, and respiratory rate, were normal. motor system examination showed widespread muscle wasting and fasciculation of the bilateral thenar and interosseous muscles. strength in the distal muscle of the upper bilateral limbs was of grade 2/5 to 3/5 and muscle strength in the lower bilateral limbs was of grade 3/5 to 4/5, graded on the medical research council muscle strength grading system. the bilateral hoffman signs were present, whereas the bilateral babinski signs were not present. tcm symptoms and signs were summarized as follows : sluggish speech, faint low voice, choke when drinking, weakness of limbs, muscle wasting of hands, muscular twitchings of upper limbs, pale facial complexion, soreness and weakness of waist and knees, excessive phlegm and saliva, constipation once every 3 days, pink tongue quality, tongue muscle atrophy and fibrillation, deep weak and thready pulse. the electromyography examination atrophy and fasciculations, the onset symptom is insidiously developing asymmetric upper limb weakness and then bulbar muscle. the clinical features are accompanied with the pathological signs : overactive tendon reflexes and clonus. based on the revisited el escorial criteria, she was diagnosed with clinically definite als, depending on the clinical manifestations and consistent electrodiagnostic studies. when the patient experienced bulbar paralysis with symptoms such as dysarthria and dysphagia, the clinical features were classified to yin syndrome in tcm. the kidney meridian dominates feet, throat, and tongue according to meridian theory of tcm. thus, the kidney deficiency leads to yinfei syndrome affecting both bulbar muscles and limbs muscles. following the tcm theory, the patient was diagnosed with yinfei syndrome because of kidney deficiency. once the patient was diagnosed with als firstly in 2002, she was started on riluzole (50 mg, twice a day) for 10 months. when she noted that the treatment can not arrest the disease condition and she can not bear the economic burden, the patient discontinued treatment because riluzole is not a cure for als. in that time modified dhyz was chosen because it has the function of replenishing kidney essence to treat yinfei syndrome. the compositions and dosage of modified dhyz are as follows : (a) radix rehmanniae preparata (shudihuang) 15 g ; (b) fructus corni (shanzhuyu) 12 g ; (c) herba cistanches (rousongrong) 15 g ; (d) herba epimedii 15 g ; epimedium herb (yinyanghuo) the dried above ground parts of epimedium brevicornum maxim.,, epimedium wushanensis t. s. ying and epimedium koreanum nakai ; (e) radix aconiti lateralis preparata (fuzi) 6 g ; (f) radix ophiopogonis (maidong) 15 g ; (g) rhizoma anemarrhenae 15 g ; common anemarrhena rhizome(zhimu), the dried rhizome of anemarrhena asphodeloides bge. ; (h) rhizoma acori tatarinowii (shichangpu) 6 g ; (i) radix polygalae (yuanzhi) 6 g ; (j) fructus trichosanthis seed 30 g ; snakegourd seed (gualouren), the dried seed of trichosanthes kirilowii maxim. or trichosanthes rosthorinii harms. ; (k) scorpio 6 g ; scorpion (quanxie), the dried body of artificial breeding of buthus martensii karsch. ; (l) agkistrodon 9 g ; long - nosed pit viper (qishe), the dried body of artificial breeding of agkistrodon acutus (guenther). the prescription was prepared from the water decoction and oral for twice daily and the patient has been using it continuously for 12 years. during the 12 years, dhyz has been modified according to the mainly accompanied syndrome or symptoms as follows (table 3) : (1) qi deficiency : plus radix astragali seu hedysari, milkvetch root (huangqin), the dried roots of astragalus membranaceus (fisch.) bge. mongolicus (bge.) hsiao or a membranaceus (fisch.) bge. ; and radix ginseng, ginseng (rensheng), the dried roots of panax ginseng c. a. mey. ; (2) obvious yin deficiency : plus radix rehmanniae recens, unprocessed rehmannia root (shengdihuang), the dried roots of rehmannia glutinosa libosch. ; fructus ligustri lucidi, glossy privet fruit (nvzhenzi), the dried ripe fruit of ligustrum lucidum ait. ; and herba ecliptae, yerbadetajo herb (hanliancao), the dried stems of eclipta prostrata l. ; (3) accumulation of phlegm - fire : minus radix aconiti lateralis preparata (fuzi) and cortex cinnamomi (rougui) ; plus concretio silicea bambusae, tabasheer (tianzhuhuang), the saps from bambusa textilis mcclure or schizostachyum chinense rendle, succus bambusae ; fresh bamboo sap (zhuli), the saps from bambusa tuldoides munro or sinocalamus beecheyanus (munro) mcclure var. henonis (mitf.) stapf ex rendle ; and rhizoma arisaematis cum bile ; bile arisaema (dannanxing), the dried roots of arisaema erubescens (wall.) schott or arisaema heterophyllum bl. or arisaema amurense maxim. ; (4) deficiency of heart blood : plus semen ziziphi spinosae, spine date seed (suanzaoren), the dried ripe seeds of ziziphus jujuba mill. spinosa (bunge) hu ex h. f. chou ; semen platycladi, chinese arborvitae kernel (baiziren), the dried ripe seeds of platycladus orientalis (l.) franco ; and caulis polygoni multiflori, tuber fleeceflower stem (yejiaoteng), the dried stems of polygonum multiflorum thunb. ; (5) constipation : optionally plus rhizoma anemarrhenae (zhimu) ; semen trichosanthis (guolouren) ; radix polygoni multiflori, fleeceflower root (heshouwu), the dried roots of p multiflorum thunb. ; semen persicae, peach seed (taoren), the dried seeds of amygdalus persica l. or amygdalus davidiana (carr.) c. de vos ex henry ; fructus cannabis, hemp seed (maziren), the dried ripe fruit of cannabis sativa l. ; radix angelicae sinensis, chinese angelica (danggui), the dried roots of a sinensis (oliv.) diels ; radix puerariae, kudzuvine root (gegeng), the dried roots of pueraria lobata (willd.) ohwi or pueraria thomsonii benth ; semen pruni, chinese dwarf cherry seed (yuliren), the dried ripe seeds of cerasus humilis (bge.) sok. or cerasus japonica (thunb.) lois. or amygdalus pedunculata pall. ; and herba cynomorii, songaria cynomorium herb (suoyang), the dried stems of cynomorium songaricum rupr. ; (6) obvious muscular twitchings : plus bombyx batryticatus, stiff silkworm (jiangcan), the dried body of bombyx mori linnaeus. ; periostracum cicadae, cicada slough (chantui), the dried shell of cryptotympana pustulata fabricius ; lumbricus, earthworm (dilong), the dried body of pheretima aspergillum (e. perrier) or pheretima vulgaris chen or pheretima guillelmi (michaelsen) or pheretima pectinifera michaelsen ; scolopendra, centipede (wugong), the dried body of scolopendra subspinipes mutilans l. koch ; (7) cervicomuscular weakness : plus radix puerariae (gegeng) ; and colla corni cervi, deerhorn glue (lujiaojiao), the glue of horn of cervus nippon temminck or cervus elaphus l. ; (8) pain of limbs : plus radix paeoniae alba, white peony root (baishao), the dried stems of paeonia lactiflora pall. ; fructus chaenomelis, common floweringqince fruit (mugua), the dried ripe fruit of chaenomeles speciosa (sweet) nakai ; and herba siegesbeckiae, siegesbeckia herb (xixiancao), the dried stems of siegesbeckia orientalis l. or siegesbeckia pubescens makino or siegesbeckia glabrescens makino. accompanied with syndrome or symptom and corresponding modified dihuang yinzi for amyotrophic lateral sclerosis after 12-year treatment and follow - up, no obvious adverse event occurred during the treatment period. in addition, the patient still survived with als and does not require permanent continuous ventilator till today (figure 1). the symptoms of choking on liquids are improved, and the utility of 30 ml water swallow test was improved with grade 2. however, muscle strength worsened slowly as follows : tongue muscle atrophy and tongue fasciculations, the slurring of speech, difficulty in communication and use of facial expression, difficulty in activity with both hands and in neck - lifting, muscle wasting of limbs, and presenting with claw hand. strength in the distal and proximal muscle of the upper bilateral limbs was of grade 0/5 and 1/5 to 2/5 respectively, and muscle strength in the lower bilateral limbs was of grade 2/5, graded on the medical research council muscle strength grading system. at last, we will follow - up the patient continuously. on 7 january 2014, the repeated emg showed motor conduction amplitude reducing gradually and velocity not changing more when compared with the initial emg (table 2). a case of amyotrophic lateral sclerosis patient was treated by using a classical chinese herbal prescription dihuang yinzi, who has survived 12 years with significant improvement in bulbar paralysis and do nt require permanent continuous ventilator till now. (a) the patient 's appearance ; (b) the patient 's tongue muscle atrophy ; (c) the patient 's feet ; (d) the patient 's palms. on 31 july 2004, a 41-year - old chinese han nationality woman was admitted to the hospital with complaints of weakened bilateral grip, slurred speech, stumbling, and muscular twitchings for 3 years. the initial symptom, the intermittent weakness of right upper limb without numbness and pain, appeared in november 2001. after 6 months, uncontrollable twitching was observed in the right upper limb and the patient 's speech became slurred. on 5 march 2002, the electromyography (emg) done at the huashan hospital affiliated to fudan university showed neurogenic injury in bilateral upper limbs and tongue (table 2). based on the revised el escorial criteria, clinically als was diagnosed. the patient was orally administrated with riluzole (50 mg, twice a day) for 10 months. she stopped the riluzole by herself because symptoms continuously deteriorated and could not bear the economic burden. on 31 july 2004, she was admitted to the hospital with weakness of limbs, dysarthria, dysphagia, and the clumsiness in daily activities. on examination, the four main vital signs, temperature, heart rate, blood pressure, and respiratory rate, were normal. motor system examination showed widespread muscle wasting and fasciculation of the bilateral thenar and interosseous muscles. strength in the distal muscle of the upper bilateral limbs was of grade 2/5 to 3/5 and muscle strength in the lower bilateral limbs was of grade 3/5 to 4/5, graded on the medical research council muscle strength grading system. the bilateral hoffman signs were present, whereas the bilateral babinski signs were not present. tcm symptoms and signs were summarized as follows : sluggish speech, faint low voice, choke when drinking, weakness of limbs, muscle wasting of hands, muscular twitchings of upper limbs, pale facial complexion, soreness and weakness of waist and knees, excessive phlegm and saliva, constipation once every 3 days, pink tongue quality, tongue muscle atrophy and fibrillation, deep weak and thready pulse. the electromyography examination the patient is a middle - aged woman. with muscles atrophy and fasciculations, the onset symptom is insidiously developing asymmetric upper limb weakness and then bulbar muscle. the clinical features are accompanied with the pathological signs : overactive tendon reflexes and clonus. based on the revisited el escorial criteria, she was diagnosed with clinically definite als, depending on the clinical manifestations and consistent electrodiagnostic studies. when the patient experienced bulbar paralysis with symptoms such as dysarthria and dysphagia, the clinical features were classified to yin syndrome in tcm. the kidney meridian dominates feet, throat, and tongue according to meridian theory of tcm. thus, the kidney deficiency leads to yinfei syndrome affecting both bulbar muscles and limbs muscles. following the tcm theory, once the patient was diagnosed with als firstly in 2002, she was started on riluzole (50 mg, twice a day) for 10 months. when she noted that the treatment can not arrest the disease condition and she can not bear the economic burden, the patient discontinued treatment because riluzole is not a cure for als. in that time modified dhyz was chosen because it has the function of replenishing kidney essence to treat yinfei syndrome. the compositions and dosage of modified dhyz are as follows : (a) radix rehmanniae preparata (shudihuang) 15 g ; (b) fructus corni (shanzhuyu) 12 g ; (c) herba cistanches (rousongrong) 15 g ; (d) herba epimedii 15 g ; epimedium herb (yinyanghuo) the dried above ground parts of epimedium brevicornum maxim., epimedium wushanensis t. s. ying and epimedium koreanum nakai ; (e) radix aconiti lateralis preparata (fuzi) 6 g ; (f) radix ophiopogonis (maidong) 15 g ; (g) rhizoma anemarrhenae 15 g ; common anemarrhena rhizome(zhimu), the dried rhizome of anemarrhena asphodeloides bge. ; (h) rhizoma acori tatarinowii (shichangpu) 6 g ; (i) radix polygalae (yuanzhi) 6 g ; (j) fructus trichosanthis seed 30 g ; snakegourd seed (gualouren), the dried seed of trichosanthes kirilowii maxim. or trichosanthes rosthorinii harms. ; (k) scorpio 6 g ; scorpion (quanxie), the dried body of artificial breeding of buthus martensii karsch. ; (l) agkistrodon 9 g ; long - nosed pit viper (qishe), the dried body of artificial breeding of agkistrodon acutus (guenther). the prescription was prepared from the water decoction and oral for twice daily and the patient has been using it continuously for 12 years. during the 12 years, dhyz has been modified according to the mainly accompanied syndrome or symptoms as follows (table 3) : (1) qi deficiency : plus radix astragali seu hedysari, milkvetch root (huangqin), the dried roots of astragalus membranaceus (fisch.) bge. mongolicus (bge.) hsiao or a membranaceus (fisch.) bge. ; and radix ginseng, ginseng (rensheng), the dried roots of panax ginseng c. a. mey. ; (2) obvious yin deficiency : plus radix rehmanniae recens, unprocessed rehmannia root (shengdihuang), the dried roots of rehmannia glutinosa libosch. ; fructus ligustri lucidi, glossy privet fruit (nvzhenzi), the dried ripe fruit of ligustrum lucidum ait. ; and herba ecliptae, yerbadetajo herb (hanliancao), the dried stems of eclipta prostrata l. ; (3) accumulation of phlegm - fire : minus radix aconiti lateralis preparata (fuzi) and cortex cinnamomi (rougui) ; plus concretio silicea bambusae, tabasheer (tianzhuhuang), the saps from bambusa textilis mcclure or schizostachyum chinense rendle, succus bambusae ; fresh bamboo sap (zhuli), the saps from bambusa tuldoides munro or sinocalamus beecheyanus (munro) mcclure var. henonis (mitf.) stapf ex rendle ; and rhizoma arisaematis cum bile ; bile arisaema (dannanxing), the dried roots of arisaema erubescens (wall.) schott or arisaema heterophyllum bl. or arisaema amurense maxim. ; (4) deficiency of heart blood : plus semen ziziphi spinosae, spine date seed (suanzaoren), the dried ripe seeds of ziziphus jujuba mill. spinosa (bunge) hu ex h. f. chou ; semen platycladi, chinese arborvitae kernel (baiziren), the dried ripe seeds of platycladus orientalis (l.) franco ; and caulis polygoni multiflori, tuber fleeceflower stem (yejiaoteng), the dried stems of polygonum multiflorum thunb. ; (5) constipation : optionally plus rhizoma anemarrhenae (zhimu) ; semen trichosanthis (guolouren) ; radix polygoni multiflori, fleeceflower root (heshouwu), the dried roots of p multiflorum thunb. ; semen persicae, peach seed (taoren), the dried seeds of amygdalus persica l. or amygdalus davidiana (carr.) c. de vos ex henry ; fructus cannabis, hemp seed (maziren), the dried ripe fruit of cannabis sativa l. ; radix angelicae sinensis, chinese angelica (danggui), the dried roots of a sinensis (oliv.) diels ; radix puerariae, kudzuvine root (gegeng), the dried roots of pueraria lobata (willd.) ohwi or pueraria thomsonii benth ; semen pruni, chinese dwarf cherry seed (yuliren), the dried ripe seeds of cerasus humilis (bge.) sok. or cerasus japonica (thunb.) lois. or amygdalus pedunculata pall. ; and herba cynomorii, songaria cynomorium herb (suoyang), the dried stems of cynomorium songaricum rupr. ; (6) obvious muscular twitchings : plus bombyx batryticatus, stiff silkworm (jiangcan), the dried body of bombyx mori linnaeus. ; periostracum cicadae, cicada slough (chantui), the dried shell of cryptotympana pustulata fabricius ; lumbricus, earthworm (dilong), the dried body of pheretima aspergillum (e. perrier) or pheretima vulgaris chen or pheretima guillelmi (michaelsen) or pheretima pectinifera michaelsen ; scolopendra, centipede (wugong), the dried body of scolopendra subspinipes mutilans l. koch ; (7) cervicomuscular weakness : plus radix puerariae (gegeng) ; and colla corni cervi, deerhorn glue (lujiaojiao), the glue of horn of cervus nippon temminck or cervus elaphus l. ; (8) pain of limbs : plus radix paeoniae alba, white peony root (baishao), the dried stems of paeonia lactiflora pall. ; fructus chaenomelis, common floweringqince fruit (mugua), the dried ripe fruit of chaenomeles speciosa (sweet) nakai ; and herba siegesbeckiae, siegesbeckia herb (xixiancao), the dried stems of siegesbeckia orientalis l. or siegesbeckia pubescens makino or siegesbeckia glabrescens makino. accompanied with syndrome or symptom and corresponding modified dihuang yinzi for amyotrophic lateral sclerosis after 12-year treatment and follow - up, no obvious adverse event occurred during the treatment period. in addition, the patient still survived with als and does not require permanent continuous ventilator till today (figure 1). the symptoms of choking on liquids are improved, and the utility of 30 ml water swallow test was improved with grade 2. the symptoms of muscle twitching of limbs were also reduced. however, muscle strength worsened slowly as follows : tongue muscle atrophy and tongue fasciculations, the slurring of speech, difficulty in communication and use of facial expression, difficulty in activity with both hands and in neck - lifting, muscle wasting of limbs, and presenting with claw hand. strength in the distal and proximal muscle of the upper bilateral limbs was of grade 0/5 and 1/5 to 2/5 respectively, and muscle strength in the lower bilateral limbs was of grade 2/5, graded on the medical research council muscle strength grading system. on 7 january 2014, the repeated emg showed motor conduction amplitude reducing gradually and velocity not changing more when compared with the initial emg (table 2). a case of amyotrophic lateral sclerosis patient was treated by using a classical chinese herbal prescription dihuang yinzi, who has survived 12 years with significant improvement in bulbar paralysis and do nt require permanent continuous ventilator till now. (a) the patient 's appearance ; (b) the patient 's tongue muscle atrophy ; (c) the patient 's feet ; (d) the patient 's palms. this is a long - term follow - up study on a case with als treated by tcm prescription. a middle - aged woman was diagnosed with als based on the revised el escorial criteria. after consuming orally administrated riluzole 100 mg daily for 10 months, she stopped this drug and then started dhyz that she has been using for 12 years. the main findings were that dhyz therapy for als may potentially improve bulbar paralysis, delay use of ventilator support, and prolong survival time ; there were fewer adverse effects. approximately 90% of als cases are sporadic, but the remaining 10% of the cases are familial. the mean age of onset is 58 to 63 years in sporadic and 43 to 52 years in familial cases of als. only 5% of the cases of als have an onset < 30 years of age. an increased risk in the sex incidence ratio was male : female = 1.5:1. in the present case, a female suffering from als was of age 38 years in sporadic who presented with limb onset and subsequently bulbar symptoms. als is primarily a clinically diagnosed disease because of the lack of an established biological marker. diagnosis of als is usually straightforward according to the progressive, generalized symptoms in the limb and bulbar regions. this can result in a delay in diagnosis because als inclines to be focal in onset presented with few clinical signs in the early disease course. the mean lag time between the onset of symptoms of als and confirmation of the diagnosis is 10 to 18 months. in 1994, the el escorial criteria for diagnosing als clinically were published by a subcommittee on als of the world federation of neurology and the revisited criteria were revised in 1999, which included the clinical neurophysiolgical measurements as diagnostic tools to exclude differential diagnosis. in 2006, the awaji criteria placed equal emphasis on both electromyographic evidence of degeneration and clinical abnormalities, thereby potentially enabling an earlier secure diagnosis of als, and this criterion has successfully increased in sensitivity to detect patients with als but additionally showed that this is achieved without increasing the number of false - positives. in 2015, an updated version of el escorial criteria for the diagnosis of als was published with the purpose of both clinical practice and clinical trial. the new diagnostic criteria of als require at least one of the following : (1) progressive upper and lower motor neuron deficits in at least one limb or region of the human body ; that is, that is meeting the revised el escorial criteria for possible als ; (2) lower motor neuron deficits as defined by clinical examination (one region) and/or by emg in two body regions (defined as bulbar, cervical, thoracic, and lumbosacral). the emg findings consist of neurogenic potentials and fibrillation potentials and/or sharp waves. in the present study, the patient was diagnosed with als according to revised el escorial criteria 2000 and confirmed both clinical and electrophysiological evidences during long - term follow - up. repeated investigations were required and the diagnosis can be confirmed with disease progression over time. in the initial emg, the damages of different extents occurred in motor nerves which were controlled by neurogenic changed muscle of the patient. motor conduction velocity in the upper and lower limbs was almost normal despite the amplitude decreased in the initial and repeated examination, because the primary abnormality of the peripheral nerve was axonal loss, rather than demyelination. it is critical that tongue innervated by a cranial nerve demonstrated evidence of acute reinnervation confirmed the diagnosis for the positive changes of resting potential, for example, fibs and wave. from the repeated emg, we can find motor conduction amplitude reducing gradually and velocity not changing more when compared with the initial emg. despite advances in the treatments and interventions, there are no medications that stop or reverse the progressive loss of motor neurons of als because of uncertainty on the pathogenic mechanisms underlying degeneration of motor neuron. riluzole remains the only available neuroprotective / disease - modifying drug for als, with only marginal effects on disease survival. although still incurable, als is not untreatable. over the past two decades, remarkable progression in integrative and aggressive supportive care in addition, emphasis has been made in therapies that may even improve the disease course of als. presently, als is considered as a complex disease with broad pathophysiological framework and numerous theories, including oxidative stress, glutamate and neuronal cytotoxicity, protein aggregation, mitochondrial impairment, cytoskeletal disruption, inflammation, apoptotic cell death, and altered regulation of gene expression. thus, combined therapies that focus on more than one pathogenic pathway may slow disease progression in multiple targets / organs interactions. impressively, the key to tcm prescription is to choose a combination of chms guided the combinatorial principle of sovereign - minister - assistant - envoy according to the patient 's syndrome in order to regain the balance state of body functions. over the past decades, many experimental and clinical studies demonstrated tcm prescriptions ; herbal components may have multiple targets and exert neuroprotection or treatment of als. for example, dhyz possesses neuroprotective and antidementia properties through preventing the loss of neural cells and synapses in rats of ischemic brain injury. many studies have demonstrated that each ingredient or its active components of dhyz exerted potential neuroprotective functions (table 1). rehmannia root and catalpol have antioxidation, antiapoptosis, antiinflammation, and other neuroprotective properties and act in neuroprotection of neurodegenerative diseases.p - coumaric acid, ursolic acid, and gallic acid from corni fructus exerted antioxidation, antiapoptosis, and antineuroinflammation against a-induced toxicity. herba cistanches and its active components possess neuroprotective function, hormone regulation, immunomodulatory, antioxidative, antiapoptotic, and antiinflammatory activities, making them potential treatment for various neurodegenerative disorders. - d - fructofuranosyl (22) -d - fructofuranosyl from radix morindae officinalis enhanced antioxidative activity, antiapoptotic activity, and energy metabolism against a-induced neurotoxicity. cortex cinnamomi has a neuroprotective effect on glutamate - induced neuronal death through the inhibition of ca influx ; 2-hydroxycinnamaldehyde has antineuroinflammatory and neuroprotective effects in the central nervous system by targeting lipoprotein receptor - related protein 1. an active compound from dendrobium and chrysotoxine exhibit antioxidant, antiapoptosis, and neuroprotective activities against neurotoxion - induced cell death.o japonicus displays antiinflammatory, antioxidative, and immunomodulative activities. schisantherin a, schisandrin b, and schisandrin c from fructus schisandrae chinensis attenuate abnormal oxidative stress and modulate the apoptotic signal pathway, exhibiting their neuroprotective function on neurodegenerative diseases. -asarone and eugenol, components of rhizoma acori tatarinowii, have neuroprotective function cells through inhibiting apoptosis against a- induced neurotoxicity. polygalae radix has neuroprotective effects against toxin - induced neuronal death through its antioxidant and antiapoptotic activities ; its ethanol extracts as a novel autophagy inducer modulate neurodegenerative disorders via reducing toxicity and clearing of mutant proteins in the cellular level.p cocos water extract has neuroprotective action against a-induced neuronal injury through suppressing the oxidative stress and the apoptosis. in conclusion, we reinforce that tcm prescription, especially dhyz, can be potentially used in als patients because of its multitargeted neuroprotection and general safety, although als has not a cure. in addition, we identified the area that is worthy of further study and dhyz as a promising candidate for further clinical application and als trials. | abstractamyotrophic lateral sclerosis (als) is a devastating progressive neurodegenerative disease with no effective treatment and death within 2 to 5 years after symptom onset. here, we reported a case of als patient using modified dihuang yinzi (dhyz), a classical traditional chinese medicine (tcm) prescription, who has survived 12 years with significant improvement in bulbar paralysis.a 41-year - old chinese han nationality woman was admitted to the hospital with complaints of weakened bilateral grip, slurred speech, stumbling, and muscle twitching for 3 years. the electromyography showed neurogenic injury in bilateral upper limbs and tongue. she was diagnosed with als according to the revised el escorial criteria. the patient was orally administrated with riluzole 100 mg daily for 10 months and then stopped. subsequently, she resorted to tcm. based on the tcm theory, the patient was diagnosed with yinfei syndrome because of kidney deficiency. dhyz was chosen because it has the function of replenishing kidney essence to treat yinfei syndrome. up to now, she has been using modified dhyz continuously for 12 years. the patient survived with als and did not require permanent continuous ventilator. in addition, the symptoms of choking on liquids are improved, and the utility of 30 ml water swallow test was improved with grade 2. the symptoms of muscle fibrillations of limbs are also reduced. however, muscle strength worsened slowly. the repeated electromyography showed motor conduction amplitude reducing gradually and velocity not changing more when compared with the initial electromyography.our findings suggested that dhyz can be potentially used in als patients because of its multi - targeted neuroprotection and general safety, although als does not have a cure. in addition, we identified the area that is worthy of further study and dhyz as a promising candidate for further clinical application and als trials. rigorous randomized controlled trials are needed in the future. |
90408028 to ct zhang and 10747150 to f gao). funding for open access charge | the genome of sorangium cellulosum has recently been completely sequenced, and it is the largest bacterial genome sequenced so far. in their report, schneiker. (in complete genome sequence of the myxobacterium sorangium cellulosum, nat. biotechnol., 2007, 25, 12811289) concluded that in the absence of the gc - skew inversion typically seen at the replication origin of bacterial chromosomes, it was not possible to discern the location of oric. in addition, the complete genome of microcystis aeruginosa nies-843 has also been recently sequenced, and in this report, kaneko. (in complete genomic structure of the bloom - forming toxic cyanobacterium microcystis aeruginosa nies-843, dna res., 2007, 14, 247256) concluded that there was no characteristic pattern, according to gc skew analysis. therefore, oric locations of the above genomes remain unsolved. using ori - finder, a recently developed computer program, in both genomes, we have identified candidate oric regions that have almost all sequence hallmarks of bacterial orics, such as asymmetrical nucleotide distributions, being adjacent to the dnan gene, and containing dnaa boxes and repeat elements. |
physarum polycephalum is a true acellular slime mold that belongs to the species of order physarales, subclass myxo - gastromycetidae, class myxomycetes, division myxostelida. the life cycle of physarum polycephalum possesses a plasmodial phase where it exists as a single cell with a large number of diploid nuclei. the plasmodium is yellow colored and moves like a giant amoeba, deploying a network of protoplasmic tubes while searching for food, which typically consists of bacteria, spores and micro - particles. in the plasmodial stage, the protoplasm is differentiated into relatively stable ectoplasm and fluid endoplasm that streams through ectoplasmic channels and tubular strands. the direction and velocity of endoplasmic flow changes according to the pressure gradient formed via ectoplasm contractions coordinated along the body. this contraction is provided by myosin oligomers interacting with actin filaments attached to the membrane. the period of force autowaves and shuttle streaming of the endoplasm varies in the range of 15 min depending on the physiological state of the plasmodium. any fragment of a plasmodium restores the integrity of the surrounding membrane and resumes the contractile and locomotive activities, therefore, fragments of standard size and shape can be used in chemotactic assays. cytoplasm streams rhythmically back and forth through a network of tubular elements, circulating nutrients and chemical signals and forming pseudopods that allow the organism to navigate around and respond to its environment. the plasmodium propagates according to the position of nutrients but also in response to external / environmental gradients in light levels and humidity. it is also well established that physarum will propagate according to gradients in certain chemical species, either chemoattractants or chemorepellents. the physarum polycephalum plasmodium is a model system for studying non - muscular motility, and its chemotactic behavior has been well documented. in particular, substances causing negative taxis (repellents) were shown to increase the period of contractility and to decrease the area of spreading when present uniformly within the substrate. the first studies on chemoattractants and their effect on physarum can be traced back over 100 y. there has been constant cyclical interest since the inception of these studies although most studies have focused on nutrient type molecules, or chemicals such as cyclic nucleotides with a known direct biological effect. a good overview of work performed on physarum polycephalum, which predates the 1960s, can be found in carlile. experimental studies confirmed that the following substances acted as chemoattractants for the plasmodium, glucose, galactose, maltose and mannose, peptones, the amino acids phenylalanine, leucine, serine, asparagine, glycine, alanine, aspartate, glutamate ; and threonine, phosphates, pyrophosphates, atp and c amp and thorium nitrate. whereas, the following compounds have been found to act as chemorepellent molecules, sucrose and inorganic salts such as the chloride salts of (k, na, nh4, ca, mg, la) and tryptophan. therefore, it is clear that the nutritional value of the substance is not paramount in determining either chemoattractant or chemorepellent properties. although recently there has been renewed interest in the question of nutritional value and chemotaxis. for some substances, the effect on the plasmodium can be determined by the proximity of the organism to the source (or the concentration of the source), meaning that some substances can act as both chemoattractant and chemorepellent molecules. an example is the sugars galactose and mannose, which are reported to act as chemoattractants and chemorepellents that inhibit motion. physarum polycephalum plasmodium has also been used in chemotactic assays to assess the biocidal and repellent effects of silver nanoparticles vs. silver ions. recently it was found that the plasmodium is strongly attracted to herbal calming/ somnipherous tablets nytol and kalmssleep. to select the principle chemoattractant in the tablets, laboratory, experiments were undertaken on the plasmodium s binary choice between samples of dried herbs / roots : valeriana officinalis, humulus lupulus, passiflora incarnate, lactuca virosa, gentiana lutea and verbena officinalis. a hierarchy of chemo - attractive force was calculated from the binary interactions and it was found that valerian root was the strongest chemo - attractant for p. polycephalum of the substances tested. valerian contains hundreds of identified, and possibly the same amount of not - yet - identified components, including alkaloids, volatile oils, valerinol and actinidine. however, actinidine is known to have a chemo - attractive effect on a number of other animal species so remains a strong candidate. kincaid and mansour found that inhibitors of the enzyme cyclic 3,5-amp phosphodiesterase act as strong or moderate chemoattractants in p. polycephalum. among the substances tested the strongest effect was observed with 4-(-3-butoxy-4-methoxybenzyl)-2-imidazolidinone and moderate effects from theophylline and other xanthine derivatives (interestingly they observed negative chemotaxis at high concentrations). in a recent study, it was found that dithiothreitol inhibits the action of camp specific phosphodiesterase, which is responsible for the hydrolysis of camp. they found that the onset of the plasmodium spreading and the transition to the stage of migration were delayed in a concentration dependent manner. they concluded that the results suggest that the autocrine production of camp and extracellular camp specific phosphodiesterase is an important constituent of the mechanism controlling the motile behavior of the physarum polycephalum plasmodium. recently, the plasmodial phase of physarum polycephalum has been used extensively as a biological computing substrate. it has been used to solve a wide range of computationally hard problems such as maze - solving, the traveling salesman problem, calculation of optimal graphs, construction of logical gates and arithmetic circuits, sub - division of spatial configurations of data points and robot control. the plasmodium s behavior is determined by external stimuli and excitation waves traveling and interacting inside the plasmodium. the plasmodium can be considered as a reaction - diffusion or an excitable medium encapsulated in an elastic growing membrane. in adamatzky a physarum machine is a programmable amorphous biological computer experimentally implemented in the plasmodium of p. polycephalum. a physarum machine on a nutrient - rich substrate behaves as an auto - wave in an excitable medium. on a non - nutrient substrate a physarum machine can be programmed by configurations of repelling (salt) and attracting (food) gradients, and localized reflectors (illuminated obstacles). gradient fields generated by discrete configurations of attractants are an important prerequisite for successful programming of physarum machines. this paper sets out to investigate the chemoattractant or chemorepellent properties of a range of simple molecules that are mainly identified as secretions from plants and/or insects. by building a database of interactions between physarum and various chemical species it should be possible to exert fine control over the movement and morphology of the slime mold. as mentioned in addition a fundamental study of the interactions of physarum with various volatile organic compounds is invaluable in elucidating the underlying metabolic processes. table 1 shows the results for the 10 replicates of the binary combinations of the 19 vocs. the result is a quaternary string a - n - ftp - b. (a) indicates the number of replicates in which physarum propagated toward substance a, (b) indicates the number of replicates in which physarum propagated toward substance b, (n) indicates the number of neutral propagation events for that binary mixture of chemicals, whereas ftp indicates the number of replicates where physarum failed to propagate from the inoculation site. format : m[a, b ] = [a : n : ftp : b ], a = number of experiments (total 10) where physarum propagates toward a in preference to b ; n = number of experiments where physarum propagates equally toward a and b (they are not chemorepellents, but neither is a strong or dominant chemoattractant), or propagates between a and b (i.e., both a and b have a chemorepellent effect). ftp = number of experiments where physarum fails to propagate from the initiation site (this is indicative that one or both of the compounds has a strong inhibitory effect on physarum, by studying the full extent of the binary interactions it becomes apparent as to the individual effect of each voc) b = number of experiments where physarum propagates toward b in preference to a ; 1 = -myrcene 2 = benzaldehyde 3 = tridecane 4 = benzylacetate 5 = eugenol 6 = benzylalcohol ; 7 = geraniol 8 = m - cresol 9 = linalool 10 = methylbenzoate 11 = cis-3-hexenyl acetate 12 = p - cymene;13 = 2-phenylethanol 14 = methyl - p - benzoquinone 15 = -farnesene 16 = -pinene 17 = limonene 18 = 3-octanone 19 = nonanal the most common result for a binary mixture of chemicals is for physarum to fail to propagate from the inoculation site in all 10 replicates (cells marked blue in the table). this indicates that many of the chemicals have an inhibitory effect on the physarum either individually or in combination. compounds that have a predominantly inhibitory effect can be deduced from the binary combinations within the table. these include in order of the greatest inhibitory effect [based on the number of experiments where all 10 replicates gave no propagation (the total number of experiments for each compound was 18) ] : nonanal (18) > benzaldehyde (17) > methylbenzoate (11) > cis-3-hexenyl acetate (10) > linalool (9) methyl - p - benzoquinone (8) > p - cymene (7) > eugenol (5) = benzyl alcohol (5) > geraniol (4) > m - cresol (3) = 2-phenylethanol (3) = benzyl acetate (3) > therefore, compounds that show no individual or cumulative inhibitory effect include farnesene, limonene, -pinene, -myrcene and 3-octanone. there were some occasions where physarum propagated toward either compound a or compound b in all 10 replicates (cells marked red in the table). the compounds observed to have a strong chemoattractive effect were -myrcene (vs. benzylacetate), tridecane (vs. benzyl alcohol, 2-phenylethanol, methyl - p - benzoquinone), p - cymene (vs. benzyl acetate), farnesene (vs. geraniol and 2-phenylethanol) and limonene (vs. geraniol and methylbenzoate). there are an additional set of experiments where at least 5 of the 10 replicates indicated a positive chemotactic response (cells marked green in the table). the compounds with the highest number of these events were as follows : farnesene (9) > -myrcene (8) > tridecane (6) > p - cymene = limonene (5) > 3-octanone (3) > -pinene (1) there were a number of experiments (14/171) where all 10 replicates resulted in a neutral outcome, indicating that neither chemical had a strong inhibitory effect or a strong chemoattractive effect (cells marked yellow in the table). in addition, there were a number of experiments (40/171) where the overall result was neutral. this is where most of the 10 replicates gave either a neutral outcome or failed to propagate, and only a limited number of replicates indicated a positive chemotactic response. we can also rank the compounds in terms of their total number of positive chemotactic events when looking at all replicates in all experiments. this gives the following ranking : (the number in brackets indicates the number of positive events out of a total of 180) farnesene (101) > -myrcene (77) > tridecane (74) > limonene (58) > p - cymene (42) > 3-octanone (41) > -pinene (30) > m - cresol (3) > benzyl acetate (2) > cis-3-hexenyl acetate (1). from these results, and the ones above, it is obvious that farnesene has the strongest chemoattractive effect on physarum polycephalum, followed by myrcene and tridecane, which have a relatively strong effect and then limonene / p - cymene/3-octanone and -pinene that have a moderate effect. if we rank the remaining compounds in terms of their total number of inhibitory events then we get the following order nonanal (180) > benzaldehyde (170) > methyl benzoate (130) > linalool (126) > methyl - p - benzoquinone (113) > eugenol (88) > benzyl alcohol (72) > geraniol (69) > 2-phenylethanol (67). this shows that both aldehydes studied have the greatest inhibitory effect with physarum failing to propagate in all replicates vs. all other compounds regardless of their chemoattractive properties. the list of inhibitory substances is also populated by all the alcohols utilized in the study, although the effect is much less marked than that observed for aldehydes. figure 1a shows selected results from the chemotactic assay between farnesene (right side of the petri dish) and geraniol (left side of the petri dish) 24 h after initiation. in all cases the physarum takes a direct horizontal path from the source of initiation toward the farnesene. the growth rates within each dish are different, but eventually all reactions proceed to the same point whereby the physarum encircles the source of farnesene. in some cases (as shown in the dish on the middle row, left side), the physarum colonizes the filter paper substrate that had been soaked in farnesene. this seems to show that there is no inhibitory effect even at close proximity to the source. figure 1b shows the results of the chemotactic assay between farnesene and geraniol after 48 h. what is interesting is that physarum remains in the localized area surrounding the source of farnesene, despite there being no additional nutrient source. often physarum may move initially toward a source, but then subsequently move away in search of nutrients. (a) showing selected results from the chemotactic assay between farnesene (right side of the petri dish) and geraniol (left side of petri dish) 24 h after initiation. (b) the results of the chemotactic assay between farnesene and geraniol after 48 h. figure 2 shows selected results of the chemotactic assay between farnesene (right side) and pinene (left side) after 48 h. again, it is clear that there is a positive chemotactic response to farnesene. however, in contrast to figure 1a (where geraniol was the other source), the growth is not as localized and does not take a direct horizontal path toward farnesene. instead, where pinene is the alternative source this probably indicates that pinene does not have a direct inhibitory effect on the growth of physarum. however, as seen with the case of geraniol, there is very limited growth on the entire side of the petri dish not containing farnesene. figure 2. selected results of the chemotactic assay between farnesene (right side) and pinene (left side) after 48 h. figure 3 shows selected results from the chemotactic assay using limonene (right side) and 2-phenyl ethanol (left side). in this case, in fact, the majority of growth is on a vertical line from the source of initiation. therefore, at least one of the compounds appears to have an inhibitory effect on the physarum, and neither has a strong chemoattractive action. selected results from the chemotactic assay using limonene (right side) and 2-phenyl ethanol (left side). figure 4 shows selected results from the chemotactic assay between limonene (right side) and 3-octanone (left side). the growth of the physarum is relatively unhindered and it is able to grow out from the point of initiation in all directions. in terms of an overall result, there is no chemotaxis toward one source in preference to another. therefore, it can be concluded that neither of the chemical substances has a strong inhibitory effect on physarum, and both appear to have a moderate chemoattractive action. selected results from the chemotactic assay between limonene (right side) and 3-octanone (left side). figure 5 shows selected results from the chemotactic assay between pinene (right side) and 3-octanone (left side). again, it is obvious that neither compound has a strong inhibitory affect on the physarum. the growth is not directly toward the source of either chemical. in some cases, the growth is in all directions, as in the example on the right side, middle row. in other cases, it appears to grow at a 45-degree angle to the source and then follow an almost circular trajectory, eventually reaching the source (this occurs for both chemicals ; see for example top left side, moving toward pinene, whereas top left side and middle row, left side, moving toward 3-octanone). actually, the movement toward 3-octanone is interesting because it takes the form of a discrete wave fragment. figure 5. showing selected results from the chemotactic assay between pinene (right side) and 3-octanone (left side). figure 6 shows selected results from the chemotactic assay between 3-octanone (right side) and benzyl acetate (left side). there is an obvious chemotaxis toward 3-octanone in preference to benzyl acetate. what is interesting is that the growth of the physarum is limited to compact fragments and many of these take different trajectories from the source and move in the general direction of 3-octanone. there is very little growth in the half of the petri dish occupied by the source of benzyl acetate. therefore, it seems likely that the growth of the physarum is altered by the long range inhibitory effect of benzyl acetate in its environment. this shows how the morphology and growth rate of physarum can be altered by its chemical environment. this wave fragment like morphology is qualitatively similar to fragments of excitation which can exist in the sub - excitable bz reaction. previously, these fragments of excitation have been utilized in collision - based computing schemes to implement ballistic gates. in this case, this allowed fine control over the trajectory and velocity vectors of the wave fragments, and for the classification of binary collisions to be undertaken. indeed, the interaction of localized waves of physarum polycephalum have been previously used for implementing arithmetic circuits. however, in this case, the physarum is confined to certain channels in order to implement computation. it is plausible that finely balanced inhibition coupled with positive chemotaxis could be used to exert fine control over the evolution and movement of these fragments enabling collision - based computing schemes to be explored. this would be in an architectureless domain, albeit it pervaded by chemical fields. it is also possible that this morphology would be useful for material transport and directed synthesis of functional materials. figure 6. selected results from the chemotactic assay between 3-octanone (right side) and benzyl acetate (left side). in the controls where a single substance was present at both sources, the predominant behavior was neutral. where the chemical was a strong attractant (farnesene, myrcene) then physarum propagated toward both sources although not necessarily to both in one replicate. where the chemical was a strong inhibitor physarum did not exhibit any chemotactic response toward distilled water. where distilled water alone was used as the two sources the propagation was predominantly neutral. the above analysis shows definite trends in terms of the types of compounds that possess inhibitory and chemoattractive responses to physarum polycephalum. in general terms oxygen functionality seems to exhibit an inhibitory affect on the propagation all the alcohols seem to populate the inhibitory group although with a lesser inhibitory effect observed than with aldehydes. most of the aromatic compounds tested also populate the inhibitory group. in terms of chemoattractive properties all the terpene derivatives without oxygen functionality populate this group with the only sesquiterpene farnesene exhibiting the highest overall effect. the fatty acid derivative tridecane also has a high chemoattractive effect while 3-octanone is unique in being the only oxygenated compound to show even a moderate chemoattractive affect. all the terpene molecules exerting a chemoattractive response share certain structural features such as the lack of oxygen functionality, unsaturation etc. however, there are distinct differences, farnesene and -myrcene are straight chain, acyclic terpenes, limonene is cyclic and pinene is bicyclic, whereas p - cymene is classed as a terpene like compound but is aromatic. these molecules do not obviously show any similarity to previously identified compounds exhibiting a strong chemoattractive response to physarum. a recent paper has described a key role of camp and extracellular camp phosphodiesterase in the motile behavior of p. polycephalum. limonene and other terpenes have been found to bind to a(2a) adenosine receptors in humans., limonene increased cytosolic camp concentration and calcium concentration, which can be achieved by the activation of adenosine a(2a) receptors. therefore, it is possible that these terpenes act directly to increase motility in physarum via enzyme inhibition. it is well documented that the hydrophobicity of certain terpene molecules contributes to their effectiveness as enzyme inhibitors. many hydrophobic compounds are associated with protein or enzyme deactivation, where acetylcholinesterase is particularly sensitive. compounds that inhibit or inactivate acetylcholinesterase cause acetylcholine to accumulate at synapses of cholinergic sites. specific acetylcholinesterase has previously been identified in the plasmodial stage of physarum suggesting that it has a role as a local mediator of motor function. 3-octanone is one of the compounds that contributes to a characteristic fungal odor, which physarum possesses, although the most obvious odor emanating from physarum is that of geosmin. gc - ms analysis was recently undertaken of the headspace above living physarum and identified geosmin as one of the major volatile components. geosmin has a very low odor threshold in humans and has a characteristic earthy flavor. in fact, many organisms use this pre - cursor in the production of terpenes, terpenoids and sterols. a number of terpene based compounds were observed in the headspace above physarum including farnesene like compounds (as there are very many terpene like molecules with similar mass and fragmentation patterns it is necessary to run standards in order to gain a positive identification) 3-octanone and other we intend to use the compounds identified in the gc - ms study to augment these experiments in terms of assessing their chemoattractive or chemorepellent effect. this study has etablished a fairly robust preference list ranked in terms of attraction and repulsion and therefore, it should be relatively facile to establish the position of new compounds such as those identified as secretions of physarum via gcms within the exisiting order. in the future experiments will be performed to ascertain any physiological effects on the slime mold by making electrical measurements of physarum with and without various chemoattractive and inhibitory compounds in the environment. using this methodology we can also control the concentration of the vocs and gauge any dose response effects or whether certain compounds exhibit both chemoattractive and inhibitory responses at different concentration levels. in addition to a fundamental study of the volatile secretions of physarum and the effect of relatively simple vocs on the propagation of the plasmodium, the aim is to use the information gained through this study and extended studies in order to exert fine control over the propagation direction / speed and the morphology of the slime mold. this work identified a number of previously unknown compounds that exert chemotactic response from the plasmodial stage of p. polycephalum. a number of relatively simple vocs were found to have either a chemoattractive or chemorepellent action when tested in binary chemotactic assays with physarum polycephalum. a number of other terpenes or terpene like molecules without oxygen functionality (-myrcene, limonene, -pinene and p - cymene) were found to exert a moderate chemoattractive response. whereas terpene compounds with oxygen functionality such as geraniol and linalool exhibited a moderate inhibitory effect. aldehydes were found to exhibit the greatest inhibitory effect on the growth / propagation of the plasmodium. a role for c - amp and c - amp phosphodiesterase enzymes in the motile behavior of p. polycephalum has been established. we can postulate based on associated evidence that the terpene like molecules (especially those with high hydrophobicity) have an inhibitory effect on enzymes linked to the motile response of physarum polycephalum thus causing a direct chemotactic effect. this work also highlighted the potential for altering the morphology of the plasmodium in response to chemical stimuli. in the presence of moderate inhibitory compounds for example it was possible to reduce the typical extended morphology of the plasmodium to compact wave fragments. it is hoped that a better understanding of the chemotaxis response of physarum polycephalum to simple vocs could provide a basis for fine control of the propagation direction, morphology and growth rate. this would be invaluable in giving a programmable substrate for applications in computation, amorphous robotics and material synthesis. from a fundamental perspective it is also invaluable to understand the semiochemicals of slime molds and their complex relationships with other organisms in their environment such as plant and insect species. the chemotaxis response of primitive eukaryotic cells is also useful as a model for pathogenesis. a recent paper by schiestl detailed the evolution of floral scent and insect chemical communication. this involved an analysis of the occurrence, commonness and evolutionary patterns of 71 floral vocs in 96 plant families and 87 insect families. the compounds were selected from each of the four chemical classes studied in the paper namely aromatic, monoterpenes, sesquiterpenes and fatty acid derivatives (fads). we aimed to get a range of different functionalities within these generic groups and also select compounds which had been identified across a high number of plant and insect families. the compounds selected were as follows : 6 aromatics (benzaldehyde, benzyl alcohol, methyl benzoate, benzylacetate, 2-phenyl ethanol and eugenol), 6 monoterpenes (limonene, -myrcene, geraniol, linalool, p - cymene and -pinene), a sesquiterpene (-farnesene) and 3 fads [(z)-3-hexenyl acetate, nonanal and tridecane ]. in addition to these 16 compounds, we also selected 3-octanone a known fungal metabolite and m - cresol and methyl - p - benzoquinone, which are known secretions of the fungus beetle bolitotherus cornutus and a range of other insect species, the true slime mold, the plasmodium of physarum polycephalum (strain hu554 hu560), was cultured with oat flakes on a 1% agar gel at 25c in the dark. to obtain large quantities of inoculated oat flakes for chemotactic assays the plasmodial phase of p. polycephalum was cultivated in large plastic containers on kitchen towels that have been wetted with 5 mls of de - ionized water. was added in the form of 50 g of rolled oats per container (organic rolled oats). these containers were covered in order to retain moisture and kept in the dark at 25c until required. sub - cultures were taken every 23 d to establish consistent cultures for ongoing experiments. sub - culturing simply involved the removal of colonized oat flakes from the main culture and addition to a separate container containing fresh rolled oats on damp kitchen paper. we use a scheme similar to that adopted by other researchers undertaking simple chemotactic assays. experiments were performed in 9 cm diameter polystyrene petri dishes. a 1% solution of agar (select agar) was added to each petri dish to give a depth of approximately 2 mm. 0.5 cm) were cut and placed on the gel surface at the furthest points from the center on a straight line. an oat flake colonized by physarum polycephalum was placed at the center of the petri dish, on a straight line connecting the two filter paper substrates and at the same distance from each substrate. the plasmodium was left on the petri dish in the dark for two hours prior to the addition of the chemicals. after two hours, the dishes were removed to a fume cupboard and 50 l of the selected chemicals were added to the filter paper substrates (in the case of methyl - p - benzoquinone, 50 mg of solid was added). all dishes were scanned in batches of six were taken using a flatbed scanner (hp scanjet 5590) attached to a pc. the assessment of the behavior in each dish was according to four classifications. if the plasmodium propagated from the inoculation site directly (approximately horizontally) to a certain chemical source, then this was counted as a positive chemotactic event (it could obviously equally indicate a strong negative chemotactic effect away from the other source). the results for each chemical source were added for each of the 10 petri dishes in order to establish whether a statistically significant trend existed for one source vs. the other. therefore, a series of preferences could be established by testing all the possible binary combinations of the 19 reactants. if physarum propagated horizontally but equally toward both sources, or simply propagated in a circular or random direction then this was classified as a balanced chemotactic response. in this case the physarum had no clear preference for either chemical source but the growth was not significantly inhibited. again, the number of this type of event from the 10 replicates of the chemotactic assay were summed to give an overall indication of the typical behavior. if physarum failed to propagate from the site of inoculation, then this was highly indicative of a direct inhibitory effect of one or both chemical sources. the number of these events was noted and summed over the replicates for that binary assay. a number of control experiments were undertaken including selected trials run with distilled water replacing one or both chemicals. we also ran a limited subset of experiments where the same compound was present at both sources on the petri dish. a recent paper by schiestl detailed the evolution of floral scent and insect chemical communication. this involved an analysis of the occurrence, commonness and evolutionary patterns of 71 floral vocs in 96 plant families and 87 insect families. the compounds were selected from each of the four chemical classes studied in the paper namely aromatic, monoterpenes, sesquiterpenes and fatty acid derivatives (fads). we aimed to get a range of different functionalities within these generic groups and also select compounds which had been identified across a high number of plant and insect families. the compounds selected were as follows : 6 aromatics (benzaldehyde, benzyl alcohol, methyl benzoate, benzylacetate, 2-phenyl ethanol and eugenol), 6 monoterpenes (limonene, -myrcene, geraniol, linalool, p - cymene and -pinene), a sesquiterpene (-farnesene) and 3 fads [(z)-3-hexenyl acetate, nonanal and tridecane ]. in addition to these 16 compounds, we also selected 3-octanone a known fungal metabolite and m - cresol and methyl - p - benzoquinone, which are known secretions of the fungus beetle bolitotherus cornutus and a range of other insect species, the true slime mold, the plasmodium of physarum polycephalum (strain hu554 hu560), was cultured with oat flakes on a 1% agar gel at 25c in the dark. to obtain large quantities of inoculated oat flakes for chemotactic assays the plasmodial phase of p. polycephalum was cultivated in large plastic containers on kitchen towels that have been wetted with 5 mls of de - ionized water. was added in the form of 50 g of rolled oats per container (organic rolled oats). these containers were covered in order to retain moisture and kept in the dark at 25c until required. sub - cultures were taken every 23 d to establish consistent cultures for ongoing experiments. sub - culturing simply involved the removal of colonized oat flakes from the main culture and addition to a separate container containing fresh rolled oats on damp kitchen paper. we use a scheme similar to that adopted by other researchers undertaking simple chemotactic assays. experiments were performed in 9 cm diameter polystyrene petri dishes. a 1% solution of agar (select agar) 0.5 cm) were cut and placed on the gel surface at the furthest points from the center on a straight line. an oat flake colonized by physarum polycephalum was placed at the center of the petri dish, on a straight line connecting the two filter paper substrates and at the same distance from each substrate. the plasmodium was left on the petri dish in the dark for two hours prior to the addition of the chemicals. after two hours, the dishes were removed to a fume cupboard and 50 l of the selected chemicals were added to the filter paper substrates (in the case of methyl - p - benzoquinone, 50 mg of solid was added). all dishes were scanned in batches of six were taken using a flatbed scanner (hp scanjet 5590) attached to a pc. if the plasmodium propagated from the inoculation site directly (approximately horizontally) to a certain chemical source, then this was counted as a positive chemotactic event (it could obviously equally indicate a strong negative chemotactic effect away from the other source). the results for each chemical source were added for each of the 10 petri dishes in order to establish whether a statistically significant trend existed for one source vs. the other. therefore, a series of preferences could be established by testing all the possible binary combinations of the 19 reactants. if physarum propagated horizontally but equally toward both sources, or simply propagated in a circular or random direction then this was classified as a balanced chemotactic response. in this case the physarum had no clear preference for either chemical source but the growth was not significantly inhibited. again, the number of this type of event from the 10 replicates of the chemotactic assay were summed to give an overall indication of the typical behavior. if physarum failed to propagate from the site of inoculation, then this was highly indicative of a direct inhibitory effect of one or both chemical sources. the number of these events was noted and summed over the replicates for that binary assay. a number of control experiments were undertaken including selected trials run with distilled water replacing one or both chemicals. we also ran a limited subset of experiments where the same compound was present at both sources on the petri dish. | the chemotaxis behavior of the plasmodial stage of the true slime mold physarum polycephalum was assessed when given a binary choice between two volatile organic chemicals (vocs) placed in its environment. all possible binary combinations were tested between 19 separate vocs selected due to their prevalence and biological activity in common plant and insect species. the slime mold exhibited positive chemotaxis toward a number of vocs with the following order of preference : farnesene > -myrcene > tridecane > limonene > p - cymene > 3-octanone > -pinene > m - cresol > benzylacetate > cis-3-hexenylacetate. for the remaining compounds, no positive chemotaxis was observed in any of the experiments, and for most compounds there was an inhibitory effect on the growth of the slime mold. by assessing this lack of growth or failure to propagate, it was possible to produce a list of compounds ranked in terms of their inhibitory effect : nonanal > benzaldehyde > methylbenzoate > linalool > methyl - p - benzoquinone > eugenol > benzyl alcohol > geraniol > 2-phenylethanol. this analysis shows a distinct preference of the slime mold for non - oxygenated terpene and terpene - like compounds (farnesene, -myrcene, limonene, p - cymene and -pinene). in contrast, terpene - based alcohols such as geraniol and linalool were found to have a strong inhibitory effect on the slime mold. both the aldehydes utilized in this study had the strongest inhibitory effect on the slime mold of all the 19 vocs tested. interestingly, 3-octanone, which has a strong association with a fungal odor, was the only compound with an oxygenated functionality where physarum polycephalum exhibits distinct positive chemotaxis. |
the first suspected case of an hpai outbreak in japan during winter 201617 was reported from akita prefecture in northern japan (figure 1). a black swan (cygnus atratus) in a zoo that died on november 15, 2016, tested positive for influenza virus antigen by a rapid diagnostic test. while this bird s specimens underwent further analysis, another influenza virus was isolated from a water sample collected at an overwintering site of migratory birds in kagoshima prefecture at the southern tip of japan on november 14, 2016 (table 1). this isolate, a / environment / kagoshima / ku - ngr - i/2016 (h5n6), was confirmed to be an h5n6 subtype having multiple basic amino acid residues, plrerrrkr / glf, at the cleavage site in the ha protein, which is characteristic of hpaivs, by conventional reverse transcription pcr and sanger sequencing. subsequently, an isolate from the first black swan, a / black swan / akita/1/2016 (h5n6), also was confirmed to be an h5n6 hpaiv, showing that all 3 chickens inoculated intranasally with 10 of 50% egg infectious dose of the virus died within 2 days. in addition, a fecal sample of a common teal (anas crecca) collected at an overwintering site of migratory birds in tottori prefecture in the middle of japan on november 15, 2016, was reported to harbor an h5n6 hpaiv, a / teal / tottori/1/2016 (h5n6) (table 1). the isolation sites of these 3 h5n6 hpaivs are distant (figure 1), although the sample collection dates were close (table 1). these 3 cases were followed by several reports of h5n6 hpaivs, including a / black swan / akita/2/2016 (h5n6), a / northern pintail / tottori / b37/2016 (h5n6), and a / crane / kagoshima / ku-4/2016 (h5n6), in japan (table 1). as of december 4, a total of 31 confirmed cases in wild birds had been reported to the ministry of environment (http://www.env.go.jp/nature/dobutsu/bird_flu/index.html), and 4 cases at poultry farms were confirmed in japan (8). locations of confirmed highly pathogenic avian influenza virus a(h5n6) infections in akita, tottori, and kagoshima prefectures, japan, 2016. to clarify the genetic background of the h5n6 hpaivs concurrently introduced into several distant regions of japan, we determined the complete genome sequences of 5 of our isolates : a / black swan / akita/1/2016 (h5n6) (genbank / ddbj / embl accession nos. lc198525lc198532), a / teal / tottori/1/2016 (h5n6) (genbank / ddbj / embl accession nos. lc199865lc199872), a / northern pintail / tottori / b37/2016 (h5n6) (genbank / ddbj / embl accession nos. lc200414-lc200421), a / environment / kagoshima / ku - ngr - i/2016 (h5n6) (gisaid epiflu [http://platform.gisaid.org/ ], genbank / ddbj / embl accession nos. epi861582epi861589), and a / crane / kagoshima / ku-4/2016 (h5n6) (genbank / ddbj / embl accession nos. these 5 isolates were almost genetically identical. even among the ha genes, which are the most frequently mutated ones among the 8 gene segments, only 38 nt mutations, including 3 nonsynonymous mutations, were detected compared with the earliest strain, a / northern pintail / tottori / b37/2016 (h5n6) (technical appendix table). the phylogenetic tree analysis of the ha gene revealed that our isolates are classified into the genetic clade 2.3.4.4c and clustered with the recent h5n6 hpaiv isolates from wild and domestic birds and humans in china, in addition to an isolate south korea, a / mandarin duck / korea / k16 - 187 - 3/2016 (h5n6) (figure 2, panel a), on the basis of a recent classification in clade 2.3.4.4 (9,10). the na genes of our isolates also form a single cluster together with the h5n6 hpaiv isolates from china into group c in the phylogenetic tree (figure 2, panel b). in addition, the remaining 6 genes were genetically close to the recent h5n6 hpaiv isolates from china in the corresponding phylogenetic trees (technical appendix figure 1), except for the polymerase basic 1 genes, which are most closely related to the counterpart of a / duck / guangdong / s4040/2011 (h4n2) that was isolated from a domestic duck at a live bird market in china (11). thus, the h5n6 hpaiv isolates would be derived from a reassortant that arose between an h5n6 hpaiv recently circulating in wild birds, poultry, or both in east asia and in low pathogenicity avian influenza virus circulating in poultry in china. the genetic background of the h5n6 hpaiv isolates in this study is similar to the recent south korea h5n6 virus collected in october 2016 and clearly different from that of recent h5nx hpaivs in russia (10), western european countries, and alaska (8). phylogenetic trees of the ha and na gene segments of highly pathogenic avian influenza virus a(h5n6) isolated in japan. the nucleotide sequences of the h5 ha (a) and n6 na (b) genes were analyzed by the maximum - likelihood method along with the corresponding genes of reference strains using mega 7.0 software (http://www.megasoftware.net/). horizontal distances are proportional to the minimum number of nucleotide differences required to join nodes and sequences. numbers at the nodes indicate the probability of confidence levels in a bootstrap analysis with 1,000 replications. the h5 ha gene sequences are classified into genetic clades as defined by lee. our putative amino acid sequence comparison revealed that a leucine residue at position 134 in the ha protein (h3 numbering) was deleted, unlike that with the closest relative a / feline / guangdong/1/2015 (h5n6) (technical appendix figure 2). our isolates have the amino acid sequence qqg at positions 226228, which are located at the receptor - binding site in the ha protein, although the corresponding amino acid sequences of the previous h5 viruses are qsg or qrg (technical appendix figure 2). these findings suggest that the receptor specificity of our h5n6 hpaiv isolates might be altered from their parental viruses (12,13). we also found 11 aa deletions in the stalk region of the na protein, unlike that of a / duck / vietnam / hu11151/2014 (h5n6), a representative virus strain of an n6 na gene - based group d (technical appendix figure 3), which belongs to a different cluster of the clade 2.3.4.4. for ha antigenic characterization, we investigated the reactivity of chicken antiserum raised against several h5 isolates to our h5n6 hpaiv isolates using the hemagglutination inhibition test (14). we selected 1 reference virus strain, a / black swan / akita/1/2016 (h5n6), and prepared single immunized chicken antiserum against the virus because of the limited variation of the nucleotide sequences in the ha genes among our 6 h5n6 hpaiv isolates. antibody titer of antiserum of a / black swan / akita/1/2016 (h5n6) were 1632-fold higher against homologous virus than against the other strains (table 2). the reactivity of the antiserum of a / chicken / kumamoto/1 - 7/2014 (h5n8), whose ha gene belongs to the genetic clade 2.3.4.4, to a / black swan / akita/1/2016 (h5n6) was 4-fold lower than that of the antiserum to the homologous combination. moreover, none of the antiserum samples tested reacted strongly with a / black swan / akita/1/2016 (h5n6) except for the homologous antiserum. these results indicate that the ha antigenicity of the h5n6 hpaivs recently introduced in japan differ appreciably from those of the previous h5nx viruses. b. swan, black swan ; ck, chicken ; hk, hong kong ; hok, hokkaido ; mal, mallard ; pf, peregrine falcon ; ws, whooper swan. low pathogenic avian influenza viruses isolated from mallard (15) and chicken in japan. we isolated 6 h5n6 hpaivs from dead birds, fecal samples of migratory birds, and environmental water sample in 3 distant regions of japan in november 2016. a genetic analysis showed that these isolates were genetically closely related to h5n6 hpaivs recently isolated in china except for the polymerase basic 1 gene segment. the ha antigenicity of our h5n6 hpaivs was demonstrated to have drifted further than viruses belonging to the same genetic clade 2.3.4.4. to prevent the spread of hpaivs by wild birds, prompt elimination of hpaivs is urgently needed in countries in asia. nucleotide and amino acid mutations in hemagglutinin (ha) genes of highly pathogenic avian influenza virus a(h5n6) isolates ; phylogenetic trees of gene segments of isolates fromin japan and reference strains ; comparison of amino acid position 134 and 227 (h3 numbering) in h5 ha ; comparison of amino acid sequence of neuraminidase stalk. | highly pathogenic avian influenza viruses (hpaivs) a(h5n6) were concurrently introduced into several distant regions of japan in november 2016. these viruses were classified into the genetic clade 2.3.4.4c and were genetically closely related to h5n6 hpaivs recently isolated in south korea and china. in addition, these hpaivs showed further antigenic drift. |
this technique requires tagging of cells with a fluorophore, which is a molecule that will fluoresce when light at appropriate wavelengths is incident on it. fluorophores should have good photostability and high quantum yield, and when used to image live cells they should be noncytotoxic and stable in vivo [2, 3 ]. they should also have low susceptibility to photobleaching without losing their sensitivity to image the cells. in order to overcome photon attenuation in living cells, fluorophores with long emission at the near - infrared (nir) region the most commonly used fluorophore to image bacterial cell is fluorescein, but owing to a variety of factors it is not a good choice. many other fluorophores are also in current use, and new fluorophores with high quantum yield and low cytotoxicity are also being continuously screened for imaging cells. porphyrin and its derivatives are nontoxic organic compounds whose fluorescent properties have been widely studied. we have investigated the use of tetra ammonium tetrakis (4-sulphonato)phenyl porphyrin (tpps) (figure 1), a water - soluble anionic compound, with multiple excitation bands and wide excitation range, as a fluorescent dye for imaging bacterial cells. in earlier studies, tpps has been used as a photosensitising agent for photo - dynamic therapy. it was found to be moderately effective against gram - positive bacteria but had no cytotoxic effect on gram - negative bacteria, even after prolonged exposure to light at high frequency. studies with tetra - cationic phthalocyanine as photosensitizer have also shown that the entry of photosensitizer into the cytoplasm increases brightness of cells, which was observed in images obtained from fluorescence microscope. the uptake of photosensitizer was further found to be an important step in affecting cells ' survival rate. these indicate that uptake of tpps into the cytoplasm by gram - positive bacteria is higher than that by gram - negative bacteria. we imaged gram - negative bacteria, pseudomonas aeruginosa, and gram - positive bacteria, bacillus cereus, on a fluorescent microscope using tpps as fluorophore and then analysed the brightness data obtained from those images. tetraphenyl porphyrin was synthesized using the protocol described by gonsalves. followed by sulphonation using chlorosulphonic acid as described by song.. purity of the compounds synthesised is verified using thin - layer chromatography, nmr, uv visible spectroscopy, and mass spectrometry. eu732606) and bacillus cereus (gram - positive bacteria) used were isolated and cultured in our laboratory. cultures of pseudomonas aeruginosa were grown overnight and inoculated in luria - bertani medium, incubated for 14 hours. 250 l of this culture was mixed with various concentrations of tpps and incubated in dark for 5 minutes. the cells were then centrifuged for 5 minutes at 5000 rpm. the cell pellet was washed and then solubilised in pbs buffer before being imaged on an olympus x171 total internal reflection fluorescence microscope in fluorescence mode. the sample was excited using light from a mercury - vapour lamp, which was passed through an olympus u - mwiba3 filter (excitation filter 460 nm495 nm and emission filter 510 nm550 nm). after that, a similar procedure as used for pseudomonas aeruginosa was followed and images were obtained. images collected from the andor ixon emccd camera were processed using andor - iq software version 1.8. all images were taken at an exposure time of 117.5 ms and real em gain of 44. another image processing software was used to measure the brightness of cells in different parts of the image. brightness was (1)v=0.299r+0.587g+0.114b, where r, g, and b are the red, green, and blue pixels, respectively. a plot was then constructed to analyse the variation of brightness with changes in concentration of tpps. the cells with tpps fluoresced, when visualised under fluorescent microscope (figure 2) with excitation at 488 nm wavelength, while control cells to which tpps was not added did not show any fluorescence. cell viability, for the pseudomonas aeruginosa cells, as indicated by their motility, was not affected by the uptake of tpps (supplementary material available online at doi:10.1155/2010/697528). image analysis revealed that the brightness increases with increasing concentrations of tpps for both pseudomonas aeruginosa and bacillus cereus before achieving saturation. the concentration at which the brightness achieves saturation is about the same for both of the bacteria (0.150.25 mg / ml), but the brightness is consistently higher for bacillus cereus (figure 3). this indicates that the uptake of tpps by the gram - positive bacteria is much higher than that of the gram - negative bacteria. high quantum yield, high solubility in water, and its nontoxic nature are properties that confer considerable advantages to tpps over other dyes in current use. the technique developed has the added advantage in that it requires a very short preparatory time. a variation in brightness was observed for the two different bacteria at the same concentrations of tpps. in conclusion, tpps can be used as a stain for fluorescent imaging and analysis of various microorganisms and could be used for identifying pseudomonas aeruginosa and bacillus cereus. | the use of tetraammonium tetrakis(4-sulphonato)phenyl porphyrin (tpps), a water - soluble anionic compound, as a stain to analyse bacterial cells using fluorescent microscopy was investigated. tpps was effectively used to analyse two different bacteria : pseudomonas aeruginosa and bacillus cereus. the variation in brightness with varying concentrations of tpps was studied. the patterns of variations for these bacteria were found to be the same, but with consistently higher brightness for bacillus cereus. |
osteoid osteoma (oo) is a benign tumor characterized by a small calcified lesion, usually less than 2 cm in diameter, with nocturnal pain alleviated by the administration of nonsteroidal anti - inflammatory drugs (nsaids). oo usually affects the long bones and mostly occurs in the first 2 decades of life. oo arising in the rib is extremely rare, and only 12 cases have been reported in detail to date. treatment for this tumor is resection of the nidus ; however, difficulty in reaching the nidus through normal tissues increases the invasiveness of the surgical procedure. in the reported cases of rib oos with surgical documentation we present the first case of oo arising in the rib treated with a computed tomography (ct)-guided procedure without en bloc resection of the affected rib. a 20-year - old male complained of nocturnal pain in his left anterior chest which had persisted for 2 years. conservative treatment was performed for a period of 22 months with administration of nsaids at a nearby hospital. therefore, he was referred to our hospital for further treatment. at the initial visit to our hospital a plain radiograph showed bulging and mild bone sclerosis of the left 8th rib (fig. ct examinations revealed a well - demarcated 5-mm nidus with peripheral bone sclerosis in this area (fig. clinical and radiological findings were compatible with an oo of the left 8th rib. because the patient refused to undergo en bloc rib resection, the operative plan was to perform resection of the nidus under ct guidance. the operative procedure was performed in the ct room under total anesthesia. after identifying the nidus with a marker, a guide pin was inserted by a power drill just adjacent to the edge of the nidus. a 5.0-mm cannulated drill was inserted over the guide pin to remove the nidus. after removing the lesion, the bone specimen which resided in the cannulated drill subsequently, heat ablation by an electrosurgical knife using a standard electrosurgical generator was performed to completely destroy any residual tumors. histopathological studies showed the characteristic appearance of an oo with differentiated osteoblasts lining the osteoid and interconnected trabeculae of woven bone (fig. complete pain relief associated with the nidus resection was achieved from the first postoperative day. the patient was free of pain at the final follow - up after 4 years, and no local recurrence was observed. oos may affect any bone, but more than half of the tumors occur in the long bones of the lower extremities. the frequency of oos affecting the ribs is extremely low (11.4%), with only 12 reported cases with surgical intervention to date [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 ]. conservative treatment consists of nsaid administration for a prolonged period of time, which reportedly leads to alleviation of pain in select cases. the common principle for operative treatment of oo is thorough resection of the nidus, and incomplete resection could lead to local recurrence. a complete resection of the nidus by open surgery may lead to further damage of bone and soft tissues around the lesion, when the affected bone is not subcutaneous or when visual localization of the nidus is technically difficult. although the ribs are easily accessed, the reported lengths of en bloc resection of the nidus ranged from 5 to 9.5 cm, which is significantly wider than the size of the nidus [4, 5, 6, 7 ]. there are reports of varying degrees of functional impairments due to rib resection. in 1990, voto. introduced the successful treatment of oos by percutaneous ct - guided resection. over the years, several percutaneous treatment methods using ct guidance have been introduced : drilling resection, thermocoagulation with radiofrequency, ethanol injection, and a combination of these methods. however, recurrence rates have been reported to be as high as 1020%, and in cases where the nidus is > 10 mm, these procedures have been reported to be more likely to fail. despite these recurrence rates, a secondary operation either by ct guidance or en bloc resection leads to good results due to the remaining trace left by the primary treatment, enabling minimally invasive en bloc resection. one drawback of ct - guided resection is that a histological diagnosis may be difficult in some cases, because the resected specimens are small in quantity. by using a cannulated drill and careful curettage, we were able to make a histological diagnosis in over 90% of the cases (data not shown). ct - guided percutaneous treatment should be considered as a first - line therapy for oo in the majority of cases, except for lesions difficult to access due to the proximity of vital organs, lesions presenting with atypical findings that are difficult to diagnose as oo, and failed primary ct - guided treatments. we were able to avoid rib resection without any recurrence of symptoms by the use of a ct - guided resection and heat ablation. in addition, we were able to obtain enough tissue for histological examination by using a 5.0-mm cannulated drill. ct - guided procedures should be considered as a treatment of choice for oo of the rib in order to minimize the loss of respiratory function. informed consent was obtained from the patient for this case report and any accompanying images. | osteoid osteoma (oo) usually occurs in the extremities of young adults. the tumor can arise in any part of the skeletal tissue ; however, it is rarely found in the rib, with limited reports to date. in this report, we present a rare case of oo arising in the rib, which was successfully treated under computed tomography guidance with minimal invasiveness. at the final follow - up after 4 years, no local recurrence was observed. |
ccdc 1435933 (1 a), 1435934 (2 a) and 1435935 (2 b) contain the supplementary crystallographic data for this paper. 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 reorganized for online delivery, but are not copyedited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors. | abstractthe synthesis of heteroatom analogues of the cyclopentadienyl anion cp is a fascinating and challenging field of research. the replacement of methine moieties by phosphorus is well investigated for the synthesis of mono, tri and pentaphospholyl ligands. on the other hand, arsenic derivatives are rare and 1,2,4triarsolyl and tetraarsolyl salts are unknown. herein, we report on the synthesis of cs[e3c2(trip)2 ] (1 a : e = p ; 1 b : e = as ; trip=2,4,6triisopropylphenyl) and cs[e4c(trip) ] (2 a : e = p ; 2 b : e = as). compound 1 b represents the first 1,2,4triarsolyl and 2 b the first tetraarsolyl anion. all salts are obtained in onepot syntheses using e(sime3)3, 2,4,6triisopropylbenzoyl chloride and csf. the products 1 a2 c4h8o2, 2 aet2o and 2 b3 c4h8o2 were characterized by xray structural analysis, which revealed planar heterocycles. nucleusindependent chemical shifts (nics) confirmed the aromaticity of these anions. notably, compound 2 aet2o is only the second tetraphospholyl ligand which is structurally characterized. |
dynamic factors have a significant role in causing cervical spondylotic myelopathy (csm). in patients with cervical ossification of the posterior longitudinal ligament (c - opll), mild symptoms of myelopathy the pathomechanism of cervical myelopathy caused by c - opll remains unknown. despite spinal stenosis (6 mm < space available for the spinal cord < 14 mm), myelopathy may not develop in patients with severe range limitations of the cervical spine. this possibility indicates that not only static factors but also dynamic factors, such as listhesis or hypermobility at the discontinuity of the ossified lesion, have important roles in the development of myelopathy, especially in mixed and segmental oplls. multidetector - row computed tomography (mdct) has given a better understanding of spinal ligament ossification. unlike the tsuyama classification based on lateral radiographs, ossification morphology and bone continuity between each vertebrae are shown more clearly by mdct. previously, we reported that ossification morphology at each disc segment was divided into 3 groups : the connection department, coating part, and non - connection department of opll. we previously used mdct to measure spinal cord cross - sectional areas (sccsas) during flexion and extension. in that report it is the possible that myelopathy is aggravated by dynamic factors, even in the connection department of opll. however, we did not investigate the sagittal range of motion (rom) and ossification of the anterior longitudinal ligaments. previously, we reported that the incidence of spinal cord injury in the stoppage department of ossification was 64% the purpose of this study was to measure rom by mdct, to investigate the influence of dynamic factors on the spinal cord of patients with c - opll. and the roms of adjacent intervertebral disc in connected vertebrae and those of others were investigated for each morphology. from january 2006 to august 2010, a total, of 110 patients (80 men and 30 women) with c - opll were enrolled in this study. in addition, 99 patients (64 men and 35 women) who had lumbar disc herniation determined by mdct were defined as normal controls. patients with opll, fused vertebrae, and spinal instability were excluded from this normal control. the aims of this study were explained to all patients before myelography and ct scanning, and all patients gave informed consent to their participation. preoperative ct scans after myelography were performed in all subjects in maximum neck flexion and extension. ct scans were obtained (1-mm - thick axial helical) with sagittal and coronal reconstruction using 64-line, multi - slice unit (light speed vct ; ge healthcare bio - sciences, piscataway, nj, usa). the rom at each disc level between c2/3 and c7/t1 in sagittal view was measured using synapse enterprise - pacs (fujifilm medical co., ltd., ossification morphology at each disc segment was divided into 6 groups : covered disc, covered vertebra, unconnected vertebra, connected vertebra (continuous), connected vertebra (localized), and others (fig. the covered disc group was defined as comprising either cranial or caudal vertebra connected by ossification, with the other intervertebral disc not completely covered by ossification. the covered vertebra group was defined as comprising either cranial or caudal vertebra connected by ossification, with the other intervertebral disc completely covered by ossification. the unconnected vertebra group was defined as coprising either cranial or caudal vertebra connected by ossification, with ossification completely covering the vertebra, including other cranial and caudal intervertebral discs or vertebrae. the connected vertebra (continuous) group was defined as comprising both cranial and caudal vertebrae connected by ossification, of the continuous type according to the tsuyama classification. in addition, the connected vertebra (localized) group was defined as comprising both cranial and caudal vertebrae connected by ossification, that was the localized type according to the tsuyama classification. the others group was defined as vertebrae with osteophytes, disc ossification, or no problematic features. the ossification morphology was defined at the biggest and longest place in much mdct slice. ossification morphology at each disc segment was divided into 6 groups : covered disc, covered vertebra, unconnected vertebra, connected vertebra (continuous), connected vertebra (localized), and others. in addition, the roms of adjacent intervertebral disc in connected vertebrae (continuous and localized) and those of others were investigated for each group. roms during neck flexion and extension were measured once on two different days by a spinal surgeon, and average values were adopted. the stat view 5.0 software (abacus, berkeley, ca) analysis of variance with a post hoc test (kruskal -wallis test) was used to perform comparisons between the groups. a p - value < 0.05 was considered to be statistically significant. the rom of the patients with c - opll was significantly smaller than that of the normal controls. there were 122 disc levels in the covered disc group, 114 disc levels in the covered vertebra group, 12 disc levels in the unconnected vertebra group, 40 disc levels in the connected vertebra (continuous) group, 20 disc levels in the connected vertebra (localized) group, and 340 disc levels in the others group (fig. 2). number of intervertebral disc according to each classification the average roms were 6.4 4.6 in the covered disc group, 4.8 4.3 in the covered vertebra group, 4.8 3.8 in the unconnected vertebra group, 2.9 2.3 in the connected vertebra (continuous) group, 2.8 2.4 in the connected vertebra (localized) group, 6.0 4.3 in the others group, and 8.1 4.2 in the normal control. the average rom of the covered disc group was significantly higher than that of the connected vertebra (continuous) group and connected vertebra (localized) group. in addition, the average rom of the normal control was significantly higher than those of the covered disc group, covered vertebra group, connected vertebra (continuous), and connected vertebra (localized) groups. the average rom of the connected vertebra (continuous) group was significantly lower than those of the covered disc group, others group, and normal control group (fig. the average rom according to each classification the average roms of the roms of adjacent intervertebral disc in connected vertebrae (continuous and localized) and those of others were investigated for each group shown in table 2 and fig. 4, were 5.9 4.8 and, 6.5 4.6 in the covered disc group, 5.5 4.3 and, 4.5 4.3 in the covered vertebra group, 3.8 1.7 and, 5.3 4.5 in the unconnected vertebra group, 2.9 2.5 and, 2.9 2.2 in the connected vertebra (continuous) group, 3.0 2.5 and, 2.5 2.3 in the connected vertebra (localized) group, and 4.8 4.7 and, 6.0 4.3 in the others group, respectively. there were no significant differences between the adjacent intervertebral disc in connected vertebrae and others. the average rom of adjacent connected vertebra (continuous and localized) and that of except adjacent connected vertebra (continuous and localized) the tsuyama classification is the most widely used criteria for classifying c - opll. on the basis of lateral radiographs alone, c - opll can be roughly classified into 4 types : (1) continuous type, which is a continuous ossified mass extending over several vertebrae ; (2) segmental type, which is a segmental ossification behind each vertebral body ; (3) mixed type, which is a mixture of these 2 types ; and (4) localized type, which includes other types, (e.g., circumscribed ossification of the ligament corresponding to the level of the intervertebral disc). in addition, mdct has enabled better understanding of ossification of spinal ligaments than provided by lateral radiographs, because the ossification morphology and bone continuity between each vertebrae have become clear. previously, we had described ossification morphology at each disc segment that had been divided into 3 groups : the connection department, coating part, and non - connection department of opll. we had used mdct to measure sccsa during flexion and extension, and sccsa did not show statistically significant differences in ossification morphology. it is possible that myelopathy is aggravated by dynamic factors, even in the connection department of opll. therefore, we reclassified these groups more finely, and examined rom at each segment using a new classification. ossification morphology at each disc segment was divided into 6 groups : covered disc, covered vertebra, unconnected vertebra, connected vertebra (continuous), connected vertebra (localized), and others. the average rom in the covered vertebra group was quite similar to that in the unconnected vertebra group. the average rom in the connected vertebra (continuous) was quite similar to that in the connected vertebra (localized) group. we used the former classification to classify the covered vertebra group and unconnected vertebra group as the coating part. similarly, we had classified the connected vertebra (continuous) group and connected vertebra (localized) group as the connection department. it is not necessary to each distribute, re - classification that we increase cases is desirable. the influence of dynamic factors on the cervical spine has been investigated by flexion extension magnetic resonance imaging (mri). matsunaga. showed that in patients with c - opll, involvement of not only chronic pathological compressive factors caused by opll but also of circulatory and dynamic factors were thought to be important in the development and aggravation of myelopathy. morio.suggested that important factors in the onset or aggravation of myelopathy are related to pathological compression by opll, cervical soft disc herniation, a developmentally narrow spinal canal, and a local or non - proportional hypermobility. previously, we reported that sccsa by mdct was measured to elucidate the influence of dynamic factors. there were no significant differences in the dynamic changes of sccsa between the connection department, coating part, and non - connection department of opll. it has also been suggested that the influence of dynamic factors is less in patients with mature continuous opll. acute spinal cord injury associated with c - opll can be induced by minor cervical trauma, and there is often radiographic evidence of trauma, termed cervical spinal cord injuries without radiographic evidence of trauma (sciworet). described the pathogenesis of central cord injury in the cervical spine as the result of a hyperextension mechanism, with subsequent compression of bony spur, a herniated disc, or a buckled ligamentum flavum. onishi. reported that sciworet with c - opll occurred at the edge of opll or oall. previously, we reported that the incidence of spinal cord injury in the stoppage department of ossification was 64% there were no significant differences between the adjacent intervertebral disc in connected vertebrae and others for each group, but the average rom of the connected vertebra (continuous and localized) group was significantly lower than that of the covered disc group and normal control group. dynamic factors were reduced at the continuous segment : therefore, frequency of sciworet at the continuous segment may be less likely to happen from other segments. the limitations of this study were that the examination of the spinal cord changes relied on mri signal intensities. second, it will be necessary to examine the differences between rom based on mdct and rom based on x - ray. we should closely observe the natural course of patients with c - opll from dynamic factors even in patients with mature c - opll, and it will be necessary to consider sciworet with respect to dynamic factors. this study was performed to measure rom in patients with c - opll by mdct, and to investigate the influence of dynamic factors on the spinal cord. and rom of adjacent intervertebral disc in connected vertebrae (continuous and localized) and those of others were investigated for each group. ossification morphology at each disc segment was divided into 6 groups : covered disc, covered vertebra, unconnected vertebra, connected vertebra (continuous), connected vertebra (localized), and others. the average rom of covered disc group was significantly higher than that of connected vertebra (continuous, localized). the average rom of connected vertebra (continuous) group was significantly lower than that of covered disc group, others group, and normal control. there was no significant difference between rom of adjacent intervertebral disc in connected vertebrae and others, but the average rom of the connected vertebra group was significantly lower than that of the covered disc group and normal control group. dynamic factor was reduced at continuous segment, but it was not increased in adjacent intervertebral disc. each author certifies that they had no commercial associations (e.g., consultancies, stock ownership, equity interest / licensing arrangement, etc.) that might pose a conflict of interest in connection with the submitted article. | abstractthe purpose of this study was to measure range of motion (rom) in patients with cervical ossification of posterior longitudinal ligament (c - opll) by multidetector - row computed tomography (mdct), and to investigate the influence of dynamic factors. the study included 101 patients with c - opll and 99 normal control patients. preoperative mdct were taken in all subjects in maximum neck flexion and extension. rom at each disc level between c2/3 and c7/t1 in sagittal view was measured. ossification morphology at each disc segment was divided into 6 groups : covered disc, covered vertebra, unconnected vertebra, connected vertebra (continuous), connected vertebra (localized), and others. the relationship between rom and the group of ossification morphology was also investigated. rom of adjacent intervertebral disc in connected vertebrae (continuous and localized) and those of others were investigated for each group. the average rom of covered disc group was significantly higher than that of connected vertebra (continuous, localized). the average rom of connected vertebra (continuous) group was significantly lower than that of covered disc group, others group, and normal control. there was no significant difference between rom of adjacent intervertebral disc in connected vertebrae and others, but the average rom of the connected vertebra group was significantly lower than that of the covered disc group and normal control group. dynamic factor was reduced at continuous segment, but it was not increased in adjacent intervertebral disc. |
the development of sustainable agriculture is a major challenge for humanity : we could eradicate hunger from the earth but still preserve our planet for coming generations, for example by using less pesticides and chemical fertilizers, reducing the greenhouse effect, maintaining small farming communities, preserving biodiversity. it is clear that legumes can provide a solution : in terms of protein content they are amongst the richest plants in the world, and they contribute to feeding the majority of the inhabitants of developing countries. thanks to symbiotic nitrogen fixation, legumes do not need nitrogenous fertilizers, production of which consumes petroleum and which contribute significantly to groundwater pollution as well as to the greenhouse effect. last but not least, legumes are very rich in molecules that have potential pharmaceutical uses. as stated by rodomiro ortiz (international institute of tropical agriculture, kampala, uganda) " food legumes are a gold - mine for the developing world ", but their productivity remains limited by a lack of access to, and control of, water resources. as a joint meeting, the conference brought together for the first time researchers working on improving grain - legume breeding and processing with those using modern genomic strategies on model plants. jean - jacques drevon (inra, montpelier, france) identified the central theme of the conference as " how to link genomics and agronomy ". over five days, 450 participants from 45 countries tried to provide answer(s) to this question. it should be noted, however, that the attendance came essentially from developed countries - fewer than 4% of participants came from africa, for example. this report focuses on a few of the presentations that illustrate the multidisciplinary nature of current research into the genetics and genomics of legumes. the large diversity of legume species initially encouraged researchers to develop two model legumes : medicago truncatula and lotus japonicus. it now appears that, contrary to early concerns, having two model legumes does not necessarily lead to a duplication of results but, in fact, often leads to an acceleration of research by creating complementary and synergistic approaches between the two research communities. paul, usa) presented persuasive evidence that the m. truncatula genome is organized into distinct regions of gene - rich euchromatin and repeat - rich pericentromeric heterochromatin. thus, sequencing the gene - space of m. truncatula can be performed efficiently using a bac - by - bac strategy, making use of bacterial artificial chromosomes (bacs) anchored in the expressed genome. young went on to describe the organization of the international sequencing consortium, which comprises groups from the usa and eu and is expected to finish sequencing the gene - space of m. truncatula by the end of 2006 (further details on this sequencing project can be found at). most plant species form symbiotic associations, known as mycorrhizae, between their roots and fungi. helge kuester (university of bielefeld, germany) reported studies using oligonucleotide microarrays to characterize the transcriptome of a particular type of mycorrhizal symbiosis - arbuscular mycorrhiza - formed by m. truncatula in response to two different species of glomus fungi. a subset of 205 genes, including several novel transcriptional regulators, was found to be induced in both endosymbioses. interestingly, in addition to these co - induced genes, several hundred genes were activated only by one or the other species of symbiotic fungus, indicating that the plant transcriptome in arbuscular mycorrhiza roots is strongly dependent on the nature of the infecting microsymbiont. root hairs are specialized outgrowths of epidermal cells of the root that represent an essential interface between the plant and the soil for nutrient and water uptake. gary stacey 's group (university of missouri, columbia, usa) was able to isolate gram quantities of soybean root hairs, and therefore open the door to future dna microarray and proteomic studies of legume root - hair infection by rhizobia. measurement and identification of the metabolome allows various issues to be addressed, such as the influence of genotype, genetic modifications and stress factors on a plant 's behavior. adrian charlton (central science laboratory, york, uk) uses nuclear magnetic resonance (nmr) spectroscopy to study the pea leaf metabolome and is able to discriminate between members of the germplasm collection maintained at the john innes centre (norwich, uk) and between plants subjected to different watering regimes. ole sgaard lund (danish institute of agricultural sciences, slagelse, denmark) described the transferal to legumes of another technology, virus induced gene silencing (vigs). his group has inoculated pea plants with constructs combining the tobravirus pea early - browning virus (pevb) with the phytoene desaturase (pds) or unifoliata (uni) genes. as would be predicted, bleaching of leaves was observed with the pds constructs and abnormal flowers with the uni constructs, whereas plants inoculated with a control construct were unaffected. the nature of molecular recognition specificity was addressed by tom ashfield (indiana university, bloomington, usa) through a study of bacterial disease resistance mediated by so - called r - genes. the rpm1 and rpg1-b genes, from arabidopsis and soybean respectively, confer resistance to pseudomonas syringae strains expressing the effector protein avrb. by comparing their sequences, ashfield discovered that the genes were not orthologous, implying independent evolution of two functionally equivalent r - alleles (' convergent evolution '). martin crespi (cnrs, gif - sur - yvette, france) reported the characterization of the m. truncatula transcriptome during root - growth accompanying adaptation to salt stress, using microarrays, subtractive hybridization libraries (ssh) and homology searches. he has identified 320 genes, of which 52 were completely unknown and 72 appear to be legume - specific, suggesting novel pathways linked to environmental stress responses in m. truncatula. several of these genes are potential regulators, for example, crespi found 11 transcription factors. christine beveridge (university of queensland, brisbane, australia) reported the integration of genetic and phenotypic data to test genetic models related to the control of pea bud outgrowth. one of seven ramosus genes, rms1, is auxin - responsive and encodes an enzyme of unknown function acting on the pathway for the biosynthesis or metabolism of a long - distance developmental signal involved in the inhibition of bud outgrowth. moreover, rms1 is regulated by auxin - independent long - distance signal(s). hans weber (institute for plant genetics and crop plant research, gatersleben, germany) showed that transgenic legumes that have incorporated metabolic pathway genes - either by expressing a bacterial phosphoenolpyruvate carboxylase gene or by the overexpression of a legume amino - acid transporter gene - have increased seed sink strength (the ability to accumulate metabolites) and protein content. these results reveal an enormous complexity and flexibility in seed development and metabolism because pleiotropic phenotypes are created even if the expression of a single gene has been altered. a general conclusion to the conference was given by marc zabeau (european plant science organisation, ghent, belgium) who developed the idea that there is an urgent need for a long - term global vision to integrate plant biotechnologies, genomics and agriculture in order to double agricultural productivity by the 2050s. only an improved understanding of plant biology, coupled with concerted international efforts, will allow us to reach the objective of an economically and environmentally sustainable agriculture. | a report on the second international conference on legume genomics and genetics, organized jointly with the fifth aep european conference on grain legumes " legumes for the benefit of agriculture, nutrition and the environment : their genomics, their products and their improvement ", dijon, france, 7 - 11 june 2004. |
obesity in childhood is strongly associated with cardiovascular risk factors (crfs) including dyslipidemia, hyperglycaemia, and hypertension. in obese children hyperinsulinaemia and other crfs are far more commonly found than in normal weight children and adolescents [24 ]. most metabolic consequences appear to be mediated through insulin resistance (ir) ; therefore improving insulin sensitivity seems even more important than weight loss. omics platforms, such as proteomics, transcriptomics, epigenomics, and metabolomics, provide insights into molecular changes and allow assessing biochemical alterations in the development of obesity and ir [7, 8 ]. while new targets or potential biomarkers are identified in humans with these approaches [9, 10 ], the role of known metabolites still needs to be evaluated. particularly, the influence of amino acid (aa) metabolism on the onset of ir still needs clarification. two recent studies have reported on the untargeted metabolomic approach to study the relation of metabolites to ir in older adults and children. untargeted metabolomics involves an unbiased screening of all metabolites present in a specimen regardless of chemical class. targeted metabolomic techniques facilitate the profiling of specific metabolites of interest in a given population, to aid in - depth analysis of metabolic processes in the context of preformed findings. thus, clinical targeted metabolomics platforms are suitable tools to reveal associations between aa and ir. different studies depicted associations between ir or type 2 diabetes mellitus (t2 dm) and branched - chain amino acids (bcaa), aromatic amino acids (aaa), sulphur containing aa, and other aas as well as short - chain acylcarnitines (carn) involved in aa metabolism in adults [1323 ]. bcaa were found to be positively associated with homeostasis model assessment (homa), an ir index, in nonobese chinese men and young finn adults. mohorko. recently reported elevated serum levels of cysteine (cys) and tyrosine (tyr) as early biomarkers for metabolic syndrome in young adults.. showed that bcaa and short - chain carn derived from bcaa contribute to the development of obesity - associated ir. however, the majority of these studies describe relations of clinical markers to metabolites in cross - sectional settings. furthermore, such associations are susceptible to confounders, like dietary protein intake that was shown to be higher in obese subjects than in normal weight subjects. a few studies describe the prediction potential of bcaa and aaa for the onset of ir [16, 18, 24 ]. although metabolomic analyses in children yield the potential to investigate the early onset of metabolic disease, studies on obese children are lacking. recently, newbern. reported an association of homa with a metabolic signature containing bcaa, uric acid, and long - chain carn in adolescent boys in a cross - sectional study. a combination of bcaa and aaa was associated with homa in obese hispanic children, but only bcaa in korean children. bcaa pattern and androgen hormone pattern were associated with childhood adiposity and cardiometabolic risk, like homa, in another recently published cross - sectional study. longitudinal studies are necessary to explore stronger association between ir and metabolic alterations. to our knowledge, two longitudinal studies in children have been published so far, showing an association of baseline bcaa with homa in healthy american children and in korean children. we embarked on a longitudinal study on obese children participating in a lifestyle intervention for inducing weight loss to explore the relationship between changes in aa metabolism and ir in the fasting state and after an oral glucose tolerance test (ogtt) in obese european children. additionally, we analysed the obesity - independent associations of changes during the intervention period in makers of ir, hemoglobin a1c (hba1c), 2 h glucose in ogtt, and changes of aa and carn. written informed consent was obtained from all parents of the participants prior to inclusion in the study. the local ethics committee of the university of witten / herdecke in germany approved the study (clinicaltrials.gov identifier nct00435734)., this outpatient intervention program is based on promoting regular physical activity, nutrition education, and behavior therapy including individual psychological care of the children and their families. the one - year training program was divided into three phases. in the first one, intensive phase (3 months), the children took part in the nutritional course and in the eating - behavior course in six group - sessions, each lasting for 1.5 hours. parents were invited to attend six evening classes. in the establishing phase (6 months), individual psychological family therapy was provided (30 minutes / month). in the last phase of the program (accompanying the families back to their everyday lives) (3 months), none of the children in the current study were smokers, took any drugs, or suffered from endocrine disorders or syndromal obesity such as prader willi syndrome. the children studied were selected at random from the obeldicks cohort reported previously choosing 40 obese children with substantial weight loss and 40 obese children without weight loss of similar age, gender, pubertal stage, and degree of overweight. we included only children who participated in ogtt both at baseline and after one year. substantial reduction of overweight was defined by a decrease in standard deviation score of body mass index (bmi - sds) 0.5 based on previous studies, whereas no reduction of overweight was defined by a decrease in bmi - sds < 0.15. the metabolomic profile of these children in respect to obesity status and weight loss was previously reported. weight was measured unclothed to the nearest 0.1 kg using a calibrated balance scale. bmi was calculated as weight in kilograms (kg) divided by the square of height in meters (m). the degree of overweight was quantified using cole 's lms method, which normalized the bmi skewed distribution and expressed bmi as a standard deviation score (bmi - sds). waist circumference was measured halfway between lower rib and iliac crest. for longitudinal analysis, blood samples were collected in the fasting state before the intervention and after 1 year. the glucose load was 1.75 g / kg with a maximum of 75 g. blood samples were taken at 8 a.m. after overnight fasting for at least 10 hours. following coagulation at room temperature, blood samples were centrifuged for 10 min at 8000 rpm at room temperature and aliquoted. glucose (boehringer, mannheim, germany), hba1c (germany tina - quant hemoglobin a1c gen), and insulin (abbott, wiesbaden, germany) were measured in serum by using commercially available test kits directly. intra - assay and interassay cvs of glucose, hba1c, and insulin were less than 5%. furthermore, serum samples were stored at 81c and thawed at room temperature for the metabolomics assay only once. metabolites were qualified and quantified with the absolute idq p 150 kit (biocrates life sciences ag, innsbruck, austria) as described previously. briefly, 10 l of blood serum was analysed with a flow injection tandem mass spectrometer (fia - ms / ms). an agilent 1200 sl series high - performance liquid chromatography system (agilent, waldbronn, germany) was coupled to a hybrid quadrupole mass spectrometer (qtrap 4000, ab sciex, darmstadt, germany). ms / ms analysis was run in multiple reaction monitoring mode with electrospray ionization used in both positive and negative modes. data acquisition on the mass spectrometer was controlled by analyst 1.5 software (ab sciex, darmstadt, germany). for raw data processing, peak integration, isotope correction, calibration, and quality control, the met iq software package (biocrates life sciences ag, innsbruck, austria) was used, which is an integral part of the absolute idq kit quantifying a total of 163 metabolites. middle- and long - chain carn, sphingomyelins (sm), acyl - linked phosphatidylcholines, ether - linked phosphatidylcholines, and lysophosphatidylcholines were not used for the data analysis of this work, since the presented study focused on alterations in aa metabolism with respect to ir. for the presented work, we analyzed 14 short - chain carn (cx : y, hydroxyl acylcarnitines cx : y - oh, oxoacylcarnitines cx : y - oxo, and dicarboxylacylcarnitines cx : y - dc), free carnitine (carn c0), and 14 aa. cx : y abbreviates the lipid side chain composition, x and y denoting the number of carbons and double bonds, respectively. the sum of leucine (leu) and isoleucine (ile) is expressed as xleu. for the data analysis performed here, only short - chain carn, carn c0, and aa are used. the sum of xleu and valine (val) the sum of phenylalanine (phe), tryptophan (trp), and tyr is expressed as aaa sum. in addition to the 29 metabolite concentrations and two sum parameters, eleven metabolite ratios were calculated resulting in a total of 42 metabolites and metabolite ratios. all statistical analyses were performed using the statistical software r (3.0.2). in a first step, we graphically screened for outliers and normality. an absolute metabolite concentration that lay greater than 1 standard deviation (sd) away from its nearest neighbor principal component analysis score plots were used as a complementary tool to ensure that no outliers remained undetected. differences in clinical parameters between baseline and follow - up were calculated using the paired wilcoxon rank test. the changes in the clinical markers, metabolite concentrations, and metabolite ratios over the one - year intervention are expressed as the relative difference of baseline and follow - up measurements (with the baseline values being the reference). for each time point (baseline and follow - up) as well as for the relative change, we calculated the following model to assess the association between the metabolites and the clinical parameters : (1) firstly, in order to account for the effect of obesity status on the metabolite level, we fitted age and sex adjusted robust regression models of the bmi on the metabolite using the m - estimator with huber bisquare weighting (r package mass) ; (2) subsequently, we regressed the obtained metabolite residuals on markers for ir with robust regression models using the m - estimator with huber bisquare weighting (r package mass). p values and estimates are taken as proxies for the strengths and directions of the associations. results of selected clinical outcomes are represented graphically in manhattan plots, where the log10(p) values are plotted and the sign is used to indicate the direction of the relationship, as assessed by the robust regression model. due to the small sample size and in order not to veil differences in p values, we will report the raw (unadjusted) p values. bonferroni corrected p values can be obtained by multiplying the reported p values with the factor 42 (number of analytes tested). characteristics of participating children are presented in table 1. in all obese children, waist circumference and 2-hour ogtt glucose decreased significantly during the intervention period. additionally, insulin levels and homa decreased in the group of 40 obese children with substantial weight loss. in contrast, insulin levels and homa increased in the group of obese children without substantial weight loss. since homa and insulin were strongly correlated (table 2) and hba1c and fasting glucose showed no changes between the two time points in any of the groups, in contrast to homa, waist circumference, and 2 h glucose in ogtt (table 1), we focused our data analysis on homa, 2-hour ogtt glucose, and waist circumference. no difference between puberty and homa status was found at baseline (p = 0.44), but after the intervention period (p = 0.036) with pubertal children having higher homa values. associations of all clinical parameters and metabolites are reported in the supplementary material (available online at http://dx.doi.org/10.1155/2016/2108909). at baseline, homa was positively associated with tyr (p = 0.004, figure 2), trp (p = 0.007), sum of aaa (p = 0.013), ornithine (orn, p = 0.026), and threonine (thr, p = 0.036) and negatively associated with carn c3-oh (p = 0.036) and the ratios of carn c5:1/carn c5 (p = 0.014) and carn c6-oxo / xleu (p = 0.044) in all obese children (figure 1). after the end of the intervention, only tyr was associated with homa in all obese children (p = 0.044, figures 2 and 3). in a stratified analysis including the 40 children with substantial weight loss, homa was negatively associated with the ratio of carn c6-oxo / xleu (p = 0.011), carn c6-oxo (p = 0.023), and carn c4 (p = 0.031) and positively associated with carn c4/carn c5-oxo (p = 0.041) and tyr (p = 0.047, figure 2) at baseline. after the intervention, only tyr was associated with homa (p = 0.041) in the children with substantial weight loss (figures 2 and 3). thr (p < 0.001) and proline (pro, p = 0.0322) were positively associated with homa, while the ratios of carn c5:1/carn c5 (p = 0.030) and carn c4/val (p = 0.033) were negatively associated at baseline. after the intervention, only the ratio of carn c5-oh / carn c5:1 was associated with homa (p = 0.048) in children without substantial weight loss (figure 3). the significant associations between the relative change of homa during the intervention period and the relative change of aa and carn are shown in table 3 for all obese children (figure 1), children with substantial weight loss, and children without substantial weight loss. the change of ratio of carn c5/carn c6-oxo was positively associated with change of homa in all three groups, while this was true for the ratio of carn c4/carn c5-oxo only in children with substantial weight loss. changes of tyr were again positively correlated with changes in homa in all children and children with substantial weight loss. two - hour ogtt glucose showed different associations compared to homa, particularly in children with substantial weight loss. at baseline, the ratios of carn c4:1/carn c4 (p = 0.011, negative), carn c4/carn c5-oxo (p = 0.023, positive), and carn c4/val (p = 0.05, positive) as well as histidine (his, p = 0.040, negative), serine (ser, p = 0.043, negative), and carn c4 (p = 0.049, positive) were associated with 2-hour ogtt glucose in children with substantial weight loss, while children without substantial weight loss showed only positive associations between arginine (arg) and 2-hour ogtt glucose at baseline. after the intervention, 2-hour ogtt glucose was associated negatively with ser (p = 0.048) and orn (p = 0.039) in children with substantial weight loss and with glutamine (gln, p = 0.011) and carn c3-oh (p = 0.012) in children without substantial weight loss. interestingly, changes of 2-hour ogtt glucose during the one - year intervention period were not significantly associated with changes of any of the measured metabolites, ratios, or sums in any of the groups. to our knowledge, this is the first longitudinal study analysing the relationships between metabolites and markers of glucose metabolism in obese children. tyr is the only metabolite which was significantly associated with homa at baseline and after intervention. changes of tyr over time were also positively associated with changes of homa in our obesity - independent model. this is in accordance with a recent study where tyr was identified as the most important metabolite in a random forest analysis in obese children. in the same study, furthermore, tyr was found to be a strong predictor for diabetes in south asian men. since phe was not associated with homa in any of the groups at any time point, we assume that there is no confounding dietary effect on tyr levels. tyr stimulates insulin secretion, but other aas are more effective in stimulating insulin release. in contrast, michaliszyn. showed a positive association of -cell function and all aas except for tyr and citrulline in adolescents. the same group reported lower aa plasma levels, except for tyr, in diabetic adolescents, which is in contrast to the role of elevated aa in ir. however, the effect on insulin secretion should not be driving the relation between tyr and homa. insulin is known to increase tyrosine aminotransferase (tat) activity in rat liver, probably by selectively slowing down the rate of degradation of tat. cys is an inhibitor of tat [19, 45 ]. in a recent study, only cys and tyr were found to be increased in nonobese adults who had one symptom of the metabolic syndrome. with further progression of the metabolic syndrome, bcaa and phe higher insulin levels in the ir state may still cover demands to ensure adequate glucose metabolism, but tyr may be affected and tyr may present an early biomarker for the onset of ir in obese children. this predictive value of tyr was shown in previous studies in adults, along with bcaa in young adults [16, 18 ]. in the presented study, we could not investigate tyr as predictor for later ir, since the obeldicks study has an interventional design not a prospective one. contrarily, lee. found bcaa, and not aaa, as predictive marker for ir in korean children. thus, further prospective, longitudinal studies are required to unravel associations between aa and ir with respect to sex and ethnicity. furthermore, tyr can affect bcaa levels, since bcaa and aa compete for the same neutral aa transporter for cellular uptake. many studies found altered levels of bcaa when studying obesity in adults, but also when looking at ir and t2 dm in adults [13, 1518, 42 ]. similar results were found in a few cross - sectional studies in children [12, 25 ]. we found no association between homa and bcaa, neither at baseline nor after the intervention. a previous cross - sectional metabolomic analysis of the presented population did not find different bcaa levels between obese and normal weight children. thus, higher bcaa levels later in life may result from competition for the neutral aas transport, higher protein intake, and/or disturbed bcaa clearance. during bcaa degradation, val, leu, and ile are first degraded to the -keto acids c5-oxo and c6-oxo by the branched - chain amino transferase (bcat). these keto acids are subsequently reduced by branched - chain -keto acid dehydrogenase (bckdh) in the rate limiting step. to identify alterations of bcaa metabolism in obesity and ir, we calculated the ratios of the different steps in the bcaa degradation pathway. we found that the ratio of c6-oxo to c5 was positively associated with homa at baseline and in the change during the intervention period in all three groups. the ratio of c5-oxo to c4 was positively associated with homa only in children with substantial weight loss only. both ratios are markers for the second, rate limiting degradation step of bcaa which is regulated by bcaa itself to keep bcaa concentrations at a constant level. thus, this pathway may have been upregulated in our study resulting in bcaa levels not associated with homa. in contrast, all other ratios showed no significant association with homa or were negatively associated with homa, particularly the first step of xleu degradation to methyl - ketopentanoate (c6-oxo) by bcat. it was shown recently that ir subjects have a significant reduction in bcat expression and other enzymes involved in bcaa metabolism in the adipose tissue compared to none - ir subjects. additionally, bcat2 (mitochondrial) was significantly downregulated, whereas bcat1 (cytosolic) was significantly upregulated in the adipose tissue of obese subjects. other mitochondrial genes of bcaa metabolism were also downregulated in adipose tissue, but not in liver or muscle tissue. our study showed that the reduction of bcaa degradation seems to precede elevated bcaa levels in ir state or obesity in children, since no elevated bcaa were found but alterations in bcaa metabolite ratios. thus, insulin negatively alters bcaa metabolism resulting in higher plasma bcaa in later life, when bcaa concentrations overcome their own degradation and contribute to the vicious circle of ir. the analysis of metabolite profiles in children allowed us to study this early development effect of ir, but far more studies in children and early adulthood are needed to investigate the molecular changes in the early state of obesity and ir. however, bcaa and bcaa metabolism are more affected by protein - rich diet than other aas, and thus diet is a known confounder, especially in obese patients with higher protein intake [15, 22 ]. after the intervention, in a state of homogenous lifestyle and diet, the bcaa ratios were no longer significantly associated with homa, except for the ratio of carn c5-oh to carn c5:1, which was positively associated with homa in children without substantial weight loss. thus, lifestyle may have strong effect on bcaa metabolism, and the results found at baseline were hidden by the homogenous lifestyle of the studied cohort. this possible confounding effect of diet and lifestyle on bcaa metabolism has to be investigated in further studies. the nonsignificant association of homa and bcaa could also be result of less power of our study. tai. depicted the same challenge when they found an association of ir with bcaa in a large group of chinese men, but not in a smaller group of indian asian men. two - hour ogtt glucose showed different associations with aa and aa derivatives compared to homa. since fewer studies exist on relations of 2-hour ogtt glucose to metabolites, we can only speculate about the underlying mechanisms. ser and glycine (gly) were found to be decreased in obese hispanic children and in obese korean children. the concentrations of gly and ser were found to be lower in diabetic than in fasted normal rats. the authors concluded that the contribution of ser to gluconeogenesis becomes proportionally higher in diabetes. thus, an increased gluconeogenesis rate in diabetic or prediabetic patients most likely leads to decreased ser levels. in this case, ser seems to be the aa which is first affected by higher gluconeogenesis rate. but to unravel this relation, further studies are needed. another explanation is the use of consumed ser for sm synthesis, since sm are elevated in ir state. furthermore, we have to recall the relatively low reliability of 2 h glucose in ogtt. additionally, elevated 2 h glucose levels in obese children tend to normalize in follow - up even without weight loss as also demonstrated in our study [57, 58 ]. all the described relations between metabolites and homa or 2-hour ogtt glucose were driven by children with substantial weight loss. thus, it is plausible that different metabolomic changes are associated with different types of ir. however, homa and 2-hour ogtt glucose did not change significantly in children without substantial weight loss during the intervention period, and thus the information in these parameters may be too little to depict associations between metabolites and clinical parameters in this group. nevertheless, estimates for the associations between metabolites and homa were different at baseline, in change, and at follow - up, assuming different metabolic pattern which could be related to ir, the intervention, or weight loss. thus, further prospective studies should focus on the relation of ir to different metabolomic patterns. however, we were not able to differentiate the effect of diet, increased physical exercise, and weight loss on metabolites and their ratio concentrations due to our study protocol. unfortunately, we could not perform stratified analyses to the influence of pubertal status on the associations of metabolites with ir. an explorative statistical approach showed no influence on the association of ir with tyr, but on carn. further studies with larger sample number are required to determine differences in ir development with respect to puberty status. although bmi is a good measure for overweight, it is not a precise measure of body fat mass. furthermore, the degree of obesity was relatively homogeneous in our obese children. additionally, the homa model is only an assessment of ir. clamp studies are actually the gold standard for analysing ir. in addition to the small sample number the relatively low sample number also reduced the statistical power of the presented data analysis, which kept us from correction for multiple testing. among the strengths of our study are the longitudinal design and the analysed children that were nave to drugs and other diseases and had similar lifestyle during the one - year intervention. the additional focus on ratios allowed for a closer insight into degradation pathways associated with obesity - related ir. thus, the influence of diet and physical activity on changes of metabolite levels should be limited. this study provides novel insights into the longitudinal interrelations of ir and obesity markers to metabolites and generates possible questions for further mechanistic studies of ir in obese children. our cross - sectional and longitudinal analyses confirm a relationship between the tyr and homa in obese children. so, tyr and the tyr metabolism should be focused more on in studies searching for early biomarkers and predictors in the switch from obesity to ir. in contrast, bcaa levels were negatively related to ir in cross - sectional analyses, while there was no significant association in the longitudinal analysis, which does not support a causal role of bcaa in inducing ir. furthermore, responders to the intervention showed different associations between homa and aa compared to nonresponders, which appears to reflect different mechanisms for the development of obesity - induced ir. further studies should also explore other analytes which were not determined in our study, such as p - hydroxyphenylpyruvate, fumarate, or acetoacetate that are involved in tyr metabolism and sulfur containing aa. | in obese children, hyperinsulinaemia induces adverse metabolic consequences related to the risk of cardiovascular and other disorders. branched - chain amino acids (bcaa) and acylcarnitines (carn), involved in amino acid (aa) degradation, were linked to obesity - associated insulin resistance, but these associations yet have not been studied longitudinally in obese children. we studied 80 obese children before and after a one - year lifestyle intervention programme inducing substantial weight loss > 0.5 bmi standard deviation scores in 40 children and no weight loss in another 40 children. at baseline and after the 1-year intervention, we assessed insulin resistance (homa index), fasting glucose, hba1c, 2 h glucose in an oral glucose tolerance test, aa, and carn. bmi adjusted metabolite levels were associated with clinical markers at baseline and after intervention, and changes with the intervention period were evaluated. only tyrosine was significantly associated with homa (p < 0.05) at baseline and end and with change during the intervention (p < 0.05). in contrast, ratios depicting bcaa metabolism were negatively associated with homa at baseline (p < 0.05), but not in the longitudinal profiling. stratified analysis revealed that the children with substantial weight loss drove this association. we conclude that tyrosine alterations in association with insulin resistance precede alteration in bcaa metabolism. this trial is registered with clinicaltrials.gov identifier nct00435734. |
pain is a complicated phenomenon often ignored in newborns. until the last decade of 1970, it was thought that infants can not feel or recall pain, so surgical procedures in infants were carried out without anesthesia or analgesia. later, research showed that the fibers that transfer the pain stimuli are organized in the fetus. the nerve pathway myelinization begins in the 2 and 3 trimesters and finishes between 30 to 37weeks of gestational age. mkoban (2003) also showed that insufficient pain control results in hypoxia and stimuli reaction. kazak compared effect of pharmacologic and non - pharmacologic treatment on pain and concluded that non - pharmacological methods are more effective than pharmacological methods for pain relief. this has helped the mother - infant bond and increased the mother 's self - esteem and skills. studies have shown that kangaroo mother care (kmc) is effective on infant pain[710 ]. kangaroo care originated in bogot, colombia because of a lack of incubators for preterm infants. during kmc the adult holds the diapered infant against his / her skin.. a breastfeeding mother may allow the infant self - regulatory access to her breast. the adult is without clothing from the waist up ; a blanket covers both the infant and adult. kmc has three provisions : 1) skin to skin contact, 2) exclusive breastfeeding, 3) support to the mother and infant. care - giving and analgesia is in the domain of nursing activities and must be a priority in nursing standards. this was a semi - experimental double blind, double group, and clinical trial study approved by the research ethics committee of mashhad university of medical sciences. sixty infants born during march to july 2006 with the following inclusion criteria : born at term with a weight between 2500 to 4000 g, type of delivery nvd, apgar score 1=7 - 10, at earliest 24 hr after birth, not fed since 30 minutes and lack of skin lesions in mother or infant, were studied. the mother was requested to put the baby under her gown between her breasts with maximum skin to skin contact. the time of skin to skin contact was 2 minutes before vaccination and 3 minutes afterwards. behavioral changes were scored according to neonatal / infant pain scale (nips) recommended for children less than 1 year old. in this scoring a score greater than 3 indicates pain.facial expression : relaxed muscles 0, grimace 1crying : no cry 0, whimper 1, vigorous crying 2breathing patterns : relaxed 0, change in breathing 1arms : relaxed / restrained 0, extended 1legs : relaxed / restrained 0, flexed / extended 1state of arousal : sleeping / awake 0, fussy 1 facial expression : relaxed muscles 0, grimace 1 crying : no cry 0, whimper 1, vigorous crying 2 breathing patterns : relaxed 0, change in breathing 1 arms : relaxed / restrained 0, extended 1 legs : relaxed / restrained 0, flexed / extended 1 state of arousal : sleeping / awake 0, fussy 1 babies in the control group were wrapped in a blanket and put near the bed of their mothers. we recorded the physiologic and behavioral reactions of these infants to pain using the same method as for the case group. data were analyzed using chi - square, fisher exact test, paired t - test and independent t - test and mann - whitney test. the case group consisted of 53% were males and 47% females whereas in controls 60% were females and 40% males ; 80% of the case group and 73.3% of the control group had 40 weeks gestational age ; 83.3% of the infants in the case group and 96.7% in the control group had a first minute apgar score of 9. mean birth weight in the case group was 3242306.6 and in the control group 3151331.5 grams. according to the results obtained from the nips score, during intervention 30% of the infants had a pain score of 6 and 70% in the case group 7, while 96.6% of the neonates in the control group had a score of 7 and 3.3% had a score of 6. comparison of pain severity before and during intervention three minutes after intervention 93.3% of the infants in the case group had pain score 0 and only 6.6 had 6 to 7 pain scores while in the control group 70% had a zero pain score and more than 26% had a 6 or 7 pain scores. this was also significant (p=0.021, table 2). mean pain intensity 3 minutes after intervention was significantly lower in the case than control group(p=0.008, table 3). according to mann - whitney test there was a significant statistical difference in the cry interval times between the 2 groups (p<0.001) during intervention. comparison of pain severity after intervention comparison of pain severity during and after intervention there was a significant statistical difference between the 2 groups in the time interval of crying after intervention (p=0.008). the preinterventional o2 saturation in the case group was 95.802.78 and 94.073.18 in the control group, during intervention 96.172.61 in the case group and 94.532.64 in control group and after intervention 95.602.19 in case group and 95.101.72 in the control group. mean pain intensity during intervention was significantly lower in the case group than in control group. mean pain intensity 3 minutes after intervention was significantly lower in the case than control group. this was compatible with the results of the gray and johnston [8, 13 ]. gray showed that pain reaction was 65% less in their case group compared to the control group. the results of johnston showed that the efficacy of the kmc was significant in relieving pain in infants of 32 weeks gestational age. the results of anderson showed that skin to skin contact is a factor that relieves infant pain and reduces behavioral and physiological reactions to painful stimulations. moreover, luedington investigated the effect of skin to skin contact on painful nursing procedures. he concluded that infants who had skin to skin contact showed less pain related reactions such as change in grimace. our study was compatible with johnson 's with regard to pulse rate in the two groups which was insignificant. gray reported that hugging neonates during vaccination decreased the crying interval compared to the control group in which the neonates were placed on a bed during vaccination. the present investigation showed that the skin to skin contact group had a crying interval time shorter than that of controls. according to the results of this study kmc decreased pain severity in neonates of the case group during the intervention and 3 minutes afterwards. | objectiveit has been demonstrated that newborns feel pain completely. thus, they should be treated with this in mind. recent research showed that non - pharmacological interventions such as kangaroo care may be useful for decreasing pain in newborns. we tried to determine the effect of kangaroo care on the pain intensity of vaccination in healthy newborns.methodsthis study was a randomized case - control clinical trial. subjects were 60 healthy full - term newborns delivered in a general hospital, in iran, from march to july 2006. they were randomly assigned to case and control groups. the case group received 30 minutes skin to skin contact, whereas infants in the control group were put, wrapped in a blanket, aside the mothers. behavioral changes of newborns were evaluated and observed 2 minutes before, during, and 3 minutes after the intervention. all procedures were filmed. an assistant who was blinded to the study, scored behavior changes using neonatal / infant pain scale. heart rate and oxygen saturation levels as displayed on the pulse monitor and duration of crying were recorded using a stopwatch.findingsmean pain intensity during the intervention v was significantly lower in the case group (p<0.006). mean pain intensity 3 minutes after intervention was also significantly lower in the case group (p<0.021). mean duration of crying was significantly lower in case group as well (p<0.001).conclusionkangaroo care may be used to decrease pain intensity in newborns undergoing painful procedures. |
replantations for major amputations of the upper limb have been widely reported and performed at many centres around the world. we report a case of successful replantation of complete avulsion amputation of the right upper limb at scapulothoracic level in a 3-year - old boy. in our literature search a 3-year - old boy was brought to the emergency room around 3 h after sustaining an injury while playing in the fields. his right upper limb accidentally got caught in the belt of a thresher machine and was avulsed from the chest wall. the child was taken to a nearby hospital with the amputated part [figures 1 and 2 ] from where he was referred to our hospital. amputated limb (dorsal aspect) amputated limb (ventralaspect) on arrival, the child was stable and the amputated limb was well preserved. the patient was evaluated to rule out other injuries and prepared for surgery while an x - ray was done for the amputated part [figures 3 and 4 ]. x - ray of the amputated part on examination, the skin disruption was found to be at axillary level while the upper limb had avulsed from the chest wall along with the scapula and its attached muscles (scapulothoracic dissociation). the deltoid muscle was disrupted from its origin and found in the amputated stump with the skin retracted distally over it. the cut ends of brachial artery and its accompanying vein were found retracted under the biceps muscle. the brachial plexus was disrupted at cord level with distal cut ends of medial, lateral and posterior cords found alongside the transected vessels. the posterior cord was found to be of longest length suggesting higher level of avulsion. after thorough cleaning of the amputated limb, the brachial artery and its accompanying vein were dissected out and tagged. the brachial artery was cannulated, and infusion of cold heparinised saline (5000 units in 500 ml) started. meanwhile, the patient was shifted to the operating room and anaesthetised. on exploration of the amputation stump, the axillary artery was found to be transected after the branch to the latissimus dorsi muscle. the posterior cord was not found in the wound, and an incision was given in the supraclavicular region for exploration of the brachial plexus. however, the proximal end of the posterior cord could not be found. since the level of transection of the axillary artery was after the take - off of the thoracodorsal and circumflex scapular arteries, the anticipated blood supply to the scapular muscles after replantation would have been doubtful. furthermore, the approach to debride the subscapularis muscle in the event of necrosis following replantation would have been cumbersome and dangerous. the supraspinatus and infraspinatus muscles were left attached to the scapula as even in the scenario of necrosis of these muscles following replantation, they would potentially be easily approached from the dorsal side. the amputated limb was then brought into the operative field [figure 5 ]. the dislocation of shoulder joint was reduced and fixed with a 1.5 mm k - wire. the fracture of glenoid neck was reduced and stabilised with two 1.5 mm k - wires. acromioclavicular joint was fixed by a k - wire, and acromioclavicular joint capsular repair was performed by figure of 8 non - absorbable sutures. the associated elbow dislocation was managed with closed reduction, and the fracture of ulna was managed by splinting. amputated limb following fasciotomy and prepared amputation stump axillary vessels were dissected to the healthy end. since no bone shortening was feasible in this case, an end - to - end vascular anastomosis was not possible. an 18 cm long saphenous vein graft was harvested from the left lower leg to reconstruct the segmental defects of the axillary artery and vein (8 cm each). the total ischaemia time was nearly 8 h (1 h warm/7 h cold). the distal end of the posterior cord was anastomosed end to side with the proximal medial cord. there was persistent ooze from the scapular side, which was packed with laparotomy sponges. intraoperatively, the patient was given unfractionated heparin and 3 paediatric units of blood were transfused. after 48 h, the sponge packs were removed from scapular site in the operating room and no further oozing was encountered. post - fasciotomy raw area was covered with split - thickness skin graft on day 20. the k - wires were removed on day 90 in the outpatient department, and the arm supported in a sling, and physiotherapy started [figures 6 and 7 ]. the last follow - up at 18 months post - operative showed evidence of recovery of crude touch sensations up to the digits. one year post - operative (frontal view) one year post - operative (oblique view) nonetheless, they are worthwhile as reported in a large series by sabapathy. in 2007 as patients with successful replants put the hand to greater use. furthermore, as reported by otto. in 2015, replantation of a traumatically amputated arm leads to good function and higher satisfaction rates than a prosthesis. although few reports are available of successful shoulder level replantation, there is none for scapulothoracic level. although venkatarama. in 2015 have reported a case of scapulothoracic avulsion amputation in a 18-year - old male, the replanted limb had to be amputated due to ensuing sepsis. hence, to our knowledge, this is the first reported case of a successful replantation of a limb separated at scapulothoracic level. literature is rife with reports of cases with scapulothoracic dissociation which is a closed avulsion injury caused by a severe traction injury and associated with massive soft tissue swelling of the shoulder, lateral displacement of the scapula and severe neurovascular injury. scapulothoracic muscles are devascularised and have to be debrided. with no muscles being available for scapulothoracic fixation bone shortening is not possible, and therefore, vascular reconstruction would always involve vein grafts. however, with the patient being a child, a reasonable degree of neural recovery is expected. furthermore, the replanted upper limb at this level would be better than any available prosthesis. the child and family have been saved the stigma of an amputation which can have serious psychosocial issues. although the child is doing fine at present, there is still a long road ahead for a reasonable functional recovery, and the secondary reconstructive procedures may be required. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. | replantations for major amputations of upper extremity have been widely performed. we report a unique case of successful replantation of scapulothoracic avulsion amputation in a child. in this manuscript, we discuss the various challenges faced during the procedure and chances of neural recovery. |
in korea, 26.5% of the total female population is obese, and women in their 30s, 40s, and 50s account for 28%, 34.4%, and 37.4%, respectively, of the total obese female population. a sharp increase in the prevalence of obesity has been particularly seen with onset of menopause, a time in which women experience many metabolic changes from female hormone fluctuations. it has been reported that women are at higher risk for obesity during this period due to increases in body fat. postmenopausal women, in particular, often develop abdominal obesity without weight change, due to excessive accumulation of visceral fat and hormonal changes in the abdomen. increased physical activity through regular exercise is very effective for mitigating obesity and improving health - related fitness at all ages [4, 5 ]. park. reported that combined exercise, resistance exercise combined with aerobic training, is more effective for mitigating obesity than aerobic training alone. recently, it was reported that gut microbiota is closely involved with obesity onset and that endotoxins residing in gut flora are also closely correlated with visceral fat increases. endotoxins promote the activation of inflammatory responses by stimulating microphages to secrete proinflammatory cytokines, including tumor necrosis factor- (tnf-). during this process the endotoxin receptor cd14 plays an important role in inflammatory and innate immune responses [1214 ]. as obesity can increase endotoxin levels, causing inflammatory responses, obese women can in turn suppress immune function. in this vein, various studies on controlling endotoxin levels and immune functions are being conducted, and interest in reducing endotoxins and improving immune functions through exercise is increasing [15, 16 ]. kim. reported that the largest immune function improvements resulted from combined exercise when aerobic, resistance, and combined exercises were compared. ng. reported that there were no changes in concentrations of serum endotoxin and tnf- after a one - time 21 km road race. reported that when 61 study participants performed high- and moderate - intensity aerobic exercise for 3040 min, 4 times a week for 12 weeks, only high - intensity exercise decreased the concentration of serum endotoxins. clearly, there have been many studies about an acute bout of exercise and aerobic exercise on physical activity and immune functions, but there is a lack of studies on endotoxin concentration changes after combined physical exercise. moreover, while there are many studies on obesity and inflammation, as well as exercise and immune functions, there are hardly any studies on the interplay between endotoxins, health - related fitness, and immune functions of postmenopausal women with abdominal obesity. accordingly, this study was conducted to examine the effects of combined exercise on health - related fitness, endotoxin concentrations, and immune functions of postmenopausal women with abdominal obesity. the participants in this study were naturally postmenopausal middle - aged women with abdominal obesity. other selection criteria included a lack of regular exercise habits, no prior or present history of genital - related diseases, and not being under treatment for any internal or gynecological diseases. as we explained above, 20 voluntary participants were randomly allocated to the combined exercise group (n = 10) or the control group (n = 10). and visceral obesity was defined as a visceral - to - subcutaneous fat ratio 0.4 based on computed tomography (ct) results. the participants ' physical characteristics are summarized in table 1. a body composition analyzer (venus-5.5, korea) was used to measure the height, weight, % body fat, body fat, and lean body mass (lbm) before and after 12 weeks of exercise. body mass index (bmi) was calculated using weight / height (kg / m). blood pressure was measured with a mercury sphygmomanometer (hico, japan) after participants were stabilized for 30 min. exercise stress testing was conducted before and after 12 weeks of exercise, using the balke treadmill protocol. maximal exercise stress was defined as the presence of any 2 of the following : (1) manifestation of steady state with oxygen intake not exceeding 150 ml / min and expected maximum heart rate (220-age) ; (2) respiratory exchange ratio (rer) 1.15 ; and (3) ratings of perceived exertion (rpe) 17. oxygen intake and heart rate during exercise were analyzed with an automatic metabolic analyzer (quarkb2, cosmed, italy). in the week prior to training, 1-repetition maximum (1rm) measurements were taken, and resistance equipment training and treadmill acclimation exercises were performed. the 1rm for resistance training was measured using the formula from the korea institute of science and technology. in weeks 16, 60% of 1rm was used for 810 repetitions. in weeks 712, 70% 1rm was used for 1012 repetitions. each session lasted for 30 min 3 times a week. for aerobic training, exercise speed and heart rate based on the maximal exercise test were used to calculate the target heart rate (thr), equivalent to 4075% of the heart rate reserve (hrr), (thr = hrr intensity (%) + resting heart rate), after which the target exercise speed was determined with a regression equation each for 40 min 3 times a week for 12 weeks. 5102). and also, total sit - ups and push - ups performed in 30 seconds were measured. all measurements were taken twice, and the highest value was recorded. to measure abdominal fat, a ct scanner (samsung ge sytec 3000 i) scanned the transverse sections one above and one below umbilical level. the area 's total abdominal fat volume was calculated by considering tissues with a hounsfield number from 250 to 50 to be fat. using abdominal and peritoneal spread as the boundary, the areas were divided into visceral fat tissue and subcutaneous fat tissue, with which the visceral fat to subcutaneous fat ratio was calculated. before and after program, 15 ml of blood was drawn from the antecubital vein under fasting, stable conditions. levels of total cholesterol (tc), triglycerides, high - density lipoprotein cholesterol (hdl - c), and low - density lipoprotein cholesterol (ldl - c) were analyzed enzymatically using an autoanalyzer (hitachi 7600 - 110/7170 analyzer, tokyo, japan). levels of tnf-, cd14, and immunoglobulins (iga, igg, and igm) were analyzed using enzyme - linked immunosorbent assay (elisa), flow cytometry, and immunoturbidimetry, respectively. the spss statistics 18.0 program was used to calculate means and standard deviations for all variables. significant differences between the exercise and control groups were determined with 2-way anova and paired t - tests. for independent correlation between changes in endotoxin and oxygen intake per weight, body composition and abdominal fat before and after the exercise program are given in table 3. in the exercise group, visceral fat and the visceral to subcutaneous fat ratio (both p < 0.01) decreased significantly compared to preprogram values. visceral fat (f = 7.619, p < 0.01) and the visceral to subcutaneous fat ratio (f = 29.894, p < 0.001) changed significantly over time and between groups. no control group variables changed after the program. in the exercise group, a significant decrease was seen in postprogram % body fat (p < 0.05) compared to preprogram values. grip strength (p < 0.05), push - ups (p < 0.05), and maximum oxygen intake per weight (p < 0.01) increased significantly. moreover, % body fat (f = 4.844, p < 0.05), grip strength (f = 4.583, p < 0.05), push - ups (f = 5.562, p < 0.05), and maximum oxygen intake per weight (f = 7.555, p cholesterol, tnf-, and c - reactive protein (crp) level changes are given in table 5. in the exercise group, hdl - c level increased significantly following the 12-week program (p < 0.05), whereas tnf- level decreased significantly (p < 0.05). moreover, hdl - c (f = 9.728, p < 0.01) and tnf- (f = 5.147, p < 0.05) levels changed significantly over time and between groups. immune functions, cd14, and endotoxin values from before and after the program are given in table 6. values of iga (p < 0.01) and igg (p < 0.05) increased significantly after program, whereas cd14 (p < 0.05) and endotoxins (p < 0.01) decreased significantly. moreover, iga (f = 8.333, p < 0.01), cd14 (f = 5.434, p < 0.05), and endotoxin (f = 8.963, p postmenopausal women often develop abdominal obesity without weight change from excessive accumulation of visceral fat in the abdomen, attributed to decreased lipolysis as a result of reduced estrogen. as a result, many postmenopausal women experience decreased lbm and increased % body fat, most frequently in the abdominal area. are also more at risk from obesity than premenopausal women. that is, more severe abdominal obesity results in greater probability of metabolic disease development. therefore, prevention and mitigation of abdominal obesity in middle - aged postmenopausal women are important. kim. reported that combined exercise and stretching (80 min sessions 5 times a week for 8 weeks) reduced visceral fat in middle - aged obese women. park. reported that an aerobic exercise regimen (60 min sessions 5 times a week for 12 weeks) reduced visceral fat in middle - aged men. choi and chun also reported that circuit training (60 min sessions 4 times a week for 12 weeks) mitigated obesity. in the present study, a 90 min combined exercise session (10 min warm - up, aerobic exercise for 40 minutes, resistance exercise for 30 min, 10 min cool - down) was performed 3 times a week for 12 weeks, resulting in reduced visceral fat (p < 0.01) and a reduced visceral to subcutaneous fat ratio (p < 0.001). in addition, a significant difference in results was seen between the control and exercise groups and over time, consistent with previous studies. these results indicate that regularly performing combined exercise helps reduce visceral and subcutaneous abdominal fat in postmenopausal women. the acsm separates motor skill - related fitness from health - related fitness and divides health - related fitness into body composition, flexibility, muscle strength, muscle endurance, and cardiovascular endurance. decline in any of these elements ' scan results in an increased likelihood of contracting a disease. flexibility can reduce muscle and joint injuries, and increased cardiovascular endurance reduces the probability of developing cardiovascular diseases. clearly, health - related fitness is very important in maintaining and improving the quality of daily life, a view supported by many studies. kim. reported that body fat, % body fat, and cardiorespiratory fitness improved in severely obese women as a result of a 12-week, low - intensity walking program. shin. reported that body fat, % body fat, muscle mass, cardiovascular endurance, muscle strength, and flexibility improved significantly in obese female college students as a result of dumbbell lifting and walking for 60 min sessions 4 times a week for 12 weeks. although exercise type and intensity influence areas of improvement, most previous studies reported that exercise improved overall health - related fitness. the 12-week combined exercise program reduced body fat and % body fat and improved cardiorespiratory fitness through aerobic exercise and also improved muscle strength and endurance by increasing lbm through resistance exercise. it is thus considered appropriate exercise to improve obese people 's overall health - related fitness. they do not exist independently in the blood but are transported combined with proteins in particle form as lipoproteins. lipoproteins are classified as hdl - c (transporting cholesterol from peripheral tissues to the liver for degradation) or ldl - c (accumulating cholesterol in the blood vessels). obesity increases the prevalence of metabolic diseases, such as cardiovascular disease and insulin resistance, by raising these serum lipid levels, and hdl - c and ldl - c, in particular, are important indicators for prediction and evaluation of coronary artery diseases. park. reported significant increases in hdl - c and significant decreases in tc among serum lipids in middle - aged women with abdominal obesity after performing a combined exercise regimen of 60 min sessions 4 times a week for 12 weeks. kang and jung also reported significant decreases in tc and significant increases in hdl - c in obese people after an aerobic exercise regimen of 40-minute sessions 4 times a week for 12 weeks. in the present study, the exercise group 's ldl - c, tc, and tg values decreased, while hdl - c values increased. although paek reported that there were no significant differences in middle - aged women 's tc values after performing aerobic dance 4 times a week for 12 weeks, most studies reported positive improvements in serum tc and tg levels as a result of regular training. in particular, hdl - c improvements, a cardiovascular risk factor, were seen in the present study. we believe that combined exercise can strengthen cardiopulmonary functions and help prevent and mitigate cardiovascular diseases and hyperlipidemia from hdl - c caused obesity. additionally, tnf-, one of the inflammatory cytokines and a primary modulator of systematic response to infectious diseases, is closely related to obesity. its serum concentrations are known to increase during high - intensity exercise, and high levels of tnf- are secreted by adipose tissues. samartn and chandra reported that tnf- and tnf- gene activations, accompanied by weight loss, were observed in obese people. conducted a 1-year weight - loss program with 56 middle - aged obese women and saw a significant tnf- decrease. kondo. reported significant decreases in tnf- in obese women after 7 months of slope jogging and walking. this study 's decrease in tnf- levels was consistent with previous studies and is believed to result from reduced adipose tissue secretion of tnf- after combined exercise decreased body fat, % body fat, and visceral fat. immunoglobulins (iga, igg, and igm) function as antibodies, carrying out humoral immune functions against antigens that invade the body. iga is found mostly in saliva and stops bacteria or microorganisms from invading the body by binding to them at sites vulnerable to bacterial invasion. the known normal values of immunoglobulins iga, igg, and igm are 160330 mg / dl, 7001400 mg / dl, and 60150 mg / dl, respectively. reported that appropriate exercise increased the serum immunoglobulin concentrations, whereas mackinnon found no effect. reported that igm decreased significantly in middle - aged women following combined exercise for 12 weeks, and tennyson and friedman reported significant increases in igg in middle - aged obese women after 12 weeks of nordic walking. in the present study, based on this study and previous studies, it is believed that the immunoglobulins concentrations differ according to exercise time and duration, intensity, and participant age. the stimulated microphages secrete proinflammatory cytokines, such as tnf-, il-6, and il-1, sometimes triggering excessive innate immune responses and causing a variety of immune responses and diseases, as well as aggravating existing diseases [9, 46 ]. in addition, when concentrations of serum endotoxin exceed 2 to 3 times the normal level, it becomes difficult to modulate the extent of inflammation, which induces weight gain. in other words, endotoxins are believed to be important obesity - related and metabolic disease risk factors. while studies on endotoxins and exercise responses are lacking, the present study showed that visceral fat and endotoxin decrease after a 12-week combined exercise program. lowered tnf- after exercise might have an important role in the obesity reduction and increased immune function. in the present study, middle - aged, postmenopausal women with abdominal obesity performed 90 minutes of combined aerobic and resistance exercises 3 times a week for 12 weeks in order to examine the effects of combined exercise on health - related fitness, endotoxin levels, and immune functions. we conclude that, as a result of the combined exercise program, abdominal fat, health - related fitness, immune response, and endotoxins all changed significantly over time and between groups. as seen in the above results, the exercise group 's abdominal obesity was mitigated due to visceral fat reduction ; grip strength, push - ups, and oxygen intake per weight improved ; and hdl - c and iga increased, while tnf-, cd14, and endotoxin levels decreased. therefore, for middle - aged postmenopausal women with abdominal obesity, a 12-week combined exercise regimen improved % body fat and health - related fitness and increased immune function, while a decrease in visceral fat and endotoxin concentrations was seen. accordingly, we believe that combined exercise is effective in mitigating abdominal obesity, preventing metabolic diseases, and enhancing immune function. additionally, endotoxins may be a potential risk factor for obesity. lowered tnf- after exercise might have an important role in the obesity reduction and increased immune function. in addition, making specific diet programs for postmenopausal woman to achieve a better control of variables on preventing metabolic diseases and mitigating obesity is urgently needed in the near future. | this study was conducted to examine the effects of combined exercise on health - related fitness, endotoxin concentrations, and immune functions of postmenopausal women with abdominal obesity. 20 voluntary participants were recruited and they were randomly allocated to the combined exercise group (n = 10) or the control group (n = 10). visceral obesity was defined as a visceral - to - subcutaneous fat ratio 0.4 based on computed tomography (ct) results. body composition, exercise stress testing, fitness measurement, ct scan, and blood variables were analyzed to elucidate the effects of combined exercise. the spss statistics 18.0 program was used to calculate means and standard deviations for all variables. significant differences between the exercise group and control group were determined with 2-way anova and paired t - tests. the exercise group 's abdominal obesity was mitigated due to visceral fat reduction ; grip strength, push - ups, and oxygen uptake per weight improved ; and hdl - c and iga level also increased, while tnf-, cd14, and endotoxin levels decreased. lowered tnf- after exercise might have an important role in the obesity reduction. therefore, we can conclude that combined exercise is effective in mitigating abdominal obesity, preventing metabolic diseases, and enhancing immune function. |
an efficient method for the construction of csp2csp3 bond in a regio- and stereoselective fashion involving 1,3-terminal dienes, enol triflates / nonaflates, and sodium formate under pd(0)-catalysis is described. the three component assembly allows trapping of a -allyl intermediate, after the initial migratory insertion of the diene, by a hydride source that leads to structurally complex and synthetically challenging tri- and tetrasubstituted alkene building blocks. |
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intranodal schawnomas are extremely rare and just a few cases have been described before. clinical presentation comprises a symptomatic mass in several anatomical regions, but they can also be found during analysis of surgical specimens resected for other reasons. the report is based on the case of an 80-year - old patient who was underwent to right hemicolectomy for an adenocarcinoma. an 80-year - old female patient, with personal history of intellectual development retardation, without other associated pathologies. besides she had been diagnosed with right colon adenocarcinoma, during the study of anemia. the extension study with thoracoabdominal computed tomography scan showed no signs of local or distant disease from the primary tumor. histopathological analysis of the surgical specimen revealed the confirmation of a low - grade adenocarcinoma, infiltrating perintestinal fat without evidence of serosa affectation (t3). for the rest of the specimen examination, 12 perintestinal lymph nodes were observed, 10 of them with normal macroscopic and microscopic characteristics. the two others instead showed whitish coloring and increased consistency, highly suspicious of involvement of primary adenocarcinoma, with a total size of 7 mm. the second, however, was microscopically constituted by a spindle cell proliferation without atypia, mitosis or necrosis, displacing the normal lymphatic tissue toward periphery (fig. we also had positive result after immunohistochemical study for vimentin and protein s100, and a negative one for actin, desminia, cd34 and cd117, all of this was compatible with intranodal schwannoma (fig. 2). figure 1:mesenteric node tumor microscopically constituted by a spindle cell proliferation without atypia, mitosis or necrosis, displacing the normal limphatic tissue toward periphery. figure 2:positive result after immunohistochemical study for vimentin and protein s100, compatible with intranodal schwannoma. mesenteric node tumor microscopically constituted by a spindle cell proliferation without atypia, mitosis or necrosis, displacing the normal limphatic tissue toward periphery. positive result after immunohistochemical study for vimentin and protein s100, compatible with intranodal schwannoma. lymphatic affectation (within lymph nodes) is extremely rare and poorly described in the literature. the lesions to beware of in the differential diagnosis are : metastatic tumor cells, including spindle cell sarcoma, spindle cell carcinoma and melanomas. among the benign lesions, the most important distinction must be made with the miofibroblastoma, whose characteristics include hemorrhage focuses and areas of starry arranged collagen. while miofibroblastomas show a positivity result for actin immunohistochemistry, they do not do it for s100 protein, whose positivity is strongly linked to the schwannomas. in our case, it was reported just one case before by piana, of mesenteric lymph node affectation by schawnnoma in a piece of colonic resection. remaining reports describe tumors located in retroperitoneal nodes, thoracic region, groin location or neck mass relative to growth lymph nodes [210 ]. in our case, as in the rest of the consulted case reports, lymph node involvement by schwannoma, could not be identified until the histopathological analysis are completed, either preoperative extension studies or macroscopic study of the surgical specimen, could suspect lymph node involvement by this tumor, because, there are not specific characteristics that could make it suspicious. it is also noteworthy that such lymph node involvement occurred in a different one from the affected by adenocarcinoma metastasis. intranodal schawnomas are extremely rare and < 12 cases have been previously described in the literature. the origin of those remains unknown ; therefore, as far as our review is concerned, there are not currently theories that could explain lymphatic localization. on the other hand, we have to notice that this type of diagnosis, although anecdotal by its infrequency, highlights the importance of correct microscopic evaluation of lymphatic tissue, included in the pieces of surgical, beyond their macroscopic appearance, because it has not only diagnostic importance, as in our case, but also implications in staging and subsequent treatment if malignant lesions are present. | abstractintranodal schawnomas are extremely rare. just a few cases have been described before. clinical presentation comprises not only symptomatic mass in several anatomical body parts, but also, they can be found during analysis of surgical specimens resected for other reasons. the report is based on the case of an 80-year - old patient who underwent to right hemicolectomy for an adenocarcinoma. the histopathologic analysis revealed one mesenteric intranodal schwannoma in the surgical specimen. the diagnosis was confirmed by immunohistochemistry with positive result for vimentin and s100 protein. less than 12 cases have been reported in the literature before. the findings pointed out our patient as, one of these few reported with such diagnosis. |
cancer an up - regulated biological process of cell growth with an ability of tumor cells to invade and metastasize. a century ago, paul ehrlich hypothesized that a magic bullet could be developed to selectively target cancer. over the past few decades, the progress in molecular biology and the understanding of malignant transformation and tumorigenesis have revealed two major classes of antitumor therapeutics : (i) application of molecularly targeted therapeutics to block major hallmarks of cancer cells, and (ii) employing drug delivery systems through tumor - targeted nanomedicines to improve the pharmacokinetics and bioavailability of vehicle - carried drugs. targeted cancer therapies can be defined as drugs developed against a specific tumor target according to its important biology function in cancer. from 1980 to 2005, a total of 205 monoclonal antibodies (mab) were studied in clinical trials [25 ]. the us food and drug administration (fda) approved the first anti - cd20 mab (rituximab) for the treatment of non - hodgkin 's lymphoma in 1997. today, twelve of these anticancer molecular - targeted mabs have been approved worldwide, eight of them were approved by us fda [35 ]. conventional anticancer drugs exhibit a lack of specificity, poor solubility and distribution, unfavorable pharmacokinetics, and high - tissue damage or toxicity. nanotechnology can bring fundamental changes to the study and understanding of biological processes in health and disease, as well as enable novel diagnostics and therapeutics for treating cancer. thus, advances made on the basis of nanotechnology could result in progress of healthcare. targeted drug delivery systems such as passive and active targeting nanoparticles or nanocarriers, with diameters ranging from 10100 nm, have been developed to improve the biodistribution, pharmacological, therapeutic and toxicity properties of agents used in cancer diagnostics and therapeutics [612 ]. the status of the development of targeting delivery systems, including targeting strategies, potential applications, and the prospects of tumor - targeted nanocarriers have been reviewed and discussed [611 ]. nanotechnology is attracting increasing attention in the biomedical community, owing to unique prospects for targeted delivery in imaging, therapy, and drug delivery. cancer nanotechnology is expected to transform current treatment systems by providing more efficient cancer diagnostics and therapeutics. today, nanocarriers are used in detecting cancer at an early stage, delivering anticancer drugs specifically to malignant cells, and determining if these drugs are killing malignant cells [913 ]. two therapeutic nanocarrier - liposomes and albumin nanoparticles have been approved by us fda for clinical practices [8, 12, 13 ]. pegylated liposomal doxorubicin represents a new class of chemotherapy delivery system that may significantly improve the therapeutic index of doxorubicin through improving therapeutic pharmacokinetics. as nanocarriers are evaluated for safety and efficacy, nanotechnology will bring with it significant advances in molecular imaging and specific targeting of tumor therapeutic agents, elevating therapeutic efficacy, and finally achieving the goal of early detection and control of cancer. customized nanoscale constructs can serve as targeted drug delivery vehicles capable of delivering large doses of radionuclide or chemotherapeutic agents into malignant cells while sparing normal tissues, greatly reducing the side - effects that usually accompany many current cancer therapies [813 ]. monoclonal antibody - guided radiation therapy, or radioimmunotherapy, demonstrated promise in preclinical and clinical anticancer applications [1419 ]. the principles and applications of molecular targeting involving radionuclide methods for tumor nuclear imaging and therapy were reviewed and discussed. two radiolabeled anti - cd20 monoclonal antibodies y - ibritumomab (zevalin) and i - tositumomab (bexxar) were approved by us fda in 2002 and 2003, respectively, for treatment of b - cell non - hodgkin 's lymphoma (nhl), which indicates the potential benefit of antibody - guided systemic radionuclide - targeted therapy [1618 ]. however, tumor targeting studies with radiolabeled mabs also showed some limitations, such as, inefficient targeting and low accumulation in tumor sites (90%) and the greatest in vivo stability for tc, in, and ga radionuclides for nuclear imaging [20, 21, 24, 30, 31, 42, 53 ]. the research on tumor - targeted diagnostic and therapeutic radionuclides is one of the potential areas of cancer drug development. normally, targeted radionuclides consist of two components, a targeting carrier and a trace amount of radionuclide with a specific radiation emitter. the tumor therapeutic efficacy and diagnostic quality are determined by the selectivity or specificity of the targeted delivery systems and the radionuclide radiation characteristics [14, 15, 20, 21 ]. the selection of potential targeted radionuclides for tumor imaging (table 1) and targeted radionuclide for internal radiotherapy (table 2) involves the physical half - life, decay mode, and the emission properties of the radionuclides. gamma emitters with energy range between 130 and 370 kev can be used for gamma imaging or single photon emission tomography (spect) [2430 ]. the high - energy positron emitters with annihilation energy at 511 kev energy can be applied for positron - emission tomography (pet) [20, 21, 31 ]. the major characteristics of nanotargeted nuclear imaging modalities are listed in table 3. in functional and molecular imaging, the in vivo radionuclide spect and pet imaging is the most sensitive with sub - nanomolar amounts of molecular probes and the highest tissue penetration range (table 3). for targeted radionuclide internal radiotherapy applications, high- and low - energy between 0.12.2 mev of -emitters the maximum tissue penetration range (110 mm) [14, 54 ] and cross - fire effects of -particles with energy range between 0.12.2 mev can kill tumor cells in close proximity to neovasculature [14, 21, 54 ]. alpha - emitters hold great promise as therapeutics for small cancer lesions and micrometastatic cancers due to the high - linear energy transfer (let, 80 kev/m) and short - range energy depositions with tissue penetration range of 50100 m. monoclonal antibody labeled with -emitters has been demonstrated to have high specific killing effects and minimal normal - tissue damage in a tumor - bearing animal model [14, 54 ]. auger electrons have an energy of < 30 kev and subcellular pathlength of 212 m. thus, auger electron emitters can exert their radiotoxic effects on cells when they are internalized into the cytoplasm [5557 ]. i - itdu - mediated nanoirradiation of dna induces efficiently death in hl60 leukemia cells and in doxorubicin, - or -radiation - resistant cell lines has been examined. the experimental findings provide evidence that ultra - selective nanoirradiation of dna through auger electron - carrying metabolic substrates offers an extremely effective strategy for inducing cell death and breaking resistance to more conventional types of irradiation or chemotherapy. the schematic illustration of tumor tissue penetration range of radiation emitters for passively and actively nanotargeted radionuclide therapy is shown in figure 1(b). typically, nanotargeted radionuclides have a two - component architecture for passive targeting imaging and radiotherapeutics for example, a pegylated nanoliposome loaded with radionuclide payloads for nuclear imaging or radiotherapeutics [20, 21, 31, 59 ]. the research and applications of selected passively nanotargeted nuclear imaging agents and radiotherapeutics are summarized in table 4. the history and progress of the preclinical development of liposome - targeted treatments for cancer before 2000 were described in detail. the clinical development of passively targeted liposomes as vehicles for targeted therapy of cancer have been summarized. in addition, the potential areas for future development of liposome - targeted strategies have also been considered. drug and radionuclides encapsulated within the liposome can occur in one of the three potential compartments : water - soluble agents are located in the central aqueous core of the liposome ; lipid - soluble agents are carried in the liposome membrane ; peptides and small proteins tend to bind to the interface between the lipid bilayer surface and the adjacent aqueous phase. the major characteristics of nanotargeted nuclear imaging modalities such as gamma imaging, spect and pet are listed in table 3. liposomes are self - assembling colloidal particles composed of a spherical bilayer of small phospholipid vesicles which is spontaneously formed when water is added to a dried lipid mixture. significant progress has been made in the use of liposome as a nanoparticle or nanocarrier for the delivery of radionuclides for imaging. selected research and applications of passively nanotargeted cancer nuclear imaging agents and radiotherapeutics delivery of tc, in, and ga radionuclides by liposomes for gamma - imaging and monitoring drug treatment have been reviewed and reported for preclinical and clinical studies [28, 29, 6063 ]. a systemic study of optimal liposome formulation and encapsulation of radionuclides was also reported [60, 61 ]. the biodistribution, pharmacokinetics, and nuclear imaging of in - dtpa - labeled pegylated liposome were studied in patients with advanced local cancer. effective targeting of solid tumors of breast (5.3 2.6 % id / kg for a tumor volume of 234.7 101.4 cm), head and neck (highest uptake of 33.0 15.8 % id / kg for a tumor volume of 36.2 18.0 cm), lung (18.3 5.7 % id / kg for a tumor volume of 114.5 42.0 cm), brain, and cervix was also observed with gamma camera and spect imaging. conventional in - based liposome (vescan) preclinical and clinical performance and evaluation of lessons learned from the formulation and process development has been discussed and summarized. in recent years, clinical studies using radiolabeled liposomes for tumor diagnostic imaging of cancer and inflammation from 1979 to 2001 have been reported. a novel amphiphilic probes for f - radiolabeling performed liposomes and determination of liposomal trafficking by pet was developed. liposomes encapsulating positron emitter f and cu were applicable for diagnostic imaging and real - time liposomal tracking in vivo [6669 ]. wang. demonstrated an intravenous administration of in - liposome by conjugating in - oxine to dtpa / peg - liposome followed by whole - body scintigraphy. images revealed that the tumor clearly accumulated in - liposome up to 48 h postinjection (p.i.). in addition to the diagnostic imaging of in - liposome, lee. demonstrated the bifunctional imaging and bimodality therapeutic efficacy of radiochemo - therapeutics of in - vnb - liposomes in ht-29/luc mouse xenografts. table 4 lists some of the selected passively nanotargeted liposomes delivery of radionuclides for nuclear imaging. the gamma scintigraphy and spect / ct image passively nanotargeted radionuclides of in - liposome (figure 2(a)), re - liposome, and re - dxr - liposome (figure 2(c)) [70, 72 ] targeting on ct-26 tumor bearing in balb / c mice animal model through the epr localization effect were illustrated. an analytical dosimetry study for the use of i, y, re, and cu radionuclide - labeled liposome for internal radiotherapy has been reported, and the analysis suggested that the optimal liposome system for radiotherapy differs from chemotherapy delivery. in previous clinical targeting tumor imaging studies, the results of the effective targeting of solid tumors in patients with advanced local cancers by radiolabeled pegylated liposomes support the possible delivery of -emitting radionuclide - loaded pegylated liposome for the treatment of solid tumors, particularly those liposomes in head and neck patients. have developed a method of labeling liposomes with radionuclides using n, n - bis(2-mercaptoethyl)-nn-diethylethylenediamine (bmeda) to after - load tc or re into liposomes [65, 75, 76 ]. in addition to therapy via intravenous administration, the intratumoral and intraoperational therapies were also investigated for the potential use of re - liposomes [7779 ]. high - resolution spect / ct images revealed the intratumoral distribution of therapeutic liposomes ; this result indicated the potential use of re - liposomes for intratumoral therapy [78, 79 ]. intraoperative passive nanotargeted re - liposome therapy showed an excellent tumor suppression and minimal side - effect profile in the head and neck squamous cell carcinoma xenograft positive surgical margin model. biodistribution, pharmacokinetics, and nuclear imaging of passively nanotargeted radio - therapeutics of in / re - liposome on c26 and ht-29 colon carcinoma - bearing animal models have been studied by our group [70, 72, 80 ]. in has a -ray with 171 kev energy for nuclear imaging and an auger electron with 0.42 mev energy in the nm tissue penetration range with specific single tumor cell or small tumor cluster killing effect (table 2 and figure 1(b)). re has a -ray with 155 kev energy for nuclear imaging and a high - energy beta emitter with 2.12 mev energy for killing nonspecific large tumor clusters. both radionuclides can be used in bifunctional nuclear imaging and internal radio - therapeutic applications. the long - circulating pegylated liposomes radiolabeled with re (re - liposomes) showed a higher uptake in the tumor as compared with re - bmeda alone. passively nanotargeted re - liposomes were found to have a 7.1-fold higher tumor - to - muscle ratio as compared with intravenously administered unencapsulated re - bmeda in a c26 murine colon carcinoma solid tumor animal model. improvement of biodistribution and therapeutic index via increase of polyethylene glycol(peg) from 0.9% to 6% on passively nanotargeted in - liposome in an ht-29/luc xenografted mouse model was observed. enhanced loading of ac and retention of three -particle - emitting daughters of ac by passively targeted liposomes have been demonstrated [8284 ]. boron neutron capture therapy (bnct) is a binary approach to cancer therapy involving the nuclear reaction that occurs when b is irradiated with thermal neutrons to yield high let of -particles and lithium nuclei (2.4 mev). these particles have a short range (< 10 m) and deposit their energy within single cells. the efficacy and successful treatment of tumors by bnct depend on the selective delivery of relatively high amounts of b to tumors. there are three important parameters for development of boron compounds : (i) achieving tumor concentration in the range of 2035 g b / g, (ii) reaching a tumor / normal tissue ratio greater than 35, and (iii) illustrating sufficiently low toxicity. application of passive stealth liposome - entrapped b delivery systems has been studied for bnct in animal models [85, 86 ]. the results of the study on b - peg - liposome through intravenous injection suggested that passively targeted delivery of sodium mercaptoundecahydrododecaborate (bsh) can increase the retention of b by tumor cells, causing the suppression of tumor growth in vivo for bnct. a high level of b concentration (22 ppm) was observed in tumor tissues at 24 h after the administration of boron liposomes, and the tumor was significantly suppressed. concomitant chemotherapy and radiotherapy has been found to improve treatment outcome in a range of solid tumors. pegylated liposome - encapsulated doxorubicin and cisplatin have shown to be the potential target drugs to tumors, showing increase in therapeutic efficacy and reduction in toxicity. trimodal cancer therapy combining antiangiogenesis, chemotherapy, and radiotherapy achieves beneficial effects when used as a clinical antitumor strategy. image - guided and passive nanocarrier - based polymeric nanomedicine for radiotherapy holds significant potential for improving the treatment of advanced solid tumors. biodistribution, pharmacokinetics, nuclear imaging, and therapeutic efficacies were investigated for nanotargeted bifunctional co - delivery radiochemotherapeutics of in / re-(vinorelbine / doxorubicin, vnb / dxr)-liposomes on colorectal carcinoma of ht-29 and c26 tumor and ascites - bearing animal models [71, 73, 9093 ]. in addition to the diagnostic imaging of in / re - liposome, the additive therapeutic efficacy was observed for the comparative co - delivery radiochemo - therapeutics of specific - killing auger electron emitters of in-(vnb)-liposomes on ht-29/luc mouse xenografts [71, 92 ]. re - dxr - liposomes could provide a beneficial and promising strategy for the co - delivery of passively nanotargeted bimodality radiochemotherapeutics in the treatment of solid tumor and ascites [73, 91 ]. the experimental results pointed to the potential benefit of the co - delivery of nanoliposome radiochemotherapeutics for adjuvant cancer treatment in oncology applications. evaluation of pharmacokinetics of in - vnb - liposome on c26/tk - luc after intraperitoneal (i.p.) and intravenous (i.v.) administration in a tumor / ascites mouse model was studied and compared, the results indicated that the i.p. previous theoretical dosimetry studies have addressed the potential use of therapeutic nanoliposomes for the treatment of tumors via intravenous injection [74, 94, 95 ]. the comparative dosimetric evaluation of nanotargeted re-(dxr)-liposome derived from the biodistribution indicated that the delivery radiation doses were safe and feasible for further clinical translation research from bench to bedside. the results for major organs doses for the re-(dxr)-liposome revealed that similar doses were received by spleen and liver, but a lower dose was given to kidney, compared with in - dtpa - octreotide therapy. lower doses were also received by total body and liver, compared with in - dtpa - human epidermal growth factor (hegf) radiotherapeutics (0.19 and 0.76 mgy / mbq, respectively). the absorbed doses for spleen, liver, kidney, and red marrow in these studies are much lower than those from y-1,4,7,10-tetraazacyclododecane - n, n,n,n-tetraacetic acid tyrosine octreotide (dotatoc) therapy. the tumor growth inhibition and therapeutic efficacy studies of passively nanotargeted radionuclides of in-(vnb)-liposome on ht-29/luc tumor bearing in scid mice animal model (figure 2(b)), and re-(dxr)-liposome on ct-26 solid tumor on balb / c mice animal model were illustrated (figure 2(d)) [71, 90 ]. the synergistic therapeutic efficacy was also demonstrated in the co - delivery of nanotargeted radiochemo - therapeutics of re - dxr - liposome. typically, nanotargeted radionuclides have a three - component architecture for active targeting therapeutics, such as pegylated nanoliposome surface bioconjugated with bioactive antibody or peptide, and encapsulated or bioconjugated with therapeutic radionuclide payloads for tumor - targeted nuclear imaging or radiotherapeutics [20, 21 ]. in addition, tumor - specific receptor targeting of nanocarriers could provide for high - antitumor therapeutic activity and imaging efficacy with low adverse side effects on healthy organs for practically any type of anticancer / imaging drug delivery systems [22, 23, 41, 42 ]. the selected research and applications of actively nanotargeted tumor nuclear imaging and radiotherapeutics are summarized in table 5. the ability to modify the surface of nanocarriers permits the improvement in the pharmacokinetics, bioavailability, toxicity, and customization of nanocarrier formulations for particular actively nanotargeted tumor imaging. enhanced tumor accumulation and visualization by -scintigraphy with in - labeled nucleosome - specific monoclonal antibody 2c5 bioconjugated immunoliposome has been studied, and the results indicated better and faster imaging in various tumor - bearing mice [4547 ]. pharmaceutical lipid - based nanocarriers modified with mab 2c5 could represent a new system for tumor - specific delivery of soluble, insoluble, and radionuclide pharmaceuticals. v3-integrin - targeted in perfluorocarbon nanoparticles have been developed and studied for the detection of rabbit vx-2 tumor angiogenesis. the circulatory half - life was estimated to be 5 h. the mean tumor uptake was 4-fold higher than the nontargeted control. the specificity activity (mci / ml) (in / np) of in to nanoparticle (np) may affect the tumor - to - muscle ratio in patients. the tumor - to - muscle uptake ratios for the nanotargeted in / np = 10 to in / np = 1 were 6.3 0.2 to 5.1 0.1, respectively. the data suggest that v3-targeted in perfluorocarbon nanoparticles may provide a clinically useful tool for detecting angiogenesis in nascent tumors. in radiolabeled soluble functionalized multifunctional drug delivery platforms of active targeting with rituximab monoclonal antibody bioconjugated on single - wall carbon nanotubes have been developed, and the selectivity of targeting disseminated human lymphoma was evaluated in vitro and in vivo. the results of the ability to target tumor specifically with prototype - radiolabeled or fluorescent - labeled, antibody - appended carbon nanotube constructs are encouraging and suggest further investigation of carbon nanotubes as a novel radionuclide delivery platform. the development of a dual - function pet / near - infrared fluorescence (nirf) molecular probe for the accurate assessment of pharmacokinetics and tumor - targeting efficacy of u87 mg human glioblastoma tumor - bearing mice has been reported. the amine - functionalized surface of quantum dot (qd) bioconjugated with arginine - glycine - aspartic acid (rgd) peptides and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (dota) for cu radiolabeled cu - dota - qd - rgd nanoconstructs with 90 rgd per qd to target angiogenesis for application in integrin-v3 pet / nirf imaging was also illustrated. this dual - function nuclear / optical in vivo molecular probe revealed a quantitative targeting ability in deep tumor lesions. dual modality optical and pet imaging of vascular endothelial growth factor receptor (vegfr) on tumor vasculature using qds of cu radiolabeled cu - dota - qd - vegf was also investigated. the u87 mg tumor uptake of active nanotargeted cu - dota - qd - vegf (1.52 0.6 % injected dose / gram (% id / g), 2.81 0.3 % id / g, 3.84 0.4 % id / g, and 4.16 0.5 % id / g at 1, 4, 16, and 24 h, respectively, postinjection) was one percentage injected dose per gram (% id / g) higher than that of passively targeted cu - dota - qd. f - labeled phospholipids quantum dot micelles for in vivo pet and optical fluorescence imaging from cells to whole body have been designed and studied. development of a bifunctional nanotargeted iron oxide (io) molecular probe for pet and magnetic resonance imaging (mri) of tumor integrin-v3 expression was reported ; this bifunctional cu - dota - io - rgd nanotargeted molecular imaging approach may allow for earlier tumor detection and may provide insight into the molecular mechanisms of cancer [32, 33 ]. the synthesis and in vivo characterization of an f - labeled trimodal (mri / pet - ct / optical) iron oxide(io) for tumor imaging, the facile conjugation chemistry, provides a simple platform for rapid and efficient io labeling. tumor targeting angiogenesis and comparison of tc - labeled peptide and tc - labeled polymer - peptide nanocarrier conjugates were investigated. specific targeting of the v3 integrin and nonspecific vascular permeability are both significant, but active specific targeting is more important than epr of the carrier molecule. nonspecific vascular permeability appears to be a major factor in reducing tumor - to - normal tissue localization ratio for the peptide molecules. biodegradable br - labeled dendritic bioconjugated rgd bifunctional nanoprobes for the noninvasive pet imaging of angiogenesis was reported. figure 3 demonstrated the in vivo actively nanotargeted radionuclides of cu - dota - qd - rgd for dual - function pet and near - infrared fluorescence (nir) imaging of a u87 mg tumor vasculature mice animal model. significant radiation - induced antisense - mediated cytotoxicity of tumor cells in vitro was achieved using an auger electron - emitting antisense antir1 messenger rna antisense morpholino (morf) oligomer administered as a member of a three - component streptavidin - delivery nanoparticle (in - morf / tat / trastuzumab). targeted angiogenesis v3 and vegfr2 with three - component actively nanotargeted radionuclides of y - liposome - ia (integrin antagonist) and y - liposome - anti - flk-1 (mab) have been reported in murine melanoma k1735-m2 and colon ct26 animal models. the results demonstrated that y - liposome - anti - flk-1 (mab) was significantly more efficacious than conventional radioimmunotherapy in the mouse melanoma model. enhanced targeting, loading and retention of ac, and three -particle - emitting daughters of ac by actively nanotargeted immunoliposomes have also been illustrated [8284 ]. the efficacy and successful treatment of tumors by bnct depend on the selective delivery of relatively high amounts of b to tumors. application of active folate - receptor targeted pamam - dendrimers and active cetuximab immunoliposome - entrapped b delivery systems has been studied for bnct applications in animal models [51, 52 ]. major challenges that have to be addressed by drug - delivery nanocarriers in cancer therapy are the low drug bioavailability of therapeutics within cancer cells and the high toxicities at normal organs due to the low tumor targeting or localization. the combination of molecular targeting of bioconjugated nanoparticles or immunoliposomes can provide targeted cell internalization and intracellular drug release to improve anticancer therapeutic efficacy and to reduce toxicity. active receptor nanotargeted polymers, dendrimers, and liposomes employed for targeting to tumor - specific receptors can prevent serious adverse side effects on healthy organs. in addition, the internalization and intracellular distribution of nanocarriers in cancer cells indicated that tumor - specific receptor active targeting of nanocarriers could provide high antitumor therapeutic activity and imaging efficacy with low adverse side effects on normal tissues. the ultimate goal in the design and preparation of multifunctional and multimodality nanoparticles in drug delivery is the creation of combined diagnostics and therapeutics (or theragnostics) and combined radiochemo - therapeutics for the targeted diagnosis and treatment of cancer [44, 97 ]. recent advances in the field of nanotechnology and nanomedicine indeed offer the promise of better diagnostic and therapeutic options. newer generation of nanoparticles has been designed and synthesized to target specific types of cell and molecule via affinity ligands from phage or small molecules, or involving antibodies or peptides for nanotargeted radionuclide or drug concurrent delivery. synergistically integrated nanoparticles with multifunctional and multimodality novel core platform for cancer nuclear imaging and radiotherapeutics have been developed. important multifunctions include imaging (single or multimodality), therapy (single drug or combination of two or more drugs), and targeting (one or more ligands) with multivalent. for example, binary nanoparticles with two functions could be developed for simultaneous molecular imaging and targeted therapy, ternary nanoparticles with three functions could be designed for simultaneous imaging, therapy and targeting, targeted dual - modality imaging, or targeted dual - modality therapy. some typical and potential nanoparticles for nuclear imaging and therapeutics are illustrated as follows : (i) radionuclide (e.g., in / re / cu)-labeled passively nanotargeted multimodel nanoliposomes [9092 ] or actively nanotargeted multifunctional and multimodal immunoliposomes [4547, 99101 ], (ii) radionuclide (e.g., f / cu)-labeled iron oxide magnetic nanoparticles for multimodal and multivalent mri - pet - optical imaging agents and therapeutics [3234 ] (iii) radionuclide (e.g.,f / cu)-labeled qds for multifunctional and multimodal imaging and therapeutics [3739, 59, 102 ] and (iv) silica nanoparticles as a platform for multimodality imaging agents and therapeutics [103, 104 ]. the simultaneous attainment of preferential localization and avoidance of the sequential biological barriers, such as res system uptake, has been studied with a multifunctional multistage delivery system of mesoporous silicon particles for imaging and therapeutic applications. development of multidrug resistance (mdr) is one of the most challenging aspects of cancer chemotherapy. bimodality codelivery chemotherapeutics in nanoemulsion formulations has shown to be very effective in enhancing the cytotoxicity in wild - type and resistant tumor cells. although nanocarriers have provided some new breakthroughs for cancer diagnosis and therapy, the potential adverse human health effects resulting from exposure to nanoparticles should also be a concern [22, 107, 108 ]. research shows that nanoparticles can stimulate and/or suppress the immune response, and that their compatibility with the immune system is largely determined by their surface chemistry. modifying these factors can significantly reduce the immunotoxicity of nanoparticles and make them useful platforms for drug delivery. the biodistribution and movements of nanoparticles through tissues and the phagocytosis and endocytosis of nanoparticles would all likely affect the potential toxicity of nanoparticles. the practical strategies for identifying and controlling interferences in common evaluation methods and the implications for regulation of nanoparticle immunotoxicity have been discussed and suggested ; the standardization of nanoparticle - tuned methods through international round robin nanocarrier systems may induce cytotoxicity and/or genotoxicity. to minimize the risks posed by nanomaterials, there are two basic avenues. many great efforts are being made to develop nanoparticles satisfactory for clinical applications, but nanoregulation is still undergoing major changes to encompass environmental, health, and safety issues [43, 107, 108, 110 ]. qds larger than the renal filtration threshold quickly accumulate in the res system following intravenous administration. great concern has been raised over the use of quantum dots in living cells and animals due to their chemical composition of toxic heavy - metal atoms (e.g., cd, hg, pb, as). radiolabeled pegylated liposomes have demonstrated effective targeting of solid tumors in patients by nuclear imaging. there were no important adverse reactions attributable to the liposome infusion, and repeated hematological and biochemical profiles performed at day 10 showed no significant changes. absorbed dose calculations provide a scientific basis for evaluating the biological effects associated with administrated radiopharmaceuticals. in cancer therapy, radiation dosimetry supports treatment planning, dose - response analyses, predications of therapy effectiveness and safety. an analytical dosimetry study for the use of radionuclide (cu, i, re, and y)-liposome in internal radiotherapy has been reported. unlike the case with radioimmunotherapy, the dose - limiting organ is likely to be the liver, and strategies intended to reduce res accumulation are needed to further improve such a tumor - targeting approach. we have studied the radiation dosimetric analysis of passively nanotargeted radiotherapeutics of re - liposome and radiochemo - therapeutics of re - dxr - liposome with olinda / exm software for system - targeted radionuclide therapy. the results showed that the red marrow was to be the critical organ in determining the maximally tolerated absorbed doses, and it was promising and beneficial to carry out further preclinical and clinical investigations. comparisons with the radiation - absorbed dose estimates for in- and y - ibritumomab tiuxetan, and the radiation absorbed per unit administered activity (mgy / mbq) for nanotargeted re-(dxr)-liposome were much lower in the major organs [90, 111 ]. recent advances in the field of nanotechnology applications in biomedicine offer the promise of better diagnostic and therapeutic options. medicine and synthetic scientists are making strides in developing nanoconstructs that can be used as core platforms for attaching different functionalities by surface conjugating or after - loading of various nanoparticles for the purposes of cancer molecular imaging and targeted drug delivery. as compared with conventional targeted radionuclide therapy or radioimmunotherapy, the use of nanocarriers can allow for specific multivalent attachment of targeted molecules of antibodies, peptides, or ligands to the surface of nanocarriers. nanotargeted radionuclide therapy can deliver a high payload of radionuclides, chemotherapeutics, and/or imaging agents to achieve multifunctional and multimodality targeting to tumor cells. the new nanocarrier drug delivery system platform can enhance the efficacy and safety of targeted therapy. future clinical trial studies are required to translate those advanced technologies to the health care of cancer patients. the optimization of the nanoparticle compositions and structure, the simultaneous attainment of preferential targeting location, reducing immunotoxic effect, and the avoidance of sequential biological barriers of the nanoparticles are the major challenges in the future research and development of passively and actively nanotargeted drug delivery systems. several passively nanotargeted radiolabeled nanocarriers have been successfully employed to image and treat tumor models both preclinically and clinically. future studies should be designed to optimize these novel approaches and to combine targeted delivery, potent radionuclides, imaging agents, chemotherapeutics and/or radiosensitizing agents. we have demonstrated that a co - delivery of radiochemo - therapeutics and simultaneous multifunctional imaging is an advantageous characteristic of nanotargeted radionuclides for cancer imaging and therapy. a good multidisciplines and multi - institutes collaboration between the academia, research institutes, and industry combing with an integrated bench - to - clinic translational approach would accelerate the progress in research of nanotargeted radionuclides toward clinical applications for the healthcare of cancer patients. | current progress in nanomedicine has exploited the possibility of designing tumor - targeted nanocarriers being able to deliver radionuclide payloads in a site or molecular selective manner to improve the efficacy and safety of cancer imaging and therapy. radionuclides of auger electron-, -, -, and -radiation emitters have been surface - bioconjugated or after - loaded in nanoparticles to improve the efficacy and reduce the toxicity of cancer imaging and therapy in preclinical and clinical studies. this article provides a brief overview of current status of applications, advantages, problems, up - to - date research and development, and future prospects of nanotargeted radionuclides in cancer nuclear imaging and radiotherapy. passive and active nanotargeting delivery of radionuclides with illustrating examples for tumor imaging and therapy are reviewed and summarized. research on combing different modes of selective delivery of radionuclides through nanocarriers targeted delivery for tumor imaging and therapy offers the new possibility of large increases in cancer diagnostic efficacy and therapeutic index. however, further efforts and challenges in preclinical and clinical efficacy and toxicity studies are required to translate those advanced technologies to the clinical applications for cancer patients. |
previous reports describe patients chewing on patches, applying more than the number of prescribed patches to the skin, and applying a patch to an area with a skin abrasion all of which can lead to fentanyl toxicity. continued vigilance among healthcare providers to detect misuse of this transdermal medication is of paramount importance to prevent untoward adverse events including death. herein, we describe a fentanyl patch complication, which meets our institutional review board standards for minimal risk reporting in a de - identified manner, as provided below, and which, to our knowledge, has not been previously reported. a 60-year - old man presented to our clinic for a follow - up visit, at which time he appeared despondent. two years prior, he had been struck by a motor vehicle and fractured his first lumbar vertebra. this fracture was treated with an l1 corpectomy and an instrumented fusion from t12 to l2. this complication coupled with the diagnosis of cancer, as outlined below, resulted in the patient being treated almost ever since with transdermal fentanyl for a chronic pain syndrome. a few months earlier, a hematologic evaluation for leukocytosis had led to the diagnosis of systemic mastocytosis with an associated clonal hematologic non - mast cell lineage disease, chronic myelomonocytic leukemia type 1 (cmml-1). the steroids were tapered slowly over months, and the patient started azacytidine in place of hydroxycarbamide to treat his cmml-1. however, during this evaluation process and treatment for mastocytosis and cmml-1, the patient remained on a fentanyl patch (100-g / h patch every 72 h). he had no history of a psychiatric disorder, but he had experimented with psychotropic substances in his youth and had a flat affect at baseline. when the patient presented for his second cycle of azacytidine, he appeared despondent and described shortness of breath. an accompanying friend reported that the patient had been less communicative over the preceding 4 days and had complained of shortness of breath. the patient 's heart rate was 96 beats / min, blood pressure 116/77 mm hg, spo2 94%, temperature 37.0c, and respiratory rate 16 breaths / min. the circumference of the left leg was 5 cm greater than that of his right leg, and his lungs were clear to auscultation. the results of this presentation raised concerns about a pulmonary embolus, and the patient was thought to be at high risk (well 's score of 7 due to clinical signs of deep venous thrombosis, likely diagnosis of pulmonary embolism, and active malignancy). he was not treated immediately with anticoagulation due to his thrombocytopenia (36 10/l) and renal insufficiency (creatinine 2.3 mg / dl). a lower - extremity doppler ultrasound did not reveal occlusive thrombus and chest radiography was unremarkable. computerized tomography (ct) of the chest with contrast was not pursued due to renal insufficiency. however, because of declining sensorium concurrent with a dropping blood pressure of 89/45 mm hg, a ct of his head was obtained. this imaging revealed an 8-mm left frontal and parietal subacute on chronic subdural hematoma without mass effect ; this finding remained stable on subsequent scans. the patient 's worsening mental status and other symptomatology in the setting of an unclear diagnosis prompted hospitalization. shortly thereafter, the patient experienced a brief tonic seizure that resolved spontaneously followed by a second more prolonged tonic seizure that involved right gaze deviation and tonic posturing of both arms. direct laryngoscopy revealed a transparent, folded, plastic - appearing item superior to the glottis. full bronchoscopy was performed to ensure that no other foreign objects had been aspirated, and none were found. the patient had no explanation for the discovery of a fentanyl patch in his larynx, but he denied any intention to harm himself. to date, it remains unclear exactly how the patch came to be positioned in this patient 's larynx. the patient was discharged from the hospital with follow - up plans for the treatment of his hematologic conditions. this case represents an unusual and unexpected complication related to the ongoing use of a fentanyl patch. this patient apparently attempted to ingest a fentanyl patch, which became lodged within his larynx. heat can increase the absorption of fentanyl, and, perhaps, the higher laryngeal body temperature resulted in increased transmucosal absorption of the medication, hence the manifestations of overdose, despite the patient 's long - standing use of this medication. this case provides further confirmation of the need for sustained, heightened vigilance for drug adherence with transdermal fentanyl. in a systematic review of the complications of opioid medications, fentanyl was the opioid most commonly implicated in such untoward events. the most frequent cases of misuse included drug - drug interactions which potentiated narcotic effects or resulted in serotonin syndrome. other problems with fentanyl relate to the acceleration of its transdermal absorption or experimentation with alternate routes of administration. although external heat sources have been used to maximize the euphoric effects of fentanyl, other patients have suffered unintentional overdoses from hyperthermia, sun tanning, and warming blankets. patients have been reported to lick and chew or rectally insert transdermal fentanyl patches, and inject or inhale volatilized extracts from the patches. inappropriate use of transdermal fentanyl patches may be more common than currently recognized and should be considered in fentanyl - treated patients who present with signs of narcotic toxicity. the patient in this report had a near fatal event after asphyxiation with a fentanyl patch. this case represents the first report, to our knowledge, of asphyxiation with transdermal fentanyl and highlights the need for an awareness for complications with the use of narcotics, particularly of transdermal fentanyl and particularly under circumstances in which the prescribed patch is absent from the skin. | narcotics are frequently prescribed to alleviate pain in patients with serious medical illnesses such as cancer. because of their nonoral route of administration, fentanyl patches are now being frequently prescribed. however, the widespread use of fentanyl patches has been associated with medication errors and misuse [butts and jatoi : j opioid manag 2011;7:3545 ]. the transdermal delivery of fentanyl may lead to unusual, unanticipated complications. herein, we describe a fentanyl patch complication, which, to our knowledge, has not been previously reported. |
seminoma is the most frequent carcinoma of the testicle in the fourth decade of life and constitutes 60% to 65% of germ cell neoplasias. several histopathological characteristics of the tumour have been evaluated and three types of pure seminoma have been described : classic, anaplastic and spermatocytic. we report a patient with seminoma arising in an undescended testis which presented as a palpable painful mass of lower abdomen. a 29 year old man with no known previous complaint presented with 4 month history of progressively enlarging mass on his left lower abdomen. the mass was slightly tender and located immediately above the left groin (figure 1). ct scan revealed a mass of 211311 cm located between the abdominal subcutaneous and external oblique fascial layers. mass was located immediately above the left groin he underwent surgery and en - bloc resection of the tumour via a left oblique flank incision was performed (figure 2). the specimen demonstrated typical characteristics of classical seminoma with a smoothly demarcated fibrous capsule, which had a vascularized outer surface, and some spermatic chord structures were also identified macroscopically. immunohistochemistry studies revealed plap (plasental alchaline phosphatase) positive (figure 3), p53 positive and cd45 negative tumour cells. tunica vaginalis was intact, however, the tunica vasculosa that was located under tunica albuginea nested tumour cell piles (positive lymphovascular invasion). plap positive tumour cells (x200) he was referred to oncology clinic after discharge. germ cell tumours (gct) of testes can be benign (teratomas) or malignant (seminoma and non - seminoma). gct most frequently occur in the gonads, only 2 - 5% of them arise in extragonadal regions such as the mediastinum, retroperitoneum, pineal gland and sacral area (1). the undescended testicles carry 20 - 48 times higher potential for malignant transformation than the normally descended testicles (2). testicular ectopia is uncommon and the most frequent ectopic location of testis are superficial inguinal pouch, infront and lateral to the external inguinal ring and very rarely in the abdomen (3). the position of the undescended testis is related to the likelihood of carcinogenesis with intra - abdominal testis having the highest malignant potential. the majority of undescended testes locate distal to the external inguinal ring and are palpable (2). exogenous estrogen administration to the mother during pregnancyhas been associated with an increased relative risk for testicular tumours in the fetus, ranging from 2.8 to 5.3 over the expected incidence. other acquired factors such as trauma and infection - related testicular atrophy have been associated with testicular tumours ; however, a causal relationship has not been established. our patient had his right testis in the scrotum and there was no palpable testis in the left scrotum. this rapidly enlarging tumour was detected in the region betwen the external oblique fascia and the abdominal subcutaneous layer, distal to the external inguinal ring (figure 4). exogenous estrogen administration to his mother during pregnancywas not reported. rapidly enlarging tumour distal to the external inguinal ring. approximately 7 - 10% of testicular tumours develop in patients who have a history of cryptorchidism ; seminoma is the most common form of tumour these patients have. however, 5 - 10% of testicular tumours occur in the contralateral, normally descended testis. the relative risk of malignancy is highest for the intra - abdominal testis (5%) and is significantly lower for the inguinal testis (1.25%) (4). orchiopexy does not alter the malignant potential of the cryptorchid testis ; however, it facilitates examination and tumour detection (5). three clinical stages for the determination of the extension of the tumour have been described. stage i is where the tumour is limited to the testis with or without invasion of epididymis or the spermatic cord. in stage the germ cell tumour often gives lymph node metastasis, except from choriocarcinoma, which is characterized by early hematogenous spread. age, tumour size, lymphovascular invasion, mitotic count, necrosis, percent of giant cell and tumour infiltrating lymphocytes are possible prognostic factors in the treatment of seminomas. in this particular case, the tumour was classified as stage i classical seminoma with positive lymphovascular invasion (tunica vasculosa nested tumour cells). unilateral rapidly enlarging tender abdominal wall mass with undescended testis should alert clinicians towards consideration of the possibility of seminoma and initiation of prompt intervention. | seminomas in undescended testes may present as abdominal wall tumours. a unilateral testis tumour in a 29 year old man with ipsilateral undescended testis is presented and relevant literature is reviewed.a 29 year old man presenting with a tender left lower abdominal mass was admitted to our clinic and initial diagnostic tests followed by abdominal computerized tomography (ct) and positron emission tomography / computerized tomography (pet / ct) were performed. abdominal ct clearly demonstrated the tumour location between the lower left abdominal subcutaneous layer and the external oblique fascia. he underwent surgery and the tumour was resected via en - bloc excision.pathological diagnosis of the resected specimen was consistent with classical seminoma and no distant metastasis was detected with pet / ct. he was referred to oncology clinic after discharge.tumours of undescended testis can present as an abdominal wall mass and clinicians must be aware of their existence. |
toxoplasmosis is caused by infection with a ubiquitous intracellular protozoan, toxoplasma gondii.1 reproduction is both sexual and asexual. sexual reproduction occurs in definitive hosts mostly involving the cat family, and occasionally mice or rats.1 asexual reproduction occurs in a wide range of animals, which includes man as intermediate host.1,2 the onset of cellular immunity against t. gondii is mediated by t - cells, macrophages, and activities of type-1 cytokines (interleukin-1 and interferon gamma). this is accompanied by the transformation of the parasite into tissue cysts resulting in chronic infection.3 humoral immune response is initiated by the production of specific toxoplasma gondii antibodies toxoplasma antibodies (igm and igg).3 igm antibody response occurs as an early event, and disappears within a few weeks or months, thus its presence in plasma indicates recent infection.3,4 igg antibody production peaks within 1 to 2 months after infection but remains elevated for life.4 several methods for diagnosis of toxoplasmosis exist and include : isolation in vivo (mice) and in vitro (tissue culture) ; detection of t. gondii by dna polymerase chain reaction (pcr) from body fluids ; computed tomography (ct) scan and magnetic resonance imaging.58 serologic testing remains the routine method of diagnosis,4 especially in resource - poor countries where it is the most available and affordable means of detecting the presence of toxoplasma gondii antibodies (toxo - igg and igm antibodies) and igm antibodies. it has been documented that more than 97% of hiv - infected individuals with toxoplasma encephalitis will test positive for toxo - igg antibodies.4 therefore, the absence of toxo - igg antibodies in plasma strongly argues against the diagnosis of toxoplasmosis.4 serological studies in many groups have shown that about 20% of people would have acquired the infection by the age of 20, and up to 50% by the age of 70.4 primary infection in a normal, immunocompetent individual is usually subclinical or associated with self - limiting nonspecific symptoms like fever and malaise.9 however, in immunocompromised patients such as hiv - infected persons, reactivation of latent disease can cause life threatening encephalitis;2 offspring of infected mothers may present with mental retardation, blindness, epilepsy, or stillbirth.10 toxoplasmic encephalitis has become one of the most frequent opportunistic infections complicating hiv infection, and the most common cause of focal brain lesion, coma, and death. in nigeria, management of patients presenting neurological symptoms poses a major clinical challenge because of the numerous possible differential diagnoses, which commonly include central nervous system lymphoma, fungal abscess, mycobacterial infection, cytomegaloviral and other direct viral infections, and kaposi s sarcoma among others for which diagnostic procedures may be cumbersome and expensive to undertake.9 this study therefore was designed to compare the pattern of seroprevalence of toxo - igg antibodies in hiv - infected persons without neurological deficits, and hiv - infected persons manifesting any form of neurological complications including motor weaknesses and speech disturbances, seizures, cranial nerve abnormalities, sensory disturbances, cerebellar dysfunction, meningismus, movement disorders, and neuropsychiatric manifestations, and to determine the proportion of these patients whose neurological deficits may be remotely attributable to t. gondii infection this study was carried out at the lagos university teaching hospital, lagos state in the south - west region of nigeria. participants were recruited from the out - patient clinic of the hospital, which had over 6000 registered hiv - positive patients from different socioethnic backgrounds. three hundred and eighty subjects who screened and confirmed positive for hiv-1 or -2 were recruited by random sampling technique at the point of registration. the 380 hiv - positive respondents were separated into 2 study groups based on the presence of clinical evidence of neurological symptoms. of the participants, 300 were without any obvious neurological symptoms while 80 of the hiv - positive participants presented with symptoms which included : neck stiffness, photophobia, tremors, irrational talk, paraesthesia, insomnia, and persistent headache, as isolated disorders or in any combination. patients with previous history of anti - retroviral drug therapy, cerebrovascular accidents, septrin prophylaxis, pyrimethamine, and chemotherapy were excluded from the study. two venous blood samples of 4.5 ml each were drawn from each subject into na - edta specimen tubes. plasma was obtained by centrifugation at 3000 rpm for 5 minutes, and stored at 20c, prior to toxo - igg assay. all collected plasma were analyzed using elisa by immuno - comb toxo - igg kit manufactured by orgenics ltd, yavne, israel, with a sensitivity of 97.2% and specificity of 93.75%. cd4 estimation was carried out with a flow cytometer (partec - model sl3). positive control must produce 2 spots on the card tooth to be valid ; negative control must produce only an upper control spot to be valid. a lower spot with an intensity higher than or equal to that of positive indicates the presence of igg antibody to toxoplasma gondii. the data were analyzed using statistical software spss version 11 (spss inc, chicago, il). the data on qualitative variables were indicated by frequency and percentages, and quantitative variables by range and mean. associations were verified by chi - square with level of significance placed at p < 0.05 (5%). this study was carried out at the lagos university teaching hospital, lagos state in the south - west region of nigeria. participants were recruited from the out - patient clinic of the hospital, which had over 6000 registered hiv - positive patients from different socioethnic backgrounds. three hundred and eighty subjects who screened and confirmed positive for hiv-1 or -2 were recruited by random sampling technique at the point of registration. the 380 hiv - positive respondents were separated into 2 study groups based on the presence of clinical evidence of neurological symptoms. of the participants, 300 were without any obvious neurological symptoms while 80 of the hiv - positive participants presented with symptoms which included : neck stiffness, photophobia, tremors, irrational talk, paraesthesia, insomnia, and persistent headache, as isolated disorders or in any combination. patients with previous history of anti - retroviral drug therapy, cerebrovascular accidents, septrin prophylaxis, pyrimethamine, and chemotherapy were excluded from the study. two venous blood samples of 4.5 ml each were drawn from each subject into na - edta specimen tubes. plasma was obtained by centrifugation at 3000 rpm for 5 minutes, and stored at 20c, prior to toxo - igg assay. all collected plasma were analyzed using elisa by immuno - comb toxo - igg kit manufactured by orgenics ltd, yavne, israel, with a sensitivity of 97.2% and specificity of 93.75%. cd4 estimation was carried out with a flow cytometer (partec - model sl3). positive control must produce 2 spots on the card tooth to be valid ; negative control must produce only an upper control spot to be valid. a lower spot with an intensity higher than or equal to that of positive indicates the presence of igg antibody to toxoplasma gondii. the data were analyzed using statistical software spss version 11 (spss inc, chicago, il). the data on qualitative variables were indicated by frequency and percentages, and quantitative variables by range and mean. associations were verified by chi - square with level of significance placed at p < 0.05 (5%). mean ages of participants (hiv - positive subjects with neurological symptoms and hiv - positive subjects without neurological symptoms) were 39.5 4.8 years and 38.4 7.2 years respectively. a higher proportion of the subjects (140 of 380, 36.7%) were between the age range of 37 to 46 years. of the respondents, 196 were males (51.6%) and 184 were females (48.4%). only 31.6% of hiv - positive subjects had a tertiary education (120 of 380). the neurological symptoms manifested by these hiv - positive respondents were : headache (20%), neck stiffness (14%), photophobia (6.3%), paraesthesia (6.3%), tremor (20%), irrational talk (20%), and insomnia (14%). of these, tremor, headache, and irrational talk constituted the majority of the clinical presentations (60%). seroprevalence of toxo - igg antibody was 40% among the hiv - positive study group with neurological symptoms and 58% among the hiv - positive study group without neurological symptoms (p = 0.00). there was a higher seroprevalence of toxo - igg antibodies among the age range of 3746 years (102 of 140) representing 72.9% of the total in that age group. of the 380 hiv - positive subjects, 206 were seropositive for toxo - igg antibodies (54.2%). however this association was only statistically significant among the study group without neurological symptoms (p = 0.00). cd4 count of all hiv - positive respondents ranged between 7 and 772 cells/l, with a median count of 320 cells/l. of the hiv - positive study group without neurological complications who had cd4 count below 100 cells/l, 79.4% were seropositive for toxo - igg antibody (statistically significant, p = 0.00), compared with 32.2% in subjects with neurological complications. the association between toxo - igg antibody and hiv infection was more significant among the hiv - positive study group without neurological symptoms (p = 0.00). seroprevalance of toxo - igg antibody in the hiv - positive study group with neurological deficit was only demonstrable in 40% of the total respondents (32 of 80), compared with 58% in subjects without neurological complications. mean ages of participants (hiv - positive subjects with neurological symptoms and hiv - positive subjects without neurological symptoms) were 39.5 4.8 years and 38.4 7.2 years respectively. a higher proportion of the subjects (140 of 380, 36.7%) were between the age range of 37 to 46 years. of the respondents, 196 were males (51.6%) and 184 were females (48.4%). only 31.6% of hiv - positive subjects had a tertiary education (120 of 380). the neurological symptoms manifested by these hiv - positive respondents were : headache (20%), neck stiffness (14%), photophobia (6.3%), paraesthesia (6.3%), tremor (20%), irrational talk (20%), and insomnia (14%). of these, tremor, headache, and irrational talk constituted the majority of the clinical presentations (60%). seroprevalence of toxo - igg antibody was 40% among the hiv - positive study group with neurological symptoms and 58% among the hiv - positive study group without neurological symptoms (p = 0.00). there was a higher seroprevalence of toxo - igg antibodies among the age range of 3746 years (102 of 140) representing 72.9% of the total in that age group. of the 380 hiv - positive subjects, 206 were seropositive for toxo - igg antibodies (54.2%). however this association was only statistically significant among the study group without neurological symptoms (p = 0.00). cd4 count of all hiv - positive respondents ranged between 7 and 772 cells/l, with a median count of 320 cells/l. of the hiv - positive study group without neurological complications who had cd4 count below 100 cells/l, 79.4% were seropositive for toxo - igg antibody (statistically significant, p = 0.00), compared with 32.2% in subjects with neurological complications. the association between toxo - igg antibody and hiv infection was more significant among the hiv - positive study group without neurological symptoms (p = 0.00). seroprevalance of toxo - igg antibody in the hiv - positive study group with neurological deficit was only demonstrable in 40% of the total respondents (32 of 80), compared with 58% in subjects without neurological complications. in this study, the data showed a toxo - igg antibody seroprevalence rate of 54.2% in hiv - infected persons. earlier serological studies in many groups showed that the prevalence varied depending on the geographical location, between 15% and 68%. a recent us survey estimated a prevalence rate of 15% in the general population11 and 40% in people with hiv.11 nissapatorn evaluated the prevalence in 505 hiv - positive patients admitted to a hospital in malaysia and reported a prevalence of 44.8%.12 wanachiwanawin estimated a prevalence rate of 53.7% in the hiv - infected population in thailand.13 meisheri in india reported a prevalence rate of 30.9% and 67.8% in the immunocompetent group and hiv - infected persons respectively.14 galva ramirez estimated a prevalence rate of 50% in the hiv - infected population in mexico.15 in africa, lindstrom evaluated 130 hiv - positive patients in uganda and estimated a prevalence rate of 54%.16 the prevalence rate in this study equates well to that seen in the african study in uganda, and that seen in mexico. the slight variations in prevalence rates obtained in this study compared with other earlier studies could be attributed to differences in geographical location. infection is more common in warm climates, and at lower altitudes than in cold climates and mountainous regions.12 however, public enlightenment / health education, and high human development index (hdi) standards could also be responsible for reduced infection. the overall seroprevalence was highest (72.9%), in the 37 to 46 year age group, which is in agreement with an earlier report by meisheri, which showed the highest prevalence within third and fourth decades of life.14 however, nissapatorn reported the highest prevalence rate within the second and third decades of life.12 the increasing prevalence rate with increasing age could be explained by increased risk of exposure to infection with increasing age. there is no statistical significant difference in age distribution between hiv - infected patients with neurological symptoms and those without. most toxo - igg antibody seropositive cases were males. the sex predilection could be attributed to the higher risk of consumption of meat (more likely to be contaminated) among nigerian males.16 there was an association between a cd4 count < 100 cells/l and toxo - igg antibody seropositivity in hiv - positive subjects without neurological symptoms : 79.4% of the hiv - positive study group without neurological symptoms who had a cd4 count < 100 cells/l were seropositive for toxo - igg antibodies (p = 0.00). but this association was not observed in subjects with neurological symptoms (32.2% ; p = 0.26). studies by eliaszewicz in france, which included 172 hiv - infected subjects with diagnosed cerebral toxoplasmosis, showed that 79% of these patients had cd4 count < 150 cells/l.17 this result is contrary to findings in this study, which may further confirm the earlier position that neurological complication among hiv - positive subjects may be significantly unrelated to toxoplasmic infections. johnson and sayles studied resistance to t. gondii infection in mice lacking cd4 expression, and concluded that cd4-deficient mice exhibited impaired resistance to a challenge infection with virulent tachyzoites;18 therefore hiv infection increases susceptibility to t. gondii infection (not necessarily with toxoplasmic encephalitis). thus cd4 serves as a reliable indicator for possible toxoplasmosis, and also contributes significantly to protection against chronic t. gondii infections via their role as helper cells for production of isotype - switched antibodies.18 however, findings from this study showed that only 40% of all respondents with neurological complications were seropositive for toxo - igg antibody compared with 58% of hiv - positive subjects without neurological complications. this implies that 60% of the subjects with neurological complications actually have other possible etiological factors ; therefore the occurrence of neurological complications among participants in this study is not associated with higher incidence of toxo - igg antibody seropositivity compared with hiv - positive subjects without neurological deficit, thus implying that neurological complications observed in this study group might be from direct invasion of the neural cells by the hiv virus, or other common differentials. in this study, the seroprevalence rate of toxo - igg antibody in hiv - infected patients, with and without neurological deficits, was evaluated. it may be concluded that though t. gondii infection is highly prevalent in our environment, it may not be responsible for the majority of cases of neurological disorders observed in our hiv - positive patients. however, extensive study in this area, using more accurate methodology including ct scan and pcr techniques, may be needed to establish the exact incidence of toxoplasmic encephalitis in these patients and evaluate differentials. | backgroundtoxoplasmosis is caused by infection with a ubiquitous intracellular protozoan parasite, toxoplasma gondii. with the advent of the hiv pandemic in nigeria, toxoplasmic encephalitis has become one of the more frequent opportunistic infections and the most commonly implicated cause of focal brain lesions complicating the course of aids.objectivesthis study was conducted to compare the pattern of seroprevalence of t. gondii (toxo - igg) antibodies among hiv - infected persons presenting with neurological complications and those without.materials and methodsplasma specimens collected from 380 subjects were tested for toxo- igg antibodies by enzyme immunoassay technique and cd4 estimation by flow cytometry. close - ended questionnaires were applied to all respondents to collect relevant data, with ethical approval from the hospital ethical committee. plasma was obtained from two study groups comprising 300 hiv - positive respondents without neurological presentations, and 80 hiv - positive respondents with neurological complications.resultsseroprevalence of toxo - igg antibodies was 58% in the hiv - positive study group without neurological complications (of these, 79.2% were males and 38.5% were females) and 40% in the study group with neurological complications (46.2% of these were males and 28.6% were females). the overall seroprevalence of toxo - igg antibodies among the hiv - positive respondents (with and without neurological complications) was 54.2% (206 of 380). seroprevalence of toxo - igg antibodies was lowest among the educated subjects (19% of the respondents with tertiary education) and among females in both study groups. a higher proportion of the subjects with neurological complications had cd4 cell count < 100 cells/l compared with respondents without neurological defects (39% vs 22.7% ; p = 0.000), but the seroprevalence of toxo - igg antibodies was higher in subjects without neurological complications (45% vs 31.3% ; p = 0.000).conclusiontoxoplasmosis, though an important opportunistic infection in our environment, may not account for the majority of neurological complications observed in patients with hiv infection in our center. |
the prevalence of antibodies in adults varies in different geographic regions, from 40%-100%, with lower rates in europe, parts of north america and australia, and higher rates in africa and asia. cmv has been detected in many body fluids, including saliva, urine, blood, cervicovaginal secretions, semen and breast milk. in children, however, the incidence and spectrum of disease in immunocompromised people establish this virus as an important human pathogen. moreover, there are number of reports of severe clinical manifestations of cmv infections in immunocompetent patients. it has been well documented that hemodialysis patients have impaired immune response, which may result in higher prevalence rates of viral infections, including cmv. infections in these patients may be due to primary infection or, more commonly, by reactivation of latent virus or re - infection with exogenous virus, which may be introduced by blood transfusion or kidney transplant. the aim of this study was to analyze the prevalence and dynamics of cmv infection among patients in end - stage renal disease undergoing chronic hemodialysis. during a 3-year period (20102012), serum samples from 162 consecutive patients undergoing chronic hemodialysis were tested for the presence of cmv - specific igm and igg antibodies. the control group consisted of 160 patients presenting for routine check - up with no symptoms of acute febrile disease (antenatal screening, couples undergoing medically assisted reproduction, elective preoperative check - up). the mean patient age was 53.7 12.4 (range 21 - 78) years, whereas the mean age of control subjects was 52.5 12.4 (range 26 - 79) years [figure 1 ]. a subset of 34 hemodialysis patients, enrolled in the transplant program, was evaluated during a period of 2 years on the request of the referring physician. age distribution of study participants anti - cmv igm and igg antibodies were detected using a commercial enzyme - linked immunosorbent assay (elisa) (eti - cytok m / eti - cytok g ; diasorin, saluggia, italy) and expressed in arbitrary units (au / ml). samples with absorbance values greater than or equal to the cut - off value + 10% are considered positive. all igm / igg - positive samples were further tested for igg avidity to confirm or to rule out acute cmv infection. the determination of igg avidity was carried out with urea as the denaturing agent using a commercial assay (avidity : anti - cmv elisa igg, euroimmun, lbeck, germany). the igg avidity index (ai) was calculated and expressed as percentage using the od values with and without urea treatment and interpreted as follows : ai 60% = high avidity antibodies indicating past cmv infection. groups of categorical variables were compared by fischer 's exact test. since distributions of numerical variables were not significantly different from normal distribution (kolmogorov smirnov test), t - test for two groups ' comparison was used. the strength of association between dependent (igg positivity) and independent variables was assessed by univariate and multiple logistic regression. the analysis was performed using software package stata / ic 11.2 for windows (statacorp lp, usa). groups of categorical variables were compared by fischer 's exact test. since distributions of numerical variables were not significantly different from normal distribution (kolmogorov smirnov test), t - test for two groups ' comparison was used. the strength of association between dependent (igg positivity) and independent variables was assessed by univariate and multiple logistic regression. the analysis was performed using software package stata / ic 11.2 for windows (statacorp lp, usa). cytomegalovirus igg antibodies were detected in 147/90.7% (95% ci = 84.494.2) hemodialysis patients and 131/81.9% (95% ci = 75.087.5) control subjects [table 1 ]. logistic regression showed that the overall cmv igg positivity in hemodialysis patients was significantly higher than in controls : crude or was 2.02 (95% ci = 1.24.35), while or adjusted for age and gender amounted 2.18 (95% ci = 1.04.25) [table 2 ]. according to age, a progressive increase in igg seroprevalence was observed in both hemodialysis patients (73.3%96.8%) and control group (42.9%89.3%). comparing seropositivity between the same age groups, patients on hemodialysis younger than 34 years had almost two times higher prevalence than control subjects (73.3% vs. 42.9%) [table 1 ]. older age group was a significant risk factor for cmv seropositivity [table 2 ]. cmv seroprevalence did not differ significantly between males and females (or = 1.22, 95% ci = 0.652.29). prevalence of cmv igg antibodies in hemodialysis patients (n=162) and control group (n=160) logistic regression analysis for the risk of cmv igg seropositivity levels of cmv igg titers in patients and controls are presented in figure 2. a nonsignificant higher proportion of hemodialysis patients (22.8%) showed a very high igg titer exceeding the upper cut - off (> 10 au / ml) compared to 16.4% of controls. the proportions of participants with lower cmv titers were similar in both groups (p = 0.219). cytomegalovirus igg antibody titers in hemodialysis patients and controls cytomegalovirus igm antibodies were detected in three (1.9%) hemodialysis patients and four (2.5%) controls. all three igm - positive hemodialysis patients showed evidence of recurrent cmv infection (igg avidity 81%, 83% and 95%, respectively). one initially seronegative hemodialysis patient (0.6%) seroconverted during the second year of the follow - up period. one of them demonstrated low igg avidity (29%) and two demonstrated borderline igg avidity (42% and 58%, respectively). cytomegalovirus is one of the most frequently encountered opportunistic viral pathogens in immunocompromised individuals, including hemodialysis patients. there are several published articles on seroprevalence of cmv among hemodialysis patients in europe in 1990s. the prevalence of cmv antibodies in this population is reported to be 67% in italy,80% in the czech republic, 83% in germany and 99.3% in serbia. a study conducted in the netherlands in 2007 showed a seroprevalence rate of 68.7% among dutch patients. in croatia, there are very few data on the prevalence of cmv infection. the only published study conducted from 2005 to 2009 among childbearing - aged women showed a seropositivity rate of 75.3%. in the croatian general population, overall cmv seroprevalence is reported to be 63% (78% in adults ; unpublished data of the laboratory for virologic serologic diagnostics, croatian national institute of public health). results of this study showed a significantly higher cmv seropositivity among hemodialysis patients (90.7%) compared to the healthy control group (81.9%). studies conducted in germany and in turkey showed similar results, while in a dutch study, the percentage of cmv - seropositive hemodialysis patients was in the range of the reported prevalence in the general population. a higher cmv prevalence in hemodialysis patients could be explained by the acquisition of cmv through repeated blood transfusions or exposure to cmv during hemodialysis procedures. in hyperendemic areas such as saudi arabia and sudan where high percentages of cmv in the population (up to 100%) were reported, the differences among hemodialysis patients and the general population were not observed. in this study, no significant difference in cmv igg antibody titers between hemodialysis patients and controls was documented. in contrast, a study conducted in thailand showed higher mean antibody titer in patients on hemodialysis compared to healthy controls. our results showed that age was a significant risk factor for cmv seropositivity. a progressive increase in seroprevalence with age age - related increase in the seroprevalence of cmv antibodies has also been shown in some other studies. females generally had higher cmv seroprevalences than males, although in most studies the differences were small. this study showed similar results (seroprevalence in women and men was 96.3% and 87.0%, respectively). among immunosuppressed individuals, reactivation of latent cmv infection seems to be more frequent than that experienced by the general population. reactivation of cmv in hemodialysis patients may be caused by the uremia - associated immunodeficiency in these patients. in this study, the prevalence of active cmv infection (detection of igm antibodies) was similar among hemodialysis patients and control group (1.9% vs. 2.5%). recurrent cmv infection was documented in all hemodialysis igm - positive patients (two of them reported recurrent infection during the second year), compared to one control subject. infection was not confirmed by nucleic acid testing since pcr is not routinely performed in asymptomatic patients. in three participants from the control group, in addition, primary infection was confirmed by seroconversion in one initially seronegative hemodialysis patient during the second year of follow - up period. the results of this study indicate that being on hemodialysis and older age are significant risk factors affecting cmv seropositivity. since primary cmv infection remains a concern due to its potential for severe disease in immunocompromised individuals, identification of seronegative individuals is important in preventing this disease. | cytomegalovirus (cmv) is an important pathogen in immunocompromised individuals. the aim of this study was to analyze prevalence and dynamics of cmv infection among patients undergoing chronic hemodialysis. from 2010 to 2012, a total of 162 patients and 160 control subjects were tested for the presence of cmv igm and igg antibodies using enzyme - linked immunosorbent assay. igm / igg reactive samples were further evaluated for igg avidity to confirm or rule out recent primary cmv infection. the overall igg seropositivity was higher in hemodialysis patients compared to controls (90.7% vs. 81.9% ; crude odds ratio [or ] = 2.02, 95% confidence interval [ci ] = 1.053.89 ; or adjusted for age and gender = 2.18, 95% ci = 1.054.55). cmv igg antibody titers were similar in both groups. there was no difference in cmv prevalence between males (87.9%) and females (96.3%). according to age, a progressive increase in seropositivity was observed in both hemodialysis patients and the control group. three hemodialysis patients (1.9%) developed recurrent cmv infection (positive igm with high avidity igg antibodies). in one patient (2.9%), seroconversion was documented during the second year of the follow - up period indicating primary infection. in contrast, in the control group, recent primary cmv infection (positive igm with low / borderline igg avidity) was demonstrated in three subjects (1.9%), whereas one (0.6%) developed recurrent infection. on multivariate logistic regression, hemodialysis and older age were significant predictors for cmv seropositivity. |
many materials can grow in multiple (meta)stable crystal structures, and phase selection is one of the most fundamental problems in material science for example, many metastable phases are only obtained by rapid quenching of a liquid into a polycrystalline multiphase solid. typical zincblende - structure (zb) iii - v semiconductors can form nanowires in the wurtzite (wz) structure as well as the zb 15. nanowires can be easily switched between structures by varying the temperature, source - material flux, or impurities 3,4,614 ; and their small diameter guarantees that they are single crystals, with phase switching occurring along the growth axis where it is easily observed. since zb and wz have different bandstructures 15, this creates unique opportunities for designing modulated nanowire structures with new electronic properties. crystal phase heterostructures are particularly interesting because they can access the electronic properties of heterostructure quantum dots for photonics and single electron transistor applications, but without the challenges of compositional control 1619. in order to take full advantage of the possibilities offered by crystal structure control, a detailed understanding of the physics behind polytype formation based on post - growth observations, different models have been proposed for the phase selection. experimental results are typically interpreted in terms of the dominant role of one of these factors 68,10,12,20,2628. here, we directly observe the dynamic processes that take place during nanowire growth for each polytype and during the switch between polytypes using in situ transmission electron microscopy. we find surprising differences in the structure and dynamics during growth of zb vs wz. most importantly, the switching process itself, and the associated changes in geometry, offer unexpected clues that allow us to develop a new model identifying the underlying mechanism driving crystal phase selection. in this model, droplet geometry is the key parameter in determining structure, but indirectly via its effect on the nanowire edge morphology. we observed the two gaas nanowire polytypes during growth in situ using a hitachi h-9000 ultra - high vacuum transmission electron microscope (uhv - tem) 29,30. si substrates were first prepared with pre - grown gaas nanowires using standard metal - organic vapor phase epitaxy (movpe) and au aerosol particles with diameters of 30 nm, 50 nm and 70 nm. such samples can be heated resistively in situ, using a pyrometer to calibrate the temperature at each heating current (see methods). pure trimethylgallium (tmga) and arsine (ash3) were used as precursor gases and were introduced close to the substrate using separate capillary tubes to a maximum total pressure during imaging of 2 10 torr. details of how the growth parameters in situ compare to conventional movpe are provided in the methods section. on heating to temperatures around 550c, a liquid auga droplet formed at the nanowire tip and growth took place at the droplet / nanowire interface. dark - field imaging conditions, as used in figure 1 below, allow the crystal structure to be distinguished, in other words the wz polytype and the two twin variants of the zb polytype (extended data figure 1). bright - field imaging conditions, as used in figure 2, allow a more accurate determination of the droplet and nanowire dimensions. we find that in situ, both zb and wz gaas can be grown by varying the precursor pressures (the v / iii ratio) while maintaining a constant temperature. within the parameter range accessible in situ, wz forms at higher v / iii ratios and zb at lower ratios at steady - state conditions ; transient conditions are discussed below. the two polytypes show striking differences in terms of their growth dynamics. for wz, growth proceeds by step flow across the droplet / nanowire interface (figure 1a and supplementary video 1). steps flow slowly with each one starting as soon as the previous one has finished its growth (extended data figure 2a). by counting the number of step flow events and correlating with the length of the nanowire (extended data figure 2b), as in ref. 31, we find that each step flow represents the addition of one wz gaas (0001) bilayer, 0.3 nm in height. the growth rates are low under the conditions accessible in situ, typically one bilayer per minute (see supporting information). growth rates are proportional to ash3 pressure (extended data figure 2c) suggesting that growth under these circumstances is limited by the arrival and incorporation of as (see supporting information). we can then understand the step flow dynamics through the solubility of as in auga. this is generally accepted to be low 32. when the droplet contains no reservoir of the rate - limiting species (in our case, as), the arriving atoms are incorporated immediately into the nanowire, leading to slow and gradual step flow 33. the growth of zb appears quite different from that of wz (figure 1b, supplementary video 2). growth also proceeds by addition of bilayers, but each bilayer flows across the growth interface too rapidly to observe. furthermore, the droplet / nanowire interface shows an oscillating geometry at the trijunction (where solid, liquid and vapor meet) similar to that seen in si, zb gap, ge and al2o3 30,31,34,35. 1b). the three dimensional geometry of this edge facet is shown schematically in extended data figure 1. material gradually adds to the edge facet to fill in the corner (panels 2 - 3). the interface jumps forwards as one step flows quickly, and the edge facet reappears (panels 4 - 5). each oscillation in trijunction geometry is correlated with the nucleation and flow of a new bilayer, as in ref. the step can move quickly, even with no reservoir of as, because it is supplied by material from the truncated volume. a rough estimate of the change in the truncated volume is consistent with it being the source for the 1 bilayer of growth. in parallel with the observed changes in the interface dynamics, the droplet geometry also changes as we vary the v / iii ratio to achieve growth of wz and zb. figure 2 and supplementary video 3 shows the effect on the droplet of changing the arsine pressure between high values, to achieve wz, and low values, to achieve zb, at constant tmga (2 10 torr). temperature was kept constant throughout the experiments (540 - 560c) to avoid introducing temperature dependencies that would obscure the observed trends (see methods). throughout the changes in arsine pressure 2b via droplet aspect ratio h / d (droplet height divided by nanowire diameter at the growth interface), or, equivalently, via the droplet angle (angle between the basal plane and the tangent to the droplet at its edge, see fig. a quasi - steady state volume is reached, depending on the v / iii ratio. these volume changes must be driven by the addition or subtraction of ga : we do not expect au to move in and out of the droplet, since its diffusion on gaas can be assumed to be negligible at this temperature 36, while as comprises only a small fraction of the volume due to its low solubility discussed above. an au - ga - as alloy with over 40% ga forms a liquid at this temperature, with no upper limit for the ga content 32, so droplets of a range of volumes are possible. furthermore, since the volume changes occur more quickly than the rate at which ga could be consumed by incorporation into the growing nanowire, the ga must be supplied or removed by surface diffusion along the nanowire. in the simplest picture 30, there is a surface reservoir of mobile ga adatoms that equilibrate with the droplet over time whenever the chemical potentials of ga in the droplet (and surface) are changed by altering the arsine pressure. (a more complete treatment would include diffusion and the effect of as flux on ga diffusion, but this would not change the general picture. 22) we have shown above that crystal structure and droplet volume both change as the v / iii ratio varies. it is possible to measure crystal structure and droplet volume by alternating between dark- and bright - field imaging conditions. the crystal structure identified during the experiment in figure 2 is shown as the red and blue data points in figure 2b. it is clear that the switch between wz and zb crystal structure occurs as the droplet passes a certain aspect ratio, under these conditions at h / d 0.95 and 125. (other experiments show a small hysteresis not visible in this data, for example fig. s3.) since the droplet takes several minutes to respond to the pressure change, it is clear that wz zb polytypic growth is correlated to the droplet h / d and rather than the instantaneous ash3 pressure. this direct correlation between crystal switch and droplet dimensions (volume, aspect ratio, angle), governed ultimately by the v / iii ratio, is a key result that provides the basis for the model we develop below. understanding that the droplet geometry is the critical parameter, rather than the gas environment itself, provides valuable guidance in growing polytype heterostructures. the length of each polytype segment depends on the time over which the droplet has the appropriate geometry. the relatively slow kinetics of the droplet volume change mean that the v / iii ratio must be designed with appropriate offsets in timing. an example is shown in figure 3. here, the v / iii ratio was set initially at a value that formed wz gaas. the arsine pressure was then decreased, for short pulses, to a v / iii ratio that would be expected to form zb gaas. the result is a series of zb inclusions in a wz nanowire with lengths that are repeatable but not directly proportional to the pulse duration. the correlation between droplet volume and crystal phase in figure 3 confirms that the droplet must reach a critical volume for the structural change to occur ; the reduction of arsine in itself may not trigger a structure change. designing a polytype heterostructure developing a framework to understand the relationship observed above between droplet volume, interface growth dynamics and crystal structure requires two additional key observations. which factor determines crystal structure, the au : ga ratio or the geometry (h / d and) ? to establish this we measured the droplet h / d and at the switch for one particular nanowire, then allowed the nanowire to increase its diameter by conformal growth on the sidewalls, and again measured h / d and while inducing a switch (extended data figure 3). as the nanowire widens, but the amount of au present does not change, relatively more ga is needed to achieve the same h / d and. we did not see any strong effect of diameter on the switch between crystal phases ; h / d and appear to be the controlling parameters. the second observation concerns the relationship between crystal phase and the dynamics at the growth front. in figure 1(c, d) and supplementary videos 2, 4 we show the crystal switch in more detail, specifically the growth of the first zb layer on wz and the first wz layer on zb. 3c) ; the droplet enlarges and the first zb layer forms as the critical h / d and are reached. as the zb step flows (too rapidly to see) across the growth interface, an edge facet appears (supplementary video 4). thus, even though steady state growth of wz proceeds without an edge facet, it is possible to form an edge facet in wz under appropriate conditions. conversely, in fig 1d, we start from a zb nanowire and increase the as pressure ; the droplet shrinks and the first wz layer grows. it grows by slow step flow and without an edge facet appearing (supplementary video 2), even though zb is exposed on the growth interface as it starts to grow. one might expect the presence of an edge facet to be controlled by the crystal structure at that facet ; but instead, figure 1(c, d) shows that it correlates with the crystal structure on the main [i.e. zb (111) or wz (0001) ] growth facet, and thus with the droplet h / d and. to untangle cause and effect, we analyze how the droplet angle will affect the morphology of the growth interface. equilibrium crystal shapes generally do not have edge angles as sharp as 90, so one would expect an edge facet in a gaas crystal. a sharp edge only exists during nanowire growth because the droplet is present, providing a capillary force that can pull on the edge facet and shrink it to zero. we assume an ideal, symmetrical nanowire where the droplet angle is the same all around the edge (see fig. comparing this ideal nanowire having an edge facet to the same geometry but with a sharp edge, the free energy differs by 31 (1)e = c1yl+12y2(cot)(cat0)l+c2y2l where l is the total length of the edge, y is the facet size and is the facet angle (see fig. 4a), cat-0 reflects the supersaturation, and c2 assembles various other second - order terms, as described in ref. 31. for a sufficiently small facet size y, this energy is dominated by the linear term c1, which reflects the capillary forces acting on the corner facet. thus for c10 we expect the edge to be sharp everywhere and have no facet. examining c1 in more detail and including all the capillary terms as described in ref. 37, we find (2)c1=e1sinvslscossin+vlsin where e is the liquid - solid interfacial energy at the edge facet, vs is the vapor - solid interfacial energy on the sidewall, ls is the liquid - solid interfacial energy at the main growth facet, vl is the vapor - liquid interfacial energy, and is the droplet angle (defined in fig. this implies that the droplet angle can alter c1 and hence change the lowest - energy state of the nanowire from one with an edge facet to one with a sharp corner. a hemispherical droplet (=90) gives the maximum possible value of c1, and so is the most favorable for eliminating the edge facet. this is shown in figure 4(b), where we calculate the size y of the edge facet as a function of angle for a symmetrical but otherwise arbitrary illustrative case. in our experiments, the droplet is never less than a hemisphere during stable growth, so the analysis in eqs. (1) and (2) predicts that a switch could occur from having an edge facet for large droplets to having a sharp edge for smaller droplets ; that is indeed what we observe. why should the presence or absence of an edge facet control the crystal phase ? without attempting to develop a microscopic model, we can understand heuristically how this would occur by considering the analysis of glas. several models have argued that the crystal structure should be determined by the location of the nucleation event on the main growth facet 68,10,13,2023,26,38. the argument is that metastable wz can only grow if it has a lower nucleation barrier than zb. with sharp edges, nucleation is expected to occur at the trijunction (rather than in the middle of the facet) so the solid - vapor interface plays a critical role. in particular, the solid - vapor interface energy is thought to be lower for wz nanowires, reducing the nucleation barrier for wz relative to zb in this geometry 20. however, when edge facets are present, nucleation on the main facet occurs away from the trijunction (presumably at position n in figure 4c 31) and the liquid - vapor interface plays no role. if we adopt this argument, it is no surprise that the radical change in the trijunction geometry can result in easier nucleation of wz than zb in one case but not the other. this is also qualitatively consistent with in - situ x - ray diffraction studies which infer (indirectly) that crystal structure in gaas nanowires is determined by geometry of the liquid - vapor interface39,40. here we have not considered effects of interlayer interactions on the nucleation barrier, which may lead to formation of higher - order polytypes under certain conditions41, since we do not observe such phases in our experiments. in figure 4b, it is intriguing to note that our analysis also suggests the possibility of an edge facet and hence zb growth at very small h / d. although we can not access such conditions in our experiments, they may occur transiently at the beginning of nanowire growth, since the droplet has much smaller h / d when sitting on a flat surface 42, and perhaps at the end of growth if material in the droplet is consumed. indeed, zb has been observed at the bases and tips of wz nanowires 20, although under growth conditions that are different enough that our model may not be applicable. recent experiments have in fact shown two transitions, from zb to wz and then back to zb, as v / iii is increased9, consistent with the model in fig. 4. however, since our experiments have only a limited v / iii ratio range, we can not observe the second transition back to zb so can not assess whether the transition is associated with interface dynamics in a way that is analogous to the switch at lower group v. the discussion above is simplified in several respects. no difference in interfacial or surface energies between zb and wz structures the small but real differences could lead to hysteresis in the switching angle as the droplet grows and shrinks. 2, with no strong hysteresis, suggests that the droplet angle has a larger effect than the differences between zb and wz interfacial energies. more importantly, our quasi-2d model treats the droplet angle as uniform all the way around the edge. for the true 3d geometry, in which the droplet sits on a hexagonal prism whose side lengths may not even be equal43, it is clear that will vary around the trijunction. at some point, will become large enough along one edge for that edge to become truncated, even though other edges remain sharp. as the droplet continues to grow we thus expect any nanowire with unequal edge lengths to show a mix of sharp and truncated edges over a range of droplet volumes. it is interesting to note that in the experiments, we observe zb once the first truncated corner appears - as in figure 1 and supplementary videos 2 and 4. since we typically stabilize conditions as soon as we see the crystal switch, the majority of nanowires presented here show a mix of sharp and truncated edges. however, we also observe symmetrically oscillating nanowires, with examples shown in supplementary video 5, presumably because the wire is more symmetrical or the droplet has grown large enough to cause all edges to be truncated. the observation mentioned above that zb grows if any edge is truncated, while wz grows only when all edges are sharp, implies that nucleation of zb at a truncated edge is actually easier than nucleation of either wz or zb at a sharp one. direct observation during growth has enabled us to probe the phenomena controlling crystal phase in nanowires. wz and zb polytypes in gaas appear strikingly different during growth, in terms of the morphology of the nanowire / droplet interface, the flow of steps, and the droplet size. the step flow kinetics can be understood as a consequence of as - limited growth, low as solubility in the droplet, and the role of the edge truncation as an alternative reservoir. examining the switch between polytypes suggests a scenario where the growth conditions (here, the v / iii ratio) determine the volume of the droplet and hence its aspect ratio and angle ; the value of determines whether an edge facet will be present ; the presence or absence of the edge facet determines the nucleation site for a new layer ; and the nucleation site determines which phase, wz or zb, is most likely to nucleate. since nanowire growth has been achieved over a wide range of parameters and growth techniques, it is possible that phase selection is controlled by different physics under different circumstances. however, the regime we analyze here, movpe under as - limited conditions, has advantages for atomic - level control and high - throughput manufacturing. this understanding of the causal sequence, in particular the changes in droplet volume with conditions and the controlling role of the droplet angle, has practical consequences. first, the dramatic changes in droplet volume as a function of conditions may be relevant to aspects of nanowire growth other than crystal phase control13. for example, kinking can be caused by depinning of droplets from the nanowire tip 37,44, and experiments such as those shown here can explore the range of conditions under which droplets attain sizes extreme enough to cause depinning. in terms of crystal phase control, since the au : ga ratio in the droplet appears not to be critical, the results may be applicable to self - catalyzed (au - free) nanowire growth (although any small difference in liquid surface energies may lead to a slightly different critical angle). a second consequence is that any means of controlling the energy balance between a truncated and sharp edge should affect the crystal phase : we have used v / iii ratio in this work, but temperature and surfactants are also possible ways to tune polytypism. we anticipate that similar behavior may occur in other iii - v semiconductors that show polytypism, although it is not guaranteed because the various interfacial energy parameters are material - specific. finally, understanding how crystal structure switching depends on the kinetics of group iii motion into and out of the droplet helps us work towards precise control of individual polytype superlattices, to enable fabrication of new types of electronic devices that make full use of the possibilities for engineering band structure that are provided by polytypic nanowires. the substrates were cut from a si(111) wafer into strips 3 mm 350m 500m, small enough to fit directly into the tem heating holder. the strips were, however, too small to be handled in the gaas growth system, so they were stacked in arrays, parallel to each other with the polished surface facing upwards, and mounted on a larger si wafer. at lund university, au aerosol particles with diameters of 30 nm, 50 nm and 70 nm were deposited onto the arrays of strips using a size - selected aerosol source at a total density of 1 particle per m. then gaas nanowires of the order of 500 nm in length were grown on the arrays using standard metal - organic vapor phase epitaxy in an epiquip system, operating at 100 mbar with ash3 and tmga as precursor gases and h2 as carrier gas. after growth, the arrays were glued to sample boxes using a small piece of sem - type double - sided carbon tape, the sample boxes were placed in a plastic bag which was vacuum sealed, then the bag was sent through air to the uhv tem at ibm. the individual strips were separated and each sample was degassed in uhv by resistive heating below 100c for 30 minutes, flowing a direct current through the si strip. the heating current required for a temperature of around 300 c was then determined in a separate uhv chamber using an infrared pyrometer. all of the strips had a similar temperature - current calibration, so it was possible to estimate the current required to heat the sample to 500 or 550c. the sample was transferred to the uhv tem column to check that the nanowires and au catalysts were still present after this process. finally, tmga was flowed to a chosen pressure around 5 10 torr as measured using a mass spectrometer, arsine was flowed to a chosen pressure around 2 10 torr as measured on the column ion gauge, the nanowires were heated to nominally 500 - 550c and gaas was grown at the nanowire tips. the crystal phase was generally controlled using arsine pressure, which was easier to measure and faster to change than tmga. after experiments on one sample were completed, the full current - temperature calibration curve was obtained for that sample. the reason for this calibration procedure was to prevent any damage (e.g. etching) of the wires by accidental overheating before the growth experiment began. due to drift of the temperature on continued heating the temperature range over which switching occurred became narrower and zb grew for all accessible pressures. above 600c the nanowires etched slowly at the au / gaas interface, presumably due to the low group v pressure. even though the conventional movpe precursor gases are used, there is no h2 carrier gas, as is typically used during movpe growth of gaas. in addition, the absolute pressures of the two precursor species are lower than typical precursor partial pressures used in movpe. the much lower partial pressures can alone account for the low growth rates observed here compared to movpe. to compare the effects of v / iii ratio, we need to consider the possible differences in more detail. the growth in this study was observed to always be group v limited, as seen in extended data figure 2(c). this is in contrast to standard movpe, where high group v flows and group iii - limited regimes are typically used. instead, one could argue that the in situ tem conditions are more similar or relevant to cbe or possibly mbe, rather than movpe. the group v - limited regime is also highly relevant to catalyst - free growth from ga droplets. one exception for movpe is a recent study 9 exploring group v - limited regimes in a standard movpe reactor to grow wz and zb gaas. in this work, it was shown that at low enough v flow to give v - limited growth, wz grows at high v / iii ratio within this regime, while zb grows when v / iii is lowered. this is in contrast to the more well - known behavior in the iii - limited regime, in which zb forms at high v / iii ratio. 9 differ is in the absolute magnitude of the v / iii ratio : here, v / iii ratios of 100 or more were found to yield v - limited nanowire growth, while in ref. 9, the v - limited regime occurred at v / iii ratios below 2. this comparison suggests that the effective as pressure at the growth front is significantly lower, relative to the ga pressure, than in typical movpe. to understand this, we first note that ash3 pyrolysis in gaas growth is generally considered to proceed heterogeneously on gaas surfaces, without interaction with the carrier gas 45. this starts with adsorption of ash3 onto the surface, followed by sequential dissociation of h atoms one by one, eventually leaving atomic as adsorbed on the surface 46. when combined with trimethylgallium (tmga), however, the two species decompose in concert via adduct formation on the surface ; this decomposition pathway does not involve hydrogen and is more efficient than the decomposition of either species alone 45. that the species decompose primarily on the surface is an important clue to the relatively inefficient supply of as in the uhv tem. first, the nanowires are grown on si substrates rather than gaas ; although there is also ample gaas surface on the pre - grown nanowire stubs, this surface is clearly different from the typical gaas substrates used for movpe nanowire growth. second, the surfaces are likely to be passivated with h when growth occurs in a h2 atmosphere ; the absence of h2 here may affect the supply in a number of ways, changing for example the decomposition process and precursor surface diffusion. finally, it must be noted that the decomposition process relies on the desorption of gas - phase as species. this adsorption process naturally depends on the partial pressure ; since as has a significantly higher vapor pressure than ga, the adsorption process of as will be reduced relatively much more by the lower partial pressure. other minor differences are expected due to the experimental setup. using needle valves rather than standard mass flow controllers to control the precursor flows, experimental parameters are less accurately controlled than in dedicated epitaxy growth systems. at the high v / iii ratios used (v / iii>100), a gauge reading the total pressure close to the sample is used to monitor the ash3 flow and provide fast feedback on the actual ash3 at the sample. to monitor tmga, a mass spectrometer is used, with a controlled, steady pressure of tmga set at the start of the experiment and generally held constant. the mass spectrometer is continuously used during the experiments to monitor any drift in the tmga pressure. a) dark field images recorded using three spots in the diffraction pattern showing how wz and the two variants of zb are distinguished in the direction. (c) image recorded during growth, also in the direction, showing the dark field contrast and the measured angle of the truncated corner. (d) a schematic diagram showing the hexagonal cross section as seen from the side (beam direction) and from above. seen from the side, the droplet contact angle, and corner - facet angle,, are shown. (a) step flow kinetics measured for the wz nanowire shown in movie 1 and figure 1(a). the gradient of the graph confirms that each step is 0.33 nm in height, i.e. a wz (0001) bilayer. (c) growth rate vs ash3 pressure, estimated by measuring length increase for the nanowire shown in figure 2 during growth intervals at different ash3 pressures. data showing that the droplet angle at the transition does not change with au composition in the droplet. (a - b) during growth, the crystal structure of a nanowire was switched back and forth several times by changing the as pressure, measuring the angles and absolute droplet volume at which the switch from wz to zb (red) and zb to wz (blue) occurred. during this time shading indicates the range of observed angles at which wz switches to zb (red) and zb to wz (blue). some hysteresis in switching is visible, perhaps because the droplet continues to change angle in the time before the switched layer grows. the data is scattered, but there is no strong dependence of angle on diameter (especially for the blue data points). for example, zb switches to wz at angles between 123 and 132 at both large and small diameter, and wz switches to zb at angles between 127 and 136. (b) switch angle versus inferred composition, as calculated from the measured h / d assuming the amount of au does not change. the data is scattered, but there is no strong dependence of angle on composition. for example, the first few data points, where the nanowire had 30 nm diameter, zb switched to wz for ga < 60%. later, at 40 nm diameter, the switch did not occur until ga 75%. the fact that droplet angles are similar in spite of the diameter change suggests that droplet geometry controls the switch while droplet volume or composition do not appear to be important. | summarycontrolled formation of non - equilibrium crystal structures is one of the most important challenges in crystal growth. catalytically - grown nanowires provide an ideal system for studying the fundamental physics of phase selection, while also offering the potential for novel electronic applications based on crystal polytype engineering. here we image gaas nanowires during growth as they are switched between polytypes by varying growth conditions. we find striking differences between the growth dynamics of the polytypes, including differences in interface morphology, step flow, and catalyst geometry. we explain the differences, and the phase selection, through a model that relates the catalyst volume, contact angle at the trijunction, and nucleation site of each new layer. this allows us to predict the conditions under which each phase should be preferred, and use these predictions to design gaas heterostructures. we suggest that these results may apply to phase selection in other nanowire systems. |
dentine sensitivity is defined as short, sharp pain from exposed dentine in response to thermal, tactile, osmotic, or chemical stimuli that can not be ascribed to any other dental defect or disease. clinically the presence of dentine sensitivity creates challenges for both patients and dental practitioners ; in addition to causing patient discomfort, dentine sensitivity can complicate the provision of restorative treatment and the treatment of periodontal tissues [3, 4 ]. dentine sensitivity is related to the exposure of dentine tubules resulting from either loss of tooth enamel or loss of periodontal tissue (gingival recession) [5, 6 ]. for dentine sensitivity to be incited, dentine tubules must be open at the dentine surface as well as remaining patent to the dental pulp [6, 7 ]. at the microscopic level dentine sensitivity is currently still described by the hydrodynamic theory proposed by brannstrom [8, 9 ], which states that following dentine exposure to stimuli such as physical touch, temperature alteration, sweet liquid, and acidic liquid, changes in intratubular fluid movement and intratubular pressure occur. these changes can cause activation of intratubular nerve fibres of pulpal origin ; this nerve excitation causes pain to be experienced [8, 10 ]. to manage dentine sensitivity products have been developed for at home use functioning primarily through two modalities : suppressing the excitability of intratubular nerve fibres or reducing the patency of exposed dentine tubules [2, 11, 12 ]. when acting to suppress the excitability of nerve fibres within dentine tubules, the most common approach is to elevate the extracellular potassium concentration within the dental pulp. this can be achieved through a patient applying a dentifrice containing potassium ions in a relatively soluble form to exposed dentine. movement of potassium ions through patent dentinal tubules will act to raise the response threshold of pulpal nociceptors and reduce their ability to fire when provoked [13, 14 ]. when considering the effectiveness of agents that reduce dentine sensitivity through reduction in the patency of exposed dentine tubules, the most successful outcomes have been achieved through application of fluoride based gels to dentine [2, 11, 12 ]. it is well documented that application of dentifrices containing stannous fluoride [1517 ] and sodium fluoride [1821 ] can promote the deposition of mineral precipitates within open dentinal tubules, thereby reducing fluid flow in dentine tubules following exposure to stimuli. a dentifrice containing functionalised tricalcium phosphate and 950 ppm sodium fluoride has been shown to enable dentine tubule occlusion in vitro ; application of this dentifrice to extracted bovine teeth following ph cycling was observed to reduce both dentine tubule opening and tubule diameter in comparison to pretreatment levels. this finding is notable, as not only there is limited data demonstrating that 1000 ppmf dentifrices can effectively reduce dentine sensitivity, but the availability of such a dentifrice could lower the cost and complexity of at home treatment undertaken by a patient suffering from dentine sensitivity, a dentifrice containing 1000 ppmf being appropriate for the control of dental caries in low caries risk patients [23, 24 ]. importantly, while assessment of the anticaries benefits of this dentifrice has been reported, no assessment of the efficacy of this dentifrice to reduce dentine sensitivity in vivo has taken place. the aim of the present study was therefore to assess the effectiveness of a dentifrice containing 950 ppm fluoride and functionalised tricalcium phosphate (ftcp) in the reduction of dentine sensitivity in vivo. the null hypothesis was that the effectiveness of the dentifrice containing ftcp and 950 ppm fluoride would not be different to the effectiveness of a dentifrice containing 1000 ppm fluoride in reducing dentine sensitivity. the present study was a single centre, parallel group, blinded (subjects and examiners) randomised controlled clinical trial conducted at the westmead centre for oral health, westmead hospital, sydney, australia. ethical approval from the western sydney local health network human research ethics committee was obtained prior to commencement of the study (sac2010/11/4.6 (3223) hrec/10/wmead/202) and the trial was registered with the australia new zealand clinical trials register. subjects included within the study were from the pool of patients eligible to receive treatment at the westmead centre for oral health. from this patient pool, during an initial off waiting list examination, eight hundred and fifty individuals indicated they suffered from dentine hypersensitivity and were willing to participate in the study. of these 850 individuals a total of 71 subjects were recruited for the study having met the strict inclusion criteria. all subjects which participated in the study consented to participation prior to the inclusion. as part of the consent process, patients were made aware of adverse effects of dentifrices, namely, abrasion and staining of hard and soft tissues and the detrimental sequelae of excessive fluoride ingestion. to be considered for this study, subjects were required to of the following inclusion criteria : being aged between 18 and 70 years;demonstrating good general health with no history of chronic illness;possession of at least 2 teeth with an exposed root surface which are responsive on probing (50 g force) or a 1 s duration cold air blast (70 psi) ; these teeth were not to exhibit diagnosed caries, defective restorations, or signs of fracture on initial assessment ; these teeth were not to have had dental restorations, periodontal surgery, or orthodontics that has resulted in postoperative pain in the immediate past 3 months;a willingness to read, understand, and sign the consent form;a capability and willingness to brush teeth at least 2 times a day for 2 minutes on each occasion. being aged between 18 and 70 years ; demonstrating good general health with no history of chronic illness ; possession of at least 2 teeth with an exposed root surface which are responsive on probing (50 g force) or a 1 s duration cold air blast (70 psi) ; these teeth were not to exhibit diagnosed caries, defective restorations, or signs of fracture on initial assessment ; these teeth were not to have had dental restorations, periodontal surgery, or orthodontics that has resulted in postoperative pain in the immediate past 3 months ; a willingness to read, understand, and sign the consent form ; a capability and willingness to brush teeth at least 2 times a day for 2 minutes on each occasion. a subject was excluded from participation in the study if any of the following conditions applied : use of a desensitising agent in the 3 months prior to the study;undertaking regular medical treatment involving anti - inflammatory or analgesic use;being pregnant or nursing;exhibiting a known allergy to any ingredients in the examined dentifrices;suffering from conditions which could increase the level of acid within the oral cavity : bulimia, gastric reflux disease;excessive dietary exposure to acids ; lemons 2 times per day, raw tomatoes 2 times per day, acidic drinks 1 litre per day (sports drinks, energy drinks, or fruit juice), wine 3 standard glasses per day;an inability to read the oral hygiene instructions provided to each participant. use of a desensitising agent in the 3 months prior to the study ; undertaking regular medical treatment involving anti - inflammatory or analgesic use ; being pregnant or nursing ; exhibiting a known allergy to any ingredients in the examined dentifrices ; suffering from conditions which could increase the level of acid within the oral cavity : bulimia, gastric reflux disease ; excessive dietary exposure to acids ; lemons 2 times per day, raw tomatoes 2 times per day, acidic drinks 1 litre per day (sports drinks, energy drinks, or fruit juice), wine 3 standard glasses per day ; an inability to read the oral hygiene instructions provided to each participant. allocation of subjects to four study groups was randomised through use of a computer algorithm to limit the impact of age, gender, diet, and current level of oral hygiene on the study results. group a. this group brushed teeth twice daily with a dentifrice containing 1000 ppm fluoride ions (mfp) : colgate cavity protection (colgate - palmolive, new york, ny, usa,). group b. this group brushed teeth twice daily with a dentifrice containing 1000 ppm fluoride ions (naf) + 19300 ppm potassium ions (kno3) : sensodyne total care (glaxosmithkline, sydney, nsw, australia). group c. this group brushed teeth twice daily with a dentifrice containing ftcp and 950 ppm fluoride ions (naf) : clinpro tooth crme (3 m espe, st. group d. this group brushed teeth twice daily with a dentifrice containing ftcp and 950 ppm fluoride ions (naf) : clinpro tooth crme (3 m espe, st. paul, mn, usa) and a directed pea sized topical application of clinpro tooth crme onto sensitive teeth before sleeping without rinsing. at no time during the trial were subjects made aware of what test group they belonged to or which product they were using ; all packaging was discarded prior to distribution and toothpaste tubes were wrapped in generic sticky white labelling. at the study commencement, all subjects were provided with a new toothbrush and dental floss and were given the same oral hygiene instructions verbally and as a take home pamphlet. subjects were also provided with a two - minute timer in a bid to maintain strict and consistent adherence to the prescribed two - minute brushing time twice daily. patients were assessed at three time points during the study : baseline, 6 weeks, and 10 weeks. the clinical assessment was completed by three senior dental officers at westmead centre for oral health to limit the effect of clinician variation on study results. standardisation was also maintained through each clinician recalibrating the process of stimulus provision to sensitive surfaces at the commencement of each assessment day. examiners remained blinded to test groups to which participants belonged at all examination points throughout the study. at each assessment, each sensitive tooth surface was exposed to three different stimuli that were applied directly onto the identified sensitive tooth. the three stimuli included an air blast, tactile stimulation, and application of a hypertonic solution. the identified tooth surface was exposed to air delivered from a standard dental unit triplex syringe from an operating distance of approximately 1 cm for a period of 1 s at an operating temperature of 21c (5c). a pressure gauge was mounted to the dental unit and calibrated prior to each assessment day to ensure air was delivered at a standard pressure of 70 psi. the identified tooth surface was stroked for 3 s using a standard dental explorer probe that was held perpendicular to the surface using a force of 50 g. hypertonic solution. a 70% hypertonic sucrose solution was applied to the identified tooth surface for 3 s. the solution was at room temperature at the time of application. following application of each stimulus patients rated the pain / sensitivity experienced using an 11-point numbered pain rating scale (nrs-11 ; figure 1). these scores were recorded on clinical test forms. following the 6-week examination patients returned the study dentifrice wash - out period to elapse before reexamination at 10 weeks to quantify any prolonged actions of each test dentifrice ; 6-week usage of a dentifrice has been shown to provide sufficient time to allow maximum benefit of a desensitising product. patient compliance in the use of the study dentifrice was established at the 6-week examination. patients were asked to return products which were issued and each tube was weighed to calculate the total amount of toothpaste used over the 6-week period. for each test group the mean pain score for each stimulus modality (air blast, tactile, and hypertonic solution) was calculated by patient and by tooth at each assessment point (baseline, 6 weeks, and 10 weeks). additionally, the mean pain scores for each test group for each stimulus were combined and divided by 3 to give a total combined modalities sensitivity (cms) score at each assessment point. the percentage change relative to baseline in mean pain score for each stimulus and the percentage change in cms relative to baseline were calculated for each test group. ancova (with baseline as the covariate) with a tukey hsd post hoc test was used for between group comparisons. of the 850 individuals from the westmead centre for oral health patient pool who indicated suffering from dentine sensitivity, 80 individuals satisfied the inclusion criteria. these 80 individuals were randomly allocated to the four study groups (a d). the study population exhibited a mean age of 40.9 and a range of 1767 years of age. seventy - one of the 80 subjects completed the 10-week clinical study and complied with the protocol given. of the participants who completed the study, there were 54 females and 17 males. no adverse soft tissue or hard tissue effects were observed by the assessing clinicians during the study ; however one participant withdrew from the study reporting an allergic reaction to colgate cavity protection. the 9 patients that did not complete the study did so as they no longer wanted to attend the recall appointments on the basis of convenience. at 6 weeks (end of the treatment phase) all groups showed a reduction in evaporative sensitivity score from baseline, with colgate cavity protection showing a 12% reduction, sensodyne total care a 19% reduction, clinpro tooth crme (brushing only) a 45% reduction, and clinpro tooth crme (brushing + topical application) showing a 43% reduction. the reduction in evaporative sensitivity scores from base line of both clinpro tooth crme groups at 6 weeks was significantly greater (p 0.05, 95% ci) than the reduction in evaporative sensitivity scores of both the positive control group (sensodyne total care) and the negative control group (colgate cavity protection). there was no significant difference (p 0.05, 95% ci) in the reduction of evaporative sensitivity scores from baseline when comparing the sensodyne total care and colgate cavity protection groups at the end of the 6-week treatment phase (tables 1 and 2 and figure 2). at 10 weeks (four weeks after cessation of treatment), all four groups demonstrated a reduction in evaporative sensitivity score from baseline, with colgate cavity protection showing a 18% reduction, sensodyne total care a 40% reduction, clinpro tooth crme (brushing only) a 24% reduction, and clinpro tooth crme (brushing + topical application) showing a 54% reduction. the reduction in evaporative sensitivity scores at 10 weeks for clinpro tooth crme (brushing + topical application) and sensodyne total care was significantly greater (p 0.05, 95% ci) than the reduction in evaporative sensitivity demonstrated by the negative control colgate cavity protection. there was no significant difference (p 0.05, 95% ci) in the reduction in evaporative sensitivity scores at 10 weeks from baseline when comparing groups using sensodyne total care, clinpro tooth crme (brushing only), and clinpro tooth crme (brushing + topical application). at 6 weeks (end of the treatment phase) all groups showed a reduction in tactile sensitivity from baseline, with colgate cavity protection showing a 20% reduction, sensodyne total care a 39% reduction, clinpro tooth crme (brushing only) a 44% reduction, and clinpro tooth crme (brushing + topical application) a 62% reduction. at 6 weeks the only group to show a significant reduction (p 0.05, 95% ci) in tactile sensitivity scores in comparison to the negative control group (colgate total protection) was clinpro tooth crme (brushing + topical application). there were no other comparisons that displayed significantly different sensitivity scores ; both clinpro tooth crme groups were not significantly different (p 0.05, 95% ci) from the positive control group, sensodyne total care (tables 1 and 2 and figure 3). at 10 weeks (four weeks after cessation of treatment), not all groups showed a reduction in tactile sensitivity from baseline, with colgate cavity protection showing a 6% increase in sensitivity. however, sensodyne total care showed a 37% reduction in tactile sensitivity, clinpro tooth crme (brushing only) a 14% reduction, and clinpro tooth crme (brushing + topical application) a 64% reduction from baseline. a significant reduction (p 0.05, 95% ci) in tactile sensitivity at 10 weeks from baseline for clinpro tooth crme (brushing + topical application) and sensodyne total care in comparison to colgate cavity protection was observed. no significant difference (p 0.05, 95% ci) in the reduction in tactile sensitivity scores from baseline to 10 weeks was observed between clinpro tooth crme (brushing only) and sensodyne total care. additionally, no significant difference (p 0.05, 95% ci) in tactile sensitivity score reduction at 10 weeks was observed between clinpro tooth crme (brushing only) and colgate cavity protection (tables 1 and 2 and figure 3). at 6 weeks all groups exhibited a reduction in hypertonic sensitivity from baseline, with colgate cavity protection showing a 32% reduction, sensodyne total care a 41% reduction, clinpro tooth crme (brushing only) a 40% reduction, and clinpro tooth crme (brushing + topical application) showing a 61% reduction. at 6 weeks the difference in % reduction from baseline was not significant (p 0.05, 95% ci) for any of the four groups (tables 1 and 2 and figure 4). at 10 weeks, all groups exhibited a reduction in hypertonic sensitivity from baseline, with colgate cavity protection showing a 42% reduction, sensodyne total care a 56% reduction, clinpro tooth crme (brushing only) a 22% reduction, and clinpro tooth crme (brushing + topical application) showing a 66% reduction. the only groups to exhibit a significant difference (p 0.05, 95% ci) in hypertonic sensitivity reduction at 10 weeks compared to baseline were sensodyne total care and clinpro tooth crme (brushing + topical application). there was no significant difference (p 0.05, 95% ci) in hypertonic sensitivity reduction at 10 weeks between the groups using colgate cavity protection, sensodyne total care, and clinpro tooth crme (brushing + topical application) (tables 1 and 2 and figure 4). at 6 weeks (end of the treatment phase) all groups exhibited a reduction in the combined sensitivity score (cms) from baseline, with colgate cavity protection showing a 20% reduction, sensodyne total care a 30% reduction, clinpro tooth crme (brushing only) a 42%, and clinpro tooth crme (brushing + topical application) showing a 52% reduction. at the end of the 6-week treatment phase, both clinpro tooth crme study groups showed a significant reduction (p 0.05, 95% ci) in cms when compared to the negative control group using colgate cavity protection. clinpro tooth crme when used with an additional topical application also demonstrated a significant reduction (p 0.05, 95% ci) in dentine sensitivity compared to the positive control, sensodyne total care, after 6 weeks (tables 1 and 2 and figure 5). at 10 weeks (4 weeks after cessation of treatment), all groups showed a reduction in combined sensitivity score (cms) from baseline, with colgate cavity protection showing an 18% reduction, sensodyne total care a 40% reduction, clinpro tooth crme (brushing only) a 24% reduction, and clinpro tooth crme (brushing + topical application) a 54% reduction. the reduction in cms for clinpro tooth crme (brushing + topical application) and sensodyne total care was significantly greater (p 0.05, 95% ci) than the reduction in cms from baseline of the negative control colgate cavity protection at four weeks after cessation of treatment. there was no significant difference (p 0.05, 95% ci) in cms reduction from baseline to 10 weeks between groups using sensodyne total care, clinpro tooth crme (brushing only), and clinpro tooth crme (brushing + topical application) (tables 1 and 2 and figure 5). the ability for fluoride ions sourced from a dentifrice to form fluoride - calcium precipitates that occlude dentine tubules with the consequence of reducing dentine sensitivity is well documented [1821, 27, 28 ]. this ability occurs due to the negative electric charge of fluoride ions, which results in their binding with calcium cations. once bound to calcium cations present within a dentifrice the fluoride ions are rendered incapable of combining with cations present at the tooth surface. to overcome the effect of reduced fluoride ion availability at the tooth surface caused by intradentifrice fluoride - calcium bonding, dentifrice manufacturers have traditionally acted to increase the concentration of fluoride ions within a dentifrice. in recent times, however, altering the chemical structure of calcium complexes within a dentifrice to reduce the bonding affinity between calcium cations and fluoride anions has been undertaken as an alternative solution to simple fluoride ion concentration increase. one such example of this has been the development and incorporation of altered calcium phosphate complexes, such as functionalised tricalcium phosphate, which is currently incorporated within clinpro tooth crme. functionalised tricalcium phosphate (ftcp) is produced through beta tricalcium phosphate (tcp) complexes being milled with sodium lauryl sulfate (sls). the tcp crystal structure exhibits several reactive sites including calcium - oxygen clusters (cao3, cao7, and cao8) and lattice defects. these reactive sites are available to undergo chemical interaction with anions such as fluoride ions. to reduce the reactivity of these sites within b - tcp complexes the incorporation of sls within the tcp structure therefore impedes fluoride ions from combining with calcium ions in the dentifrice, so in turn potentially increasing the concentration of both calcium and fluoride to tooth surfaces. [25, 3234 ] have reported a synergistic relationship between ftcp and fluoride with regard to enamel remineralisation ; enamel that is remineralised through a fluoride / ftcp combination demonstrates significantly greater surface and subsurface rehardening following ph cycling compared to that achieved by fluoride application alone. notably, the results of the present study suggest that this remineralisation synergy between fluoride and ftcp may also produce a benefit in terms of enhancing dentine tubule occlusion ; the two test groups that utilised clinpro tooth crme (950 ppm f) demonstrated a greater level of sensitivity relief for all stimuli except the hypertonic test at 10 weeks (group c) in comparison to the group using colgate total, despite the fact that colgate total contains a greater concentration of fluoride ions (1000 ppm f). previous microscopic analysis showing the ability of clinpro tooth crme to reduce the diameter of tubule openings following ph cycling to a greater degree than sensodyne nupro 5000 and topex renew supports this possibility. within the present study a greater reduction in dentine sensitivity was observed when an additional topical application of clinpro tooth crme was placed to supplement application provided through brushing alone. this finding is in contrast to reports in the literature stating that there is no evidence to suggest that additional topical application increases the effectiveness of a desensitizing dentifrice. first an intentional topical application of a dentifrice potentially allows a greater concentration of fluoride ions to be applied to a tooth surface in comparison to that applied through unintentional brushing alone. secondly, through an additional topical application, fluoride ions can remain at a sensitive surface for a longer duration than following brushing and rinsing and thirdly through increasing the duration of fluoride ion presence upon a sensitive surface, the propensity for fluoride migration and tubule occlusion is raised. the greater success of clinpro tooth crme when applied as an additional topical application rather than brushing alone may also be a result of greater salivary fluoride concentration as a by - product of topical application and no rinsing following application. this possibility is consistent with studies that have examined the effect of patient activity following dentifrice application on the level of salivary fluoride. nordstrm and birkhed determined that a topical application of dentifrice to a tooth surface combined with regular brushing resulted in a greater concentration of salivary fluoride than when compared to brushing alone. sjgren and melin identified that a single post - brushing rinse with fluoridated water decreased fluoride concentration by a factor of two when compared to no rinsing, while rinsing with fluoridated water two times following brushing decreased salivary concentration by a factor of 5 when compared to no rinsing. the reduction in cms of the negative control group over the study duration can be in part attributed to the placebo effect which is well documented in dentine sensitivity studies [7, 38, 39 ]. additionally the hawthorne effect, which describes a positive response to noninterventional treatment, should also be assumed to have had some impact on the study results. improved oral hygiene in patients enrolled in the study could have also reduced perceived dentine sensitivity across all groups as a reduction in tooth surface plaque facilitated increased dentifrice access to dentine tubules. these reasons may also account for the antihypersensitivity effects continuing for the month following the cessation of the use of the treatment dentifrices. an identified limitation of the study was the numbers of patients enrolled in each study group. due to the breadth of the potential patient pool, patients eligible for treatment at westmead centre for oral health, it was anticipated that including 40 subjects per group would be an achievable goal over a 3-year study period. as a result of the very strict exclusion criteria, especially the requirement that the use of a desensitising agent in the 3 months prior to the study excluded an individual from being a subject, patient recruitment was extremely difficult. however despite the smaller numbers than originally forecast per group, the results of the present study provide useful information. not only are the numbers sufficient to demonstrate statistical significance, but when tooth number rather than patient number is used for analysis the subject number exceeds 50 for each group and the statistical outcomes remain the same. the addition of ftcp to a dentifrice suitable for daily oral hygiene can enhance the ability of dentifrice fluoride to reduce dentine sensitivity. in the present study twice daily brushing with clinpro tooth crme resulted in a similar reduction in dentine sensitivity to that achieved through brushing with a dentifrice containing kno3 + f (sensodyne total care). if clinpro tooth crme is used twice daily for brushing in combination with a nightly topical application, it can be more effective in reducing dentine sensitivity than twice daily brushing with sensodyne total care. | background. to assess the clinical efficacy of a dentifrice containing fluoride and functionalised tricalcium phosphate (ftcp) in reducing dentine sensitivity. methods. a 10-week parallel blind randomised control trial was conducted. subjects were assigned to one of four groups and instructed to brush twice daily : a : colgate cavity protection (1000 ppmf - mfp) ; b : sensodyne total care (1000 ppmf - naf + 19300 ppmk+-kno3) ; c : clinpro tooth crme (950 ppmf - naf + ftcp) ; and d : clinpro tooth crme (brushing + additional topical application). seventy - one patients were assessed at baseline, 6 weeks, and 10 weeks for cold, tactile, and hypertonic sensitivity using the nrs-11 pain rating scale. a combined modalities sensitivity score (cms) was calculated. results. at 6 weeks, patients reported the following reduction in cms : a (20%) ; b (30%) ; c (42%) ; d (52%). at 10 weeks, patients reported the following reduction in cms : a (18%), b (40%), c (24%), and d (54%). the only cms comparisons to show a significant difference (p < 0.05) were between groups a and d (6 and 10 weeks). conclusions. addition of ftcp to a dentifrice enhances the ability of dentifrice fluoride in reducing dentine sensitivity. using clinpro tooth crme twice daily for brushing can be as effective to reduce dentine sensitivity as twice daily brushing using sensodyne total care. however, additional nightly topical application of ftcp, in addition to twice daily brushing, showed an enhanced reduction in dentine sensitivity. |
spinal tumors are known to be relatively rare among neoplastic diseases. but as the expected length of life extends every year and due to the rapid development in diagnostic tools, the incidence of spinal tumors is increasing markedly. as a result, many studies on spinal tumors are under progress. spinal tumors often do not accompany symptoms, but as it progresses, it may accompany muscle weakness, pain, or even serious disability such as quadriplegia. therefore, like other tumors, early discovery and treatment may have an absolute impact on the prognosis of the patient2,6). generally, for spinal tumors, metastatic spinal tumor is known to be much more common than primary spinal tumor. therefore, once a spinal tumor is identified on imaging studies such as ct scan or mri, full metastasis work up for finding a probable primary location of the tumor is essential. especially more in case of patients with a previous cancer history (lung cancer, breast cancer, or else). it is easier to regard the spinal tumor as metastatic lesion from the existing tumors of other organs. an abdominal ct scan was taken of a 57-year - old male patient who had sacral and right buttock and thigh pain, and revealed with a tumor - like lesion at the t12 vertebrae. previously in 2007, the patient had been diagnosed with colon cancer, confirmed as adenocarcinoma by open biopsy. targeting the cancer lesion, only endoscopic mucosal resection was executed at that time and was considered as a cure. after a period of 3 years, on a follow up large intestine endoscope, a recurred lesion was identified, thus abdominal low anterior tumor resection with colostomy was performed immediately. the patient was also diagnosed with liver cancer in 2009, which was confirmed as hepatocellular carcinoma. about this hepatic lesion the t12 level tumor mass was first discovered in the abdominal ct which was taken to confirm the hepatic lesion, and in the follow - up abdominal ct executed 6 months later, the diameter of tumor mass was increased from 1.5 cm to 4 cm (fig. 1, 2). first, it was presumed that the lesion might be a metastatic lesion originated from the primary colon or liver cancer considering the previous cancer history of the patient. as a treatment for the vertebral lesion, total laminectomy, right hemi - vertebrectomy of t12 and tumor resection was performed, and pedicle screw fixation of t10, t11, l1, and l2 vertebral bodies were done. observed intra - operatively, the tumor was found to be a brownish, hypervascular, and friable mass compressing the thecal sac on right side of t11 - 12 level, and additional erosion of the t12 lamina was also identified. on the final pathologic report, however, it was confirmed as plasmacytoma (fig. 3, 4), which was completely different pathologically from previously diagnosed colon cancer or liver cancer. on additional immunostaining, the tumor cells also showed a monoclonality for lambda immunoglobulin in its light chain. in conclusion, the patient was finally diagnosed to have triple primary - origin tumors of adenocarcinoma, hepatocellular carcinoma, and vertebral plasmacytoma. generally, spinal tumors are mostly metastatic tumors, and those that often cause metastasis to spine include lung cancer, breast cancer, and prostate cancer. on the contrary, primary spinal tumor is very rare so that among the most representative of such, multiple myeloma is found in 1 - 2 persons per 100,0001), and chordoma is found in 0.5 persons per 100,0003). therefore, when a patient is found with a lesion suspicious of a tumor in the spine, the expectation is that it is a metastatic lesion and the metastatic examination and treatment are conducted, and in most cases the future biopsy results are consistent. under such circumstances, when a lesion suspicious of spinal tumor is found in a patient already diagnosed with tumors in other organs, the probability and the suspicion that this lesion is a spinal metastasis from an existing tumor are bound to be even greater. as in our case above, however, there clearly is the case where the spinal lesion is primary and a completely different type of tumor is diagnosed despite having two different types of tumors in different organs, and the treatment directions may differ completely depending on this. for example, when a lesion suspicious of tumor is found in spine from the imaging study executed with the backache as the main symptom for a lung cancer patient, generally it is considered the spinal metastasis of the lung cancer and only palliative radiation therapy may be executed. if the actual spinal lesion was chordoma, however, the treatment method must be completely different - it is resistant to general radiation therapy, so the treatment direction should be surgical removal and proton - beam radiation4,5). for this reason, it is emphasized that even if the discovered spinal lesion is suspicious of being a metastatic lesion, histological confirmation should be executed when possible. triple primary - origin tumor is a very rare case across the world ; this report intended to cover the case report and also the necessity of histological confirmed diagnosis of spinal tumor. so far, it is difficult to make a perfect differential diagnosis with imaging study alone without a biopsy for modern technology, and this may be resolved with development of imaging and interpretation technologies in the future. with this report, we wish to emphasize the necessity of pathologic confirmation and adequate further treatment even in a patient with known malignancies. | generally, among the extradural spinal tumors, metastatic spinal tumor is much more common than primary spinal tumors. thus, in the case of a spinal tumor patient with cancer history (such as lung cancer, breast cancer, etc.), we used to infer that the spinal lesion is the metastasis from, primary malignancy. we introduce an experience of a case of triple primary origin tumor in a 57-year - old man. when the spinal lesion was found on the abdominal computed tomography scan, he already had a history of colon cancer and liver cancer. initially, it was thought that the lesion would probably be a metastatic tumor from the liver or colon cancers, and the operation was performed accordingly. in the pathologic final report, however, the mass was proven to plasmacytoma - the third primary lesion. the patient underwent chemotherapy after surgery. globally, the triple primary origin tumor has been reported very rarely. with this report, we wish to emphasize the necessity of pathologic confirmation and adequate treatment even in a patient with known malignancies. |
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