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the disease anisakiasis was first recognized by van thiel in 1960.1 it is caused by an infestation of the third - stage larvae of anisakis simplex. a. simplex is distributed worldwide and is frequently found in a large variety of fish consumed by humans. eating uncooked fish or seafood containing the resulting disease, known as anisakiasis or anisakidosis, is due mainly to two mechanisms : allergic reaction and direct tissue damage. the former ranges from isolated urticaria and angioedema to life - threatening anaphylactic shock associated with gastrointestinal symptoms (gastroallergic anisakiasis). allergic reactions can occur after primary infection, solely through the presence of anisakis allergens in food.1,2 tissue damage is due to invasion of the gut wall and development of eosinophilic granuloma, ulcer, or perforation.2,3 cases of gastric anisakiasis (95%) are more common than those of enteric anisakiasis, and the latter is rarely reported.4 anisakis larvae can be seen directly by esophagogastroduodenoscopy or colonoscopy and can be removed with biopsy forceps. only one similar report exists in which an anisakis larva was found with a duodenal ulcer, but it was not reported in detail.3 herein, we report a case of a 47-year - old man who had duodenal anisakiasis with a duodenal ulcer. a 47-year - old otherwise healthy man visited our hospital with a complaint of sharp epigastric pain lasting 2 days. the patient did not have h. pylori and had no abnormal findings on an esophagogastroduodenoscopy (egd) conducted 6 months previously. additionally, he had no recent travel history and no significant medical history ; however, the patient had eaten a raw flatfish 3 days before admission. he had increased bowel sounds and direct tenderness in the epigastric area but did not have abdominal distention or a palpable mass. laboratory tests revealed normal biochemical and hematologic blood indexes, except for an elevated white blood cell count of 15,500/mm with a normal eosinophil count and c - reactive protein level of 3.63 mg / dl. on admission, egd revealed a round to oval active duodenal ulcer, about 10 mm in diameter, with a sharp margin and marginal elevation at the duodenal bulb. at 5 mm away from the ulcer margin, a whitish linear worm was found with half of its body penetrating the duodenal mucosa (fig. 2). additionally, a rapid urease test showed a negative result. a proton pump inhibitor (pantoprazole 40 mg, 1 tablet qd) was administered to treat the duodenal ulcer. the patient 's symptoms rapidly improved after removal of the duodenal anisakis, and the patient was discharged on the third day after egd. the follow - up egd after 4 weeks of treatment for the duodenal ulcer revealed complete healing at the ulcer site without anisakis (fig. anisakiasis, which was first reported by van thiel in 1960,1 is caused by the third - stage larvae of anisakis species, mainly a. simplex, and to a much lesser extent a. physeteris, after the ingestion of raw or undercooked fish or seafood. the adult anisakis lives in the stomach of marine mammals such as whales and dolphins. humans are only accidental hosts.2 anisakiasis is classified as either the luminal or the invasive form, according to the presence of bowel wall invasion by anisakis larvae.2 the luminal form does not cause major clinical symptoms, but the invasive form can. the invasive form is subdivided into gastric and intestinal types, according to the penetration site. cases of gastric anisakiasis (95%) are more common than are those of enteric anisakiasis, and the latter is rarely reported.2,3 anisakiasis is caused by two main mechanisms : allergic reactions and direct tissue damage. the former ranges from isolated urticaria and angioedema to life - threatening anaphylactic shock associated with gastrointestinal symptoms. allergic reactions can occur after the primary infection of anisakis with exposure to allergens in the food. direct tissue damage is due to parasite invasion of the gut wall, development of eosinophilic granuloma, or perforation.4 sudden abdominal pain occurs within 12 to 72 hours after the ingestion of raw fish in cases of acute gastric anisakiasis. in these instances, the larvae can be seen directly by egd and can be removed with biopsy forceps. chronic anisakiasis of the stomach may mimic a peptic ulcer, chronic gastritis, or gastric cancer. there are occasional reports of gastric anisakiasis with a morphology that resembles the borrmann type 2 advanced gastric cancer or a type iia + iii early gastric cancer.5 there are also a few reports of the endoscopic discovery of a worm in the base of a gastric ulcer.6 patients with enteric anisakiasis have diffuse and colicky abdominal pain within several days after ingesting raw fish. it has been reported that anisakiasis is usually a self - limiting disease cured by conservative management for 1 to 2 weeks after the onset of symptoms. a positive result on a serological test has been shown to be helpful when diagnosing ; however, positive ige values against anisakis in subjects without symptoms of allergy can not be considered a reliable indicator. this antibody has been detected in 25% of healthy controls and lacks specificity because of cross - reactivity with other parasite antigens. additionally, it is not generally available and is therefore of limited benefit in early diagnosis.7 increased eosinophil levels are observed in less than half of the patients with anisakiasis, and when the eosinophil levels are increased, they tend to be normal on the first hospital day and gradually rise later.4,7 this case showed a normal eosinophil level ; the eosinophil count is therefore not likely to be a useful marker in diagnosing small - intestinal anisakiasis. intestinal anisakiasis (including duodenal anisakiasis) causes direct damage to the gut wall, whereas the role of an allergenic response in the pathogenesis is not clear. the diagnosis of intestinal involvement is still difficult, and often it is only possible with a histologic specimen. a history of raw fish ingestion within 3 days is the only specific clinical clue to the diagnosis, but it may be missed because the patient is often unaware of the risk.7 imaging may be useful in guiding the diagnosis. plain x - ray is not specific, whereas small - bowel x - ray series may show a localized stenosis due to focal edema.8 sonography provides an easy, noninvasive, inexpensive alternative with high sensitivity, if guided by history and clinical suspicion. a common sonographic finding is a relatively large amount of ascitic fluid and dilatation of the small intestine and marked localized edema of kerckring 's folds.8 serology, if specific ige is used, could be helpful for the diagnosis. in contrast, other techniques (latex - based agglutination procedures, ouchterlony tests, immunoelectrophoresis, immunofluorescence, indirect hemagglutination, complement fixation, immunoblotting, and enzyme - linked immunosorbent assay [elisa ]) are of limited benefit in early diagnosis, because they lack specificity as a result of cross - reactivity with other parasite antigens and are generally unavailable.9 microscopically, a definite diagnosis is confirmed by the recognition of anisakis larvae. the lesions range from diffuse interstitial edema to phlegmonous infiltrate with numerous eosinophils and epithelioid cell granulomas with foreign body giant cells.7 in addition, capsule endoscopy recently revealed small intestine anisakiasis beyond the reach of fiber endoscopy.10 the best treatment for anisakiasis is prophylaxis. the larvae can not survive in temperatures above 60 for 10 minutes or at -20 for 24 hours. if an egd exam reveals that the larvae have invaded the intestine or the stomach wall, then endoscopic removal consists of grasping the body of the worm and slowly pulling it away.5 the patient in our case had duodenal anisakiasis with duodenal ulcer, which is a rarely involved site. additionally, this patient did not have h. pylori or other factors leading to duodenal ulcer, and anisakis did not seem to produce the duodenal ulcer. in the present case, we believe that the possible mechanisms of duodenal anisakiasis accompanied by duodenal ulcer without gastric anisakis were increased gastric acid output, increased gastric emptying, and easily penetrable inflamed mucosa adjacent to the duodenal ulcer. in summary, we experienced a rare case of a 47-year - old korean male who had duodenal anisakiasis accompanied by duodenal ulcer without gastric anisakis, which was successfully treated by endoscopic removal of anisakis with biopsy forceps. | humans can be incidentally parasitized by third - stage larvae of anisakis species following the ingestion of raw or undercooked seafood. acute gastric anisakiasis is one of the most frequently encountered complaints in korea. however, duodenal anisakiasis with duodenal ulcer had not been reported in korea, despite the habit of eating raw fish. in this case, a 47-year - old man was hospitalized because of sharp epigastric pain and repeated vomiting after eating raw fish 3 days previously. on admission, esophagogastroduodenoscopic examination revealed an active duodenal bulb ulcer. at 5 mm away from the ulcer margin, a whitish linear worm was found with half of its body penetrating the duodenal mucosa. herein, we report this case of duodenal anisakiasis accompanied by duodenal ulcer. |
use of allogeneic stem cell transplant (allo - sct) as a therapeutic option for otherwise lethal diseases is continuously increasing. however, graft - versus - host disease (gvhd) remains a major complication of allo - sct, affecting up to 4060% of allo - sct patients. gvhd occurs when immune competent cells, namely, t - lymphocytes, recognize membrane antigens on the donor cells. these membrane antigens include a set of host polypeptides such as major and minor histocompatibility antigens displayed by the human leukocyte antigen system. the polymorphism of these polypeptides triggers t - cell activation and ultimately tissue injury through a variety of cellular effector mechanisms. the activation of the donor immune cells is augmented also by cytokines released from the site of tissue injury associated with the intense conditioning regimen (cytokine storm). acute gvhd (agvhd) usually occurs in the first 100 days after transplantation, whereas onset of chronic gvhd (cgvhd) is observed later. changes in the onset period of both acute and chronic gvhds have been observed, with acute cases occurring 100 days after transplantation and chronic cases noticed earlier than usual. these changes from traditional patterns of acute and chronic gvhd were observed especially in the context of reduced conditioning intensity and use of peripheral blood as a stem cell source [46 ]. over the years, several methods for gvhd prophylaxis and treatment, such as immunosuppressive medications, graft engineering, and cellular therapies, have been explored [714 ]. calcineurin inhibitors and methotrexate (mtx) combination therapy was used successfully to reduce the incidence and severity of gvhd and is the standard of care for gvhd prophylaxis [15, 16 ]. successful gvhd prophylaxis was also reported using combinations with mycophenolate mofetil, sirolimus, and other immunomodulatory agents. other approaches used to reduce the risk of gvhd include use of nonmyeloablative conditioning and gut decontamination to reduce tissue damage and cytokine storm that might trigger gvhd and biological agents that can modify the cytokine and immune response after allo - sct [1824 ]. glucocorticoids are the mainstay of agvhd therapy due to their lymphocytic and anti - inflammatory properties, and these compounds have been the primary treatment of gvhd for more than 3 decades. complete responses to steroid therapy are usually expected in almost one - third of agvhd cases after human leukocyte antigen (hla) matched sibling donor allo - sct, and partial response is anticipated in another one - third of patients [25, 26 ]. however, a lower response rate is observed after unrelated donor allo - sct. currently, there is no standard second - line therapy for acute or chronic gvhd, and several candidate drugs were tested. supplementation of steroid therapy with agents such as antibodies against il-2r, antithymocyte globulin (atg), etanercept, and infliximab did not significantly improve survival over single agent steroids. subsequently, corticosteroids remain the standard of care for initial treatment of agvhd [2730 ]. steroid refractory acute and or chronic gvhd still represents a major therapeutic challenge. furthermore, development of steroid sparing agents to be used either in combination with steroids or in salvage therapy is a top priority in the treatment of acute or chronic gvhd due to the short- and long - term complications associated with steroids use. mtx, one of the earliest drugs used for gvhd prophylaxis, inhibits dihydrofolate reductase and production of thymidylate and purines, thereby suppressing t - cell response and proliferation as well as expression of adhesion molecules [3133 ]. although mtx was widely applied for gvhd prophylaxis, only few published trials addressed its efficacy in the treatment of acute and chronic gvhd [3441 ]. in this review, we pooled data from existing clinical trials to determine safety and efficacy of mtx in the treatment of acute and chronic gvhd. we searched the medline database from january 1985 to april 2014 using the following keywords : graft - versus - host disease treatment and methotrexate, steroid refractory acute gvhd, and steroid refractory chronic gvhd. in addition, reference lists from review articles and selected papers were hand searched. only peer - reviewed original articles written in english language, reporting at least five or more cases of either chronic or agvhd treated with mtx with a description of detailed diagnostic and response criteria as well as clinical endpoints, were included. the objective of this study was to review all the available published clinical trials on the efficacy and safety of low dose mtx for management of acute and chronic gvhd. two trials were excluded (one in chinese language and one on a canine model). eight studies were included (4 single arm prospective phase i / ii studies and 4 retrospective studies). the studies included 238 patients, and the related clinical data are listed in table 1. data from 17 patients were shared between two publications by the same group, and data from the more recent study were used. two reviewers using a standardized approach independently conducted data extraction. data for authors names, journal, year of publication, sample size, mtx dose and duration of treatment, median age of patients, sex, gvhd subtype, additional immunosuppressive drugs used, response to mtx therapy in different gvhd subtypes, numbers of patients assessable for 1 year overall and disease - free survival, and information pertaining to study design were collected. evaluation of response to mtx treatment in steroid refractory or nave acute and chronic gvhd was the primary endpoint, whereas mtx toxicity in these patients was the secondary endpoint. the studies reported a consecutive series of 238 adult and pediatric patients with both acute and chronic gvhds after allo - sct between 1995 and 2013, and a summary of demographic data is provided in table 2. the diagnosis and grading of both agvhd and cgvhd were based on established clinical criteria and were uniform among all studies. diagnosis of agvhd was reported for 113 patients ; 59 patients were refractory to steroid therapy, whereas the remaining 54 were steroid nave at the time of mtx therapy. although most of the patients had low - grade agvhd, comprised between grades i and ii (n = 67), a more severe agvhd, classified as grade ii or iv, was present in 46 patients. the skin, gastrointestinal tract (git), and liver were involved in 89, 57, and 21 patients, respectively. diagnosis of cgvhd was reported for 125 patients ; 39 patients were refractory to steroid therapy, whereas 86 patients were steroid nave. extensive cgvhd was present in 85 patients, and 40 patients exhibited limited cgvhd symptoms. organs involved were the skin (n = 78), oral mucosa (n = 40), git (n = 8), liver (n = 69), and lungs (n = 2). all studies with the exception of those carried out at the university of beijing were conducted with weekly administration of mtx, either orally or intravenously. the median dosage was 7.5 mg / m / week (range 315 mg) for a median of 4 doses (range 350) for treatment of cgvhd. identical dosage for a median of 3 doses (range : 260) was used in agvhd treatment. at the university of beijing, two initial doses of mtx were administered in the first week, followed by weekly doses administered until response ; a patient was defined as nonresponsive after unsuccessful administration of 6 doses. in all studies, mtx was administered in combination with steroids or following treatment with other immunosuppressive drugs. a complete response (cr), partial response was defined as incomplete disappearance of symptoms, with a grade decrease of a minimum of one stage in at least one target organ. for cgvhd, a partial response (pr) was defined as a change from extensive to limited stage or a higher than 50% improvement in objective parameters of cgvhd manifestations, such as surface area involvement of skin and transaminase or bilirubin level. patients were considered to have no response (nr) or treatment failure, if gvhd progressed or failed to improve (stable disease) or disease progressed on treatment. the common terminology criteria (ctc) for adverse events, version 3.0, were used to grade the severity of side effects. the results and discussions of each study were reviewed for the use / tapering off of other immunosuppressive agents after start of mtx therapy whenever available. an or to mtx treatment in agvhd was reported in 79 out of 113 patients (69.9%). cr was observed in 67 out of 113 patients (59.2%) while pr was observed in 35/113 patients (10.6%). the response was better in patients with lower grade agvhd (grades i - ii) as compared to patients with grades iii - iv (or 50/58 (86%) and 11/20 (55%), resp.). response was observed at a higher frequency in skin involvement where or was observed in 72/89 patients (80.6%), whereas or was observed in 35/57 and in 11/23 patients with git or liver involvement, respectively (table 3). the or of cgvhd to mtx treatment was reported in 97 out of 125 patients (77.6%) ; 62/125 patients showed cr (49.6%), whereas 35/125 patients (28%) showed pr. the response was better in 33/40 (82.5%) patients with limited than in 62/85 (72.9%) patients with extensive cgvhd. the response was also variable at different sites of cgvhd involvement, and the best response was observed in cutaneous (60/78 patients (77%)) than in mucosal lesions (17/40 (42.5%)), git involvement (4/8 (50%)), and hepatic involvement (50/69 (72%)). the response in patients with single organ involvement was better than in patients with multiple organ involvement. an or was observed in 47/60 (78.3%) and 61/73 (83.5%), and among these patients cr was observed in 47/60 (78.3%) and 49/73 (67.1%) of patients with single organ involvement agvhd and cgvhd, respectively. agvhd patients with multiorgan involvement exhibited or of 24/38 (63.1%), and among these patients, those with cr were 19/38 (50%). patients with cgvhd and multiorgan involvement presented an or of 32/41 (78%), and cr was observed in 13/41 (31.7%) patients. overall, the low dose of mtx used in these studies was well tolerated with low incidence of grades iii - iv toxicity. hematologic toxicity of grades iii - iv was observed in 47/113 agvhd patients (41.5%) and in 22/125 cgvhd patients (17.6%). in one study, grade iii elevation of transaminases was observed in a small number of patients with agvhd (4/113 (3.5%)). although no grades iii - iv toxicity was observed for other organs, the hematologic, hepatic, mucosal, and pulmonary tissues presented frequently with grades i - ii toxicity. mild impairment of renal functions was reported in only 6 patients whom remained stable without further intervention. although median survival varied among studies, we attempted to calculate survival at 1 year following mtx treatment for both forms of gvhd. the or of agvhd and cgvhd patients after one year of mtx therapy was 84/113 (74.3%) and 115/125 (92%), respectively. no patient with cgvhd died from progression of gvhd (no response), whereas progression of agvhd while receiving mtx therapy was the cause of death in 11/113 (10%) patients. other causes of death were disease relapse, observed in 10/113 and 5/125 patients with agvhd and cgvhd, respectively, and infections, observed in 7/113 and 5/125 patients with agvhd and cgvhd, respectively. an additional 3 patients with agvhd died because of hemorrhage, heart failure, and multiorgan failure. steroid refractory gvhd is a critical medical condition that carries higher risks of morbidity and mortality than common gvhd. to date, there is no sufficient evidence to guide the choice of a second - line treatment for steroid refractory gvhd. selection of a second - line treatment regimen should take into consideration the overall health of the patient, the previous immune suppression regimen, and the expected toxicity of the chosen regimen. mtx is a safe and effective prophylactic treatment for early prevention of gvhd after allo - sct. recent studies suggested that low doses of mtx exhibit antimitotic effects and can induce a sustained suppression of t - cell activation, supporting use of mtx as a gvhd therapy. the safety of mtx for gvhd treatment was confirmed in this report, since most of the toxicities observed in the literature were of grade i or ii. hematologic cytopenia was the most common complication, whereas mucosal, git, hepatic, and pulmonary involvement was reported sporadically. cytopenia of grades iii to iv was reported less frequently than its milder forms and occurred mainly in agvhd, possibly because of the vulnerability of the newly engrafted hematopoietic tissue. in cgvhd, no grades iii - iv hematologic toxicity was observed, and the most reported complication was the suppression of blood counts. in agreement with other studies reporting mtx use for gvhd prophylaxis hepatic grades iii - iv toxicity was reported in only 3.5% of agvhd patients, reflecting the relative safety of mtx in the context of agvhd [43, 44 ]. although immune reconstitution was not assessed uniformly, the relatively low incidence of fatal infectious complications, especially in the context of severe immune suppression dictated by gvhd and its treatment, denotes that mtx did not impair immune reconstitution in those patients. reported that maintenance therapy of childhood acute lymphoblastic leukemia with mtx can cause a profound and rapid short - term drop in t - cell subsets, which reverted to pre - mtx levels one week after administration. the reported t - cell suppression might be responsible for the rapid response of gvhd to mtx, which is followed by early immune reconstitution and is characterized by fewer fatal complications due to infections. mtx was effective in treatment of both agvhd and cgvhd with overall response rates of 69.9% and 77.6%, respectively. better responses were observed in certain groups of patients, including those with lower grade gvhd, cutaneous involvement, and isolated organ involvement. in the multivariate analysis conducted by huang. on 86 cgvhd patients treated with mtx, the better response in these patients might be related to less severe tissue damage and the higher regenerative potential of skin in comparison to other organs, such as liver and lungs. however, this conclusion is speculative due to the lack of histopathology of affected tissues before and after treatment. cr was observed more frequently in agvhd (59.2%) than in cgvhd (49.6%) patients, and this event may be explained by the higher proportion of the more aggressive disease in cgvhd (68%) than in agvhd (40.7%) patients. the high response rate to mtx treatment translated into better utilization of immunosuppressive agents, so that 69.2% and 79.2% of agvhd and cgvhd patients, respectively, were able to either discontinue treatment with or reduce the dose of concomitant immunosuppressive agents such as corticosteroids. this is an additional advantage of mtx therapy for gvhd that is expected to reduce long - term complications and increase the quality of life of affected individuals. patient selection criteria were not uniform, so that the studies reviewed presented high variability in factors such as initial treatment, use of different conditioning regimen, pre- and posttransplant immune suppression protocols, and graft source and hla matching. furthermore, there is no objective way to quantitatively assess and compare responses across the studies and the relatively small patient numbers did not allow subgroup analysis and generation of more robust data. in conclusion, pooling of data from published studies reporting use of mtx for treatment of agvhd and cgvhd highlights the potential of mtx as a gvhd therapy and as a steroid sparing agent. however, prospectively randomized studies with higher patient numbers are needed to confirm the value of mtx for gvhd treatment. | glucocorticoids have been the primary treatment of graft - versus - host disease (gvhd) over the past decade. complete responses to steroid therapy are usually expected in almost one - third of agvhd cases and partial response is anticipated in another one - third of patients. however, for those patients not responding to corticosteroid treatment, there is no standard second - line therapy for acute or chronic gvhd. methotrexate (mtx) for treatment of steroid refractory gvhd has been evaluated in a number of studies. results from peer - reviewed original articles were identified and the pooled data analyzed. despite several limitations in data collection and analysis, weekly administration of methotrexate at a median dose of 7.5 mg / m2 seems to be safe with minimal toxicities in the context of both agvhd and cgvhd treatments. the observed overall response (or) in patients with agvhd to mtx treatment in the published studies was 69.9%, with complete response (cr) in 59.2% and pr in 10.6%. in cgvhd the or was 77.6%, with cr reported in 49.6% and pr in 28% of patients. predictors of better responses were lower grade gvhd, cutaneous involvement, and isolated organ involvement. mtx as a steroid sparing agent might reduce long - term complications and improve the quality of life of gvhd affected individuals. |
many postmenopausal women use long - term hormone replacement therapy (hrt) and for most of them the potential benefits greatly outweigh its possible disadvantages or risks. irregular bleeding occurs in up to 60% of hrt recipients which leads to discontinuation of the therapy in up to one in three users (13). the management of hrt - associated bleeding problems is invariably unsatisfactory, since there are no established methods for regulating or reducing the bleeding. first, the potential health advantages of hrt, apart from the meno - pausal symptom relief, require longer use to be of benefit, although expectation of unscheduled vaginal bleeding may deter many women from starting hrt. second, erratic bleeding in post - menopausal women may be a presenting symptom of malignancies of the cervix, endometrium or ovaries ; therefore, current management protocols which involve invasive and costly investigations, may further result in anxiety. following rapid conversion to 3- and 3-hydroxy - metabolites and the 4-isomer, tibolone may engage the er-, expressing estrogenic actions or the progesterone and androgen receptors and act as a progestogen - androgen (4). tibolone has been administered in europe as an effective alternative to continuous hrt in treating climacteric symptoms and vaginal atrophy, in improving libido and in preventing bone loss (5) with minimal stimulatory effect on the breasts (6, 7) or endometrium (8). these data, however, mostly result from observational studies, while results from randomized controlled trials are highly needed to reach a common conclusion. this randomized controlled trial was undertaken to assess the effects of continuous hrt and tibolone regimen on the frequency of spotting / bleeding episodes and vaginal maturation value (vmv) in menopausal women. a total of 150 healthy postmenopausal women, aged 4560 years, whose last menstrual period was more than a year ago and had a plasma 17-estradiol < 35 the subjects were referred from fadjr hospital and a private clinic in tehran, when they had attended to seek medical advice about their menopause. none of the participants had been treated with hormones known to influence vaginal bleeding during the preceding six months before the study. the initial screening included taking a medical history, and doing physical and gynecological examinations. patients were randomly allocated into three groups, according to a computer - generated list of random numbers, and were followed for six months. india) plus one cal - d tablet (calcium 500 mg and vitamin d 200 iu), (pharmachemi ltd., iran) on daily bases, 50 women received 0.625 mg conjugated equine estrogen plus 2.5 mg medroxyprogesterone acetate (cee / mpa), (aboreihan ltd., iran) plus a daily cal+d tablet, and the remaining 50 women received only a cal+d tablet and served as the control group. symptoms were recorded using a questionnaire that assessed vaginal bleeding or spotting, vaginal dryness and intention to continue the medications. the following definitions were used for bleeding / spotting recordings : bleeding : any vaginal flow requiring more than one sanitary napkin per day. spotting : any vaginal flow requiring not more than one sanitary napkin per day. estrogenisation of the vagina was assessed by performing vaginal smears before and after the treatment by drawing the rounded part of a spatula from the upper portion of each lateral vaginal wall to the lower part of the organ. the resulting specimens were thinly smeared onto a slide and immediately fixed by 95% ethyl alcohol. vaginal smears were examined by a skilled pathologist. vaginal maturation value (vmv) was calculated by multiplying the percentage of cell types by a weighting factor and then adding up the total. the weights applied were : superficial cells 1.0 ; intermediate cells 0.5 ; para basal cells 0. blood chemistry tests were performed for sex hormone binding globulin (shbg) and the free estradiol index (fei = estradiol 100/ shbg). the levels of shbg and estradiol were determined using elisa (manufactured by ibl and drg kits, respectively). they were analyzed in triplicate and in random order so as to reduce system bias and interassay variations. all the measurements were repeated after six months of commencing the study. for a better evaluation of vaginal bleeding, the endomerium was assessed by sonography, and when endometrial double - wall thickness was more than 5 mm, additional biopsies were performed. this study was approved by the ethic committee of tarbiat modares university and the participaants signed an informed consent form. the, kruskal wallis and wilcoxon signed rank tests were used for the analysis of non - parametrical data. for the analysis of parametric data, tukey adjustment for the, kruskal wallis and wilcoxon signed rank tests were used for the analysis of non - parametrical data. for the analysis of parametric data, tukey adjustment for the comparison of means (anova) and paired t - test were used. the, kruskal wallis and wilcoxon signed rank tests were used for the analysis of non - parametrical data. for the analysis of parametric data, tukey adjustment for the comparison of means (anova) and paired t - test were used. four women withdrew from the study in the first month due to breast tenderness in three women and fear of breast cancer in another. during the study two women in the tibolone group, six women in the continuous hrt group and one in the control group interrupted the treatment before the third month due to vaginal bleeding or prolonged vaginal spotting. finally, 47 women in the tibolone group, 42 women in the cee / mpa group, and 48 women in the control group completed the six- month period of the study. no individual with bleeding or spotting was found to have endometrial hyperplasia in endometrial biopsy. there were no statistically significant differences among the three groups for parameters including age, age at menopause, gravidity, bmi and vmv shown in table 1 before the treatment. comparing the demographic data among the three groups of menopausal women on hrt, tibolone and placebo (m sd or n (%)) the cee / mpa group had higher incidence of bleeding and spotting episodes than the tibolone and control groups during the first three months (table 2). bleeding / spotting (b / s) episodes among the three groups of menopausal women on hrt, tibolone and placebo (m sd or n (%)) pa, p - value between tibolone and cee / mpa groups, pb, p - value between tibolone and control groups, pc, p - value between cee / mpa and control groups test, kruskal wallis test wilcoxon signed rank test there were no statistically significant differences in the initial data (table 3) among the three groups. in the tibolone group, vaginal maturation value (vmv) and free estradiol index (fei) increased while the number of women with vaginal dryness and sex hormone binding globulin (shbg) decreased significantly (p < 0.05). in the cee / mpa group, vmv and shbg increased and the number of women with vaginal dryness decreased significantly upon treatment (p < 0.001). there were significant differences between the tibolone and cee/ mpa groups for shbg, fei and the number of women with vaginal dryness (p < 0.001). in comparison with the control group, cee / mpa and tibolone increased vmv and decreased the number of women with vaginal dryness (p < 0.01). women in the tibolone group were more willing to continue the medication in comparison with the cee / mpa and the control groups (p < 0.01). comparing the three groups of menopausal women before and after treatment with hrt, tibolone and placebo (m sd or n (%)) pa, p - value between tibolone and cee / mpa groups, pb, p - value between tibolone and control groups, pc, p - value between cee / mpa and control groups paired t test, test, wilcoxon signed rank test anova (tukey) test, kruskal wallis, test the purpose of administering continuous hrt is to prevent scheduled bleeding and improve compliance among women wishing to remain amenorrheic. however, women on continuous hrt may still develop unscheduled bleeding, a serious and disturbing adverse event which may influences their decision to whether terminate or adhere to the treatment. accepted treatment modalities have been re - evaluated in varying estrogen - progestin dose ratios and alternative therapies have been considered in an attempt to alleviate their side - effects (9). furthermore, investigators have focused on identifying the biological mechanisms responsible for the occurrence of unscheduled bleedings. unscheduled bleedings may be influenced by factors such as menopausal age, bmi, the level of endogenous estrogens and its corresponding effect on mucosal changes in the genital tract (2). the present study was designed to examine the effects of tibolone on the genital tract in postmen - pausal women compared with that of continues hrt over a 6-month period. in the present study, the prevalence of bleeding / spotting episodes in continues hrt was significantly higher as compared to tibolone and the controls. this study supports the results of studies that depict a lower bleeding/ spotting episodes for tibolone intake, than continues hrt (2, 10, 11). in order to acquire objective data vaginal maturation, as assessed by vmi, vaginal dryness and fei are highly reliable predictors of genital tract mucosal changes following different treatment regimens. significant differences in mean vmi, the number of women with vaginal dryness and fei were observed in women in hrt and tibolone groups in comparison with the control group as similarly reported by several other investigators (12, 13). we have shown here by cytological analysis that six months of tibolone therapy produces a significant, sustained oestrogenic effect on the vagina and tibolone in comparison to cee / mpa significantly had lower bleeding / spotting episode rate. tibolone decreased vaginal dryness, as seen in our previous study and improved sexual dysfunction in comparison with cee / mpa (14). as sexuality is an important aspect of health that affects the overall well - being of postmenopausal women, tibolone can be an appropriate choice for hrt in postmenopausal women as it has low bleeding/ spotting episodes. tibolone increased fat free mass without any increase in weight or fat mass in postmenopausal (15). as adipose tissue has adverse effects on vaginal bleeding due to undesirable hormonal changes, this beneficial effect may cause low vaginal bleeding in individuals taking tibolone. the limitation of this study was lack of endometrial assessment by regular sonography, unless vaginal bleeding or spotting was the case. in osteoporosis prevention and arterial effects of tibolone study (opal), endometrial thickness measured by transvaginal ultrasound increased slightly during the first year on tibolone and cee / mpa, but no further progression occurred after three years (11). the rate of bleeding/ spotting episodes decreased progressively in cee / mpa group after three months of treatment. it seems that tibolone can be an appropriate choice for hrt due to lower rates of bleeding/ spotting episodes and higher acceptance rate in postmenopausal women. | introductionmany postmenopausal women who are on hormone replacement therapy discontinue medications due to vaginal bleeding. tibolone, a synthetic steroid, has minimal stimulatory effect on the endometrium. the aim of this study was to assess the effects of continuous hrt regimen and tibolone on the onset of vaginal bleeding and vaginal maturation value.materials and methodsa total of 150 healthy women in postmenopausal period were randomly enrolled in this controlled clinical trial. patients were randomly allocated into three groups, and were followed for six months. the first 50 women received 2.5 mg tibolone plus a cal+d tablet (500 mg calcium and 200 iu vitamin d) daily, the second 50 women received 0.625 mg conjugated equine estrogen and 2.5 mg medroxyprogesterone acetate (cee / mpa) plus one cal+d tablet daily, and the remaining 50 received only one cal+d tablet per day and served as the control group. symptoms were recorded using a questionnaire that assessed vaginal bleeding or spotting, vaginal dryness and intention to continue the medications. vaginal maturation value was assessed by examining vaginal smears before and after the treatment. the results for the three groups were analyzed using statistical methods.resultsin comparison with the control group, cee / mpa and tibolone increased vaginal maturation value and decreased the frequency of vaginal dryness (p < 0.01). women in tibolone group were more likely to continue the treatment regimen than those in the cee / mpa or the control groups (p < 0.01).conclusiontibolone can serve as an appropriate choice for hrt as it has low rates of vaginal bleeding/ spotting episodes and high acceptance rate in postmenopausal women. |
men worry over the great number of diseases, while doctors worry over the scarcity of effective remedies. 500 bc a recurring theme in treating the critically ill, and indeed in all of medicine, is the search for evidence - based practice. as we all know, however, application of such principles is somewhat curtailed by the paucity of data even when considering aspects of treatment that to the ill - informed would seem fundamental. the study by dubois and colleagues in critical care medicine addresses a fundamental question : the use of albumin in the critically ill. one may think this is yet another study adding to the confusion regarding the use of albumin when compared with other colloids as resuscitative fluids. but no, this prospective, randomised controlled study on 100 patients in a mixed intensive care unit (icu) setting crudely examined the effect of albumin on organ function in hypoalbuminaemic patients. g / l were eligible provided that they had an expected length of stay > 72 hours, a life expectancy > 3 months, needed full active treatment and had not undergone albumin administration in the previous 24 hours. the primary endpoint was the effect of albumin administration on the delta sequential organ failure assessment score from day 1 to day 7. three hundred millilitres of 20% albumin was given on day 1, followed by 200 ml / day providing the serum albumin remained below 31 interestingly, nearly 2,000 consecutive patients were screened before an adequate cohort was achieved, with almost 50% of patients not being hypoalbuminaemic on admission or during admission. the results, in brief, showed a greater change in sequential organ failure assessment score between the two groups : 3.1 1.0 in the albumin group compared with 1.4 1.1 in those patients with no albumin administration. the major effects were seen in the cardiovascular, neurological and respiratory components of the sequential organ failure assessment score. potential benefits, however, were seen in those patients receiving albumin ; for example, the mean daily caloric intake was higher and the mean daily volume intake was lower both variables associated with improved outcome. we know albumin is ' safe ' but we do not know whether its usage confers an additional benefit in our critically ill patients, and in fairness the authors do not make any extravagant claims. dubois and colleagues point out that this is a pilot study and will hopefully pave the way for further work that will aid our future decision - making. another intravenous treatment that has sparked much debate over the past few years is that of red blood cell transfusion. anaemia is common in the critically ill, with between 40% and 45% of our patients receiving at least one red blood cell transfusion. a recent prospective study reported in this journal demonstrated that up to 11% of survivors from the icu required red blood cell transfusion within 7 days of icu discharge, in keeping with other studies. unsurprisingly, associated factors for the need for transfusion included the haemoglobin level at discharge, sepsis and unresolved organ failure. there are potential negative outcomes associated with repeated blood transfusion in the critically ill patient, and an alternative approach is the use of erythropoietin. a recent study in critical care medicine examined the use of recombinant erythropoietin in patients in post - icu long - term acute care to assess the impact on the need for transfusion. a total of 84 patients in two centres were randomised to treatment or to placebo in a double - blind trial for a period of 12 weeks, or until death / discharge. the transfusion threshold was a haematocrit level less than 24% together with the clinician 's discretion. patient groups were well matched, including biochemical parameters such as iron stores and serum creatinine (although the starting haemoglobin level was higher in the treatment group). the primary end point was the number of red cell units transfused. at 40 days there was a significant reduction in the number of units transfused between the groups (p < 0.006) ; at 84 days the reduction was still evident but less marked (p < 0.05), reflecting the increased use of red cells in the initial study period. beyond this it is difficult to draw too much more from the paper by silver and colleagues. the patient numbers were small with a relatively short follow - up period, and also the study was powered for the primary endpoint with 86 participants ; however, there were 23 patients who were withdrawn from the study. analysis was on an intention - to - treat basis, but there appears to be a degree of post - hoc analysis in an attempt to add strength to the conclusions provided ; examining mortality / morbidity, the effect on mechanical ventilation and the length of stay in hospital revealed no significant differences between the actively treated and placebo groups. as clinicians we require robust data that we can then apply to our daily practice, and it is difficult to take a message from this study that supports any such change. as concluded by silver and colleagues, larger studies are required to take the investigation of the use of recombinant erythropoietin forward in this patient population. clinical studies with a more robust design are required to investigate safety, efficacy, and potential economic advantages. one area of critical care management that continually provokes debate is the modality of choice for the treatment of acute renal failure in the setting of the icu. the study by vinsonneau and colleagues appears at first glance to answer this burning question ; but although the title promises much, one is left a little dissatisfied. this was a large, prospective, randomised multicentre study in 21 icus over a 3.5-year period, and the organisation involved was impressive. the primary end point was the 60-day mortality following the randomisation of 360 patients to either continuous venovenous haemodiafiltration or intermittent haemodialysis (ihd) in centres familiar with both techniques. as with all studies on acute renal failure, the eligibility criteria suffer from a lack of consensus with regard to a definition for acute renal failure. this is exemplified by the need to change the criteria for entry into this study after 8 months due to the inclusion rate being too low. vinsonneau and colleagues fail to demonstrate any differences in 60-day mortality between the two groups and conclude that all patients with acute renal failure as part of multiple - organ dysfunction syndrome can be treated with intermittent haemodialysis. a problem when comparing any two treatments is ensuring both adequate and comparative dosing regimens, and herein lies a major flaw in this study. the study started more than 7 years ago, during which time the practices in both continuous venovenous haemodiafiltration and ihd have changed considerably. as conceded by vinsonneau and colleagues, this may have lead to changes in investigator practices during the study period, particularly with respect to the delivered dose of renal support. this possibility, however, is hard to ascertain given that the dialysis dose in the ihd group is not stated. interestingly the mortality decreased in the ihd arm of the study over the time of recruitment, which reflected a change in practice towards an increase in dialysis prescription. given the lack of control regarding the dosage in both arms of the study, definitive conclusions are hard to make regarding treatment. furthermore, doubts exist regarding the statistical power of the study a point raised in the excellent accompanying clinical comment, which concludes that whether ihd is as good as or even better than continuous renal replacement therapy can not be answered by this study. the debate surrounding the use of albumin in the critically ill will continue to run until a well - designed, adequately powered study to definitively answer this question is undertaken. this study would need strict adherence to the delivered dose, would need to assess the timing of initiation of treatment (perhaps employing the risk, injury, failure, loss and end - stage kidney classification : rifle criteria) and should examine more secondary endpoints. so what is the answer ? perhaps the best practice is to offer the patient the technique with which the unit is most familiar until we have a definitive answer. | further work on the use of albumin in the intensive care unit is discussed. the interesting pilot study by dubois and colleagues examines the potential benefits for albumin supplementation in the hypoalbuminaemic critically ill patient. maintaining the fluid theme, we discuss recent work on factors influencing post - intensive care unit blood transfusion as well as another study on erythropoietin. finally, a large multicentred trial comparing continuous venovenous haemofiltration with intermittent haemodialysis is outlined, the results of which pose more questions than answers. |
a 35-year - old female patient presented with recent - onset of pain, redness, and lid swelling in the right eye. four years earlier she had undergone bone marrow transplantation (bmt) for aml, with successful remission. she was under treatment with oral steroids for graft - versus - host - reaction (gvh). her best - corrected vision in the right eye was counting fingers and in the left eye was 20/20. the right eye showed lid edema, conjunctival and ciliary congestion, chemosis, a paracentral corneal epithelial defect, 3 + cells in the anterior chamber (ac) with fibrin strands and hypopyon and a posterior subcapsular cataract. applanation intraocular pressure (iop) was 34 mmhg in the right eye and 30 mmhg in the left eye. right fundus could not be seen clearly due to media opacities produced by a combination of corneal epithelial defect, ac reaction, and cataract. initial diagnosis of a corneal epithelial defect in the right eye, and bilateral glaucoma was made. she was started on topical antibiotics (gatifloxacin), ocular lubricants (carboxy - methylcellulose), and mydriatic - cycloplegics (1% atropine) for the right eye and topical glaucoma medications (0.5% timolol) bilaterally. on follow - up, the epithelial defect had healed and the lid edema, redness, and iop had reduced. she was prescribed topical steroids (1% prednisolone acetate) and was reviewed at close intervals. nevertheless, the hypopyon was not responsive even after a week of treatment [fig. 1 ]. lid edema and congestion have reduced after treating glaucoma and ocular surface disease due to graft - versus - host disease (gvh) due to failure of conventional treatment, masquerade syndrome was suspected and investigated. b - scan ultrasonography showed a few intravitreal echoes, choroidal thickening, and exudative retinal detachment [fig. 2 ]. b - scan shows choroidal thickening and elevated retina inferiorly suggestive of choroidal infiltration and exudative retinal detachment aspiration of the ac infiltrate was performed under aseptic conditions. cytology of anterior chamber (ac) aspirate shows malignant cells confirming the rare location of ocular relapse in acute myeloid leukemia (aml) based upon the cytology and ultrasonography, we arrived at a diagnosis of relapse of aml involving the ac and choroid. it is extremely rare for relapse of aml to present with anterior segment manifestations. in a prospective 2-year study of 53 patients undergoing treatment of aml, no patient presented with hypopyon uveitis. our patient presented with multiple possible causes for visual loss and pain including corneal epithelial defect, ac reaction, and posterior subcapsular cataract and glaucoma. the patient was taking oral steroids for gvh, which confounded her initial clinical picture. in leukemic patients who have undergone bmt, the occurrence of gvh can further mask a classic presentation of masquerade syndrome by causing a congested, painful eye. increase in iop can to be due to the presence of tumor cells in the ac. however, in this patient, iop was high in both eyes at initial presentation. the secondary glaucoma was possibly due to a combination of tumor infiltration affecting the right eye and systemic steroid therapy affecting both eyes. posterior segment infiltration could not be visualized due to a combination of corneal, lenticular, and ac pathology. a high index of suspicion is necessary in patients with leukemia who are in remission. in such patients, mumps uveitis and chemical reiter 's syndrome may further complicate the diagnosis. in patients with a malignant cause of masquerade syndrome, cytologic analysis of intraocular fluids has been reported to be an essential diagnostic procedure with positive yield in 64% of cases. although cd56 expression of tumor cells is believed to be associated with cns involvement, immunophenotyping could not be done in our case. since there are few references of immunophenotyping of hypopyon cells in aml, this association with cd56 is yet to be confirmed. it is postulated that the blood - ocular barrier may be responsible for creating a pharmacological sanctuary, resulting in suppression, but not eradication of malignant cells by chemotherapeutic agents. patients with aml treated with radiotherapy and chemotherapy developed leukemic hypopyon 3 months to 9 years after initial diagnosis. all reviewed patients in this report died within 1 year of developing leukemic hypopyon. although the treatment of aml has changed since, their conclusion that leukemic hypopyon is associated with systemic leukemic relapse even if systemic examination reveals no leukemia, except for that in the ac, remains valid today. patients with hypopyon uveitis may present with or without the classical clinical signs of masquerade syndrome depending upon coexisting ocular pathology. judicious use of cytology was useful in differentiating it from true inflammation and confirming this rare complication of aml. timely diagnosis is essential, particularly if the suspicion of ocular or systemic malignancy needs to be validated. | anterior segment infiltration in acute myeloid leukemia (aml) presenting as hypopyon uveitis is very rare. we report this case as an uncommon presentation in a patient on remission after bone marrow transplant for aml. in addition to the hypopyon, the patient presented with red eye caused by ocular surface disease due to concurrent graft - versus - host disease and glaucoma. the classical manifestations of masquerade syndrome due to aml were altered by concurrent pathologies. media opacities further confounded the differential diagnosis. we highlight the investigations used to arrive at a definitive diagnosis. in uveitis, there is a need to maintain a high index of clinical suspicion, as early diagnosis in ocular malignancy can save sight and life. |
back pain can occur due to excessive lordosis and degeneration and may involve vertebral bodies, intervertebral discs, facet joints including all the spinal elements such as flaval ligaments, interspinous ligaments, and posterior vertebral elements. mechanical pressure to the back can cause repetitive strains of the interspinous ligament with subsequent degeneration and collapse of vertebral body. in addition, degeneration of a specific spinal element or a group of elements can result in further degeneration elsewhere in the spine. correct diagnosis and treatment of spinal pain requires a combination of clinical examination and imaging studies. till date, clinical examination and computed tomography (ct) or magnetic resonance imaging (mri) were the only tools available to the clinician. now, with the wider availability of positron emission tomography (pet) systems, fluorodeoxyglucose (fdg) serves as a robust marker to track infection and inflammation. pet / ct combination reveals the source of pain so that proper treatment to be selected. the close approximation of adjacent spinous processes with resultant further degeneration and inflammation was named after christian ingerslev baastrup, a danish radiologist (18551950) in 1933. a 66-year - old adult male presented with renal derangement, anemia, and recent onset of moderate to severe back pain. patient was clinically evaluated and underwent mri of lumbar spine. mri of lumbar spine [figure 1 ] reported t1-weighted and t2-weighted hypointense lesions of lumbar vertebrae along with degenerative changes. based on mri findings patient was referred for a whole body fdg - pet / ct to rule out multiple myeloma. magnetic resonance imaging (sagittal view) of lumbar spine showed ill - defined t1-weighted and t2-weighted hypointense lesions in lumbar vertebrae suspicious for marrow infiltrative disease. cord signal appears normal whole body fdg - pet / ct [figure 2 ] from head to mid - thigh showed abnormal increased fdg uptake in interspinous ligament between l3-l4 vertebrae and the l3-l4 spinous processes (standard uptake value, max 6.2). fdg uptake is appreciated in the apposing surfaces of the spinous processes on transaxial images that show enlargement, flattening, and sclerosis [figure 3 ]. degenerative changes of spine were noted apart from the mild irregularity of the spinous process of l3 with minimal enhancement of the interspinous ligament at l3-l4 level raising the possibility of baastrup 's syndrome. there were no other abnormal sites of fdg uptake in spine, other bones, lymph nodes, and organs. whole body flurodeoxyglucose - positron emission tomography / computed tomography images showed abnormal increased flurodeoxyglucose uptake in interspinous ligament between l3-l4 vertebrae and the l3-l4 spinous processes (standard uptake value, ma 6.2). flurodeoxyglucose uptake is appreciated in the apposing surfaces of the spinous processes on transaxial images that show enlargement, flattening, and sclerosis. degenerative changes of spine were noted apart from the mild irregularity of the spinous process of l3 with minimal enhancement of the interspinous ligament at l3-l4 level raising the possibility of baastrup 's syndrome. there were no other abnormal sites of flurodeoxyglucose uptake in spine, other bones, lymph nodes, and organs. flurodeoxyglucose - positron emission tomography / computed tomography ruled out any flurodeoxyglucose avid marrow involvement (a) high - resolution computed tomography images of the lumbar spine illustrating close approximation and contact of spinous processes at l4-l5 level with sclerosis, flattening, and enlargement of the articulating surfaces (white arrow). (b) axial image in the same patient illustrating normal architecture of l2 spinous process (white arrow) baastrup 's disease usually affects elderly, over 70 years of age ; commonly involves the lumbar spine, especially at l4-l5 level. it usually starts as back pain with midline distribution that worsens during extension, is relieved during flexion and is exaggerated on local application of pressure at the affected spinal level. this disease process usually results from excessive lordosis causing mechanical pressure and repetitive strains of the interspinous ligament with subsequent degeneration and collapse. posterior ligaments are the first to be involved (sprained) as a result of extreme forward flexion followed by the interspinous ligaments. there may be spur formation and further inflammation of the adventitious bursa which is present in the interspinous space. pain may be related to the impingement of nerves due to the inflammation and edema. in patients with baastrups disease, fdg uptake may also be related to the development of highly vascularized granulation tissue / inflammation in response to injury at the junction of the interspinous ligament and spinous process (due to the inherent active metabolism of this region). in < 10% of the patients with symptomatic baastrup 's disease, mri reveal lumbar interspinous bursitis, which is depicted as a fluid - like signal located between the pathological adjacent spinous processes. the hallmark of imaging is the close approximation and contact of adjacent spinous processes, with all the subsequent findings including edema, cystic lesions, sclerosis, flattening and enlargement of the articulating surfaces, and bursitis. the interspinous bursa may extend between the ligamentum flavae in the midline forming an epidural cyst and further contributing to the already existing canal stenosis. our patient showed minimal enhancement of the interspinous ligament at l3-l4 level on the ct part of pet / ct with corresponding focal fdg uptake suggesting local inflammation ; however, there was no evidence of any localized fluid or cystic collection. our patient underwent a 4-week trial of a nonsteroidal anti - inflammatory medication and stabilization exercises for the lumbar spine and was symptomatically better at 2 month follow - up. baastrup 's disease may occur in association with other degenerative factors such as loss of disc height, spondylolisthesis, and spondylosis with osteophyte formation. however, baastrups disease as a single entity is also reported in the absence of the predisposing degenerative factors. apart from the conservative management with analgesics and nonsteroid anti - inflammatory drugs, the other therapeutic strategies include percutaneous infiltrations with long - acting corticosteroids mixed to local anesthetics or surgical therapies such as excision of the bursa or osteotomy. specifically for the percutaneous infiltrations, imaging guidance (pet or fluoroscopy) ensures accurate needle positioning with resultant increase of technical and clinical efficacy and at the same time decrease of potential complications rate. surgery with either partial or total excision of the spinous processes does not always result in pain alleviation. utility of fdg - pet / ct in inflammatory diseases is well known. here is another case illustrating its additional benefits in identifying back pain due to baastrups disease ; a disease due to close approximation of adjacent spinous processes resulting in inflammation of interspinous ligaments and bursa. | baastrup 's disease is a benign condition, which presents as chronic low back pain. it is also known as kissing spine syndrome and refers to close approximation of adjacent spinous processes producing inflammation and back pain. this condition is often misdiagnosed, resulting in incorrect treatment and persistence of symptoms. diagnosis of baastrup 's disease is verified with clinical examination and imaging studies. conventionally, clinicians resort to magnetic resonance imaging (mri) of spine rather than x - ray or computed tomography (ct) in the evaluation of back pain. mri can additionally identify flattening, sclerosis, enlargement, cystic lesions, and bone edema at the articulating surfaces of the two affected spinous processes. studies have reported that 18fluorine fluorodeoxyglucose - positron emission tomography / ct (fdg - pet / ct) can detect a bursitis or an inflammation as a form of stress reaction despite a negative mri and 99mtc methylene diphosphonate (mdp) bone scan. pet / ct is usually not a recommended investigation for this condition. however, this case report highlights the benefit of fdg - pet / ct in identifying the site of inflammatory pathology. it is also known to identify the exact site of inflammation where steroid or local anesthetic injection can be administered to alleviate pain, especially in patients with multilevel vertebral involvement. |
chronic obstructive pulmonary disease (copd) is now considered as a respiratory disease with systemic manifestations.1 copd first affects the lungs primarily, and then structural and functional changes also take place in other organs. the degree of airflow limitation as defined by forced expiratory volume in 1 second (fev1) has been used as an index of disease severity, partly because of its significant relationship with mortality.2,3 guidelines indicate that some multidimensional staging systems including systemic components in addition to airflow limitation may be used to assess disease severity,1,4 although the definition of disease severity remains elusive. the bode index, incorporating measurements of nutrition, airflow limitation, dyspnea, and exercise capacity, has been established for multidimensional staging.5 it is useful in predicting mortality and hospitalizations and in reflecting disease modification and progression.6 more simplified indices such as the ado index, composed of age, dyspnea, and airflow limitation,7 and the dose index, composed of dyspnea, airflow limitation, smoking status, and exacerbation frequency,8 have been developed recently and have been validated. in two european cohorts, both the ado index and the updated bode index significantly predicted the 3-year mortality risk after the first hospital admission due to an exacerbation of moderate - to - severe copd, although the original bode index did not accurately predict such a risk.7 the dose index was related to a range of clinically important outcomes of copd such as health status, hospital admission, or respiratory failure, and was predictive of exacerbations.8 although some multidimensional staging systems have been developed recently, these indices have rarely been compared. more information about the properties of these indices or relationships is needed so that they can be better used in clinical practice or trials and, furthermore, be adopted concretely in the guidelines in future. we hypothesized that the bode, ado, and dose indices would be useful as predictors of mortality in copd, but that they would have some different characteristics. we previously examined the factors related to 5-year mortality using multiple measurements in patients with copd.9 in the present study, by reviewing those data, we analyzed the discriminative and predictive properties of those multidimensional indices in patients with copd. we previously recruited 150 consecutive male outpatients with copd and analyzed factors related to mortality.9 entry criteria included (1) smoking history of more than 20 pack - years, (2) maximal fev1/forced vital capacity ratio of less than 0.7 and prebronchodilator fev1 of less than 80% of the predicted value, (3) regular attendance at kyoto university, kyoto, japan, outpatient clinic over 6 months, (4) no uncontrolled comorbidities, and (5) no copd exacerbations over the preceding 6 weeks. pulmonary function tests were performed at least 12 hours after the withdrawal of inhaled bronchodilators.9 patients performed spirometry at 15 and 60 minutes after inhaling salbutamol (400 g) and ipratropium bromide (80 g) using a metered - dose inhaler with a spacer device. exercise tests were performed on symptom - limited progressive cycle ergometry 60 minutes after the inhalation of bronchodilators as described in detail previously.911 patients wore a face mask and began unloaded pedaling for 3 minutes, after which the workload was increased progressively by an increment of 1 w every 3 seconds to the limit of tolerance. predicted values were calculated using the standard equation.12,13 dyspnea was evaluated by the japanese version of the modified medical research council (mrc) dyspnea scale, which is a 5-point scale (04) based on degrees of various physical activities that precipitate dyspnea.14,15 higher scores on the mrc indicate more severe dyspnea. health status was assessed by the japanese version of the st. george s respiratory questionnaire (sgrq).9,16,17 the sgrq consists of 50 items divided into the three components of symptoms, activity, and impacts, and the total score was calculated with scores ranging from 0 to 100. the bode index was principally based on the original report5 whose score comprises the body mass index (bmi), percentage of the predicted fev1, dyspnea by the mrc, and 6-minute walking distance (6mwd) as an exercise capacity component. scores ranged from 0 (best status) to 3 (worst status) for fev1 and mrc, and 0 or 1 for bmi. in the present study, we employed the modified bode index (mbode), which used vo2 as % predicted, as tested by the progressive cycle ergometry instead of the 6mwd, and was previously developed and validated.18,19 the % predicted was scored as follows : score 0 for > 70% predicted, score 1 for 60%69% predicted, score 2 for 40%59% predicted, and score 3 for 40 70% predicted, score 1 for 60%69% predicted, score 2 for 40%59% predicted, and score 3 for 40<% predicted. points for each component were added together so that the mbode index ranged from 0 to 10. the ado index was calculated based on the original report,7 with the score comprising age, dyspnea by the mrc, and fev1. scores ranged from 0 (best status) to 5 (worst status) for age, 03 for mrc, and 02 for fev1. points for each component were added so that the ado index ranged from 0 to 10. the dose index was calculated primarily based on the original report,8 with scores comprising dyspnea by the mrc, fev1, smoking status, and exacerbation frequency per year. scores ranged from 0 (best status) to 3 (worst status) for mrc, 02 for fev1 and the exacerbation frequency, and 0 or 1 for smoking status. in the present study, the modified dose (mdose) index was calculated using the question during the last year, how many severe or very unpleasant attacks of chest trouble have you had ? on the sgrq, instead of exacerbation frequency over the previous year as indicated in the original manuscript.8 points for each component were added so that the dose index ranged from 0 to 8. results were expressed as mean standard deviation, unless otherwise stated. relationships between the mbode, ado, and mdose indices were analyzed by spearman s correlation coefficient tests. univariate and multivariate cox proportional hazards analyses were performed to investigate the relationships between the clinical measurements and mortality. results of regression analyses were presented in terms of estimated relative risks (rr) with corresponding 95% confidence intervals. baseline characteristics of the 150 male patients with stable copd are shown in table 1 and elsewhere.9 their average age was 69 7 years and the postbronchodilator fev1 was 47.4% 17.4% predicted. the frequency distributions of mbode, ado, and mdose scores are shown in figure 1. the ado scores were more narrowly distributed than the mbode scores, with 55 patients (37%) and 42 patients (28%) scoring 3 and 4, respectively. the median of the mdose score was 2, with the distribution of scores tending to skew toward the lower scores. there were significant relationships between the three indices (mbode vs ado, correlation coefficients [rs ] = 0.60, p < 0.0001 ; ado vs mdose, rs = 0.55, p < 0.0001 ; and mdose vs mbode, rs = 0.64, p < 0.0001). of the 150 patients enrolled, six were unavailable for follow - up and 31 patients died.9 causes of death were as follows : 20 due to copd or copd - related diseases, four due to malignant disorders (two lung cancer cases), one due to myocardial infarction and hepatic failure, respectively, and five due to unknown reasons. univariate cox proportional hazards analyses were performed to investigate the relationship between clinical indices and mortality (table 2 and elsewhere9). age, bmi, fev1, peak vo2, mrc, and the sgrq total score were all significantly related to mortality. all the mbode, ado, and mdose indices were strongly related to mortality (rr = 1.618, p < 0.0001 ; rr = 2.342, p < 0.0001 ; and rr = 1.521, p = 0.0002, respectively). to investigate the abilities of the aforementioned three multidimensional staging methods to predict mortality as compared with airflow limitation, exercise capacity, dyspnea, and health status, multivariate cox proportional hazards analyses were performed (table 3). model 1 includes three analyses investigating the relationships between each index and fev1 with mortality. the mbode and ado indices were more significantly related to mortality than fev1, but mdose index was not. model 3 includes three analyses investigating the relationships between each index and mrc with mortality. these results indicate that predictive properties of mbode and ado indices might be stronger than those of fev1 and mrc as well as mdose index. in contrast, model 2 includes three analyses investigating the relationships between each index and peak vo2 with mortality. peak vo2 was more significantly related to mortality than all three indices (p < 0.0001), indicating that the predictive property of exercise capacity might be stronger than different multidimensional staging. model 4 includes three analyses investigating the relationships between each index and sgrq with mortality. in contrast with model 2, all three indices were more significantly related to mortality than the sgrq total score. to investigate which of the mbode, ado, and mdose indices was the most significantly correlated with mortality, stepwise multivariate cox proportional hazards analysis was performed (table 4). both the mbode and ado indices were significantly related to mortality (rr = 1.351, p = 0.027 ; and rr = 1.653, p = 0.031) but not the mdose index (p = 0.87). we evaluated the predictive and discriminative properties of different multidimensional staging systems in patients with copd. among the mbode, ado, and mdose indices, the mbode index seemed to have the best discriminative property. we found that all three indices were significantly predictive of mortality, although the predictive ability of the mdose tended to be inferior to the other two. in contrast, values for the peak vo2 on progressive cycle ergometry were more significantly correlated with mortality than any of the three multidimensional staging systems. we previously examined predictors of mortality in patients with copd and reported that exercise capacity, dyspnea, and health status were significantly related to mortality independently of airflow limitation.9,20,21 these results suggested the need to incorporate these systemic elements in addition to fev1 in evaluating patients with copd. among the three indices, the mbode and ado indices were more strongly predictive of mortality than fev1, mrc, or sgrq. thus, these multidimensional staging systems seem to be more useful in prognostic assessments than airflow limitation, dyspnea, or health status alone in patients with copd. after evaluating their properties well in different study situations the relationships between the mbode, ado, and mdose indices were moderate, indicating that they do not necessarily evaluate the same aspects of copd, although some components are common to all three indices. in fact, the stepwise multivariate cox proportional hazards analysis showed that both the mbode and ado indices were independently related to mortality in patients with copd. thus, using more than one index might have some complementarily or additive advantage in prognostic assessment. there were differences in the discriminative properties among the mbode, ado, and mdose indices. the mbode score was more widely and normally distributed than the scores of the other indices examined, indicating its superior discriminative property. in our single population sample, the age component of the ado index might have varied less widely than the original report of the ado index,7 as shown here that about 65% of our patients scored 3 or 4. regarding the mdose index, firstly, its range from 0 to 8 is narrower than the bode and ado indices ranging from 0 to 10. secondly, in the present study, the median score on the mdose index was low at 2, and its distribution was skewed toward lower scores, which were consistent with the original report.8 thus, although the ado and dose indices are simple and easy assessments for use in primary care settings, as they do not include an exercise capacity component, their discriminative properties may be limited. in the present study, peak vo2 was more strongly related to mortality than the mbode, ado, and mdose indices, indicating that peak vo2 is an important factor in copd in relation to mortality. in addition to studies examining predictors of mortality in copd,9 peak vo2 is reported to be a useful prognostic factor in other disorders22,23 such as interstitial lung diseases,24 primary pulmonary hypertension,25 cyctic fibrosis,26 or chronic heart failure.27 cardiopulmonary exercise testing has some utility in diagnosis and functional evaluation, and prognostic stratification is considered to be a major indication for cardiopulmonary exercise testing in patients with pulmonary or cardiac disorders.23 in the present study, we compared different multidimensional staging systems from the viewpoint of the relationship of their scores with mortality. although these or similar indices are sometimes referred to as disease severity indices, strictly speaking, disease severity in copd has not been defined. when disease severity is considered according to the closeness of death, however, there are other ideas about defining disease severity, such as its relationship with health status or exacerbations. it seems that multidimensional staging is superior to the current means of disease staging based on the level of fev1.28,29 a standardized definition of disease severity is needed to eliminate confusion and to officially establish a novel disease severity staging system not based solely on fev1 in future. as a limitation, although several multidimensional staging systems in addition to the bode, ado, and dose indices have been developed in recent years,30,31 we did not assess them in the present study. we demonstrated that the multidimensional staging systems of the mbode, ado, and mdose indices were significant predictors of mortality in patients with copd. an understanding of the properties or characteristics of the indices is important when using them in clinical practice, and, at the same time, a definition of disease severity needs to be established in order to propose the indices assessed in this report as a new disease severity index. | backgroundchronic obstructive pulmonary disease (copd) is considered to be a respiratory disease with systemic manifestations. some multidimensional staging systems, not based solely on the level of airflow limitation, have been developed ; however, these systems have rarely been compared.methodswe previously recruited 150 male outpatients with copd for an analysis of factors related to mortality. for this report, we examined the discriminative and prognostic predictive properties of three copd multidimensional measurements. these indices were the modified bode (mbode), which includes body mass index, airflow obstruction, dyspnea, and exercise capacity ; the ado, composed of age, dyspnea, and airflow obstruction ; and the modified dose (mdose), comprising dyspnea, airflow obstruction, smoking status, and exacerbation frequency.resultsamong these indices, the frequency distribution of the mbode index was the most widely and normally distributed. univariate cox proportional hazards analyses revealed that the scores on three indices were significantly predictive of 5-year mortality of copd (p < 0.001). the scores on the mbode and ado indices were more significantly predictive of mortality than forced expiratory volume in 1 second, the medical research council dyspnea score, and the st. george s respiratory questionnaire total score. however, peak oxygen uptake on progressive cycle ergometry was more significantly related to mortality than the scores on the three indices (p < 0.0001).conclusionthe multidimensional staging systems using the mbode, ado, and mdose indices were significant predictors of mortality in copd patients, although exercise capacity had a more significant relationship with mortality than those indices. the mbode index was superior to the others for its discriminative property. further discussion of the definition of disease severity is necessary to promote concrete multidimensional staging systems as a new disease severity index in guidelines for the management of copd. |
the present study was designed to study the feasibility of single miniplate osteosynthesis in the fracture of angle of mandible. in this study 110 patient were included and treated with single miniplate osteosynthesis at upper border along champy 's line of osteosynthesis. all the cases were treated successfully, common complications which we have observed in this study, cosmetic disfigurement, delayed union, infection, wound dehiscence and paresthesia. single miniplate fixation in unfavorable fracture is questionable and hence these fracture require some alternative method (locking plate, etc.) for fixation. mandibular fractures make 80% of all facial trauma and in 20 - 30% cases the fracture occurs in the site of the angle. certain structural and functional peculiarities- thinner compact plate, shape changes during life and frequent impacted or partially erupted teeth, bilateral muscle cover and endosseous and extra osseous blood circulation- condition the peculiarities of the treatment of fracture. the present study was aim to determine the effectiveness and the advantages of non compression miniplate osteosynthesis at upper border of mandible along champy 's lines of osteosynthesis. present study included 150 patients of maxillofacial injuries either reported to trauma center of c.s.m. medical university, lucknow or were referred from other hospitals for specialized treatment between the period jan 2008 and july 2009 at this center. patients detailed history including age, sex, number and location of fractures, time between trauma and surgery were recorded. ninety - eight (98) were men and 52 were women with a mean age of 29 and 26 years, respectively [table 1 ]. patients were operated under local anesthesia/ general anesthesia by open reduction with single miniplate fixation with mmf. data related to the clinical follow - up of wound, occlusion and suture removal, paresthesia and esthetics and radiological observations were recorded at regular intervals upto 3 months. road traffic accidents (70%), assault (12%), fall (11.6%), gunshot injury (3%) and sports (0.3%) were responsible for most of the mandibular injuries. adekeye (1980) reported 76% and ellis, reported only 15% with vehicular accidents. this difference in etiological factors may be explained by the environmental and social characteristics of the locality under study. sex distribution (n=150) there were 92 (61%) fractures of the mandible associated with middle third, 36(24%) isolated mandibular fractures only and 22 (15%) isolated middle third region fractures only [table 2 ]. all the 128 cases of fracture mandibles have total 175 fracture anatomical sites the incidence of mandibular fractures anatomical location wise were as condylar fractures 44 (25%), coronoid process 02 (1%) and fracture of the ramus 3 (2%), fracture of the angle 51(29%), body fractures 40 (23%) and symphysis and parasymphysis 30 (17%) [table 3 ]. all the 51 (29%) patients who had participated in this study, of the mandibular angle fractures were treated with single noncompression miniplate at upper border along champy 's line of osteosynthesis. out of 150 patients of pan facial trauma only 36 patients [table 4 ] had isolated mandibular angle fracture, majority of the patients referred to maxillofacial surgery opd after neurosurgery clearance, postorthopedics treatment. patients usually operated (under la / ga) for angle fracture average 2 - 7 days postinjury. patients were treated intraoral open reduction and 2.0 mm noncompression miniplate fixation at superior border (champy 's technique) and supplemented with mmf for 7 - 14 days. fracture distribution (n=51) fracture of the mandibular condyle is the commonest site for mandibular fracture, the angle fracture is the most frequent site when only one fracture is present (ellis, moos and el attar, haugh). seat belt law have resulted in a dramatic decrease in injury in general and severe injury in particular (thomas 1990), similar trend has been reflected in the incidence of facial injury. ewers and harle (1985) quote the first description of mandibular fracture fixation using plate and screws by hausmann in 1886. there is still debate in the management of angle fracture about the convenience of traditional treatment with mmf versus surgical treatment. spiessl and schroll, described a plate - fixation system which was applied to the lower border of the mandible, the biomechanically most unfavorable site, although used dynamic compression plate and bicortical screws which resulted in rigid fixation to the fracture segments. the ao / asif recommends that sufficient internal fixation hardware be applied to resist the maximum forces of mastication. and to restore the compression and tension trajectories by the application of two compression plates, one along the superior and one along the inferior margin of the buccal cortex but to malunion and malocclusion. ao reconstruction plate should be used in cases of infected and comminuted fractures or bone loss and in oblique fracture also. these plates are thicker stronger and available in 6 - 24 holes length. in 1973, michelet, described the treatment of mandibular fracture using small, easily bendable noncompression miniplates placed transorally and anchored with monocortical screws. champy later performed a series of experiments with miniplate that delineated ideal lines of osteosynthesis within the mandible. plates placed along these lines were thought to provide optimal fixation and stability [figure 1 ]. ideal plate placement for angle fractures was along the superior border of the mandible above or just below the superior oblique ridge. because these plates were small and monocortical screws, placement was possible without damaging the tooth roots [figure 2 ]. champy states that this miniplate system also gives sufficient support and stability to the bone fragments to allow immediate function subsequent clinical studies corroborated the effectiveness of the champy technique.schierle, reported that two - plate fixation may not offer advantages over single - plate fixation in general. champy 's line of osteosynthesis champy technique - single miniplate placement on superior border of an angle region in the present study 8 out of 51 cases (16%) reported postoperative infection ; this observation is probably due to delay in treatment as already mentioned that patients were treated as secondary referrals after postorthopedic treatment and neurosurgical clearance with a mean delay of 2 - 7 days [table 5 ]. another reason to this finding may also be attributed to the poor oral hygiene due to stasis of saliva owing to mmf. other causes that may be reasoned are strong distractive forces at the mandibular angle region and concomitant fractures at other sites of mandible both leading to instability of so called attempted reduction and fixation of fracture fragments and presence of impacted / partially erupted tooth. our results are in corroboration with other studies, quoted as follows fracture of the angle are associated with the highest incidence of postsurgical infection of all mandibular fractures reported by lizuka and lindqvist it is the angle region where the horizontal and vertical rami of the mandible of the mandible joint and where the powerful elevator muscles attached to the ramus transfer their force to the body of the mandible. this creates great demands of fixation if rigidity under functional loads is to be maintained the forces that must be countered in fracture at the mandibular angle have been derived from maximum voluntary bite force measurements, which in a healthy adult male may be in order of 50 kilo pounds (kp). complications associated with methods of fixation (n=51) ellis, examined various treatments modalities for angle fractures. they showed a significantly higher complication rate using compression plates on both mandibular borders intraorally in comparison to the champy technique. thirteen percent rate of infection using a solitary lag screw (ellis and ghali) and a 17% rate of infection when the fracture was treated by mmf with or without placement of a positional wire or plate. iizuka, also noted infection as an important complication and suggested that it can be reduced by experience of the operator. anderson and alpert (1992) observed that infection could be avoided by avoiding a treatment delay. the effects of a tooth in the fracture line as a cause of infection can be minimized by preoperative antibiotic therapy. ezsias and sugar (1994) reported a high incidence of 18 and 25% infection with miniplate fixation at the angle. kuriakose, reported a better treatment outcome for angle and comminuted fractures with rigid plates., published results 110 of 127 potential responses were received (87%). among 104 surgeons who treated mandible fractures, 86 (83%) treat more than 10 mandibular fractures per year the preferred techniques for simple, non comminuted mandibular angle fractures in this group were : single miniplate on the superior border (champy technique) with or without arch bars (44 surgeons, 51%) ; tension band plate on the superior border and nonlocking, bicortical screw plate on the inferior border (11 surgeons, 13%) ; dual miniplates (nine surgeons, 10%) ; a locking screw plate on the inferior border only (six surgeons, 7%), and three - dimensional plates (five surgeons, 6%). a single miniplate on the superior border of the mandible has become the preferred method of treatment among ao faculty. when using large, inferiorly based plates more surgeons are now favoring neutral rather than eccentric screw placement. barry cp, presented 50 patients of isolated angle fractures treated with superior border plating and reported 12% experienced complications requiring plate removal, 8% patients experienced superficial soft tissue infections associated with bone plate, treated with oral antibiotics, 2% experienced plated exposure and a further patient 2% presented with a fractured bone plate. all six (12%) patients were treated by bone plate removal under general anesthesia. permanent inferior alveolar nerve sensory deficit (< 12 months) was present in 8%. nineteen percent patients with normal postinjury / preoperative sensory function had a postoperative sensory deficit. the incidence of complications is one of the criteria of the evaluation of the efficacy of treatment. our observations have shown the following one or other type of complications (dictated in terms of infection, delayed healing / union, wound dehiscence, malocclusion, paresthesia and disfigurement) delayed union in four out of 51 cases (8%) owing to instability of fracture fixation. wound dehiscence was reported in six out of 51 cases (12%) that too due to infection. malocclusion in four out of 51 cases (8%) were again attributable to instability because of strong distractive forces overcome with weak osteosynthesis fixation device. paresthesia was reported in five out of 51cases (9.8%) reason may again be instability and distraction at the fracture site leading to infection inflammation causing further nerve injury. as an individual_complication disfigurement ranked highest with 12 out of 51 cases (23%) possible explanation may be that when the fixation of fracture at angle region was done using single miniplate at superior border then flaring at lower border at angle region and increase in intergonial distance occurs which on subsequent healing / union results into disfigurement. gutwald r studied the comparison of the different osteosynthesis techniques showed that in the case of miniplate fixation torsion and gapping of the bone fragments occurred following plate application and screw tightening when the plates were pressed onto the bone, so last incongruence 's between bone surface and plate were transferred to the mobile bone fragments resulting in more extended gaps and torsion. this was only observed to a much lesser extent with the mini - locking - system due to the fixation principle avoiding pressure to the bone. during functional loading the mini - locking - system showed also a significant higher stability in comparison to conventional miniplates in literature regarding cases of mandibular angle fractures, the incidence of complications varies : feller, state that healing complications occur in 2.3% of cases, according to dhariwal, such percentage is 7.3%, lamphier. 13.3%, atanasov 25.2%, etc. such huge difference between the findings presented by different authors exists because some authors attribute bleeding, hematomas, infections, neural damage and postoperative calluses to complications, while others think that complications include fracture fragments adhesion failure, damage to the lower alveolar nerve, osteomyelitis and malocclusion. osteomyelitis ranks among the most common and most severe complications of fracture healing. according to the findings presented by fox and kellman, inflammatory complications in cases of mandibular angle fractures treated with two monocortical miniplates fixation amount to 2.9%,which is comparable to or better than the infection rate reported with the single miniplate fixation technique in other studies and according to ellis to 15.8%. | aim : the present study was designed to study the feasibility of single miniplate osteosynthesis in the fracture of angle of mandible.materials and methods : in this study 110 patient were included and treated with single miniplate osteosynthesis at upper border along champy 's line of osteosynthesis.results:all the cases were treated successfully, common complications which we have observed in this study, cosmetic disfigurement, delayed union, infection, wound dehiscence and paresthesia.conclusion:single miniplate fixation in unfavorable fracture is questionable and hence these fracture require some alternative method (locking plate, etc.) for fixation. |
bacille calmette guerin (bcg) vaccination is recommended under expanded program for immunization by world health organization at birth in developing countries. it is a live vaccine which is contraindicated in immunodeficiency states like acquired immunodeficiency syndrome (aids) and severe combined immunodeficiency disease (scid). skin manifestations in disseminated bcg lesions in infants with scid have been reported in the past. we report disseminated bcg vaccine disease with skin lesions in one of the twins suffering from scid. a 7-month - old first born male twin infant, adopted by his uncle, presented with loose stools for 3 days in a private clinic. after 48 hours loose stools improved but he developed fever, cough and fast breathing. a diagnosis of viral or nosocomial infection was made and child was transferred to pediatric intensive care unit. the child was brought back to the clinic after 3 days with history of fever, cough, fast breathing and skin rashes. past medical history revealed frequent infections since birth, including episode of documented rota virus diarrhea, bronchiolitis and oral thrush. he was an adopted child from his paternal uncle and product of in vitro fertilization and twin gestation (first twin). the infant was on formula feeds and few sessions of breastfeeding daily by his biological mother since birth who was living in the same premises. infant received hepatitis b, bcg and oral polio vaccine (opv) vaccine at birth followed by three doses of dpt, hepatitis b and opv at 6, 10 and 14 weeks age. on examination child had fever (102f), respiratory rate 56 breaths per minute, mild pallor, oral thrush and red to purple, papulo - nodular, fixed and non - itching subcutaneous lesions, 0.5 - 2 cm in size, over neck, back and abdomen [figure 1a ]. the differential diagnosis for the rash was drug reaction, panniculitis and vitamin k - dependent bleeding (vkdb) from prolonged use of broad spectrum antibiotics. a skin biopsy was taken from three sites. an abnormal prothrombin time (pt) 16 sec (12 sec control) suggested vkdb and was treated with vitamin k (5 mg intravenously for 3 days). investigations showed anemia (hb 10.0 g / dl), positive c - reactive protein (8 mg / dl), and normal total leukocyte count (6200 cells / mm : neutophils 42%, lymphocytes 48%, monocytes 9%, eosinophils 1%, basophils 0), platelets (280 10/mm) and sterile blood and urine cultures. in view of non - improvement and persistent pneumonitis after 72 hours of intravenous antibiotics and supportive treatment (repeat chest x - ray revealed bilateral infiltrates) infant was investigated for tuberculosis. mantoux test was non - reactive and gastric aspirates were negative for acid fast bacilli. a skin biopsy showed sheets of macrophages with neutrophilic micro abscess ; species stain was acid fast bacilli (afb) strongly positive (6 +) with infiltrate of macrophages laden with bacilli (globii) [figure 1b ]. a diagnosis of disseminated tuberculosis was made and antitubercular therapy (isoniazid, rifampicin, pyrazinamide and ethambutol) with prednisolone (2 mg / kg) was started. skin lesions in infant modified ziehl nelson staining of skin biopsy revealed numerous acid fast bacilli with globi formation. zn stain 400 in view of non - resolving pneumonia, lymphopenia (2976/mm) and history of recurrent past infections immunodeficiency was suspected. an immunological profile showed low immunoglobulins, normal b lymphocytes and decreased t cells and natural killer (nk) cell counts [table 1 ] suggestive of scid. a skin biopsy was sent for culture and polymerase chain reaction (pcr) analysis. culture rapid (automated bact alert 3 d system ; biomerieux, france) after 3 weeks was positive for acid fast bacilli and immune chromatographic test mpt64 implying mycobacterium tuberculosis complex (mtb) which was further confirmed by oligonucleotide probe liquid hybridization protection assay (accuprobe, genprobe, sa). pyrazinamidase screening results showed resistance, and further molecular typing and biochemical tests confirmed mycobacterium bovis. the infant deteriorated and on day 10 of admission, was put on mechanical ventilator and died after 5 days. family was counseled and the mother and another twin offered to be screened for immunodeficiency. there was no history of recurrent infections and failure to thrive in parents or other twin. the facial features and blood groups of both the twins were different suggesting dizygosity. immunological profile of the infant disseminated bcg disease is a rare complication (approximately 1/million) of bcg vaccination in immunocompetent children. talbot. and loette. have reported the occurrence of disseminated bcg disease in 86% and 100% children with immune defects, respectively. disseminated bcg disease generally manifests as fever, malnutrition, and pneumonitis 3 - 6 months after receiving bcg vaccination. skin manifestations like subcutaneous nodules, rash, ulcers, and erythema in scid infants with disseminated bcg disease have been reported by some authors. more than 30 genetic defects are known in scid which may be inherited as x - linked recessive or autosomal recessive. x - linked scid in 50% and adenosine deaminase (ada) deficiency (autosomal recessive) in 15 - 20% cases of scid are most common disorders. unless treated scid is uniformly fatal in first 2 years of life. disseminated bcg infection also carries very high fatality (1) in children with scid. routine newborn screening by t - cell receptor excision circles (trecs) by pcr, using dna extracted from newborn dried blood spots (guthrie cards), has been recently implemented in wisconsin (usa). as routine bcg vaccination is recommended at birth to all newborns in most developing countries and children with immunodeficiency are symptomatic afterwards, most of these children remain at risk of fatal disseminated bcg disease. | fatal - disseminated bacillus calmette guerin (bcg) disease is well known in infants with severe combined immunodeficiency after bcg vaccination. we report a 7 month male infant delivered as a product of in vitro fertilization and twin gestation that presented with fever, cough and multiple nodular skin lesions. a biopsy of skin lesions revealed the presence of acid fast bacilli. mycobacterium bovis infection was confirmed by polymerase chain reaction (pcr) and molecular studies. immunological profile confirmed the diagnosis of severe combined immunodeficiency. only few reports of similar case exist in the literature. |
participants were drawn from the framingham heart study. for details regarding the study sample, outcome ascertainment, and diabetes diagnosis, all participants gave written informed consent, and the study was approved by the institutional review board of the boston medical center. participants who developed diabetes during follow - up were allowed to contribute information to both nondiabetic and diabetic categories (transition in this direction was permitted). short- (10-year), medium- (20-year) and long - term (30-year) risks of developing cvd were calculated for participants with and without diabetes. the 30-year risk estimate approximates the lifetime risk in individuals aged 50 years or older. the original cohort was further stratified by bmi category ; this was not done on the offspring cohort due to fewer numbers of events. the inclusion of the offspring cohort allows for the analysis of more contemporary data. for computing the 10-, 20-, and 30-year risks (4) based on a method proposed by gaynor. (5) as previously described (6). this approach uses age as a time scale of analysis and adjusts for the competing risk of death to avoid risk inflation introduced in standard survival methods that do not fully account for individuals who die during follow - up (and, therefore, can no longer develop the event of interest). participants were drawn from the framingham heart study. for details regarding the study sample, outcome ascertainment, and diabetes diagnosis, all participants gave written informed consent, and the study was approved by the institutional review board of the boston medical center. all analyses were sex specific. participants who developed diabetes during follow - up were allowed to contribute information to both nondiabetic and diabetic categories (transition in this direction was permitted). short- (10-year), medium- (20-year) and long - term (30-year) risks of developing cvd were calculated for participants with and without diabetes. the 30-year risk estimate approximates the lifetime risk in individuals aged 50 years or older. the original cohort was further stratified by bmi category ; this was not done on the offspring cohort due to fewer numbers of events. the inclusion of the offspring cohort allows for the analysis of more contemporary data. for computing the 10-, 20-, and 30-year risks this approach uses age as a time scale of analysis and adjusts for the competing risk of death to avoid risk inflation introduced in standard survival methods that do not fully account for individuals who die during follow - up (and, therefore, can no longer develop the event of interest). table 1 presents the number of individuals, bmi categories, and cvd cases over the duration of follow - up stratified by diabetes status. overall, in the original cohort, the lifetime risk of cvd was 38.7% in women and 55.4% in men. in the offspring cohort, the lifetime risk of cvd in women was 27.2% and 39.8% in men. over 30 years, the lifetime risk of developing cvd in the original cohort was 38.0% among women without diabetes, whereas it was 67.1% among women with diabetes (table 1, middle panel). among men, the lifetime risk of cvd without diabetes was 54.8% and with diabetes 78.0%. similar patterns were observed in the offspring cohort and with 10- or 20-year risk data (table 1, middle panel). over 30 years, the lifetime risk of cvd among normal - weight women without diabetes was 34.3% (table 1, lower panel), whereas it was 46.7% among obese women without diabetes. among women with diabetes, the 30-year risk was 54.8% among normal weight women and 78.8% among obese women with diabetes. similar patterns were observed among men, and the lifetime risk of cvd approached 90% among men with both obesity and diabetes. over 30 years, the lifetime risk of developing cvd in the original cohort was 38.0% among women without diabetes, whereas it was 67.1% among women with diabetes (table 1, middle panel). among men, the lifetime risk of cvd without diabetes was 54.8% and with diabetes 78.0%. similar patterns were observed in the offspring cohort and with 10- or 20-year risk data (table 1, middle panel). over 30 years, the lifetime risk of cvd among normal - weight women without diabetes was 34.3% (table 1, lower panel), whereas it was 46.7% among obese women without diabetes. among women with diabetes, the 30-year risk was 54.8% among normal weight women and 78.8% among obese women with diabetes. similar patterns were observed among men, and the lifetime risk of cvd approached 90% among men with both obesity and diabetes. the lifetime (i.e., 30-year) incidence of cvd adjusted for mortality among participants with diabetes was two - thirds to three - quarters in the original cohort, and roughly one - half to two - thirds in the offspring cohort. lifetime risk varied according to bmi category, with participants with obesity and diabetes having the highest risk of developing cvd. the lifetime risk of diabetes has been estimated at 32.8% for men and 38.5% for women (7). the lifetime risk of diabetes increases in proportion to bmi, ranging from 7.6% among underweight individuals to as high as 74.4% among individuals with stage 2 obesity (8). similarly, our findings demonstrate that the lifetime risk of cvd is higher among individuals with both obesity and diabetes, with the lifetime risk of cvd approaching nearly 80% in obese women and nearly 90% in obese men. given the recent increases in both the prevalence and incidence of diabetes, projections for the burden of diabetes in the u.s. by 2050 we have already demonstrated that the attributable risk of cvd due to diabetes has increased (10) ; this trend may continue to worsen if current trajectories do not change. limitations of our study include the selection of all white individuals, which potentially limits generalizability. we did not exclude individuals with type 1 diabetes ; however, there are less than 10 individuals in our sample and, therefore, this is unlikely to have affected the results. lastly, given the shorter follow - up time in the offspring, we note that these are more long - term than lifetime risk estimates. | objective we assessed the lifetime risk of cardiovascular disease (cvd) among individuals with and without obesity and diabetes.research design and methods participants were drawn from the original and offspring cohorts of the framingham heart study. lifetime (30-year) risk of cvd was assessed using a modified kaplan - meier approach adjusting for the competing risk of death, beginning from age 50 years.resultsover 30 years, the lifetime risk of cvd among women with diabetes was 54.8% among normal - weight women and 78.8% among obese women. among normal - weight men with diabetes, the lifetime risk of cvd was 78.6%, whereas it was 86.9% among obese men.conclusionsthe lifetime risk of cvd among individuals with diabetes is high, and this relationship is further accentuated with increasing adiposity. |
molecular chaperones are essential for the maintenance of cellular homeostasis by facilitating various functions including degradation of proteins, translocation, folding of co - translational products, and protein complex assembly. many molecular chaperones are also known as heat shock proteins (hsp) and their expression is upregulated in response to stress. the large and varied heat shock protein family has usually been classified into major classes defined by molecular weight. the 70 kda family of proteins is the most abundant and thoroughly studied family of heat shock proteins. hsp70 functions both as a holdase (binding and holding onto unfolded polypeptides by withdrawing aggregation - prone species) as well as a refoldase (assisting non - native proteins to fold to the native state). the highly conserved hsp70 structure is typically composed of an n - terminal atpase domain (44 kda), a c - terminal domain containing a -sandwich subdomain, which is the substrate binding domain (1518 kda,) followed by an -helical subdomain (10 kda) [7, 8 ]. hsp70 protein activity occurs through the interaction of hydrophobic peptide segments of other proteins with its substrate binding domain (sbd) in an atp - dependent manner. the hsp70 atpase cycle switches between the low affinity atp - bound state and rapid substrate - exchange rates and the high affinity adp - bound state with low substrate - exchange rates. hsp40 is a co - chaperone and is required to stimulate the atpase activity of hsp70 and results in hsp70-adp with a high affinity for substrate. in order for the nucleotide binding cleft to be opened, the release of adp is facilitated by a nucleotide - exchange factor, such as bag-1 protein. the atpase cycle is completed when atp binds the atpase domain of hsp70 resulting in a conformational change in the substrate binding domain and bound substrates are released. the eevd motif present at the end of the c - terminus of hsp70 members is involved in binding to the tetratricopeptide repeat (tpr) domains of hsp70/hsp90 organizing protein (hop) and carboxy terminus of hsp70 interacting protein (chip). the eevd motif has been suggested to recruit hsp40 as it has been shown to bind and regulate the function of hsp40 [12, 13 ]. all hsp40s contain a j - domain that is required for facilitating the cellular activity of hsp70 proteins through its interaction with their partner hsp70s. the j - domain has a conserved histidine - proline - aspartic acid (hpd) tripeptide ; variations within the hpd motif are known to abolish the stimulation of hsp70 atpase activity by hsp40 [14, 15 ]. type i hsp40s are highly conserved and contain a glycine - phenylalanine (g / f) and cysteine rich region that contains four motifs of cxxcxgxg, a glycine / methionine rich region, a carboxy - terminal peptide binding domain and a dimerization domain [16, 17 ]. type i hsp40s also have a peptide - binding region at the c - terminus which aids in the binding to non - native polypeptides, in turn allowing hsp40 proteins to transport substrates to hsp70 proteins for folding [18, 19 ]. type ii hsp40s contain the j - domain and the g / f rich region, along with the peptide - binding region at the c - terminus [2022 ]. the highly specialized type iii hsp40s possess only the conserved j - domain which can occur anywhere on its sequence and primarily functions to recruit hsp70 to a particular location. the african trypanosome, trypanosoma brucei, is a blood - borne unicellular parasitic protozoan and the etiological agent of both human and animal african trypanosomiasis. african trypanosomiasis is described as one of the neglected tropical diseases crippling economic development and causing death in africa 's poorest and most marginalized communities. due to a lack of new treatments against human african trypanosomiasis and emergence of resistance to older drugs, human treatments currently available are connected with high levels of toxicity and resistance. heat shock proteins have been emerging as prospective drug targets (hsp70s as drug targets reviewed in). drugs causing cellular stress resulting in the induction of heat shock proteins anti - heat shock protein drugs used in combination with current drugs could therefore synergistically improve the effectiveness of available drugs. parasitic heat shock proteins have been revealed as drug targets, including malarial drug target p. falciparum hsp90 and trypanosomal t. evansi hsp90 [29, 30 ]. amongst the known hsp90 proteins, pfhsp90 was shown to have the highest atpase activity, and its inhibition by geldanamycin (ga) was stronger than seen for human hsp90. semi - synthetic inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-aag) has been shown to curb growth of the p. falciparum parasite and to inhibit the growth of the t. evansi parasite by specifically binding and inhibiting hsp90. 17-aag could likely be effective against infection caused by t. brucei due to the sequence similarity between tbhsp90 and tehsp90. sera from patients infected with trypanosomes were screened to identify diagnostic antigens, and although tbhsp70 was identified as a candidate, by itself, it demonstrated inadequate specificity and sensitivity in diagnosis of trypanosomiasis. it may however be useful as a diagnostic antigen if used in conjunction with other immunogenic proteins. of the leishmanial and trypanosomal hsp70s and hsp40s, a number of important and well - known studies have been performed on t. cruzi. cytoplasmic tchsp70b demonstrated high atpase activity in comparison to human hsp70 and was shown to be heat inducible. in addition to stimulating the immune response, tchsp70b is one of the immunodominant antigens in t. cruzi infected individuals [31, 33 ]. a type i hsp40, tcj2, was able to stimulate the already high basal atpase activity of tchsp70b by 1.5 fold, whilst the type iii hsp40 tcj1 showed no significant stimulation. furthermore, tcj2 was shown to complement the well - characterized saccharomyces cerevisiae type i hsp40, ydj1, and was subsequently suggested to be involved in cytoprotection. tchsp70b, displaying typical chaperone properties, is homologous to t. brucei hsp70, and tcj1 and tcj2 are homologous to tbj1 and tbj2, respectively. genome annotation revealed t. brucei to possess a complement of 65 hsp40 proteins and 12 hsp70 proteins. in silico analysis of tbhsp70.c indicated the presence of atypical acidic residues in the substrate binding domain as well as in the substrate binding cavity of tbhsp70.c and these may play a role in substrate discrimination. however, the residues required for association with hsp40 proteins within both the atpase and substrate binding cavity, as well as the hsp70 phosphorylation site, are well conserved. in addition the c - terminal eevd motif is absent from tbhsp70.c and it contains the sequence rieainante at the end of the protein. tbhsp70.c was predicted to be cytoplasmic. to date, tbhsp70.c has not been biochemically characterized and co - chaperones have not been identified. of the 65 hsp40s, only one of the type iii t. brucei hsp40 proteins, tbj1, has been expressed, purified, and biochemically characterized to date. no in vitro characterization of the type i t. brucei hsp40 protein, tbj2, has been completed ; in vivo analysis revealed that tbj2 is essential to the survival of the cell [38, 39 ]. tbj2 was demonstrated to be upregulated upon inducing heat shock and to localize in the cytoplasm. according to tritrypdb, tbhsp70.c is localized in the cytoplasm and tbj2 was selected as a probable partner of tbhsp70.c. this research aims to functionally characterize tbhsp70.c and tbj2 as its potential co - chaperone. we successfully expressed and purified tbhsp70.c and revealed for the first time that tbhsp70.c and tbj2 can prevent thermal aggregation of malate dehydrogenase (mdh) and rhodanese and that tbj2 stimulates the atpase activity of tbhsp70.c.. a greater understanding of the hsp70-hsp40 partnerships is important as molecular chaperones have been implicated in parasite survival and growth. louis, mo usa), merck chemicals (darmstadt, germany), biorad (usa), or roche molecular biochemicals (indianapolis, in, usa). the t. brucei treu927 strain was a kind donation from professor george cross (rockefeller university, new york, usa). e. coli strain bb1994 (mc4100 dnak52 sidb1::tc pdmi,1::cmr kanr) was kindly provided by dr. prior to phylogenetic tree analysis, trypanosomatid amino acid sequences were obtained from the genedb database and other hsp70 protein sequences were retrieved from the national centre for biotechnology information (ncbi), both sets of sequences in fasta format. the phylogenetic tree was generated using molecular evolutionary genetics analysis (mega5) version 5.2.2 maximum likelihood analysis. the coding regions of full length tbhsp70.c and tbj2 were pcr amplified from t. b. brucei treu927 genomic dna. forward primer (5-ggt acc atg acc tac gaa gga-3) with a kpni restriction site (underlined) and reverse primer (5-gtc gac tta ctc tgt gtt tgc-3) with a sali restriction site (underlined) were used for tbhsp70.c. the forward (5-gaattcggatcccatatggtgaaagaaacaaaatac-3) (ecori, bamhi, ndei) and reverse (5-aagcttctcgaggtcgacctattgctgcgtacacg-3) (hindiii, xhoi, sali) primers were used for pcr amplification of tbj2. the n - terminal his - tagged pqe80-tbhsp70.c and pet28a - tbj2 constructs were verified by restriction digestion analysis and dna sequencing (data not shown). an overnight culture was prepared by inoculating e. coli bb1994 [pqe80tbhsp70.c ] in 25 ml 2 yt broth supplemented with 100 g / ml ampicillin and 50 g / ml kanamycin at 30c with shaking. the overnight culture was transferred to 225 ml 2 yt broth and grown with shaking to midlog phase (a600 0.6) before inducing tbhsp70.c protein expression by the addition of isopropylthiogalactoside (iptg) to a final concentration of 1 mm. cells were harvested (5000 g ; 15 min) 3 hours after induction and resuspended in lysis buffer (100 mm tris - hcl, ph 8, 300 mm nacl, 10 mm imidazole). cells were stored at 80c overnight and thawed with the addition of 1 mg / l lysozyme and 1 mm pmsf culminating in lysis. cellular debris was removed by centrifugation (12000 g ; 40 min ; 4c) and the supernatant was incubated with nickel - charged sepharose beads in lysis buffer overnight at 4c. the bead - suspension was centrifuged (5000 g ; 1 min ; 4c) and three washes were performed with wash buffer (100 mm tris - hcl, ph 8, 300 mm nacl, 20 mm imidazole). purified protein was subsequently dialysed overnight [100 mm tris - hcl, ph8, 100 mm nacl, 5% (v / v) glycerol, 50 mm kcl, 2 mm mgcl2, 0.5 mm dithiothreitol ]. both e. coli bl21(de3) [pet28a - tbj2 ] and bl21(de3) [pet14b - tchsp70b ] cells were grown at 37c, maintaining a selective pressure using kanamycin (50 g / ml) and were harvested 5 hours after induction. to remove the presence of co - purified dnak, tbj2 and tchsp70b bound to the ni - nta column were washed 5 times with the addition of 20% glycerol and 10 mm atp to the wash buffer. all of other steps of the purification were performed in the same manner as for tbhsp70.c. sodium - dodecyl sulphate - polyacrylamide gel electrophoresis (sds - page) analysis was used to evaluate the purity of the protein. anti - tbhsp70.c was produced in rabbit using the c - terminal antigen, cqrgrgvtegsgrpp at residues 648662, from the tbhsp70.c protein sequence. western analysis was used to verify the integrity and identity of tbhsp70.c using affinity - purified anti - tbhsp70.c peptide antibody. western analysis was performed using anti - his antibody to target tbj2 and tchsp70b and anti - dnak antibody to detect dnak. antibodies were detected using the ecl advance blotting detection kit and viewed using the chemidoc eq (biorad, usa). the ability of tbhsp70.c and tbj2 to suppress aggregation of bovine rhodanese (sigma - aldrich) was assessed spectrophotometrically. rhodanese was denatured overnight at 30c ; rhodanese denatured in denaturing buffer (6 m guanidine hydrochloride, 50 mm hepes, ph 7.0, 100 mm nacl) was added to assay buffer (50 mm hepes, ph 7.0, 100 mm nacl) to a final concentration of 1.5 m was monitored at 300 nm over a period of 40 min at room temperature using a kc junior microplate reader (bio - tek instruments, usa). molecular chaperone proteins of interest were added at various concentrations to measure their ability to suppress rhodanese aggregation. tbhsp70.c, tchsp70b, and tbj2 are not aggregation prone as no significant increase in turbidity was observed when these proteins were assayed in the absence of rhodanese ; protein samples that had been inactivated by boiling for 20 minutes also displayed no chaperone activity (data not shown). absorbance was plotted as percent rhodanese aggregation over 40 min subsequent to normalizing against assays with rhodanese alone. the abilities of tbhsp70.c and tbj2 to prevent thermal aggregation of mdh were analysed by spectrophotometry. the assay was initiated by adding 0.72 m mdh and the molecular chaperone proteins to assay buffer (50 mm tris - hcl, 100 mm nacl ; ph 7.4) heated to 48c. aggregation of the protein substrate was monitored by measuring the scatter of light at 360 nm over 30 min at 48c in a helios alpha db spectrophotometer with a peltier - controlled cell. hydrolysis of atp by tbhsp70.c was assessed using a modified version of the ascorbic acid / ammonium molybdate colorimetric assay to measure the release of inorganic phosphate during the reaction. the assay was performed as described in with a few modifications. tbhsp70.c and control protein tchsp70b (0.4 m) were equilibrated to 37c in atpase buffer (25 mm hepes, ph 7.4, 2 mm mgcl2, 50 mm kcl, 0.5 mm dtt). stimulation of the basal atpase activity of both tbhsp70.c and tchsp70b by co - chaperone tbj2 was assessed at equal concentrations (0.4 m) and in a 2-fold molar excess to tbhsp70.c (1 m) and tchsp70b (1 m). samples were taken in triplicate at regular time intervals (0, 30, 60, 120, 180, and 240 minutes). all assays were corrected for the spontaneous breakdown of atp observed in a control experiment in the absence of protein. any background atp hydrolysis observed for tbj2 was corrected for by subtracting this activity from the reactions containing this protein. statistical analysis was performed on data generated from both the aggregation suppression and atpase activity assays. the relationship between two variables was analysed using one - way analysis of variance (anova). wild type lister 927 variant 221 t. brucei brucei bloodstream form lysates (10 cells / ml) were used for the heat stress inducibility experiment. bloodstream form t. brucei lister 927 variant 221 strain trypanosome parasites were cultured in filter sterilized complete iscoves modified dulbeccos media (imdm) based hm1 - 9 medium [imdm base powder, 3.6 mm sodium bicarbonate, 1 mm hypoxanthine, 1 mm sodium pyruvate, 0.16 mm thymidine, 0.05 mm bathocuprone sulphate acid, 10% (v / v) heat inactivated foetal bovine serum, 1.5 mm l - cysteine, 0.2 mm -mercaptoethanol, ph 7.5 ] in a humidified chamber at 37c with an atmosphere of 5% co2. separate 25 ml culture of cells were exposed to heat shock at 42c for a period of 60 min in plugged flasks, allowing no entry of co2. cell lysates were harvested by centrifugation at 800 g for 10 min, washed twice in pbs buffer (10 mm na2hpo4, 2 mm kh2po4, 137 mm nacl, 2.7 mm kcl), and repelleted prior to resuspension in sds - page loading buffer to a final cell count of 5 105 cells/l. the lysates (5 10 cells per lane) were resolved on a 10% sds - page gel. differences in tbhsp70.c protein expression were detected using polyclonal rabbit anti - tbhsp70.c peptide antibody (1 : 5000) and goat anti - rabbit igg hrp - conjugated secondary antibody (1 : 5000) in subsequent western analysis. bioinformatic analysis revealed t. brucei hsp70.c to be a eukaryotic isoform that may represent a novel family of hsp70 proteins as it did not cluster phylogenetically with any of the other primary hsp70 proteins. phylogenetic tree analysis was used to establish orthology between the novel hsp70.c proteins within the tritryps and to compare their phylogenetic relationships with cytoplasmic hsp70s from the tritryps and the hsp70s from the t. brucei complement along with well - characterized eukaryotic and prokaryotic canonical hsp70 proteins (figure 1). tbhsp70.c may prove to be an anti - parasitic drug target as phylogenetic analysis suggests that tbhsp70.c clusters only with hsp70 proteins from the tritryps. not surprisingly, the hsp70.c proteins clustered together phylogenetically, tbhsp70.c formed a monophyletic clade with tbghsp70.c and a close link with tcohsp70.c, suggesting possible paralogy. the hsp70.c cluster formed a closer phylogenetic relationship with the cytoplasmic hsp70s, including inducible human hsp70b (hshspa6), constitutively expressed human hsc70 (hshspa8), bovine hsc70 and plant hsp70 than with mitochondrial hsp70s, the nef - acting hsp110s, or the dnaks (prokaryotic hsp70 homologue) (figure 1). recombinant tbhsp70.c was expressed and purified from an e. coli dnak - minus strain (figure 2(a)). the e. coli dnak - minus strain was used to eliminate endogenous dnak, the bacterial homologue of hsp70. soluble tbhsp70.c was successfully purified by nickel - affinity chromatography under non - denaturing conditions and anti - tbhsp70.c peptide antibodies recognized full - length tbhsp70.c at 73 kda, confirmed by western analysis (figure 2(a)). 600 mg of protein was purified from 1 l of e. coli cells and was of sufficient purity for subsequent in vitro assays. an alternative strategy was adopted for the purifications of recombinant tchsp70b and tbj2 to eliminate co - purifying dnak due to a lack of an e. coli dnak - minus strain compatible with the pet vectors. modifications to the nickel - affinity chromatography protocol included the addition of 20% glycerol and 10 mm atp to the wash steps. the non - denaturing purification resulted in the removal of co - purified dnak, confirmed by western analysis, from both the tchsp70b (figure 2(b)) and tbj2 (figure 2(c)) purifications. for the tchsp70b and tbj2 purifications, 1176 mg and 900 mg of protein were purified from 1 l of e. coli cells, respectively, and the elimination of dnak warranted the use of tchsp70b and tbj2 in subsequent in vitro assays. tbhsp70.c was able to suppress the protein aggregation of chemically denatured rhodanese in a dose - dependent manner (figure 3). tchsp70b, an orthologue of bos taurus hspa8, was used as a positive control. in addition to demonstrating typical chaperone properties [32, 34 ], the previously characterized tchsp70b, also homologous to tbhsp70, contains an intact c - terminal eevd motif. the addition of tbhsp70.c and tchsp70b resulted in a marked increase of aggregation suppression in a dose - dependent manner (figure 3). tbj2 (0.5 m, 0.7 m, 1 m) resulted in 4.1%, 8.3%, and 14.2% suppression of rhodanese aggregation, respectively. to investigate the ability of tbj2 to enhance the holdase function of tbhsp70.c, the addition of tbj2 at a submolar concentration to tbhsp70.c resulted in 27.7% suppression of rhodanese aggregation (figure 3). equal concentrations of tbj2 and tbhsp70.c resulted in 37.5% suppression of rhodanese aggregation, which was comparable to the 41.2% suppression produced by tchsp70b at equimolar concentrations to tbj2. tbj2 in partnership with tbhsp70.c demonstrated increased aggregation suppression. at equimolar concentrations tbj2 demonstrated a synergistic effect and thus the ability to function as a co - chaperone to both tchsp70b and tbhsp70.c. mdh was used as a second substrate to establish whether tbhsp70.c and tbj2 could suppress aggregation of mdh. bsa was used as a negative control and had no effect on mdh aggregation (data not shown). a dose response was evident upon the addition of various concentrations of tbhsp70.c as turbidity levels decreased with an increase in chaperone concentration (figure 4). a similar result was obtained for tbj2 where increased chaperone concentrations caused decreased turbidity and a dose - dependent suppression of mdh aggregation (figure 4). the ability of tbj2 to enhance the holdase function of tbhsp70.c was assessed by maintaining constant tbhsp70.c concentrations and varying those of tbj2 (figure 4). tbhsp70.c (0.5 m) resulted in 61.8% suppression of mdh aggregation (figure 4). the addition of tbj2 at a submolar concentration (0.3 m) to tbhsp70.c resulted in 66.9% mdh aggregation suppression and tbj2 at molar excess (0.7 m) resulted in 85% suppression of mdh aggregation (figure 4). tbj2 concentrations of 0.3 m and 0.7 m enhanced tbhsp70.c chaperone activity ; however, the increase in turbidity was not enough to be an additive effect. equal concentrations of tbj2 and control protein tchsp70b resulted in 93.9% suppression of mdh aggregation, which appeared to be an additive effect (figure 4). tbj2 did not demonstrate the ability to co - chaperone either tbhsp70.c or tchsp70b using mdh as a substrate. tbj2 may have been binding as substrate to tbhsp70.c, thus resulting in the decreased suppression of mdh aggregation that was observed. the ability of tbj2 to stimulate the basal atpase activity of tbhsp70.c and tchsp70b was investigated. tbhsp70.c was determined to have a basal atpase activity of 7.6 nmol pi / min / mg and tchsp70b a basal atpase activity of 18.3 nmol pi / min / mg. the basal atpase activity of tbhsp70.c and tchsp70b was represented as 100% (figure 5). equal concentrations of tbj2 and tbhsp70.c resulted in a significant 1.62-fold stimulation of the basal atpase activity of tbhsp70.c (figure 5). equal concentrations of tbj2 and tchsp70b caused a slightly lower but still significant 1.39-fold stimulation of tchsp70b basal atpase activity (figure 5). tbj2 at 2-fold molar excess to tbhsp70.c and tchsp70b resulted in a significant 2.88-fold enhancement of the basal atpase activity of tbhsp70.c and a significant 2.62-fold stimulation of tchsp70b atpase activity (figure 5). tbhsp70.c, tchsp70b, and tbj2 denatured by boiling displayed no atpase activity (data not shown). under conditions of stress, a cell responds by increasing the synthesis of heat shock proteins to manage the elevated levels of denatured proteins. t. b. brucei 427 v221 cells were exposed to heat shock by incubating them at 42c for one hour. recombinant tbhsp70.c purified from e. coli bb1994 cells was used as a positive control. even though there was no notable difference in the tbhsp70.c protein levels on the sds - page gel, tbhsp70.c protein expression proved to be inducible by heat stress, as shown by an increase in the top band in the western analysis (figure 6). two bands were detected by the anti - tbhsp70.c peptide antibody. the top band at 73 kda increased in intensity from 37c to 42c indicating that at 37c tbhsp70.c was expressed in bloodstream form cells and upon exposure to heat stress tbhsp70.c was upregulated. interestingly, the expression level of the non - specific 68 kda band, detected by anti - tbhsp70.c peptide antibodies, remained constant upon heat shock, suggesting it to be a separate protein and not a degradation product of tbhsp70.c (figure 6). a study of the evolutionary relationships between kinetoplastid hsp70 proteins and typical hsp70 proteins has revealed a novel and divergent group of proteins, the orthologues tbhsp70.c, tchsp70.c, and lmhsp70.c. tbj2 was selected as a probable co - chaperone of tbhsp70.c due to the presence of its functional j - domain and because of its predicted cytosolic localization (data not shown). when studying molecular chaperones, the presence of dnak is a major concern due to the possibility that basal expression of bacterial or co - purified dnak could mask the chaperone activity of the t. brucei heat shock proteins under investigation. the co - purification of dnak is likely as a result of denatured exposed surfaces of the target protein interacting with dnak by acting as a substrate. using an e. coli dnak - minus strain and modifications to the purification protocol, dnak was eliminated from the tbhsp70.c and the tbj2 and tchsp70b purifications, respectively. amongst the various roles of hsp70 proteins demonstration of the holdase - function, by suppressing protein aggregation, is used to display chaperone activity [48, 49 ]. mdh aggregation suppression assays are often performed in the absence of atp [50, 51 ]. the addition of atp decreases the affinity of an hsp70 protein for mdh, resulting in its release and subsequent re - aggregation. a loss of affinity for substrate in the presence of atp has been demonstrated for p. falciparum hsp70, e. colihsc66, dnak and bovine hsp90. the model thermolabile substrate mdh was used in this study to investigate the ability of tbhsp70.c and tbj2, in isolation and in partnership, to suppress the heat - induced aggregation of mdh. a second substrate, rhodanese, was selected to investigate whether the substrate binding specificity of tbhsp70.c for rhodanese would differ considering the presence of fewer hydrophobic residues within rhodanese than in mdh. a recent in silico study using an e. coli dnak l484w mutant predicted that an increase in hydrogen bonds and hydrophobic interactions allows an enhanced interaction between chaperone and substrate. tbhsp70.c was shown to bind and suppress aggregation of both rhodanese and mdh, thereby displaying typical chaperone activity. however, tbhsp70.c suppressed aggregation of mdh to a greater degree than was observed for rhodanese. hsp70s generally function in cooperation with hsp40s, where hsp40 either mediates the interaction of hsp70 with its substrate or holds the substrate and recruits hsp70 to the unfolded polypeptide. hsp40 proteins are able to bind unfolded substrates independently of hsp70 proteins and prevent their aggregation [18, 58 ]. once bound, the substrate is transferred to hsp70 by the j - protein ; at the same time hsp40 stabilizes the hsp70-substrate association by stimulating the atpase activity of hsp70. a similar result was seen for the homologue of tbj2, tcj2, in its ability to independently suppress aggregation of mdh. the reduced chaperone activity observed in this study using rhodanese could furthermore be as a result of non - productive and aggregation - prone intermediates formed whilst rhodanese refolds. in the presence of tbhsp70.c the effect of tbj2 on both tbhsp70.c and tchsp70b was additive rather than synergistic when using mdh. a similar result was observed during the suppression of aggregation of mdh by plasmodium falciparum hsp40 and hsp70. the ability of a type iii hsp40 protein, tbj1, to function as a co - chaperone was supported by the findings that tbj1 assisted trypanosoma cruzi hsp70 and medicago sativa hsp70 in suppression of mdh aggregation. suppression of protein aggregation assays has also been used to successfully determine the effect of small molecule modulators on the abilities of pfhsp70 proteins to suppress the aggregation of alcohol dehydrogenase and mdh. the ability of tbhsp70.c to successfully suppress aggregation of two individual substrates was not surprising. even though some conservation has been lost in the tbhsp70.c hydrophobic arch, some residues are replaced by acidic and aliphatic residues and the hydrophobic pocket, represented by the valine residue, is well conserved (data not shown). enhanced hsp70 atpase activity is specific and is modulated through the interaction of an hsp40 partner. this data would suggest that tbj2 acts as a co - chaperone by means of its j - domain interacting with the atpase domain of tbhsp70.c, either recruiting substrates to the hsp70 or merely mediating the interaction of the hsp70 with its substrate and stimulating the atpase activity of the hsp70. however, the level of atpase activity stimulation by tbj2 is low in comparison to what has previously been observed for hsp70-hsp40 partnerships. yeast hsp40, ydj1p, was shown to stimulate the atpase activity of yeast hsp70, ssa1p, by 6 - 8-fold [18, 61, 62 ]. tbj2, homologue of tcj2, demonstrated a greater stimulation of tbhsp70.c atpase activity than was observed for tcj2-stimulated atpase activity of tchsp70b. the low stimulation of the atpase activity of tchsp70 by tcj2 was suggested to be as a result of a masking effect of the already very high basal atpase activity of tchsp70b, reported to be 100 times greater than that observed for human hsp70. further characterization of the atpase activity of tbhsp70.c would entail investigating the activation of the atpase activity of tbhsp70.c by various substrates. maximal stimulation of the atpase activity of hsp70 proteins has been demonstrated to take place in the presence of both co - chaperone and substrate. to date, very little research has been performed on the heat shock response within t. brucei. the focus has been on investigating the effect of heat stress on t. cruzi and/or its vector, panstrongylus megistus as well as on molecular chaperones belonging to the t. cruzi parasite. a study in which t. b. brucei bloodstream form cultures were exposed to heat shock resulted in the upregulation of the expression levels of tbhsp70.c. this finding would imply that tbhsp70.c is required for protein quality control not only under normal conditions but also when the cell is placed under thermal stress. this is the first report of data showing that the expression levels of tbhsp70.c are increased by heat stress. tchsp70 mrna levels showed a very significant four - fold upregulation upon heat stress at 37c when compared to mrna levels at 28c. the ability of tbj2 to significantly enhance the basal atpase activity of tbhsp70.c suggests that tbj2 functions as a co - chaperone of tbhsp70.c. however, further experimental analyses including binding studies and co - localization will need to be performed to conclusively confirm tbj2 as co - chaperone of tbhsp70.c. furthermore, the possibility of other cytoplasmic tbj proteins functioning as co - chaperones of tbhsp70.c will need to be investigated. due to its atypical features, tbhsp70.c is likely to interact with a specific set of protein substrates in the cell. tbhsp70.c is the first t. brucei hsp70 protein to be functionally characterized, along with its probable partnership with co - chaperone tbj2. this research opens up prospects for further studies of the yet unexplored multiple hsp70/hsp40 partnerships in t. brucei. future research would entail isolating and characterizing the remaining 11 hsp70 proteins from t. brucei to enable differentiation between unique and canonical features and furthermore investigating which of them form partnerships with the 65 hsp40 proteins. characterization of these and other chaperone / co - chaperone interactions could further enhance understanding of the cell biology of t. brucei. | the neglected tropical disease, african trypanosomiasis, is fatal and has a crippling impact on economic development. heat shock protein 70 (hsp70) is an important molecular chaperone that is expressed in response to stress and hsp40 acts as its co - chaperone. these proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. a novel cytosolic hsp70, from trypanosoma brucei, tbhsp70.c, contains an acidic substrate binding domain and lacks the c - terminal eevd motif. the ability of a cytosolic hsp40 from trypanosoma brucei j protein 2, tbj2, to function as a co - chaperone of tbhsp70.c was investigated. the main objective was to functionally characterize tbhsp70.c to further expand our knowledge of parasite biology. tbhsp70.c and tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile mdh and chemically denatured rhodanese. atpase assays revealed a 2.8-fold stimulation of the atpase activity of tbhsp70.c by tbj2. tbhsp70.c and tbj2 both demonstrated chaperone activity and tbj2 functions as a co - chaperone of tbhsp70.c. in vivo heat stress experiments indicated upregulation of the expression levels of tbhsp70.c. |
angiotensin ii (angii) is the biologically active peptide of the renin - angiotensin system, a well - known regulator of cardiovascular homeostasis and brain functions [1, 2 ]. over the past ten years, a large number of studies have unraveled an additional role for the renin - angiotensin system in proliferative pathologies such as hyperplasia and cancer [38 ]. it is generally admitted that at1 receptor activation is responsible for most of the reported effects of angii, whereas the at2 receptor behaves as a negative regulator of at1 signaling pathways [911 ]. indeed, functional negative crosstalk between at1 and at2 receptors has been largely described in several physiopathological conditions including hypertension [1, 10, 11 ]. activation of the at1 receptor triggers a large number of intracellular kinases leading to modulation of cell proliferation, migration, and inflammation, three processes closely associated with tumor progression. accordingly, several groups have shown that blocking at1 receptors using specific receptor antagonists (arbs) is effective in reducing tumor growth and metastasis in preclinical models [1216 ]. studies using knockout animals have further pointed out a role for stromal at1 receptors from the host in tumor - associated macrophages infiltration and in cancer - related angiogenesis [17, 18 ]. a recent study showing a dramatic overexpression of at1 receptors in a subpopulation of invasive breast tumors has highlighted the potential use of arbs as novel therapeutic agents against breast cancer. indeed, it has been shown that losartan, an at1 receptor blocker, was able to inhibit breast tumor growth and invasion, suggesting that effective treatments for breast cancer may be developed using drugs already used in clinics with few side effects. these exciting findings have, however, been challenged by a recent meta - analysis that suggested that arbs medication may be associated with increased risk of cancer. these provocative results have not been further validated by other groups, which rather found no effect of arbs related to the risk of cancer [21, 22 ]. nevertheless, the beneficial effects of angiotensin receptors blockade in cancer still remain a matter of debate and need to be better explored, possibly with the use of new models. to date, most studies have mainly focused on the role of the at1 receptor in cancer, while the role of the at2 subtype has been largely neglected. discrepancies in meta - analysis studies might reflect variable levels of expression of at2 receptors in different tumors. it is important to keep in mind that antagonizing the at1 receptor by arbs leaves the at2 receptor fully available for activation by local angii. thus, it is essential to determine whether the at2 receptor antagonizes, or mimics, the effects of the at1 subtype on cancer cell proliferation and invasion. expression of at2 receptors from either the tumor or stroma has been shown to attenuate the growth of pancreatic carcinoma, lung adenocarcinoma, and pheochromocytoma. in contrast, other studies indicate that at2 receptor expression associates with poor prognosis of astrocytomas and that its deletion or blockade delays tumor vascularization and progression. expression of at2 receptors in luminal epithelial cells of the normal breast has been shown to be significantly increased in breast hyperplasia and carcinomas. at2 may thus represent a new therapeutic target against breast cancer, and elucidating the functional role of these receptors in breast cancer is of major importance. the aim of the present study was to provide a cellular model for proper investigation of the effects of the at2 receptors in breast cancer progression. the d3h2ln cancer cell line (obtained from caliper 's) is a luciferase - expressing cell line that was derived from a spontaneous lymph node metastasis of mda - mb-231 human breast adenocarcinoma cells. d3h2ln cells were grown in dmem 4.5 g / l glucose supplemented with 10% fetal calf serum (fcs), 1% nonessential amino acids, and 1% penicillin streptavidin and were maintained at 37c in a 5% co2 atmosphere. the tripu3-ef1-flag - hat2-ires - gfp lentiviral vector (figure 1) was constructed from a modified trip lentiviral vector [30, 31 ] by inserting the entire coding sequence of flag - tagged human at2 receptor (flag - hat2) between bamhi and xhoi restrictions sites upstream of the internal ribosomal entry site (ires) gfp cassette. the human flag - hat2 sequence was amplified by pcr using the plasmid pbc - sf containing flag - hat2 cdna (a generous gift of dr. laurent daviet), the forward primer 5-gccggatccatgaagacgatcatcgccctgagc-3 containing the flag initiating codon, and the reverse primer 5-cgctcgagttaagacacaaaggtctccatttc-3 containing the stop codon of human at2. production of infectious recombinant lentiviruses was performed by transient transfection of 293 t cells as described in. for infections, 3 10 d3h2ln cells in 35 mm dishes were transduced with 5 g / ml of viral p24 (hiv-1 capsid protein). the resulting stable cell line d3h2ln - at2 was grown in complete medium as described above for parental d3h2ln. for facs analysis, cells were detached with pbs - edta 1 mm, and 10 cells were fixed in paraformaldehyde 4% for 10 min. after 3 washes in pbs, gfp - positive cells were analyzed on fc-500 facscalibur flow cytometer using cytomics rxp software. cells were lysed one hour in ice with ripa buffer containing 50 mm tris ph7.5, 100 mm nacl, 50 mm na fluoride, 0.1% sds, 0.5% na deoxycholate, and 1% triton x-100 and extemporaneously supplemented by protease inhibitors (1 mm pmsf, 1 g / ml aprotinin, 5 g / ml leupeptin, and 1 g / ml pepstatin). the cell lysate was directly used for western blotting or incubated with 2 g of anti - flag - m2 antibody (sigma) overnight with rotation at 4c for flag - at2 immunoprecipitation. protein g sepharose beads (50% slurry, roche) were then added for an additional hour at 4c. the immunocomplexes were washed three times with lysis buffer and eluted in laemmli 's sample buffer containing urea 6 m. after one hour denaturation at 60c, proteins and immunocomplexes were resolved on 10% sds - page and transferred to pvdf membranes. flag - hat2 expression was revealed using flag - m2-hrp antibodies and enhanced chemiluminescence (ecl+, ge healthcare). 10 cells / well) were transferred to 12-well culture plates 24 h before binding assays. one day after plating, cells were washed briefly in 25 mm tris - hcl buffer, ph 7.4 containing 140 mm nacl, 5 mm mgcl2, and 0.1% bsa. binding experiments were performed at 4c to avoid any functional interference such as receptor activation, phosphorylation, or internalization. binding was initiated by the addition of h - angii (4 pm) and increasing concentrations of nonradioactive angii (10 m to 10 m) as competitor, in a 500 l volume of binding buffer comprising 25 mm tris - hcl, ph 7.4, 5 mm mgcl2, 0.1% bsa, and 100 mg / ml bacitracin. selective at1 or at2 receptor antagonists, losartan (galena), and pd123319 (sigma), respectively, were used at 10 m. the aim of this study was to generate a useful cellular model for studying the role of at2 receptors in breast cancer. to this end, we constructed and characterized a human breast cancer cell line stably expressing high amounts of human at2 receptors at the plasma membrane. as a recipient for at2 receptor expression, we chose the highly aggressive d3h2ln subline derived from the well - known metastatic and triple - negative breast cancer cells mda - mb-231. d3h2ln cells are of particular interest for the study of breast cancer cell progression both in vitro and in vivo since they are highly invasive and metastatic. after intracardiac injection into nude mice, these cells rapidly disseminate and colonize distant organs including the brain, the lungs, and the bones which are the major sites of metastasis in human breast cancer. in addition, d3h2ln cells constitutively express high levels of firefly luciferase, which will be most convenient for future in vivo bioluminescence analysis of tumor progression and metastatic dissemination in response to at2 receptor activation. preliminary experiments indicated that d3h2ln cells express very low levels of endogenous at2 receptor transcripts as assessed by rt - pcr (data not shown) which was a prerequisite for our study. we thus designed a human at2 receptor - containing expression vector with the objective to reach high levels of expression of the at2 receptor and easy detection of the receptor at the cell membrane. first, to facilitate at2 receptor detection, we used a flag - tagged human at2 receptor (flag - hat2), which can be revealed by immunofluorescence and immunoprecipitation using anti - flag antibodies. we reasoned that by tagging the receptor at the extracellular n - terminus, we would also be able to easily detect its expression at the plasma membrane. to fulfill the other criteria and maximize the expression efficiency, the flag - hat2 receptor sequence was cloned into a modified trip lentiviral vector containing ires - gfp (figure 1). this lentiviral vector is of great interest since it allows high levels of at2 receptor expression, together with concomitant expression of the green fluorescent protein (gfp) that will serve as a positive control for infection efficiency. gfp expression will also be a valuable tool for the sensitive detection of the infected cells by facs and immunofluorescence studies. lentiviral particles containing flag - hat2 were thus produced and used to transduce d3h2ln cells for 48 h. stably infected cells maintained in culture were thereafter designated transduction efficiency was evaluated by flow cytometry measuring gfp - positive cells. as shown in figure 2(a), 99.5% of the cells transduced with the at2 lentiviral vector were positive for gfp expression, indicating that virtually all infected cells had incorporated the construct. we then evaluated whether d3h2ln cells also expressed detectable amounts of the at2 receptor. to this end, we performed western blotting and immunoprecipitation analyses using anti - flag antibodies. as shown in figure 2(b) (left panel), anti - flag - hrp antibodies revealed a major polypeptide at 45 kda corresponding to the molecular weight of unglycosylated flag - hat2 receptor, in d3h2ln - at2 but not in parental d3h2ln cells. additional polypeptides of higher molecular weights (80 and 110 kda) immunoprecipitated from d3h2ln - at2 cells (figure 2(b), right panel) might illustrate receptor dimerization or the presence of different glycosylated forms of the at2 receptor. in conclusion, in the present study, we successfully isolated a stable cell line (d3h2ln - at2) constitutively expressing the flag - tagged human at2 receptor and concomitantly the gfp. to note, these cells remained stable in culture after more than 15 passages (data not shown). to further characterize the d3h2ln - at2 cells, culture dishes were placed under a phase contrast microscope, and pictures were taken at low (100) and high (400) magnification. as shown in figure 3, there was no clear morphological differences between parental and d3h2ln - at2 cells, indicating that overexpression of at2 receptors in d3h2ln breast cancer cells does not significantly alter cell structure, shape, or organization. we next evaluated whether the ectopically expressed flag - hat2 receptor was localized at the cell surface of d3h2ln - at2 and able to bind angii with high affinity. to address these questions, competition binding experiments were performed on intact cells with tritium labeled angii (h - angii) in the presence of increasing concentrations of unlabelled angii. results revealed a classical competition binding profile in d3h2ln - at2 cells (figure 4(a)), indicating the presence of a single population of receptors with an ic50 of 1.55 0.45 nm (n = 3) for angii, as expected for a bona fide angii receptor. in contrast, no specific angii binding could be detected by binding assay in parental d3h2ln cells (data not shown), indicating that parental d3h2ln cells do not express detectable levels of at1 and at2 receptors in our experimental conditions. as shown in figure 4(b), total binding of 4 pm radiolabelled angii to d3h2ln - at2 cells could be displaced (75%) by adding an excess (1 m) of the selective at2 receptor antagonist pd123319 but not in the presence of an excess of the at1 receptor antagonist losartan. these results indicate that at2 is the major angii binding site in d3h2ln - at2 cells. they also suggest that overexpression of at2 in breast cancer cells does not modulate levels of membrane at1 receptors. thus, ectopically expressed flag - hat2 receptors in d3h2ln breast cancer cells are correctly folded at the plasma membrane and are able to bind the natural agonist with the expected high affinity. total receptor density at the cell surface was quantified to 65 pmol of receptor per 10 d3h2ln - at2 cells, which corresponds to a high level of receptors. in conclusion, we report here the generation and characterization of a novel model of human invasive breast cancer cells (d3h2ln - at2) that express high amounts of flag - tagged human at2 receptor at the plasma membrane. these cells also express gfp and luciferase, which makes them suitable for fluorescence and bioluminescence studies in vitro and in vivo. of interest, d3h2ln - at2 cells do not express detectable at1 binding sites, as evaluated by radioligand binding assay, therefore allowing the characterization of at2 functions independently of those related to at1 receptor activation, which is of great interest in the context of at1 blockade by arbs. breast cancer is a leading cause of death by malignancy in women worldwide, and identifying new personalized therapeutic targets to fight this disease is a challenge for the coming years. angiotensin receptors, being exposed at the plasma membrane, are easily targetable by selective receptor agonists or antagonists that may represent new and potent anticancer drugs. with the emergence of novel nonpeptidic selective agonists of the at2 receptor such as compound 21 [34, 35 ], reliable tools are now available to evaluate the effects of this receptor in vitro and in vivo. there is urgent need to determine the effects of the at2 receptor subtype in breast cancer. the cellular model presented here offers a unique opportunity to evaluate the consequences of at2 receptor activation and blockade on breast cancer proliferation, invasion, and migration, as well as on tumor growth and metastasis formation. | recent studies have highlighted the at1 receptor as a potential therapeutic target in breast cancer, while the role of the at2 subtype in this disease has remained largely neglected. the present study describes the generation and characterization of a new cellular model of human invasive breast cancer cells (d3h2ln - at2) stably expressing high levels of flag - tagged human at2 receptor (flag - hat2). these cells exhibit high - affinity binding sites for angii, and total binding can be displaced by the at2-selective antagonist pd123319 but not by the at1-selective antagonist losartan. of interest, high levels of expression of luciferase and green fluorescent protein make these cells suitable for bioluminescence and fluorescence studies in vitro and in vivo. we provide here a novel tool to investigate the at2 receptor functions in breast cancer cells, independently of at1 receptor activation. |
the uvb - driven conversion of cholesta-5, 7-diene-3-ol (7-dehydrocholesterol, 7dhc) to (3, 5z, 7e)-9, 10-secocholesta-5, 7, 10(19)-trien-3-ol (cholecalciferol, vitamin d3) is one of the most fundamental reactions in photobiology. in addition to vitamin d3, the reaction also generates tachysterol (6e-9, 10-secocholesta-5(10), 6, 8-trien-3-ol, t), lumisterol (9, 10-cholesta-5, 7-diene-3-ol, l), as well as iso - tachysterol. the conversion of 7dhc to its analogs has been demonstrated by the holick group as a two - step process (fig. 1). the first step is the formation of pre - d (unless specified, the term d used in this paper refer to the d3-like, d2-like, or pd analogs with short chains). the second step is the conversion of pre - d to the d, t, and l analogs. tremendous efforts have been made to investigate the photosynthesis and biological activity of many d analogs with different side chains on the 17-position. the effect of potential interactions of previtamin d3 with intracellular proteins and/or lipids in the skin on the kinetics and thermodynamics of the thermal isomerization to form vitamin d3 has been established. however, to the best of our knowledge, the effects of the sidechain length and composition on the real time conversion kinetics from pre - d to d have not been reported in the literature. uvb induced transformation of 5,7-dienes to the d, t, and l - like analogs. to determine the kinetics of the conversion of pre - d to d and to search for additional biologically active 5,7-dienes, a series of pregna-5, 7-dienes with different side chains were synthesized and subjected to uvb irradiation. the conversion from pre - d to d after uv initiation in addition, the distribution of each product from the uvb irradiation of novel 5,7-dienes synthesized was also described in this report to provide potential insights in optimizing yields of future organic synthesis for desired secosteroids and minimize undesired side products. the kinetics of the conversion from pre - d to d is shown in figure 2 using compound 4a - s as an example. before irradiation, only proton nmr peaks corresponding to the 5,7-diene were observed (peaks 2 and 6, figure 2a). after uvb irradiation for 5 min, many new peaks appeared corresponding to different initial photolysis products (fig. intensities of proton nmr peaks (peak number 5, 9 and 10, figure 2b and c) corresponding to pre - d decreased over time, while intensities of peaks (peak number 1,12,13, figure 2b and c) corresponding to d increased over time, clearly indicating that the thermodynamically unstable pre - d isomerized to the more stable d structure. there were no changes in the amounts for all other products (t, isot, and l) based on the peak intensities corresponding to those products once uvb irradiation was terminated. figure 2d shows the real - time monitoring of changes of pre - d and d over time at 40 time points (30 min / time point). the rate of conversion was calculated by fitting the corresponding peaks at those time points. 1.8 mm of each compound solution in methanol - d4 was irradiated for 5 min by uvb (280315 nm). each irradiated sample was monitored by proton nmr at 37c immediately after the irradiation in a time course manner. data was recorded for each time point of 0 min, 10 min, and every 30 min thereafter. relative rates of conversion for the irradiated products (pre - d - like to d - like) based on the slope (a) of the equation : y = ax+b ; solvent : cd3od ; concentration : 1.8 mmol. analysis of the compound structures and their relative conversion rate reveal two interesting trends associated with side chain length and composition. first, with a few exceptions, compounds with a longer (or larger) side chain (e.g., 7cc, 7cb, 7cd, 8a) generally have a faster conversion rate than compounds with a shorter (or smaller) side chain (e.g., 4a - s, 4c, 4a - r, 5c and 4b). second, all compounds having a tertiary alcohol moiety at the c20 position (e.g., the top four ranked compounds 7cc, 7cb, 7ca, and 7cd) have higher conversion rate than those not having this tertiary alcohol moiety. this may suggest that pre - d compounds with tertiary alcohol side chains are more prone to undergo isomerization to form d analogs, therefore leading to the higher conversion rate than the non - tertiary moieties. figure 3 shows the comparison of changes of d - like analogs over time using compounds 4a - r, 8a, and 7cc as examples. overall, compound 7cc has the highest conversion rate while compound 4a - s showed the lowest conversion rate. comparison of conversion from pre - d analogs to d - like analogs over time for 4a - r, 8a, and 7cc. the isomerization of pre - d to form d is an antarafacial -sigmatropic hydrogen shift from c19 to c9, one of processes defined by the woodward - hoffmann rule. this process requires a large rotation around c6-c7 bond which intuitively means a pre - d analog with a larger sidechain (e.g., 7cc) may convert to the d analog slower than an analog with a shorter sidechain (e.g., 4a). however, several previous report have shown that addition of a single hydroxyl group either on the sidechain (e.g., 25-hydroxylation) or on the ring systems (e.g., 11-hydroxylation) can significantly affect the kinetics of this conversion process. since most of the analogs reported in the current study have a hydroxyl group at c20, it is possible that 20-hydroxylation also exerts its influence to the overall kinetics. it may also be worthwhile to point out that the only reaction observed in this process (proton nmr monitoring after irradiation) is the conversion of pre - d to d analogs. the characteristic peaks for d kept increasing over time while the characteristic peaks for pre - d kept decreasing. this observation is consistent with the fact that the conversion from pre - d to d is a thermodynamic process, while the conversion from the 5,7-diene precursors to pre - d and from pre - d to tachysterol, lumisterol and iso - t is a photochemical process which requires uvb irradiation. the distribution of the products after 5.5 h post uvb irradiation is shown in table 2. we selected the 5.5 h as the ending point because after this time the kinetic curves from pre - d analogs to d analogs have significant deviation from linearity, although after a long time (5~7 d), virtually all pre - d analogs are converted to their corresponding d analogs. as we discussed earlier, except d and pre - d, the amounts of other products from uvb irradiation of the 5,7-dienes remains unchanged during the proton nmr monitoring process. the percentage of the parent compounds is generally reversely proportional to the relative conversion rate, although there are some exceptions. this can be confirmed by 4a - s, which had the lowest conversion rate but with the highest percentage of its parent compound (30%). compounds with the highest conversion rates (7cc, 7cb, 7ca ranked 1st, 2nd, and 3rd, respectively) had relatively lower percentages of their parent compounds (15%, 14%, 10% for 7cc, 7cb, 7ca, respectively). compounds 4b, 5c had both low percentages of their parent compounds (17%, 9% for 4b, 5c, respectively) and low conversion rates (4b, 5c ranked 9th, 10th, respectively). there does not appear to have an identifiable correlations between photochemical product distributions and the length or composition of the side chains in these 5,7-dienes, suggesting these two factors are not the dominant in this photochemical process. percentages of lumisterol - like (l - like) and iso - tachesterol - like (iso - t - like) compounds are similar for all tested compounds (49% and 611% for l - like and iso - t - like compounds, respectively). it should be of particular interest to note that several compounds tend to form more t - like compounds (48%, 30%, 35%, 43%, 37% for 7cc, 7cb, 7ca, 4b, 5c, respectively) during photolysis. this may suggest that the side chain of these molecules may accelerate the conversion of pre - d to t - like compounds. on the other hand, it might be attributed to the high stability of the t - like analogs of these compounds under uvb irradiation. the fact that compound 8a had only 1% of t - like remaining but with a relatively higher amount of iso - t (11%) may indicate that the t - like analog of 8a is extremely unstable in the presence of uvb irradiation and converted to the corresponding iso - t analog very quickly. the characteristic peak of each specific product (d, t, and l) was integrated 5.5h and divided by the sum of integrations of all products. na, not available ; d, vitamin d ; l, lumisterol ; t, tachysterol ; pre - d, pre - vitamin d ; iso - t, iso - tachysterol. even though no obvious correlation between the side chain and product distribution can be identified at this stage, these studies do suggest that they have certain influences on the product distribution. accumulating evidence in the literature clearly indicated that unique nonhypercalcemic d analogs possessing beneficial biological activities (e.g., antiproliferation and anti - inflammation) exist either endogenously produced or newly synthesized. such compounds likely have significantly improved toxicity profiles compared with 1,25-dihydroxy vitamin d3 (the biologically active form of d3). at least currently, making these compounds using the classical photochemistry method is much easier than total organic synthesis. therefore, understanding and proper use of the effects of the side chain and other factors on the photochemical product distributions may provide very useful controls when optimizing the yields of desired products while minimizing the undesired side products. the kinetics of the conversion from pre - d to d is shown in figure 2 using compound 4a - s as an example. before irradiation, only proton nmr peaks corresponding to the 5,7-diene were observed (peaks 2 and 6, figure 2a). after uvb irradiation for 5 min, many new peaks appeared corresponding to different initial photolysis products (fig. intensities of proton nmr peaks (peak number 5, 9 and 10, figure 2b and c) corresponding to pre - d decreased over time, while intensities of peaks (peak number 1,12,13, figure 2b and c) corresponding to d increased over time, clearly indicating that the thermodynamically unstable pre - d isomerized to the more stable d structure. there were no changes in the amounts for all other products (t, isot, and l) based on the peak intensities corresponding to those products once uvb irradiation was terminated. figure 2d shows the real - time monitoring of changes of pre - d and d over time at 40 time points (30 min / time point). the rate of conversion was calculated by fitting the corresponding peaks at those time points. 1.8 mm of each compound solution in methanol - d4 was irradiated for 5 min by uvb (280315 nm). each irradiated sample was monitored by proton nmr at 37c immediately after the irradiation in a time course manner. data was recorded for each time point of 0 min, 10 min, and every 30 min thereafter. relative rates of conversion for the irradiated products (pre - d - like to d - like) based on the slope (a) of the equation : y = ax+b ; solvent : cd3od ; concentration : 1.8 mmol. analysis of the compound structures and their relative conversion rate reveal two interesting trends associated with side chain length and composition. first, with a few exceptions, compounds with a longer (or larger) side chain (e.g., 7cc, 7cb, 7cd, 8a) generally have a faster conversion rate than compounds with a shorter (or smaller) side chain (e.g., 4a - s, 4c, 4a - r, 5c and 4b). second, all compounds having a tertiary alcohol moiety at the c20 position (e.g., the top four ranked compounds 7cc, 7cb, 7ca, and 7cd) have higher conversion rate than those not having this tertiary alcohol moiety. this may suggest that pre - d compounds with tertiary alcohol side chains are more prone to undergo isomerization to form d analogs, therefore leading to the higher conversion rate than the non - tertiary moieties. figure 3 shows the comparison of changes of d - like analogs over time using compounds 4a - r, 8a, and 7cc as examples. overall, compound 7cc has the highest conversion rate while compound 4a - s showed the lowest conversion rate. comparison of conversion from pre - d analogs to d - like analogs over time for 4a - r, 8a, and 7cc. the isomerization of pre - d to form d is an antarafacial -sigmatropic hydrogen shift from c19 to c9, one of processes defined by the woodward - hoffmann rule. this process requires a large rotation around c6-c7 bond which intuitively means a pre - d analog with a larger sidechain (e.g., 7cc) may convert to the d analog slower than an analog with a shorter sidechain (e.g., 4a). however, several previous report have shown that addition of a single hydroxyl group either on the sidechain (e.g., 25-hydroxylation) or on the ring systems (e.g., 11-hydroxylation) can significantly affect the kinetics of this conversion process. since most of the analogs reported in the current study have a hydroxyl group at c20, it is possible that 20-hydroxylation also exerts its influence to the overall kinetics. it may also be worthwhile to point out that the only reaction observed in this process (proton nmr monitoring after irradiation) is the conversion of pre - d to d analogs. the characteristic peaks for d kept increasing over time while the characteristic peaks for pre - d kept decreasing. this observation is consistent with the fact that the conversion from pre - d to d is a thermodynamic process, while the conversion from the 5,7-diene precursors to pre - d and from pre - d to tachysterol, lumisterol and iso - t is a photochemical process which requires uvb irradiation. the distribution of the products after 5.5 h post uvb irradiation is shown in table 2. we selected the 5.5 h as the ending point because after this time the kinetic curves from pre - d analogs to d analogs have significant deviation from linearity, although after a long time (5~7 d), virtually all pre - d analogs are converted to their corresponding d analogs. as we discussed earlier, except d and pre - d, the amounts of other products from uvb irradiation of the 5,7-dienes remains unchanged during the proton nmr monitoring process. the percentage of the parent compounds is generally reversely proportional to the relative conversion rate, although there are some exceptions. this can be confirmed by 4a - s, which had the lowest conversion rate but with the highest percentage of its parent compound (30%). compounds with the highest conversion rates (7cc, 7cb, 7ca ranked 1st, 2nd, and 3rd, respectively) had relatively lower percentages of their parent compounds (15%, 14%, 10% for 7cc, 7cb, 7ca, respectively). compounds 4b, 5c had both low percentages of their parent compounds (17%, 9% for 4b, 5c, respectively) and low conversion rates (4b, 5c ranked 9th, 10th, respectively). there does not appear to have an identifiable correlations between photochemical product distributions and the length or composition of the side chains in these 5,7-dienes, suggesting these two factors are not the dominant in this photochemical process. percentages of lumisterol - like (l - like) and iso - tachesterol - like (iso - t - like) compounds are similar for all tested compounds (49% and 611% for l - like and iso - t - like compounds, respectively). it should be of particular interest to note that several compounds tend to form more t - like compounds (48%, 30%, 35%, 43%, 37% for 7cc, 7cb, 7ca, 4b, 5c, respectively) during photolysis. this may suggest that the side chain of these molecules may accelerate the conversion of pre - d to t - like compounds. on the other hand, it might be attributed to the high stability of the t - like analogs of these compounds under uvb irradiation. the fact that compound 8a had only 1% of t - like remaining but with a relatively higher amount of iso - t (11%) may indicate that the t - like analog of 8a is extremely unstable in the presence of uvb irradiation and converted to the corresponding iso - t analog very quickly. the characteristic peak of each specific product (d, t, and l) was integrated 5.5h and divided by the sum of integrations of all products. na, not available ; d, vitamin d ; l, lumisterol ; t, tachysterol ; pre - d, pre - vitamin d ; iso - t, iso - tachysterol. even though no obvious correlation between the side chain and product distribution can be identified at this stage, these studies do suggest that they have certain influences on the product distribution. accumulating evidence in the literature clearly indicated that unique nonhypercalcemic d analogs possessing beneficial biological activities (e.g., antiproliferation and anti - inflammation) exist either endogenously produced or newly synthesized. such compounds likely have significantly improved toxicity profiles compared with 1,25-dihydroxy vitamin d3 (the biologically active form of d3). at least currently, making these compounds using the classical photochemistry method is much easier than total organic synthesis. therefore, understanding and proper use of the effects of the side chain and other factors on the photochemical product distributions may provide very useful controls when optimizing the yields of desired products while minimizing the undesired side products. we have synthesized several novel 5,7-dienes and examined the real - time conversion kinetics from pre - d analogs to their corresponding d analogs. the rate of this conversion was found to be correlated with side chain length and composition. photochemical product distributions from these 5,7-dienes are also documented under the experimental conditions, although no significant correlations to the side chain was observed. to the best of our knowledge this is the first time that the kinetics for conversion in real time from pre - d to d is described. we are well aware the limitation of our current study performed in an isotropic solution environment that is too simple compared with the complex biological environment existing in the human skin. the interactions between pre - vitamin d3 and the surrounding biological environment have profound effects to the kinetics and thermodynamics of vitamin d3 formation. therefore, the future direction of this study will be to evaluate this thermal isomerization process of pre - d to d under a more physiologically relevant condition by using either human skin or its mimic such as a liposomal model. nevertheless, the current study could still serve as step - stones for future kinetic studies of novel 5,7-dienes under more physiologically relevant ex vivo or in vivo conditions, as well as providing insights in controlling yields of the biologically active products such as nonhypercalcemic d analogs from the photochemical transformation of novel 5,7-dienes. compound 8a (7dhc) was purchased from sigma - aldrich and recrystallized from methanol to remove impurities. compound 8b (ergosterol) was also purchased from sigma - aldrich and used for nmr experiment without further purification. compounds 4a - c, 5c, 7cc, 6d and 6da were generated following a previously reported procedure. generally, compound 1 was protected by an acetyl group at the 3-position to give compound 2. compound 2 was then brominated at the 7-position followed by dehydrobromination to afford compound 3 with an extra double bond at the 7 position. compounds 7ca, 7cb, and 7cd are new molecules that have not been reported previously. the synthesis of 7ca - cd was performed by reacting 3c with appropriate alky lithium or grignard reagents to build up the tertiary alcohol moiety in the structure (fig. alkyl lithium reagents for the synthesis of 7ca and 7cd were commercially available from sigma - aldrich. grignard reagents used for the synthesis of 7cb and 7cc were freshly prepared by reacting the corresponding alkyl bromide with magnesium in anhydrous thf. nmr and mass spec data : 20-oh-20-methyl-7dhp (7ca) : h nmr (500 mhz, cd3od) : 5.555.58 (m, 1 h), 5.395.42 (m, 1 h), 3.493.57 (m, 1 h), 2.402.46 (m, 1 h), 2.202.31 (m, 2 h), 1.312.01 (m, 24 h), 1.30 (s, 3 h), 1.23 (s, 3 h), 0.97 (s, 3 h), 0.80 (s, 3 h). esi - ms : calculated for c22h34o2, 330.3, found 353.3 [m+na ]. 20-oh-20-hexyl-7dhp (7cb) : h nmr (500 mhz, cd3od) : 5.545.59 (m, 1 h), 5.385.42 (m, 1 h), 3.483.57 (m, 1 h), 2.402.47 (m, 1 h), 2.252.30 (m, 1 h), 2.162.25 (m, 1 h), 1.302.00 (m, 24 h), 1.28 (s, 3 h), 0.97 (s, 3 h), 0.93 (t, j = 6.0 hz, 3 h), 0.82 (s, 3 h). esi - ms : calculated for c27h44o3, 400.3, found 423.3 [m+na ]. 20-oh-20-phenyl-7dhp (7cd) : h nmr (500 mhz, cd3od) : 7.46 (d, j = 8.0 hz, 2 h), 7.29 (t, j = 8.0 hz, 2 h), 7.18 (t, j = 7.5 hz, 1 h), 5.535.56 (m, 1 h), 5.355.39 (m, 1 h), 3.493.56 (m, 1 h), 2.392.45 (m, 1 h), 2.222.30 (m, 1 h), 1.682.06 (m, 8 h), 1.66 (s, 3 h), 1.251.65 (m, 7 h), 0.96 (s, 3 h), 0.83 (s, 3 h). esi - ms : calculated for c27h36o2, 392.3, found 415.3 [m+na ]. the synthesis and chromatographic data for 7cc, 6d, 6da, 4b, 4c, 5c, 4a - r, 4a - s were described in our previous publications. figure 4. step a is for 1a, 1d only ; step d is for 3(a c) only ; step 3 is for 3c only ; step f is for 3d only ; step g is for 3c only. reagents and conditions : (a) ac2o, microwave, p - toluenesulfonic acid monohydrate ; (b) dibromantin, 2,2'-azobisisobutyronitrile, benzene / hexane (1:1), 100c, reflux ; (c) bu4nbr, bu4nf, thf, rt ; (d) lialh4, thf, 0c ; (e) k2co3, meoh - thf, argon bubbling, rt ; (f) k2co3, meoh - thf, air, rt ; (g) r - li (7ca,7cd) or r - mgbr (7cb - ml), thf ; (h) dbu, thf, mei. each of these compounds was dissolved in methanol - d4 to make a 1.8 mm solution and subsequently transferred to a quartz nmr tube. a standard proton nmr at 37c was run for each sample before irradiation by uvb. each sample was then irradiated for 5 min using a biorad uv transilluminator 2000 (biorad, hercules, ca). the spectral characteristics of the uvb (280320 nm) source were published previously and its strength (4.8 0.2 mw cm) was measured routinely using a digital uvb meter model 6.0 (solartech inc. each irradiated sample was monitored by proton nmr at 37c immediately after the irradiation in a time course manner. data was recorded for each time point of 0 min, 10 min, and every 30 min thereafter. analysis of data was accomplished by using acd software (advanced chemistry development, toronto, on, canada). the percentage of products (d, t, l, iso - t) at each time point is determined by the peak areas calculated from the proton nmr spectra. the characteristic peak of each specific product (d, t, and l) was integrated at each time point and plotted against time to produce a kinetic curve (supporting information). the equation generated from this curve is expressed as follows : y = ax+b, where a stands for the slope, b stands for the intercept of the curve in y - axis. the relative accuracy of the equation is judged by r (the closer to 1, the better). | novel pregna-5, 7-dienes were synthesized and subjected to uvb irradiation to generate the corresponding pre - d intermediates, tachysterol and lumisterol analogs. the kinetics of the conversion from each of the pre - d intermediates to the corresponding novel d analogs was investigated by using real time 1h nmr measurements inside the nmr magnet. both the length and composition of the side chains were found to affect the rate of the kinetic conversion from pre - d intermediates to the thermodynamically more stable d analogs. compound 7cc which has both a long side chain and a tertiary alcohol moiety showed the highest conversion rate, while compound 4a - s which has a very short side chain without the tertiary alcohol had the lowest conversion rate among the 13 tested compounds. we also determined product distributions for these 5,7-dienes upon uvb irradiation followed by thermodynamic equilibration. no clear correlations between product distribution and side chain length or composition were identifiable under the current experimental conditions, suggesting there are other factors affecting the kinetics during the photochemical reactions for these 5,7-dienes. to the best of our knowledge, this is the first time the influences of side chain length and composition on the real time conversion kinetics from pre - d to d are studied. this study could serve as step - stones in future kinetic studies of novel biologically active 5,7-dienes and their corresponding d analogs under more physiologically relevant ex vivo or in vivo conditions, as well as providing important insights into optimizing yields of the desired active products during their organic syntheses. |
glioblastoma (gbm) is the most frequent primary central nervous system malignancy, with a peak incidence between 75 and 84 years of age [1, 2 ]. temozolomide (tmz) given concurrently with radiation therapy followed by the same agent as monotherapy for 6 months to 1 year is the current standard of care for patients with newly diagnosed gbm [3, 4 ]. although grade 34 absolute leukopenia and neutropenia rarely occur, almost 80% of patients receiving tmz are at risk of severe lymphopenia [3, 4, 5 ]. most importantly, impaired cellular immune response resulting from selective cd4 + t - cell depletion has been described in patients receiving tmz for the treatment of different malignancies [6, 7, 8 ]. the occurrence of opportunistic infections during treatment with tmz is a well - documented complication, especially those caused by pneumocystis jiroveci, which led the us food and drug administration (fda) to recommend the use of routine prophylaxis. other pathogens, including cytomegalovirus and varicella zoster virus, have also been anecdotally reported in the context of tmz use [10, 11 ]. here we report the case, the first to our knowledge, of invasive pulmonary aspergillosis in a patient receiving tmz for treatment of newly diagnosed gbm. a 78-year - old woman was diagnosed 4 months before admission with a right frontal gbm with extension to the basal ganglia and temporal lobe. the patient underwent a frontal craniotomy and partial resection of the lesion, followed by hypofractionated radiation therapy (16 fractions of 250 cgy) with concomitant daily tmz (75 mg / m). during the concurrent phase, she developed grade 1 lymphopenia and grade 2 nausea, but the treatment was otherwise well tolerated. two days after completing chemoradiation, the patient was admitted for dyspnea and asymmetrical lower extremity edema. at admission, she denied cough, sputum, hemoptysis or thoracic pain. medications included omeprazole, amlodipine, metformin, gliclazide, sitagliptin and prophylactic sulfamethoxazole - trimethoprim. she was also taking dexamethasone 4 mg / day on a tapering scale. upon physical examination, she had normal vital signs, with a respiratory rate of 18 breaths / min and oxygen saturation of 95% on room air. she had asymmetric edema of the lower limbs and, on auscultation, fine respiratory crackles bilaterally. blood counts were : hemoglobin 11.9 g / dl, platelets 67,000/mm and leukocytes 7,030/mm, with an absolute lymphocyte count of 800/mm. a venous duplex - scan of the lower extremities showed an extensive deep vein thrombosis involving the left common femoral vein. an angiotomography of the thorax diagnosed an acute pulmonary embolism and also revealed scattered lobulated nodules with areas of cavitation, measuring up to 2.7 cm, located predominately in the upper lobes, some with a surrounding ground - glass halo. serum galactomannan levels, determined using an enzyme - linked immunosorbent assay, were elevated (optical density index = 6.7, optical density index 0.5 is considered a positive result). blood samples obtained for culture and cryptococcus neoformans and histoplasma antigen assays were negative. a ct - guided pulmonary biopsy was then performed. histochemical analysis (gomori 's silver stain) showed septate hyphal elements with acute - angle branching (fig. the patient was promptly started on antifungal therapy with voriconazole and was discharged to complete the treatment as an outpatient. a control ct scan done approximately 30 days after admission revealed a reduction of the nodular lesions and ground - glass component previously identified (fig. written informed consent for publication of the clinical details and/or clinical images was obtained from the guardian / relative of the patient. gbm is the most frequent primary central nervous system malignancy [1, 2 ]. surgery followed by concurrent radiation, tmz and adjuvant tmz is the current standard of care for patients with newly diagnosed gbm [3, 4 ]. in this report, we present the case of a 78-year - old female diagnosed with pulmonary aspergillosis shortly after completing concurrent chemoradiation. the patient was treated with voriconazole, which is the drug of choice for the treatment of invasive aspergillosis according to published guidelines. in most patients, tmz is well tolerated, and major adverse events include nausea, vomiting and myelotoxicity, mostly thrombocytopenia and lymphopenia. lymphocyte count depletion may result in an increased risk of opportunistic infections, and selective cd4 + t - cell depletion was reported in patients receiving tmz for the treatment of melanoma and neuroendocrine tumors [7, 8, 9 ]. albeit uncommon, the association of tmz with p. jiroveci pneumonia is well described in the literature, and the use of prophylaxis with smx - tmp is currently recommended by the fda [3, 6, 10 ]. among 64 patients accrued in the phase 3% of patients receiving radiotherapy plus tmz had severe infections, but only 2 cases of proven opportunistic infections occurred [3, 4 ]. scarce reports in the literature also suggest an increased risk of infection by other agents, including cytomegalovirus and varicella zoster virus [10, 11 ]. pulmonary aspergillosis is an uncommon condition in healthy individuals and occurs most frequently in the setting of immunosuppression, including patients with acquired immunodeficiency syndrome and those undergoing treatment for hematological malignancies associated with prolonged neutropenia and high doses of corticosteroids. aspergillus is a ubiquitous fungus that is often identified in cultures of respiratory fluids even in the absence of infection. drugs that result in impaired cellular immune response mechanisms could result in an increased risk of pulmonary aspergillosis. the classic triad of pulmonary aspergillosis characterized by hemoptysis, chest pain and fever was absent in our patient. however, the combination of the radiological findings, elevated galactomannan antigen levels, histopathological presentation and a positive culture for a. fumigatus confirmed the diagnosis. galactomannan is a constitutive antigen of the aspergillus cell wall released during growth of hyphae that can be measured in the serum or bronchoalveolar lavage fluid. in invasive aspergillosis, the sensitivity and specificity of the method range from 61 to 71 and 89 to 93%, respectively. it is important to stress that the galactomannan assay exhibits cross - reaction with other agents, including histoplasma. in addition, false - positive results may occur in the presence of antibiotics, particularly piperacillin / tazobactam. although the first line of defense against inhaled conidia is represented by alveolar macrophages that trigger a neutrophil - mediated innate response, antigen - presenting cells play a key role in the activation of cellular adaptive immunity, in a process that involves cd4 + t - cell differentiation. therefore, lymphopenia and, more importantly, cd4 + t - cell depletion, represent major predisposing host factors for invasive aspergillosis [13, 15 ]. as previously mentioned, selective cd4 + lymphopenia is a well - described adverse effect induced by tmz. in a series of 97 patients with melanoma treated with tmz 75 mg / m daily for 6 weeks, 60% of patients developed an absolute lymphocyte count < 800/mm, with a clear predominance of cd4 + t - cell depression. in a recent publication by grossman., cd4 + t - cell counts of individuals with newly diagnosed high - grade gliomas receiving tmz were prospectively evaluated. cd4 + t - cell counts lower than 300/mm occurred in 73% of patients, and survival was influenced by lymphocyte toxicity grades. in addition to lymphopenia, tmz could also impair lymphocyte function through blockage of intracellular signaling pathways. the possibility that prolonged use of corticosteroids could have contributed to the onset of the invasive infection must be taken into account ; however, the immunosuppressive effect of tmz has been described in subsets of populations for whom continuous use of corticosteroids is seldom recommended, including patients with melanoma and neuroendocrine tumors [6, 7, 8 ]. the case reported herein adds to the available evidence suggesting that patients receiving tmz are at risk for aspergillosis, even though these events are rare. since there is no recommendation for prophylaxis against opportunistic agents other than p. jiroveci, individuals in this situation should be closely monitored for infections, and even unspecific symptoms should prompt an immediate and thorough investigation. | leukopenia and selective cd4 + lymphopenia represent major adverse events associated with the use of temozolomide (tmz), an oral alkylating agent incorporated in the treatment of glioblastoma (gbm). the increased risk of opportunistic infections, including those caused by pneumocystis jiroveci and cytomegalovirus, has been previously described in the literature. here we report the case, the first to our knowledge, of a patient with pulmonary invasive aspergillosis immediately after the completion of chemoradiation with tmz for gbm. diagnosis was confirmed through a ct - guided lung biopsy, and the patient had excellent response to systemic voriconazole. this case illustrates that tmz can be associated with severe opportunistic infections, presumably associated with t lymphocyte immune dysfunction, and patients exposed to this agent should be carefully monitored. |
a hydatid cyst contains the larval stage of the tapeworm, echinococcus granulosus, which is a parasite responsible for the zoonotic illness, echinococcosis. it has previously been demonstrated that some parasitic infections induce antitumor activity against certain types of cancers [16 ]. in addition, mice immunized with toxoplasma gondii tachyzoites, or toxocara canis egg antigens, that were subsequently challenged with fibrosarcoma cells, showed a reduction in solid tumor growth compared with mice that were not immunized, yet were challenged with the same cells. an inhibition of tumor cell proliferation has also been associated with certain types of parasites. these findings support the hygiene theory whereby a decrease in exposure to infectious agents early in life is predicted to increase susceptibility to allergies, and perhaps autoimmune diseases [911 ]. protoscolices are small masses with four suckers and a double row of hooks and each is capable of developing into an adult worm in the intestine of the final host. akgl. showed that in a large retrospective study of patients with hydatid disease, the prevalence of cancer was significantly lower than in normal subjects. in another study, it has been shown that cancer - associated mucin - type o - glycosylated antigens such as tn and sialy - tn antigen were presented in hydatid cyst protoscolices and adult stage of echinococcus granulosus. these common antigens may have a role in induction of immunological cross - reactions between cancers and hydatid cyst. to explore the anticancer activity of this parasite, the effects of hydatid cyst protoscolices on the proliferation and death of mouse fibrosarcoma cells, and baby hamster kidney (bhk) fibroblast cells, was investigated. in this experimental study, echinococcus granulosus hydatid cysts were collected from sheep or cattle from a slaughter house in isfahan, iran. protoscolices were then centrifuged at 2000 g, for 2 min, and the supernatant was discarded. 10 ml saline was added to the remaining protoscolices in a test tube and centrifuged as above, and the supernatant was discarded. two cell lines including bhk fibroblast cells, as well as wehi-164 and balb / c mouse fibrosarcoma cells, were provided by the pasture institute (tehran, iran). cells were cultured in dulbecco 's modified eagle 's medium (dmem ; sigma) supplemented with 20 mm hepes, 0.2 mm l - glutamine, 50 m 2-mercaptoethanol, 0.15% sodium bicarbonate, 50 g / ml gentamicin, and 10% fetal calf serum (fcs), as reported previously. the in vitro lysis of wehi-164 tumor cells by parasites was assayed by detecting the release of ldh as reported previously. briefly, 1 10 wehi-164 cells were incubated with hydatid cyst protoscolices at 37c in 5% co2 and 90% relative humidity. at the indicated time points the amount of ldh present in each sample was estimated using the kit (parsazmoon, tehran, iran) according to the manufacturer 's instructions. for each experiment, six culture flasks containing 10 ml culture medium with 1 10 freshly prepared and viable wehi-164 or bhk cells were prepared. flasks a, b, and c were treated with 10, 50, or 100 live hydatid cyst protoscolices, respectively, while the remaining three flasks were left untreated as control samples. cell count and ldh concentrations were determined for each flask after 48 h. each experiment was performed in triplicate. the proliferation and viability of wehi-164 fibrosarcoma cells treated with echinococcus granulosus hydatid cyst protoscolices (figure 1) were compared with untreated wehi-164 fibrosarcoma cells (control group). in these assays, however, when 50 or 100 protoscolices were added, cell proliferation was inhibited and cell lysis increased. the cell counts for these assays are presented in table 1 and figure 1, while the ldh assay results are presented in figure 2. in similar experiments, the effects of hydatid cyst protoscolices on the proliferation and viability of bhk fibroblast cells were evaluated in comparison with untreated bhk cells (control group). a significant difference between the number of viable cells detected in treated and control groups was observed. however, the difference in the number of dead cells associated with each group was not significant (table 2 and figure 3). the results of this study indicate that hydatid cyst protoscolices inhibit the proliferation of wehi-164 and bhk cells and have the capacity to induce cell death in wehi-164 cells in vitro. these results are consistent with previous studies that have shown some parasitic and microbial infections interfere with tumor growth to mediate anticancer activities [18 ]. for example, different strains of trypanosoma cruzi produce a reagent that selectively damages human cancer cells in vitro and reduces tumor growth in vivo [2, 4, 15 ]. therefore, the anticancer activity observed for hydatid cyst protoscolices is consistent with these previous studies. in turkey, 1/1200 patients with differing hematologic neoplastic diseases have experienced acute leukemia and liver hydatidosis concomitantly. moreover, given that hydatid cysts are endemic in turkey, this rate of incidence is also consistent with the findings of the present study. in another investigation, akgl. showed that in a large retrospective study of patients with hydatid disease, the prevalence of cancer was significantly lower than in normal subjects. the anticancer activity of hydatid cyst protoscolices may be due to surface antigen activity, or the excretory - secretory products of the parasite. in work by alvarez errico., the carcinoma - associated tn antigens were detected in echinococcus granulosus protoscolices and circulating tn antigens in hydatid patients. these antigens have been implicated in metastasis of tumor cells, and a direct link has been shown between carcinoma aggressiveness and the density of them. however, the mechanisms involved in the inhibition of cell proliferation and induction of cell lysis mediated by hydatid cyst protoscolices are not clear and require further investigation. | both in vitro and in vivo models have demonstrated that some parasites can interfere with tumor cell growth. the present study investigates the anticancer activity of hydatid cyst protoscolices on wehi-164 fibrosarcoma cells and baby hamster kidney (bhk) fibroblast cells in vitro. those above two cell types were treated with live hydatid cyst protoscolices or left untreated for control groups. after 48 h, lactate dehydrogenase (ldh) and cell counts were assayed for both treated cells and control groups. following treatment with hydatid cyst protoscolices, cell proliferation of both cell types was inhibited, and lysis of fibrosarcoma cells increased. based on these results, it appears that hydatid cyst protoscolices have strong anticancer activity, and additional studies are needed to further clarify the mechanisms of this activity. |
the association between diabetes mellitus (dm) and cancer has recently received significant attention due to increases in the prevalence of both diseases. dm is not a single disease, but a group of metabolic disorders characterized by a series of potential confounding factors (obesity, varying levels of metabolic control, profiles of antidiabetic treatment and possible chronic complications or comorbidities) that may influence the association between diabetes and cancer. therefore, the characteristics of cancer and the metabolic abnormalities of their host may influence cancer cell survival, proliferation, and spread. upper tract urothelial carcinoma (utuc), histologically similar to bladder tumor, is less common than bladder cancer. from this perspective, there is little clinical evidence of oncological outcomes and prognostic factors in utuc after radical nephroureterectomy (rnu), and most well - known prognostic factors are related to tumor factors such as stage, grade, and tumor multifocality. therefore, the preoperative prognostic factors related to utuc patients should be identified. several studies have demonstrated that patients with dm have greater cancer mortality compared with non dm patients, and published studies reporting evidence linking dm and bladder cancer showed that dm has a negative effect on bladder cancer prognosis. however, to the best of our knowledge, there is a lack of data regarding the prognostic significance of preoperative glycemic control in surgically treated patients with utuc who have dm. therefore, in the current study, we examined the impact of dm and glycemic control on the prognosis of utuc after rnu. data from 597 utuc patients who underwent rnu between 2004 and 2014 were collected from six tertiary medical centers in korea. patients with a previous history of bladder cancer, regional lymph node metastasis or distant metastasis (the lymph node status was only purely based on preoperative radiologic findings), or received preoperative chemotherapy were excluded. patients who had dm (all type2 dm), but in whom the preoperative hemoglobin a1c (hba1c) level was not checked, were also excluded. finally, 566 patients were reviewed retrospectively. the patient demographics, perioperative data, pathologic findings, and clinical outcomes, including survival data, were collected retrospectively using a prespecified template for consistent data collection for an electronic medical record review. preoperative radiological evaluations included abdominal computed tomography and chest x - ray or computed tomography and (when clinically indicated) positron emission tomography or bone scan. for the analysis, the patients were divided into three groups : patients without a history of dm, patients with well controlled dm (hba1c 10 mm that was previously undetected on a computed tomography scan in the operative field, regional lymph nodes, and/or distant organs. recurrence of bladder cancer was not considered as disease recurrence. a biopsy for tissue confirmation all surgical specimens were processed according to the standard pathological procedures and were reviewed by uro - pathologists. tumors were staged according to the american joint committee on cancer seventh edition tnm staging system. the tumor grade was assessed according to the 1998 world health organization / international society of urologic pathology consensus classification. demographic, clinical and pathological data were compared using the kruskal - wallis test for continuous variables and the chi - square test for categorical variables among the three groups. survival analyses were performed using the kaplan - meier method with the log rank test. cox proportional hazard regression analysis was used for identification of independent prognostic factors for each dependent variable. all p - values were two - sided and p 10 mm that was previously undetected on a computed tomography scan in the operative field, regional lymph nodes, and/or distant organs. recurrence of bladder cancer was not considered as disease recurrence. a biopsy for tissue confirmation all surgical specimens were processed according to the standard pathological procedures and were reviewed by uro - pathologists. tumors were staged according to the american joint committee on cancer seventh edition tnm staging system. the tumor grade was assessed according to the 1998 world health organization / international society of urologic pathology consensus classification. demographic, clinical and pathological data were compared using the kruskal - wallis test for continuous variables and the chi - square test for categorical variables among the three groups. survival analyses were performed using the kaplan - meier method with the log rank test. cox proportional hazard regression analysis was used for identification of independent prognostic factors for each dependent variable. all p - values were two - sided and p < 0.05 was considered statistically significant. the median age of enrolled patients was 70.0 years (interquartile range [iqr ], 13 years), with a median follow - up period of 33.8 months (iqr, 41.4 months). the clinicopathological characteristics were similar among the no - dm group (n=431), well controlled dm group (n=68), and poorly controlled dm group (n=67) except for recurrence rate, cancer death rate, and rate of death from any cause. the recurrence rate, cancer death rate, and rate of death from any cause were high in the poorly controlled dm group compared to the non - dm and well controlled dm groups (p < 0.05) (table 1). the median time to recurrence was 17.7 months (iqr, 28.5 months) ; 92 patients (16.3%) had disease recurrence after rnu. of these, 21 patients (3.7%) had local recurrence and 71 patients (12.5%) experienced distant metastasis. compared to the non - dm and well controlled dm patients, poorly controlled dm patients showed significantly shorter rfs (no dm vs. hba1c 7, p=0.011 ; hba1c < 7 vs. hba1c 7, p=0.001) (fig. 1a). however, no difference was observed between non - dm patients and well controlled dm patients (p=0.05) (fig. in univariable and multivariable cox regression analyses, poorly controlled dm was associated with increased risk of disease recurrence (univariable : hazard ratio [hr ], 1.96 ; 95% confidence interval [ci ], 1.15 to 3.34 ; p=0.013 ; multivariable : hr, 2.26 ; 95% ci, 1.31 to 3.90 ; p=0.003) (tables 2 and 3). during follow - up, the median time to cancer - specific death was 30.4 months (iqr, 39.3 months). the css at 3 and 5 years was 85.5% and 76%, respectively. compared to the non - dm and well controlled dm patients, poorly controlled dm patients showed significantly shorter css (no dm vs. hba1c 7, p=0.001 ; hba1c < 7 vs. hba1c 7, p=0.001) (fig. 1b), and no significant difference was observed between non - dm and well controlled dm patients (p=0.418) (fig. poorly controlled dm was associated with increased risk of cancer - specific mortality (univariable : hr, 2.93 ; 95% ci, 1.79 to 4.78 ; p=0.001 ; multivariable : hr, 2.96 ; 95% ci, 1.80 to 4.87 ; p=0.001) (tables 2 and 3). with respect to overall mortality, the median time to death from any cause was 30.0 months (iqr, 39.0 months). similar to rfs and css, poorly controlled dm patients had significantly shorter os compared to non - dm and well controlled dm patients (no dm vs. hba1c < 7, p=0.075 ; no dm vs. hba1c 7, p=0.001 ; hba1c < 7 vs. hba1c 7, p=0.001) (fig. 1c). in univariable and multivariable cox regression analyses, poorly controlled dm was associated with increased risk of overall mortality (univariable : hr, 2.10 ; 95% ci, 1.41 to 3.12 ; p=0.002 ; multivariable : hr, 2.13 ; 95% ci, 1.40 to 3.22 ; p=0.001) (tables 2 and 3). of note, well controlled dm showed borderline significance in univariable analysis for os, but lost its significance in multivariable analysis. the median age of enrolled patients was 70.0 years (interquartile range [iqr ], 13 years), with a median follow - up period of 33.8 months (iqr, 41.4 months). the clinicopathological characteristics were similar among the no - dm group (n=431), well controlled dm group (n=68), and poorly controlled dm group (n=67) except for recurrence rate, cancer death rate, and rate of death from any cause. the recurrence rate, cancer death rate, and rate of death from any cause were high in the poorly controlled dm group compared to the non - dm and well controlled dm groups (p < 0.05) (table 1). the median time to recurrence was 17.7 months (iqr, 28.5 months) ; 92 patients (16.3%) had disease recurrence after rnu. of these, 21 patients (3.7%) had local recurrence and 71 patients (12.5%) experienced distant metastasis. compared to the non - dm and well controlled dm patients, poorly controlled dm patients showed significantly shorter rfs (no dm vs. hba1c 7, p=0.011 ; hba1c < 7 vs. hba1c 7, p=0.001) (fig. 1a). however, no difference was observed between non - dm patients and well controlled dm patients (p=0.05) (fig. 1a). in univariable and multivariable cox regression analyses, poorly controlled dm was associated with increased risk of disease recurrence (univariable : hazard ratio [hr ], 1.96 ; 95% confidence interval [ci ], 1.15 to 3.34 ; p=0.013 ; multivariable : hr, 2.26 ; 95% ci, 1.31 to 3.90 ; p=0.003) (tables 2 and 3). during follow - up, 82 patients (14.4%) died from utuc. the median time to cancer - specific death was 30.4 months (iqr, 39.3 months). the css at 3 and 5 years was 85.5% and 76%, respectively. compared to the non - dm and well controlled dm patients, poorly controlled dm patients showed significantly shorter css (no dm vs. hba1c 7, p=0.001 ; hba1c < 7 vs. hba1c 7, p=0.001) (fig. 1b), and no significant difference was observed between non - dm and well controlled dm patients (p=0.418) (fig. poorly controlled dm was associated with increased risk of cancer - specific mortality (univariable : hr, 2.93 ; 95% ci, 1.79 to 4.78 ; p=0.001 ; multivariable : hr, 2.96 ; 95% ci, 1.80 to 4.87 ; p=0.001) (tables 2 and 3). with respect to overall mortality, the median time to death from any cause was 30.0 months (iqr, 39.0 months). similar to rfs and css, poorly controlled dm patients had significantly shorter os compared to non - dm and well controlled dm patients (no dm vs. hba1c < 7, p=0.075 ; no dm vs. hba1c 7, p=0.001 ; hba1c < 7 vs. hba1c 7, p=0.001) (fig. 1c). in univariable and multivariable cox regression analyses, poorly controlled dm was associated with increased risk of overall mortality (univariable : hr, 2.10 ; 95% ci, 1.41 to 3.12 ; p=0.002 ; multivariable : hr, 2.13 ; 95% ci, 1.40 to 3.22 ; p=0.001) (tables 2 and 3). of note, well controlled dm showed borderline significance in univariable analysis for os, but lost its significance in multivariable analysis. a previous study reported that preexisting dm increases the risk of several cancers, including cancer of the breast, colorectum, endometrium, liver, and pancreas. in american cancer society cancer prevention study ii, adults with dm were at increased risk for cancer related mortality. other studies have reported a possible association of dm with mortality from cancer of the liver, pancreas, colorectum, lung, and breast. similarly, diabetes was associated with a statistically significant 1.3- to 2.5-fold increased risk of bladder cancer in previous cohort studies, and studies on cancer mortality have found that bladder cancer patients with diabetes have greater cancer mortality compared with their nondiabetic counterparts. utuc is relatively rare and shares many characteristics with urothelial cancer of the bladder, therefore most decision making regarding utuc is extrapolated from evidence in bladder cancer. however, there are anatomical, biological, and practical differences between bladder cancer and utuc. compared with other malignancies, fewer studies on the potential impact of dm on css in patients with utuc have been reported in the literature. rieken. reported that diabetic patients with utuc who did not use metformin were at significantly higher risk of disease recurrence and cancer - specific death compared to nondiabetic patients and diabetic patients with utuc who used metformin. hwang. reported that dm was an independent risk factor for rfs in utuc, but they did not report on the relationship between glycemic control status and long - term prognosis such as css and os. in the current study, we found that diabetic utuc patients with poor glycemic control showed shorter median rfs, css, and os compared with diabetic utuc patients with good glycemic control and non - diabetic patients. there was no significant difference between non - dm and well controlled dm patients. in our study, we used the hba1c level, which is a more informative measure compared with a simple dm history or a single serum glucose test. our results showed that glucose regulation status using hba1c was a clinically significant prognostic factor for predicting the survival of patients with utuc. previous studies reported association of poor glycemic control according to the hba1c level with worse outcomes in other cancers.. reported that poor glycemic control was related to disease progression in renal cell carcinoma and proposed that stricter glycemic control would contribute to improved outcomes. siddiqui. also reported an association of elevated hba1c with aggressive clinical behavior in patients with colorectal cancer. an informative measure such as hba1c which can reflect the glycemic control status rather than a simple dm history or a single glucose test is warranted in patients with utuc. in addition, our study suggests that even diabetic patients could have long - term survival comparable with non dm patients through strict glucose control. the mechanism by which dm contributes to cancer mortality has not been fully elucidated, however plausible explanations have been suggested to explain the relationship between dm and cancer. hyperglycemia can provide more glucose to tumor cells, and hyperinsulinemia elicited by hyperglycemia could lead to activation of insulin / insulinlike growth factor 1, which can influence cancer progression. in addition, hyperglycemia activates various signaling pathways that cooperate to control cancer cell behavior, including proliferation, migration, invasion, and recurrence. the biological mechanism underlying the relationship between dm and its potential promoting effect on urothelial cells is under investigation. in an in vitro experiment, expression of igf - receptor i, which can promote cell growth and antiapoptosis, has been reported in invasive urothelial carcinoma of the bladder. in addition, increased advanced glycosylated end products due to poor glycemic control may lead to structural changes such as reduced expression of the subtype e - cadherin, which has been associated with poor oncologic outcome in patients with bladder cancer, and chronic inflammation and often accompanying obesity may lead to the release of cytokines which can enhance cancer growth. further research is warranted for a better understanding of the effect of dm on the development and progression of cancer. this study also included other prognostic factors in utuc in addition to glucose control status. the most well - established prognostic factors, including tumor stage, grade, and lymphovascular invasion, were also independent prognostic factors in our study. in addition, adjuvant chemotherapy was found to be an adverse prognostic factor for css and os but not with rfs in our multivariate analysis. the reason for this finding may be that because the adjuvant chemotherapy was administered in advanced disease (pathologic stage t3 or t4), it might affect the poor css and os. a recent meta - analysis demonstrated that os and disease - free survival benefit is only obtained with cisplatin - based combination chemotherapy (cbcc) but not with non - cisplatin based regimens. another study reported that only 22% of the patients were eligible to receive cbcc after rnu and/or approximately 40% of patients did not receive a cbcc regimen at all. unfortunately, information about the chemotherapeutic regimen was not considered in our study, but we may speculate that cbcc regimen could not be administered to a considerable number of patients after rnu. we suggest that because patients will lose their kidney after rnu, prudent preoperative evaluation is necessary to predict which patients will benefit from neoadjuvant or adjuvant chemotherapy. first, the study was conducted with a retrospective design which warrants further prospective study. second, data from multiple institutions could have several limitations, including variations among several surgeons and pathologists. third, we did not examine the effect of lymph node status (pn0/pn+) which could have affected the oncologic outcomes. fourth, we did not evaluate the hba1c level in patients without a history of dm. in this perspective, our results might have been affected by selection bias because of the possibility of undiagnosed dm among patients classified as not having dm. in addition, we did not investigate the dose, type, duration of anti - diabetic medication, and the glycemic control status from rnu to recurrence and/or death which would affect the oncological outcomes, and there were no available data on the type of chemotherapeutic regimen. further investigations with a prospective design, including the type and dose of anti - diabetic drug and type of chemotherapeutic regimen are needed to confirm our result. despite these limitations, this is the first study to evaluate the impact of glucose control status using hba1c on oncologic outcomes of patients with utuc. further study is necessary to elucidate the mechanism for the adverse effect of poor glycemic control status on utuc patients. in our study diabetic utuc patients with poor preoperative glycemic control had significantly adverse oncologic outcomes compared with diabetic utuc patients with good preoperative glycemic control and non - diabetics. css and os of non - diabetic patients did not differ significantly from that of patients with good preoperative glycemic control. rigorous diabetes control and monitoring using hba1c is necessary to improve the prognosis of patients with dm and utuc. | purposethe purpose of this study is to evaluate the effect of diabetes mellitus (dm) and preoperative glycemic control on prognosis in korean patients with upper tract urothelial carcinoma (utuc) who underwent radical nephroureterectomy (rnu).materials and methodsa total of 566 patients who underwent rnu at six institutions between 2004 and 2014 were reviewed retrospectively. kaplan - meier and cox regression analyses were performed to assess the association between dm, preoperative glycemic control, and recurrence - free, cancer - specific, and overall survival.resultsthe median follow - up period was 33.8 months (interquartile range, 41.4 months). a total of 135 patients (23.8%) had dm and 67 patients (11.8%) had poor preoperative glycemic control. patients with poor preoperative glycemic control had significantly shorter median recurrence - free, cancer - specific, and overall survival than patients with good preoperative glycemic control and non - diabetics (all, p=0.001). in multivariable cox regression analysis, dm with poor preoperative glycemic control showed association with worse recurrence - free survival (hazard ratio [hr ], 2.26 ; 95% confidence interval [ci ], 1.31 to 3.90 ; p=0.003), cancer - specific survival (hr, 2.96 ; 95% ci, 1.80 to 4.87 ; p=0.001), and overall survival (hr, 2.13 ; 95% ci, 1.40 to 3.22 ; p=0.001).conclusiondiabetic utuc patients with poor preoperative glycemic control had significantly worse oncologic outcomes than diabetic utuc patients with good preoperative glycemic control and non - diabetics. further investigation is needed to elucidate the exact mechanism underlying the impact of glycemic control on utuc treatment outcome. |
3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors (hmg - coa reductase inhibitors ; statins) were introduced in clinical practice in 1976. statins have drastically changed not only the treatment regimen for hypercholesterolemia but also the treatment strategy for preventing cardiovascular disease. then, nearly 25 million people received statin therapy worldwide. however, there were limited data regarding racial differences in response to statins. in addition, mechanisms regarding racial differences are not sufficiently elucidated. in this review, several large - scale clinical trials have demonstrated the safety and efficacy of statins in reducing cardiovascular events. the cholesterol treatment trialists collaboration conducted a meta - analysis of five randomized trials of statins that compared more intensive (higher dose or more powerful statin) and less intensive (lower dose or less powerful statin) regimens in 39 612 patients with coronary artery disease (cad). intensive statin treatment was associated with a greater reduction in major vascular events compared with less intensive therapy and a 1 mmol / l (39 mg / dl) decrease in low density lipoprotein cholesterol (ldl - c) was associated with a reduction in all - cause mortality, cardiac events, and coronary events by 19, 21, and 23%, respectively. all of these data have updated the clinical guidelines of the european society of cardiology, the american college of cardiology / american heart association, the japanese cardiology society, and the japan atherosclerosis society. however, there is a wide variation in inter - individual or inter - racial differences in response to statins. in particular, asians and westerners are reported to have different responses to statin from each other. a lower dose of statins has demonstrated similar relative risk reduction of cardiovascular events to a higher dose of statins in western patients. in fact, the maximum dose of atorvastatin in clinical practice was 40 mg per day in japan, while the dose is 80 mg per day in the united states. the maximum dose of available statins in japanese and westerners is shown in table 1. discovery (the direct statin comparison of ldl - c values : an evaluation of rosuvastatin therapy) programs consisted of a series of trials incorporating 14,000 patients from several countries, which investigated the impact of rosuvastatin on cardiovascular risk reduction. also, clinical outcomes in a number of different populations worldwide and differences in lipid responses to rosuvastatin and atorvastatin between chinese and caucasian were examined. this study compared the percentage change in ldl - c in response to rosuvastatin or atorvastatin in patients with type iia or iib hypercholesterolemia between chinese and caucasian, which was performed using the following studies : discovery - hong kong, dicovery - asia, dicovery - alpha, dicovery - netherlands, dicovery - penta, dicovery - uk, dicovery - triple country, and other databases. the ldl - c reduction with rosuvastatin (10 mg) in chinese patients was significantly greater than in westerners (52.8% versus 40.9 to 49.7%) while no significant difference was observed in the ldl - c response to atorvastatin (10 mg). a meta - analysis reported that the dose - response relationship between statin dose and ldl - c reduction by rosuvastatin and atorvastatin was similar in western and asian populations, whereas a three to four - fold greater dosage of statins was administered to the western vs the asian population. in the report, a > 40% reduction in ldl - c required atorvastatin 80 mg or rosuvastatin 40 mg in the western population, whereas the dose of atorvastatin or rosuvastatin required to achieve the same result in asians was 18.9 mg or 14.1 mg, respectively. additionally, the required duration of statin administration for lipid - lowering was significantly longer in the western vs the asian population. in concrete terms, the duration over which reduction in ldl - c was exhibited by rosuvastatin was 24.0 and 10.3 months in westerners and asians, respectively. similarly, the duration was 22.0 and 7.8 months for reduction in ldl - c by atorvastatin in westerners and asians, respectively (table 2). yang. examined the dose - response of rosuvastatin to ldl - c reduction in their meta - analysis including 36 randomized trials. there was no significant difference in the dose - response relationship for ldl - c reduction by rosuvastatin between westerners and asians. for atorvastatin, no difference was also observed in pharmacokinetics of the drug between asians and caucasians. a paucity of data regarding the difference in the dose - response relationship between asians and westerners for other statins has been reported. for simvastatin, ldl - c reduction by simvastatin of 5 mg daily was 26.0% in the japan lipid intervention trial. the magnitude of the reduction was similar to the results of other simvastatin studies using higher doses (2040 mg daily) conducted in western countries. for pitavastatin, the pharmacokinetics and dose - response relationship in ldl - c reduction were not different between japanese and caucasian in an open - label, single - dose, two - way crossover pharmacokinetic study, resulting in the recommended dose by the regulatory authorities being similar between the two countries. in summary, the differences in response to statins between asians and westerners were observed for all statins except for pitavastatin. it was speculated that the differences in the ldl - c response to rosuvastatin between chinese and caucasian was yielded by the different pharmacokinetics. pharmacokinetics is the study of the time course of drug absorption, distribution, metabolism, and excretion. in clinical practice, pharmacokinetics is applied to achieve both a safe and effective therapeutic range of drugs in an individual. a population pharmacokinetic analysis revealed no clinically relevant differences in pharmacokinetics among caucasian, hispanic, and african american or afro caribbean groups, while other pharmacokinetic studies of rosuvastatin have demonstrated an approximate two - fold elevation in median exposure (maximum plasma concentration and the area under the plasma concentration curve) in asian populations compared with westerners. in both populations, rosuvastatin showed dose - dependent reductions in ldl - c. yang and colleagues also examined racial differences between asian and western populations in rosuvastatin pharmacodynamics in which an indirect comparison was performed. pharmacodynamics assesses the relationship between the drug concentration at the site of action and the resulting effect quantitatively, which includes both therapeutic and adverse effects. the pharmacodynamics was examined by assessing a relationship between the dose of rosuvastatin and ldl - c reduction, which showed no significant difference between westerners and asians. based on these data, the differences in response to statins between westerners and asians detailed mechanisms of the differences in response to statins between asians and westerners are not fully elucidated. to date, several studies have reported that genetic factors were related to differences in reactions to statin and statin - related side effects, which could potentially explain the racial differences between asians and westerners. a genome - wide study in patients treated with simvastatin found a significant association between single - nucleotide polymorphisms (snps) located within the slco1b1 gene on chromosome 12 and muscular side effects of statins. slco1b1 encodes an organic anion transporting polypeptide 1b1 (oatp1b1) that is expressed on the basolateral membrane of hepatocytes and can facilitate hepatic uptake of certain clinically relevant drugs such as statins except for fluvastatin. slco1b1 polymorphisms included two snps (388a > g, 521t > g) and four haplotypes (slco1b1 1a, slco1b1 1b, sloco1b1 5, and slco1b1 15). slco1b1 1a is a wild type, slco1b1 1b has one snp (388a > g), slco1b1 5 has the other snp and slco1b1 15 has both snps. the transport activity on hepatic cells was upregulated in people with slco1b1 1b, while the activity was downregulated in people with slco1b1 5. in people with slco1b1 the frequency of the four important haplotypes was different among different races (table 3), which could affect racial differences in response to statin. major genetic determinants of rosuvastatin pharmacokinetics is the 421c > a polymorphism in the drug efflux transporter atp - binding cassette g2 gene (abcg2). subjects with the variant allele have plasma rosuvastatin concentration twice as high as those with the wild - type genotype. the abcg2 polymorphism is more common in east asians than in westerners, which might contribute to the difference in pharmacokinetics and lipid response to rosuvastatin between the two ethnic groups. a large - scale genetic analysis among 148 snps within 10 genes participating in cholesterol biosynthesis, cholesterol transport, and statin metabolism was conducted. this analysis assessed lipid reductions in response to pravastatin therapy in 1536 individuals (caucasian : 88.7%, african american : 6.5%, hispanic : 2.9%, asian : 1.2%, and others : 0.7%) in which two snps (snp 12 and snp 29) in the gene coding for hmg - coa reductase were significantly associated with efficacy of pravastatin in ldl - c. in individuals with a minor allele of each snp, pravastatin reduced total cholesterol by 22% (absolute difference : 9.2 mg / dl) compared with those without the snps. ldl - c was also reduced by 19% (absolute difference : 6.4 mg / dl). the association between the snps and the lipid - lowering effect of pravastatin was observed after adjusting for the other 33 snps evaluated in the hmg - coa reductase gene as well as for 148 snps in 10 genes evaluated in the study. in addition, the association between snp 29 and the lipid - lowering effect of pravastatin was more profound in westerners compared with the other ethnic groups. candidate genes by which statins influence ldl - c reduction are shown in table 4. genetic effects, not only on efficacy but also on adverse effects of statin, have been previously reported. in the study of the effectiveness of additional reductions in cholesterol and homocysteine (search) and in the heart protection study, rs4363657 c and rs4149056 c alleles in slco1b1 had markedly elevated risks of myopathy, which was also found in the statin response examined by genetic hap markers (strength) trial. in the strength trial, carriers of 2 alleles and 1 allele of the rs4149056 had a 2.6- and 1.4-fold higher incidence of adverse effects by simvastatin, while the ldl - c - lowering effect of simvastatin was similar between carriers and non - carriers. in the subjects treated with atorvastatin and pravastatin, no statistically significant difference was observed in the incidence of adverse effects between those with at least 1 allele and those without. in the search trial, participants with rs4363657c and rs4149056 alleles had a 4-fold higher risk of severe myopathy and a 17-fold higher risk when comparing the participants with and without both alleles. for patients treated with pravastatin, no excess risk was observed in carriers of rs4149056. in the justification for the use of statins in prevention : an intervention trial evaluating rosuvastatin (jupiter), the effect of rs4363657c and rs4149056c in slco1b1 on clinically reported myalgia was assessed. in the rosuvastatin - treated group, the rate of myalgia was 4.1 events per 100 person - years, which was comparable with the rate in the placebo group. among those on rosuvastatin, there were no differences in the rate of myalgia in subjects with each allele compared with those with neither allele. the hazard ratio for myalgia of the subjects with an rs4363657c or rs4149056c allele compared with those without neither allele was 0.95 (95% confidence interval (ci) 0.791.14) and 0.95 (95% ci 0.791.15), respectively. taken together, these lines of evidence indicate that the effect of the rs4363657c and rs4149056c alleles on the risk of myalgia was different between populations treated with rosuvastatin and simvastatin. carriers of this polymorphism would be expected to have reduced hepatic uptake of statins, resulting in higher circulating statin concentrations and an increased risk of myopathy. it is possible that increased circulating statin levels as a result of the sclo1b1 polymorphism are less toxic to muscle cells for hydrophilic agents, such as rosuvastatin and pravastatin, compared with more hydrophobic statins such as simvastatin. in this text, the differences in statin response between asians and westerners and genetic influences on the differences were described. however, genetic effects on statin response are still controversial. in addition, the lipid - lowering effect of statins involved several processes to exhibit lipid - lowering, which include absorption of the drug, transportation to hepatic cells, inhibition of hmg - coa reductase, nuclear translocation of srebp-2, increased synthesis of ldl receptor, and endocytosis of ldl - c by the ldl receptor. because each process is affected to some extent by different genetic factors, known genetic variants per se could not fully explain inter - individual or inter - racial differences in response to statins. therefore, future research is needed to clarify which gene polymorphisms are related to the processes that act to exhibit lipid - lowering effects and to what extent the genetic factors affect the response to statins. in addition, we should recognize that not only genetic factors but also non - genetic factors, such as body surface area, dietary style, and adherence to drugs play important roles in different responses to statins between different races. racial differences exist in the response to statins between asians and westerners through different pharmacokinetics, which is partially explained by genetic factors. future research is required to elucidate to some extent the gene factors that are associated with racial differences in statin response. h.d. has received speakers ' bureau / honoraria from msd, astrazeneca, kowa pharmaceutical, sanofi - aventis, glaxosmithkline, shionogi, daiichi - sankyo, takeda pharmaceutical, mitsubishi tanabe pharma, pfizer, and astellas pharma and research funds from takeda pharmaceutical, bristol - myers squibb, nippon boehringer ingelheim, astellas pharma, novartis pharma, msd, sanofi - aventis, otsuka pharmaceutical, dainippon sumitomo pharma, pfizer, kowa pharmaceutical, shionogi, astrazeneca, teijin, and morinaga milk industry. k.m. has received speakers ' bureau / honoraria from msd, astrazeneca, kowa pharmaceutical, sanofi - aventis, shionogi, daiichi - sankyo, takeda pharmaceutical, pfizer, astellas pharma, and novartis pharma. | statin treatment to reduce low - density lipoprotein cholesterol (ldl - c) is associated with the prevention of cardiovascular events in western patients. similar results have been reported in studies conducted in japan. however, the dose of statins and the degree of ldl - c reduction achieved with statins are different between asian and western patients. in addition, there are limited data regarding racial differences in response to statins. in this review, racial differences between asians and westerners in response to statins are described. |
a 66-year - old man admitted to our hospital because of limb and gait ataxia. his mental status had been improved slowly, but he developed limb and gait ataxia. cerebellar function test showed remarkable dysmetria and dysdiadochokinesia especially, in the left upper and lower extremities. laboratory studies including electrolyte profile and liver function tests were within normal limits except for slightly elevated aspartate aminotransferase (47 iu / l) and alanine aminotransferase (92 iu / l). an erythrocyte sedimentation rate (24 mm / h) and c - reactive protein (0.89 mg / dl) were slightly increased. the serological tests for detection of epstein - barr virus, varicella - zoster virus, herpes simplex virus, cytomegalovirus, and human immunodeficiency virus were normal. cerebrospinal fluid examination revealed a cell count of 38 white blood cells with lymphocytes predominant, protein 72 mg / dl, glucose 37 mg / dl (serum glucose 95 mg / dl). technetium-99 m hexamethyl propylene amine oxime - single photon emission computed tomography (hmpao - spect) showed markedly cerebellar asymmetry, suggesting hypoperfusion in the right cerebellum (figure 1a). hmpao - spect performed 4 months later demonstrated complete resolution of the cerebellar asymmetry (figure 1b). we constructed the subtraction ictal spect coregistered to mri (siscom) image by subtraction between two spects, and siscom image also showed hypoperfusion in the right cerebellum (figure 1c). acute cerebellitis is rare but well recognized complication of the central nervous system characterized by cerebellar dysfunction, the frequency of which was estimated to be 0.4% of all patients evaluated for neurological problems at a child s hospital.1 most often, the main symptoms are headache, vomiting, and disturbance of consciousness varying from somnolence to coma.2 although the pathogenic mechanisms remains uncertain, direct invasion of an etiologic agent or autoimmune demyelinating process similar to acute disseminated encephalomyelitis and multiple sclerosis has been suggested.3 varicella - zoster virus, herpes simplex virus, epstein - barr virus, rotavirus, echovirus, coxsackie virus, mumps, measles, lyme disease, rubella, borrelia brugdorferi, coxiella burnetii, salmonella typhi, and bordetella pertussis are known as causative agents of ac in child or young adult.1,2,4,5 influenza virus can also cause ac.6,7 to our knowledge, ac after influenza vaccination in an elderly patient has not yet been reported. only one case of ac after influenza vaccination has been reported, but the patient was 5 years of age.6 although ac is more common in children, adult cases of cerebellitis have been also reported, but causative agents of these cases are not influenza virus.811 we were not able to determine the definite etiology of our case, close temporal relation between influenza vaccination and an evolution of symptom suggests that influenza vaccination may cause ac in an elderly patient. brain mri in ac demonstrated bilateral or unilateral diffuse cerebellar hemispheric abnormalities in t2-weighted images, and pial enhancement in contrast enhanced t1-weighted images.1 however, brain mri demonstrated no abnormality in the majority of ac patients.12 in such cases, brain spect might demonstrate regional per - fusion abnormalities despite of normal brain mri findings.12 nagamitsu.13 stress the importance that cerebellar hypoperfusion is a more sensitive marker than mri in ac. the decreased cerebellar blood flow could be explained by a post - infectious demyelinating autoimmune response.1 whereas, gruis.14 reported on a case with increased cerebellar blood flow, reflecting the inflammatory component of cerebellitis. in our case, brain hmpao - spect showed marked cerebellar asymmetry, despite of normal brain mri findings, and after the acute phase, the regional blood flow returned to be normal. traditional side - by - side visual comparison of the spect images can be difficult to detect subtle abnormality. siscom has the potential to overcome many of the limitations associated with the traditional side - by - side inspection of the images.15 in fact, we do nt know exactly why the left extremities showed dysmetria and dysdiadochokinesia despite of the right cerebellar hypoperfusion in the initial spect. however, one conceivable explanation is the possibility of bilateral manifestation with unilateral cerebellar pathology. on the literature,16 the unilateral cerebellar infarction produced limb ataxia bilaterally. furthermore, stimulation of the unilateral deep cerebellar nuclei in monkey showed a bilateral movement representation.17 although our patient demonstrated that the left extremities were more affected than the right extremities, the fundamental pathology on neuroimaging existed on one cerebellum. the siscom analysis for our patient showed definitely right cerebellar hypoperfusion on the initial spect. so, it is possible that unilateral cerebellar pathology could be manifestated with bilateral cerebellar dysfunction such as our patient. we conclude that influenza vaccination can cause ac in the elderly and brain spect imaging is more useful than mri in identifying patients with ac, especially when mri is normal. | acute cerebellitis (ac) is a benign and self - limiting inflammatory disease. it typically occurs as a primary infectious or postinfectious disorder. although ac mostly presents in early childhood, it can appear in adult. a 66-year - old man admitted to our hospital because of limb and gait ataxia. three weeks ago, he took an influenza vaccination. there was no abnormality on brain mri with contrast enhancement, but technetium-99 m hexamethyl propylene amine oxime - single photon emission computed tomography (hmpao - spect) showed markedly cerebellar asymmetry, suggesting hypoperfusion in the right cerebellum. influenza vaccination can cause ac in the elderly and brain hmpao - spect imaging is more useful than mri in identifying patients with ac. |
the wnv neutralization titers of several us plasma derived igiv products (gammagard liquid / kiovig ; gammagard s / d/ polygam s / d ; iveegam en [baxter healthcare corporation, westlake village, ca, usa ]) and plasma samples obtained from us blood donors after a nat - confirmed wnv infection were determined by an infectivity assay as earlier described (2), adapted to a classical microneutralization format (3). wnv neutralization titers (i.e., the reciprocal dilution of a 1:2 series resulting in 50% neutralization [nt50 ; detection limits 21 by the current assay) (2), 40% of the 2008 igiv lots had higher titers. west nile virus (wnv) neutralization by us plasma - derived immune globulin intravenous (human) (igiv) released in 2008 and plasma from donors with past wnv infection (past wnv), confirmed by nucleic acid testing. wnv neutralization titers are shown as the mean sem (limit of detection < 0.8 for undiluted igivs and < 7.7 for prediluted sera). plasma obtained from persons with nat - confirmed wnv infection had even higher titers ; mean sem titer was 208 40 for 30 persons available for testing. when results were corrected for the immunoglobulin (ig) g concentration in plasma (1%), compared with the 10% igiv preparations, the mean neutralization titer of the plasma samples was 100 higher than that of the igiv lots tested (2,080 vs. 21). the most comprehensive collation of information about the incidence of wnv infection in the united states is available from arbonet. when that information is combined with information obtained from the nationwide screening of the blood supply for wnv rna by nat (1,4,5), the current prevalence of past wnv in the us population is estimated to be 1%. busch. has noted that large - scale, community - based serologic surveys are hardly feasible because of their expense and because wnv elisa assays are possibly biased by cross - reactions with other flaviviruses (1). cumulatively, 5,503 persons were tested for wnv infection by elisa, and the results have shown highly divergent seroprevalence rates ranging between 1.9% (6) and 14.0% (10). the use of igiv lots, each representing the serostatus of several thousand donors in 1 sample, makes seroepidemiology practical (13) because it allows a large donor population to be surveyed by analyzing comparably few samples. the use of a more complex yet functional virus neutralization assay minimizes concerns about cross - reactivity with flaviviruses of other serocomplexes (e.g., dengue virus) that occasionally circulate in the us population. also, epidemiologic considerations render interference by st. louis encephalitis virus, a flavivirus within the same serocomplex, highly unlikely (2). the specificity of the neutralization assay was confirmed by testing igiv lots manufactured from european - derived plasma against tick - borne encephalitis virus, a flavivirus closely related to wnv and circulating in europe. although these lots contained high neutralization titers against tick - borne encephalitis virus, only 1 of 20 had a detectable neutralization titer of 5 against wnv (unpub., we determined that the mean titer of samples obtained during 20032008 from persons with a confirmed diagnosis of wnv infection was 100 higher than the mean titers of igiv lots produced in 2008. this determination provides an independent experimental measure of the frequency of past wnv infection in the general us population, as reflected by the plasma / blood donor community, and the results correlate well with results of previously published theoretical extrapolations (1), which estimated that 1% of the population has already been infected with wnv. the increasing levels of wnv neutralizing antibodies in igiv lots from us plasma and the particularly high titers in donors who have had a wnv infection suggest the possibility of preparing igiv products with sufficiently high titers to be useful for wnv prophylaxis or treatment. several ongoing or imminent wnv vaccine clinical trials stress the practical value of an independent confirmation of extrapolations that estimate the percentage of the us population with past wnv infection. knowing the percentage of preexisting wnv seroprevalence as well as estimates of the mostly asymptomatic incidence rates (14) can be of vital importance in designing vaccine trials. | this study investigated the association of ongoing west nile virus (wnv) infections with neutralizing antibody titers in us plasma - derived intravenous immune globulin released during 20032008. titers correlated closely with the prevalence of past wnv infection in blood donors, with 2008 lots indicating a prevalence of 1%. |
endometriosis is an inflammatory pathology in which there is an increase in positivity for nuclear factor - kappa b (nf-b), cyclooxygenase (cox)-2, and aromatase expression in the eutopic endometrium.1 these functional changes create a vicious cycle of increased inflammation and local estrogen production that helps perpetuate the survival of endometrial cells in ectopic locations by blocking their phagocytosis via activated macrophages.1,2 progression of endometriosis depends on continuous seedling of aromatase - positive cells carried to the pelvis by retrograde menstruation.3 in patients submitted to endometrial resection following laparoscopic treatment for endometriosis, the recurrence rate of the disease has been reported to be much lower.4 these results suggest that the presence of an intact endometrium rather than completeness of surgery may be a key factor in the success of treatment. therefore, inducing long - lasting amenorrhea is a necessary step in the treatment of endometriosis and involves use of medication over prolonged periods of time.1 one promising approach is the use of oral contraceptives, since they are not only extremely effective in controlling pain and curbing inflammation, but they also prevent the recurrence of endometriosis following surgical treatment.5 the mechanism of action of oral contraceptives is complex and involves a central ovulation - blocking effect in addition to local effects on the endometrium and endometriosis lesions.2 this local action involves blockade of several enzymatic pathways, such as cox and aromatase p450, that are necessary for the survival and progression of endometriosis.1 progesterone and oral contraceptives containing gestodene have been reported to obstruct nf-b activation and its translocation to cell nuclei where it would activate the genes responsible for the inflammatory cascade.1,6,7 however, not every woman using oral contraceptives in a continuous regimen for the treatment of endometriosis becomes amenorrheic, and the occurrence of breakthrough bleeding is associated with activation of nf-b in the endometrium, initiating a cascade of inflammatory events and leading to persistent cox-2 and aromatase expression in this tissue.1,6 unabated aromatase expression leading to an enhanced local inflammatory, prostaglandin - mediated response may be the underlying mechanism behind the persistent pain and bleeding in endometriosis patients using oral contraceptives.8 these hypotheses may also explain the greater efficacy of the combination of oral contraceptives and aromatase inhibitors in managing endometriosis - related pain in patients who fail to respond satisfactorily to hormone therapy alone.9 resveratrol, a polyphenolic compound isolated from the skin of red grapes, is a natural aromatase inhibitor. in addition to its anti - inflammatory properties, at pharmacologic doses resveratrol also inhibits aromatase activity at both the enzyme and gene transcription levels.8,10 it was recently reported that resveratrol exerts a potentiating effect on pain management when used in association with oral contraceptives in patients with endometriosis.8 these preliminary results suggest that the association of oral contraceptives with plant - derived anti - inflammatory drugs may improve the efficacy of oral contraceptives for the treatment of endometriosis - related pain by increasing amenorrhea rates without exacerbating the side effects of the treatment.1,8 another promising compound is pycnogenol (horphag, geneva, switzerland), a complex mixture of procyanidins and polyphenol components harvested from the bark of the french maritime pine (pinus pinaster). pycnogenol has been shown to exert anti - inflammatory and antithrombotic effects11 by inhibiting both cox-1 and cox-2 enzymatic activity.12 suppression of nf-b - dependent gene expression, which activates the inflammatory cascade, also contributes to the pain - ameliorating effects of pycnogenol.13,14 since endometriosis is an inflammatory pathology in which expression of these inflammatory markers is exacerbated, combining oral contraceptives with this natural nf-b inhibitor would appear logical.1 in the present report, the effects of an association of pycnogenol and oral contraceptives on pelvic pain were investigated in patients with endometriosis. this open, office - based study was conducted to evaluate the effect of pycnogenol on the management of endometriosis - related pain in patients using postoperative hormonal therapy. forty - five patients of reproductive age (range 2237 years) with a laparoscopically confirmed diagnosis of endometriosis and whose main indication for laparoscopy was pelvic pain were treated at the outpatient clinic of the instituto da mulher with oral contraceptives in extended regimens, either alone or in association with pycnogenol. laparoscopy was performed at the itaigara memorial day hospital by the same team of surgeons (hm, ch) in all cases. the diagnosis of endometriosis was made visually during laparoscopy, the lesions were digitally photographed for medical records, and the extent of endometriosis was staged according to american society for reproductive medicine guidelines.15 in accordance with this staging system, 26 patients were classified as having stage i or ii, while the remaining 16 were classified as having stage iii or iv of the disease. before beginning hormonal treatment at the instituto da mulher, the patients were interviewed to determine the severity of dysmenorrhea and at that time were randomly allocated to one of the four treatment groups according to the week of the month (weeks 14) in which they were first seen. in group 1 (n=7) the patients were treated with an oral contraceptive containing gestodene 75 g and ethinylestradiol 30 g, administered in a continuous regimen. in group 2, the same oral contraceptive regimen was used in association with 100 mg of pycnogenol (n=14). in group 3 (n=13), the patients were treated with an oral contraceptive containing drospirenone 3 mg and ethinylestradiol 30 g (n=13) administered in a continuous regimen and in group 4 (n=11), patients followed the same contraceptive regimen used by the women in group 3 but in association with a daily dose of 100 mg of pycnogenol. pycnogenol (pinus pinaster extract), prescribed at a dose of 50 mg every 12 hours, was obtained either from a compound pharmacy (fagron, rotterdam, the netherlands) or in the form of a commercially available product (flebon, farmoquimica, rio de janeiro, brazil). in all cases, the study was approved by the internal review board of the instituto da mulher and the itaigara memorial day hospital. neither flebon nor the oral contraceptive pills used in extended regimens are new investigational drugs. flebon is approved by the brazilian regulatory authorities (national health surveillance agency) for the treatment of varicose veins and there is no contraindication to its use by oral contraceptive users. contraceptives containing either gestodene or drospirenone (libbs farmacutica, so paulo, brazil) are already approved by the national health surveillance agency for use in extended regimens ; therefore, this indication is not off - label in brazil. because this study involved no new investigational drugs and since all the drugs used in the study are readily available at any dispensing pharmacy in brazil, the institution s internal review board dispensed with the need for the patients to sign an informed consent form. however, the attending physician discussed the medication with each patient individually and the patients verbally agreed to use the treatment selected. for this reason, neither the physician nor the patients were blinded to the treatment. during office evaluation conducted prior to initiation of treatment and after 3 months use of the medication, the patients were asked to grade the severity of their pelvic pain, including dysmenorrheal, in accordance with a visual analog pain scale graded from 0 to 10, on which 0 was indicative of no pain and 10 the most excruciating pain. this evaluation was carried out by the same investigators (hm, ch) prior to oral contraceptive use and after 3 months of pill use. statistical analysis was performed using the student s t - test, with p - values<0.05 being considered statistically significant. there was no statistically significant difference in mean pain score prior to initiation of treatment between the four groups. the use of oral contraceptives alone, both those containing gestodene (group 1) and those containing drospirenone (group 3), used in a continuous regimen, resulted in a significant decrease in menstrual - related pain (dysmenorrhea) at the end of the third month of treatment when compared with pretreatment values. however, in patients using oral contraceptives together with pycnogenol (groups 2 and 4), the reduction in mean pain score was more pronounced and significantly greater than that found in groups 1 and 3 in which the oral contraceptives were used alone. on the other hand, there were no statistically significant differences in post - treatment pain scores between groups 1 and 3 or between groups 2 and 4. in users of pycnogenol + oral contraceptives, 8/14 (57%) of patients in group 2 and 6/11 (54%) of those in group 4 reported complete resolution of pain, while none of the patients in groups 1 and 3 had become completely pain - free by the end of the third month of treatment. no additional side effects were observed with the use of pycnogenol therapy compared with those recorded with the use of oral contraceptives alone. there was no statistically significant difference in mean pain score prior to initiation of treatment between the four groups. the use of oral contraceptives alone, both those containing gestodene (group 1) and those containing drospirenone (group 3), used in a continuous regimen, resulted in a significant decrease in menstrual - related pain (dysmenorrhea) at the end of the third month of treatment when compared with pretreatment values. however, in patients using oral contraceptives together with pycnogenol (groups 2 and 4), the reduction in mean pain score was more pronounced and significantly greater than that found in groups 1 and 3 in which the oral contraceptives were used alone. on the other hand, there were no statistically significant differences in post - treatment pain scores between groups 1 and 3 or between groups 2 and 4. in users of pycnogenol + oral contraceptives, 8/14 (57%) of patients in group 2 and 6/11 (54%) of those in group 4 reported complete resolution of pain, while none of the patients in groups 1 and 3 had become completely pain - free by the end of the third month of treatment. no additional side effects were observed with the use of pycnogenol therapy compared with those recorded with the use of oral contraceptives alone. based on the results of this preliminary observational study, the use of pycnogenol combined with oral contraceptives in a continuous regimen appears to be more effective than oral contraceptives alone for the treatment of endometriosis - related pain. this increased efficacy likely results from the more effective suppression of nf-b activation at both the pre and post translational level following combined use of these compounds.13,14 the greater reduction in pain scores during use of oral contraceptives combined with pycnogenol when compared with oral contraceptives alone is therefore the consequence of more effective blockade of nf-b activation in endometriosis lesions and in the eutopic endometrium because of a synergetic effect of the combined therapy on both pre and post dna binding of this transcription factor.1,6 the combination of hormone therapy with plant - derived nf-b blockers may represent a viable alternative for the treatment of endometriosis - related pain, since these agents may increase the efficacy of oral contraceptives in controlling this symptom without augmenting the incidence of undesirable side effects.1 previous reports showed that pycnogenol alone was as effective as gonadotropin - releasing hormone analogs for the treatment of endometriosis after the second month of treatment.16 pycnogenol affects the transactivation capacity of nf-b, thus reducing expression of the inflammatory genes regulated by this transcription factor.17 however, pycnogenol does not prevent nf-b from binding to dna, suggesting that its mechanism of action is different from that of progesterone, which acts by preventing translocation of the activated nf-b subunit to the cell nucleus and its consequent binding to dna, where it will activate translation of genes such as cox-2 that are involved in the inflammatory cascade.17 cox-2 will stimulate prostaglandin production, which in turn will activate the aromatase gene, thus stimulating local estrogen synthesis in the endometrium and enhancing the inflammatory reaction, as occurs in the breast.1,18 although both progestins and pycnogenol reduce inflammation by modulating nf-b - induced gene transcription, they act on different steps in this mechanism, which explains the greater efficacy of the combination therapy for the control of endometriosis - related pain. when given together with an oral contraceptive, not only is the dna binding activity reduced but also the trans - activation capacity of the bound nf-b.13,14,17 this distinctive effect on the nf-b activation pathway may provide a plausible explanation at the molecular level for the results reported here. it is also noteworthy that the increased efficacy in terms of pain control in endometriosis is not accompanied by any increase in incidence of side effects. the disappearance of dysmenorrhea in almost 50% of the patients as reported here also confirms previous observations that use of pycnogenol alone is effective in treating this ailment.1921 however, these findings, albeit encouraging, should be considered as preliminary, and larger, well designed, blinded clinical trials are required to confirm these initial observations. the idea of combining a progestin with a post - translation inhibitor of nf-b action may constitute a new approach that will not only increase the efficacy of hormonal treatment in inducing long - lasting amenorrhea but will also reduce endometriosis pain scores. | endometriosis is a chronic disease in which a persistent state of heightened inflammation is maintained by nuclear factor - kappa b (nf-b) activation. the progestins present in oral contraceptives are potent inhibitors of nf-b translocation to cell nuclei, while pycnogenol (pinus pinaster) acts by blocking post - translational events. in this study, the effects of pycnogenol on pain scores were investigated in patients with endometriosis using oral contraceptives containing either gestodene or drospirenone in extended regimens. pain scores were determined using a visual analog scale before and after 3 months of treatment. oral contraceptives, used alone (groups 1 and 3) or in association with pycnogenol (groups 2 and 4), resulted in significant decreases in pain scores after 3 months of treatment ; however, this reduction was significantly greater in the groups using oral contraceptives + pycnogenol (groups 2 and 4) compared with those using oral contraceptives alone (groups 1 and 3). in the groups using oral contraceptives alone, 50% of patients became pain - free by the end of the third month of treatment. these results suggest that pycnogenol increases the efficacy of oral contraceptives for the treatment of endometriosis - related pain. |
epigenetics is described as inherited alterations in gene expression or silencing that take place without changes in dna sequence (jaenisch and bird, 2003 ; bird, 2007). most epigenetic studies are focused on the characterization of covalent and noncovalent modifications of dna and histones and the mechanisms by which such modifications influence chromatin architecture and subsequent gene expression (goldberg., 2007). thus, we sought to review the critical role of the histone code in the context of intestinal physiology. the histone code is defined by posttranslational modifications, such as acetylation and methylation of key residues of core histone tails, which can synergize or antagonize each other and influence chromatin accessibility (active vs. silenced) and transcription during cell fate determination and tissue renewal (jenuwein and allis, 2001) (fig. it is known that core histone nterminal chains can impact transcriptional activity via different types of multivalent modifications, including methylation and acetylation, but also ubiquitination, phosphorylation, and sumoylation (kouzarides, 2007 ; ruthenburg., 2007). in addition to their role as transcriptional regulators (koch., 2007 ; pandian., 2014), histone modifications can influence other dnarelated processes, including dna replication, doublestrand break reparation, and retrotransposable element silencing (groth., 2007 ; kinner., 2008 ; di giacomo., 2014). methylation / acetylation of histone tails is one of the most important functional characteristics of euchromatin and heterochromatin patterning in the nucleus. mono, di, and trimethylation of specific histone lysine and arginine residues are mostly associated with a tightly compacted and transcriptionally repressed form of chromatin called heterochromatin. histone lysine acetylation however confers to dna a relaxed and accessible (rna pol ii, transcription factors) conformation called euchromatin. the concerted action of histone deacetylases (hdacs) and methyltransferases is generally responsible for heterochromatin domain organization, while the activity of histone acetyltransferases and demethylases is associated with transcriptionally active euchromatin regions. methylation and acetylation of histone lysine tails constitute the most important posttranslational modifications occurring in heterochromatin and euchromatin organization (fig. 2) (grunstein, 1997 ; jaenisch and bird, 2003 ; kim and kim, 2012). due to their compact assembly, heterochromatin structures limit the access of transcriptional machinery to the more packaged dna molecules which results in gene silencing (li and zhang, 2012). in contrast, euchromatin conformation is associated with the recruitment of transcription factors and rna polymerase, resulting in gene expression (fig. deregulation of the physiological balance between the formation of euchromatin and heterochromatin structures can result in different malignancies such as colorectal cancer (chen., 2010). for example, histone h3 lysine27 trimethylation (h3k27me3), h4k16 methylation, or h4k20 deacetylation have all been associated with transcriptional aberrations observed in multiple human cancers, including colorectal tumors (fraga., 2005 ; hammoud., 2013). interestingly, pharmacological strategies targeting key chromatin editors, including histone methyltransferases and histone deacetylases (hdac) have recently been proposed to fix aberrant cell growth and survival in these human neoplasms (benoit., 2013a,2013b ; carson., 2015 although epigenetic modifications have been widely studied in different biological contexts, there is still a lack of comprehensive understanding of the role played by epigenetics and chromatin remodeling on critical gene expression patterns regulating differentiation and proliferation of intestinal progenitors. so far, our group and others have highlighted that a tightly regulated cooperation between chromatin marks, such as histone methylation and acetylation have indispensable roles in the control of proliferation and differentiation of absorptive cells in the ta zone of the intestinal crypt (suzuki., we will focus on recent discoveries on the fundamental roles of chromatin regulation in human intestinal tissue homeostasis. methylation of specific lysines or arginines on histone tails creates a platform for transcriptional repression or activation depending on the exact position of the lysine residue in the histone protein. for example, methylation of histone h3k4, h3k36, and h3k79 gives rise to a transcriptionally active region of chromatin while h3k9, h3k27, and h4k20 methylation are usually associated with gene silencing (sims., 2003). the core catalytic subunit which controls histone lysine methylation is the set domain, the active functional unit of many protein methyltransferases responsible for the addition of methyl groups to histones (greer and shi, 2012). one important example of setcontaining proteins is the polycomb group (pcg) proteins which are a protein family implicated in transcriptional repression during tissue formation and development (lewis, 1978). the core prc2 ultrastructure contains the methyltransferase unit enhancer of zeste homolog 2 (ezh2), the stabilization protein suppressor of zeste12 (suz12) (margueron and reinberg, 2011), as well as eed which is acting as a bridge between prc2 and prc1 (cao., 2014). pcgs have been known to maintain pluripotence in embryonic stem cells (boyer., 2006 ; lee., 2006) to control somatic cell differentiation (caretti., 2004 ; ezhkova., 2009 ; benoit., pcgs play a pivotal role in intestinal tissue development by controlling cell fate determination of absorptive cells, which are the predominant cell type in the intestinal epithelium with the important function of absorbing nutrients. specifically, recent characterization of the role of prc2 in intestinal cells (benoit., as aforementioned, the suz12 subunit is essential for proper functioning of the prc2 complex which consists of the addition of three methyl groups to lysine 27 of histone h3 (h3k27) at target loci, leading to a transcriptionally silent chromatin state (cao., 2002 ; martinezgarcia and licht, 2010 ; margueron and reinberg, 2011). in the ta zone of the intestinal crypt compartment, pcg proteins catalyse trimethylation of histone h3 (h3k27me3) and this process coincides with the repression of terminal differentiation genes such as sucraseisomaltase (si), a specific marker of mature absorptive cells (benoit., 2012). incidentally, expression of the si protein was found to be inversely correlated with the presence of suz12, supporting the inhibitory influence of pcgs on cell differentiation in the ta zone (benoit. the possibility that the activity of pcg proteins could allow ta cells to preserve their proliferative state while repressing the expression of intestinespecific differentiation genes was then tested using human cell models. abolition of prc2 activity in colon carcinoma (caco2/15) and human normal nonimmortalized intestinal crypt (hiec) cells resulted in the acceleration of absorptive cell terminal differentiation, manifested by a robust expression of the si protein (benoit., in these cell models, the prc2catalyzed h3k27me3 mark (martinezgarcia and licht, 2010 ; margueron and reinberg, 2011) was significantly downregulated, confirming the essential implication of pcgs and histone methylation in the regulation of absorptive cellspecific gene expression. our laboratory has recently found that inhibition of src family kinases (sfks) is associated with a decrease of h3k27me3 mark as well as a significant increase in intestinal cell differentiation markers. these data suggest that sfks, which are negative regulators of the differentiation of intestinal cells, could act upstream of the prc2 complex (seltana., 2013). a detailed characterization of these sfk regulatory mechanisms needs further investigation thus, the elevated expression of pcgs in the ta zone of the cryptvillus axis, as well as their impact on cell statespecific gene expression provides compelling evidence on the role of pcg proteins in the maintenance of cellular proliferation and suppression of differentiation. the pcg proteins are best known for their repressive action on the differentiation of various tissuespecific stem cells such as mesenchymal stem cells, hematopoietic stem cells and skeletal muscle stem cells (chen., 2012). deregulation of pcg gene expression has also been reported in many cancers including colon cancer (sauvageau and sauvageau, 2010 ; benoit., 2012). in human colon adenocarcinoma, an upregulation of suz12 expression and distribution throughout the tissue has been observed, confirming the implication of pcgs in the preservation of cancer progression (benoit., 2012). interestingly, pharmacological inhibition of the catalytic subunit of prc2 (ezh2) has shown impaired growth and survival properties in human colorectal cancer cell lines and patientderived colon cancer stem cells (benoit., 2013a,2013b). in each case, a knockdown of suz12 was sufficient to phenocopy the inhibition of ezh2, further supporting a critical involvement of the prc2 in colorectal tumorigenesis. histone acetylation is another set of epigenetic mechanisms able to regulate cell proliferation and differentiation in the intestinal ta zone. indeed, acetylation of histone tails counterbalances the histone net charge in order to induce a more relaxed chromatin conformation facilitating access of transcriptional machinery to the dna promoter and resulting in derepression of gene expression associated with cellular differentiation (fig. however, histone deacetylases (hdacs) counter this effect by removing acetyl groups from lysine residues within dna bound core histones (haberland., 2009). histone deacetylation is a fundamental phenomenon that increases dna interactions with histones, and as a result, downregulates gene expression by inhibiting the recruitment of rna polymerase, transcription factors and cofactors at promoter regions and other regulatory elements (fig. 2) (marks., 2000 ; glozak and seto, 2007). human hdacs are grouped into 4 classes : hdaci, hdacii, hdaciii, and hdaciv based on their sequence homology to the original yeast enzymes and domain organization. class i includes hdac1, 2, 3, 8, class ii includes hdac4, 5, 7, and 9, while hdac6 and 10 constitute class iii and hdac11 is known as class iv (marks, 2007 ; witt., 2009). out of the four hdac classes, classes i and ii have been show to play pivotal roles in the maintenance of normal intestinal physiology and are overexpressed in colon tumors (mariadason, 2008). in 2008, suzuki. (2008) demonstrated that during differentiation of absorptive cells, the promoter of the sucraseisomaltase (si) gene, a hallmark of terminal differentiation of absorptive cells (beaulieu, 1997), was highly acetylated (suzuki., 2008). previous studies have tested hdac inhibitors (hdaci) to directly investigate the role of histone acetylation in the regulation of the physiology of intestinal cells. for example, the hdaci sodium butyrate has been used to assess differentiation and proliferation of caco2 cells (mariadason., 2000, 2001). although sodium butyrate has been shown to induce expression of some absorptive cell differentiation markers such as dpp4 and alkaline phosphatase, it does not upregulate si expression (mariadason., 2000). newly confluent caco2/15 cells represent a relevant model of absorptive cell progenitors in their last dividing cycles in the ta zone since their proliferation rate diminishes while a differentiation process begins (beaulieu and quaroni, 1991 ; vachon and beaulieu, 1992). treatment of these cells with hdaci suberanilohydroxamic acid (saha) which suppresses the activity of both class i and ii hdacs resulted in a dramatic upregulation of histone acetylation and si expression at both mrna and protein levels (roostaee., in addition, in newly confluent caco2/15 cells incubated with saha, the colonic enterocyte maturation marker slc26a3 (down regulated in adenoma, slc26a3/dra) was significantly upregulated. if histone acetylation accelerates intestinal cell maturation, hdac inhibition should result in an early expression of intestinespecific markers in the crypts as shown previously in mouse fetal gut explants (tou., 2004). to test this possibility, mice were treated with saha and the expression of differentiation markers in intestinal crypts was investigated. the results showed that overall expression of si and slc26a3 was significantly enhanced while the si and slc26a3 proteins were detected in the upper part of ileal (fig. 3a, b) and colonic crypts, respectively, in mice receiving saha, suggesting that histone acetylation participates in the repression of absorptive cell differentiation in the ta zone (roostaee., 2015). (a and b) representative immunofluorescence illustrations showing that histone acetylation promotes expression of enterocyte specific differentiation protein si (green) in the mouse ileum treated with saha (b) compared to the control (ctrl) mouse (a) (c f) saha treatment has no significant effect on the differentiation of goblet cells. (c and d) alcian blue staining of mouse ileum for the detection of goblet cells in the ctrl mice treated with dmso (c) or saha (d). (e and f) counting of goblet cells in the intestinal crypts (e) and villi (f) demonstrates that there is no significant difference in goblet cell number between mice treated with saha or control mice. the use of genetically modified mouse models has also contributed to our understanding of the involvement of hdac in intestinal homeostasis. ablation of hdac1 and hdac2 in the mouse intestinal epithelium, early in development, was reported to promote expression of absorptive cell differentiation markers such as si, intestinal alkaline phosphatase and fatty acid binding protein but this phenomenon was accompanied by a loss of differentiation of secretory lineages and a stimulation of cell proliferation (turgeon., 2013). however, some of these observations were attributed to the only partial deletion of the target genes since a complete and simultaneous deletion of hdac1 and hdac2 in the adult intestine leads to a rapid loss of proliferative crypt cells in vivo and in intestinal organoid cultures (gonneaud., 2015 ; zimberlin., 2015), a phenomenon also observed in caco2/15 cells and in the intestine of mice treated with the hdaci saha (roostaee., 2015). consistently, enhanced histone acetylation has been related to elevated p21 cellular levels, resulting in cell cycle arrest and apoptosis (xiong., 1993 moreover, evaluation of goblet and paneth cell number in the intestine of mice treated with saha suggests that hdac inhibition does not influence sp cell differentiation (fig. further studies in murine models knocked down for either hdac1 or hdac2 as well as those expressing low levels of hdac1 and in the absence of hdac2 and vice versa disclosed the existence of dosagedependent specific and similar effects on intestinal epithelial cells (gonneaud. further work is thus needed to more precisely assess the respective roles of these hdacs. the presence of other hdacs in intestinal epithelial cells that may participate to the phenomenon should also be considered in future studies. for instance, hdac3 and hdac4 which were both characterized as repressors of p21 expression in colorectal cancer cell lines have been reported to be predominantly expressed in the proliferative cells of the crypt in the normal intestine (wilson., 2008, 2006). the data reported above indicate a role for both pcg proteins and hdacs for promoting cell proliferation and repressing absorptive cell differentiation in the ta zone of the intestinal crypt. transition of cells from the proliferative to the terminal differentiation state requires global changes in histone modifications which influence the expression of multiple gene targets (glozak and seto, 2007). this process involves removal of methyl groups of h3k27me3 and subsequent acetylation of h3k27 to form an acetylated histone h3k27 (h3k27ac) complex (ong and corces, 2011). in this context, the study of suzuki. indeed, di/trimethylation levels of histone h3 at lysine 9 on the promoter region of the si gene were found to be associated with low si expression in the crypt while its change to diacetylation at lysine 9/14 was linked to the upregulation of si expression at the cryptvillus junction (suzuki., 2008). the transition of specific histone tail residues from a methylated to an acetylated state to impact proliferation rate versus differentiation and apoptosis is not limited to the intestine. for instance, human acute myeloid leukemia cells treated with the dna methyltransferase inhibitors 5azacitidine and decitabine showed exchanges of methyl groups for acetyl functions on histone h3 lysine 27 that were associated with interleukin 3 expression (buchi., 2014). furthermore, fiskus. (2009) reported that dznep and panobinostat, histone methyltransferase and hdac inhibitors respectively, acted synergistically to induce antiproliferative and proapoptotic gene expression in human acute myeloid cells. cooperation between transcription factors, signaling pathways and epigenetic mechanisms is essential for the tight control of the constant renewal of intestinal tissue. based on the recent data discussed in the previous sections, we propose an updated model (fig. 4) to illustrate major mechanisms involved in the regulation of proliferation and differentiation of absorptive intestinal cells in the crypt. on one hand, the prodifferentiation factors cdx2, hnf1, and gata4, which are necessary for the induction of si expression and the control of absorptive cell proliferation and differentiation, are expressed in all epithelial cells of the ta zone and above (escaffit., 2006 ; benoit., 2010). on the other hand, epigenetic mechanisms act upstream of these prodifferentiation transcription factors to maintain cell proliferation and transiently prevent differentiation in the ta zone. interestingly, abolition of prc2 activity or hdacs has no significant effect on the expression of cdx2 or hnf1 (benoit., 2012 ; roostaee., therefore, transcription factors and histone modifications under the regulation by hdac and pcg proteins appear as two sets of counteracting mechanisms that exceptionally integrate in the ta zone to maintain rapid and proper renewal of the intestinal epithelium. however, these mechanisms are transient so that after a few rounds of cell division, stimulation of cell proliferation and transcriptional repression of cell differentiation are halted through a yet unknown mechanism causing histone demethylation and acetylation both occurring in the upper crypt and corresponding to absorptive cell terminal differentiation (fig. 4). this model provides a more detailed understanding of the crucial regulatory role of epigenetic mechanisms in the ta zone, in proper and constant maintenance of intestinal tissue renewal. this information may also have important implications in the comprehension of gastrointestinal disorders such as colorectal cancer in which perturbations in epigenetic mechanisms have been observed (hammoud., 2013). the intestinal crypt compartments are defined as the stem cell, ta, and td zones. the ta zone is composed of quickly dividing cells issued from the stem cell zone. for absorptive cells, experimental evidence suggests that the prc2 complex and hdacs maintain cellular proliferation and restrain differentiation. removal of methyl groups from h3k27me3 and acetylation of histones eliminates transcriptional repression, and the cell differentiation process starts (benoit., 2012 ; roostaee., some studies have also reported a conversion of methylatedtoacetylated histones (suzuki., 2008 ; ong and corces, 2011). the prodifferentiation factors cdx2 and hnf1 are expressed by all epithelial cells including the cells residing in the ta zone and can upregulate expression of intestinespecific genes such as si. epigenetics is a fastgrowing field, which opens new avenues for fundamental research on the regulation of cellular proliferation, differentiation and subsequent generation of intestinal epithelial tissue under normal and diseased conditions. in this review, we discussed recent findings concerning epigenetic mechanisms that regulate intestinal cell fate in the ta zone. however, further research is still needed to explain how these epigenetic mechanisms are regulated at the cellular and molecular level under physiological and cancer conditions. | a controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the cryptvillus axis. one important checkpoint in this process occurs in the transit amplifying zone of the intestinal crypts where different signaling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. however, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. there have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. in this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development, and tissue renewal. j. cell. physiol. 231 : 23612367, 2016. 2016 the authors. journal of cellular physiology published by wiley periodicals, inc. |
chronic biofilm infections play an important role in otitis media,1 cholesteatoma,2 sinusitis,3 osteoradionecrosis,4 tracheitis,5 and tonsillitis,6 making these infections difficult to eradicate. in nature, bacteria have been observed to exist in both planktonic or biofilm forms (large, complex, multicellular communities encased in a dynamic extrapolysaccharide matrix composed of extracellular dna, polysaccharides, and proteins).7, 8, 9, 10, 11 bacteria within biofilms are highly tolerant to antibiotics and host defenses, making them a significant problem in healthcare today. healthcareassociated infections are responsible for 1.7 to 2 million infections per year in the united states.12, 13 according to a report by the national institutes for health (bethesda, md), biofilms account for over 80% of infections in the body,14, 15 and it is estimated that 60% to 70% of nosocomial infections in the united states specifically are associated with implanted devices.16 the economic burden of healthcareassociated infections is considerable ; the overall direct costs to hospitals in the united states range from $ 28 billion to $ 45 billion.13 biofilms are thus an important area of study given their impact on morbidity, mortality, and annual healthcare expenditure. perhaps the biggest challenge that bacteria within biofilms present is their ability to evade antibiotics and the host immune defenses. this unique ability of biofilms has been attributed to stationary phase physiology, horizontal exchange of antibacterial resistance genes, tolerance, adaptive resistance, and efflux pumps.7, 17 resistance to host immune defenses still is being investigated ; recent studies have suggested that biofilms trigger impaired neutrophil activity and use neutrophils to enhance initial biofilm development.18, 19 these factors make biofilms a particularly unique therapeutic challenge. for this reason, there has been interest in investigating molecules and surfaces that can disperse or inhibit biofilm formation and eradicate persister cells. recently, kolodkingal. reported that damino acids inhibited and disassembled biofilms formed by bacillus subtilis.20 however, this group later discovered that the strain had a mutation in the gene dtd, which prevents misincorporation of damino acids into proteins.21 when that gene was repaired, b. subtilis biofilms were not susceptible to inhibition or dispersion by damino acids.21 since then, there have been several other reports that damino acids inhibit biofilm formation in various bacterial species, including s. aureus, s. epidermidis, and other pathogens. however, numerous inconsistencies exist between these studies.20, 21, 22, 23, 24, 25 in addition to b. subtilis, kolodkingal. reported that damino acids inhibit biofilm formation by p. aeruginosa.20 p. aeruginosa is an important pathogen in opportunistic infections and cystic fibrosis and has become a major cause of nosocomial infections worldwide (10% of nosocomial infections in the european union).26 the ability of p. aeruginosa to cause these infections, colonization of biomedical implants, and infection in chronic wounds is dependent on biofilm formation by this pathogen. therefore, we reexamined the ability of damino acids (leucine, methionine, alanine, tryptophan, and tyrosine) to inhibit biofilm formation in two commonly studied wild type strains of p. aeruginosa : pao1 and pa14. our pao1 strain was obtained from colin manoil 's laboratory, uw genome sciences, at the university of washington (seattle, wa) ; pa14 was obtained from stephen lory 's laboratory, department of microbiology and immunobiology, at harvard university (boston, ma). both strains were stored at 80c in 20% glycerol and plated onto luriabertani (lb) agar to obtain single, isolated colonies, which were then inoculated into lb broth for planktonic growth to log phase. planktonic cultures were grown at 37c, with shaking at 225 rotations per minute (rpm) for 16 to 18 hours (new brunswick scientific c24 classic benchtop incubator shaker, edison, nj). the absorbance of an overnight culture of pao1 and pa14 grown in lb broth (described above) was measured at an optical density of 600 nanometer (nm) using a 1:10 dilution (barnstead thermolyne turner spectrophotometer model 340, dubuque, ia). all cultures were adjusted with sterile lb to a uniform od of 0.3 at 600 nm. eight damino acid conditions and eight lamino acid conditions were tested : 10 mm d / lleucine, 10 mm d / lmethionine, 10 mm d / lalanine, 10 mm d / ltryptophan, 1 mm d / ltyrosine, 10 um d / ltyrosine, d / lamino acid mix 1 (mixture of 10 mm leu, met, ala, trp, and 10 um tyr), and d / lamino acid mix 2 (mixture of 10 mm leu, met, ala, trp, and 1 mm tyr). the amino acids and concentrations tested were chosen based on previously published studies.20 planktonic cultures were diluted 1:1000 in sterile m63 minimal media supplemented with mgcl2 (1 mm), dglucose (0.2%), casamino acids (0.5%),20 and the d or lamino acid condition described above. one milliliter of this mixture was then distributed into each well of a sterile, untreated, 24well polystyrene cell culture plate (cellstar 24w suspension multiwall plate, greiner bio one, austria) and incubated at 30c for 24, 48, and 72 hours (fisher isotemp incubator model 255d, waltham, ma). pa14 biofilm formation in d / lleucine, methionine, mix1, mix2, and tryptophan were incubated for 7 days. pao1 biofilm formation in d / lmix1 and mix2 was also incubated for 7 days. prior to incubation, each plate was sealed with a gaspermeable membrane to allow for a high, uniform rate of gas exchange in all wells while maintaining a contaminantfree environment within each well (breathe easy gas permeable sealing membrane for microtiter plates, diversified biotech, dedham, ma). at each time point (24, 48, 72, and 168 hours), the plates received a gentle wash in tap water. the remaining adherent biofilms were dried for at least 48 hours and then stained with 0.1% crystal violet and rinsed. quantitative measures of remaining crystal violet were not performed in this part of the study (in 24well plates) because of the variability of adherence of these large biofilms. alternatively, this measure was performed in 96well plates (see next section, 96well plate quantitative biofilm assay). the bacterial strains were prepared in the same manner as in the 24well plate biofilm assay section. after diluting the bacterial cultures in 1:1000 m63 media supplemented with the appropriate single amino acid or amino acid mixture, 100 l of this dilution was distributed into each well of a 96well plate (corning 96 well clear flat bottom polyvinyl chloride (pvc) not treated microplate corning inc., corning ny) with 16 replicates for each bacterial strain / amino acid condition to control for welltowell variation. an additional condition was tested ; biofilms were grown in the presence of 20 minimal inhibitory concentration (mic) ciprofloxacin as a comparison. a gaspermeable membrane was applied to each plate and incubated for 6, 12, 18, 24, 36, 48, 72, and 96 hours at 30c. to assess biofilm formation, each plate was washed twice by submerging and gently shaking the plate back and forth in tap water. the plates were then dried for at least 48 hours. after drying, 125 microliters of 0.1% crystal violet was added to each well and incubated for 10 minutes at room temperature to stain for adherent biofilm. after 10 minutes incubation, the plates were washed in a tap water basin four times by complete submersion of the plate with gentle shaking. when the plates were dry, the crystal violet was extracted by solubilization in 200 l of 33% glacial acetic acid for 15 minutes on an orbital shaker at 140 rpm (lab line junior orbit shaker, melrose park, il). the amount of crystal violet eluted was then measured using a microplate reader (595 nm) (biotek synergy ht, winooski, vt). our pao1 strain was obtained from colin manoil 's laboratory, uw genome sciences, at the university of washington (seattle, wa) ; pa14 was obtained from stephen lory 's laboratory, department of microbiology and immunobiology, at harvard university (boston, ma). both strains were stored at 80c in 20% glycerol and plated onto luriabertani (lb) agar to obtain single, isolated colonies, which were then inoculated into lb broth for planktonic growth to log phase. planktonic cultures were grown at 37c, with shaking at 225 rotations per minute (rpm) for 16 to 18 hours (new brunswick scientific c24 classic benchtop incubator shaker, edison, nj). the absorbance of an overnight culture of pao1 and pa14 grown in lb broth (described above) was measured at an optical density of 600 nanometer (nm) using a 1:10 dilution (barnstead thermolyne turner spectrophotometer model 340, dubuque, ia). all cultures were adjusted with sterile lb to a uniform od of 0.3 at 600 nm. eight damino acid conditions and eight lamino acid conditions were tested : 10 mm d / lleucine, 10 mm d / lmethionine, 10 mm d / lalanine, 10 mm d / ltryptophan, 1 mm d / ltyrosine, 10 um d / ltyrosine, d / lamino acid mix 1 (mixture of 10 mm leu, met, ala, trp, and 10 um tyr), and d / lamino acid mix 2 (mixture of 10 mm leu, met, ala, trp, and 1 mm tyr). the amino acids and concentrations tested were chosen based on previously published studies.20 planktonic cultures were diluted 1:1000 in sterile m63 minimal media supplemented with mgcl2 (1 mm), dglucose (0.2%), casamino acids (0.5%),20 and the d or lamino acid condition described above. one milliliter of this mixture was then distributed into each well of a sterile, untreated, 24well polystyrene cell culture plate (cellstar 24w suspension multiwall plate, greiner bio one, austria) and incubated at 30c for 24, 48, and 72 hours (fisher isotemp incubator model 255d, waltham, ma). pa14 biofilm formation in d / lleucine, methionine, mix1, mix2, and tryptophan were incubated for 7 days. pao1 biofilm formation in d / lmix1 and mix2 was also incubated for 7 days. prior to incubation, each plate was sealed with a gaspermeable membrane to allow for a high, uniform rate of gas exchange in all wells while maintaining a contaminantfree environment within each well (breathe easy gas permeable sealing membrane for microtiter plates, diversified biotech, dedham, ma). at each time point (24, 48, 72, and 168 hours), the plates received a gentle wash in tap water. the remaining adherent biofilms were dried for at least 48 hours and then stained with 0.1% crystal violet and rinsed. quantitative measures of remaining crystal violet were not performed in this part of the study (in 24well plates) because of the variability of adherence of these large biofilms. alternatively, this measure was performed in 96well plates (see next section, 96well plate quantitative biofilm assay). the bacterial strains were prepared in the same manner as in the 24well plate biofilm assay section. after diluting the bacterial cultures in 1:1000 m63 media supplemented with the appropriate single amino acid or amino acid mixture, 100 l of this dilution was distributed into each well of a 96well plate (corning 96 well clear flat bottom polyvinyl chloride (pvc) not treated microplate corning inc., corning ny) with 16 replicates for each bacterial strain / amino acid condition to control for welltowell variation. an additional condition was tested ; biofilms were grown in the presence of 20 minimal inhibitory concentration (mic) ciprofloxacin as a comparison. a gaspermeable membrane was applied to each plate and incubated for 6, 12, 18, 24, 36, 48, 72, and 96 hours at 30c. to assess biofilm formation, each plate was washed twice by submerging and gently shaking the plate back and forth in tap water. the plates were then dried for at least 48 hours. after drying, 125 microliters of 0.1% crystal violet was added to each well and incubated for 10 minutes at room temperature to stain for adherent biofilm. after 10 minutes incubation, the plates were washed in a tap water basin four times by complete submersion of the plate with gentle shaking. when the plates were dry, the crystal violet was extracted by solubilization in 200 l of 33% glacial acetic acid for 15 minutes on an orbital shaker at 140 rpm (lab line junior orbit shaker, melrose park, il). the amount of crystal violet eluted was then measured using a microplate reader (595 nm) (biotek synergy ht, winooski, vt). we began by examining biofilm formation by two strains of p. aeruginosa : pao1 and pa14. the strains were grown in lb media, diluted into m63 minimal media, and added to 24well plates. a diffusion membrane was placed over the wells, and the plates were incubated at 30c for up to 7 days. biofilm formation was assayed visually, and photographs were taken. in the absence of amino acids, there was increased biofilm formation with increased incubation time as seen in both the dark field and crystal violet stains (fig. addition of l versus damino acids (alanine, leucine, tyrosine, methionine, or tryptophan) did not have an inhibitory effect on formation of biofilms at 24, 48, and 72 hours (fig. mixes of lamino acids also had no effect, whereas mixes of damino acids delayed biofilm formation at 24, 48, and 72 hours. however, this effect dissipated by 168 hours, indicating that damino acids do not prevent biofilm formation (fig. sidebyside comparison of biofilms grown in the absence or presence of d and lamino acids at 24, 48, 72, and 168 hours. mixture 1 contains 10 mm concentration of alanine, leucine, methionine, tryptophan, and tyrosine at 10 um. (b) biofilm grown in the presence of amino acids at 24, 48, and 72 hours. we then examined biofilm formation by a second p. aeruginosa strain : pa14. in the absence of amino acid supplementation, pa14 formed biofilms similarly to pa01 (fig. in contrast to pa01, individual damino acids delayed biofilm formation up to 72 hours (fig. thus, for both pa01 and pa14, damino acids do not prevent biofilm formation. sidebyside comparison of biofilms grown in the absence or presence of d and lamino acids at 24, 48, 72, and 168 hours. mixture 1 contains 10 mm concentration of alanine, leucine, methionine, tryptophan, and tyrosine at 10 um. biofilm grown in the presence of amino acids at 24, 48, and 72 hours. although it was clear that damino acids do not prevent biofilm formation, there was some inconsistency in the crystal violet staining in the 24well plates (fig. 1 and fig. this may have been due to the loose adherence of the biofilms to the surface of the plate, resulting in loss of material during the wash prior to staining with crystal violet (compare dark field photographs and crystal violet stains in fig. 1 and fig. 2). because of this inconsistency, we quantitatively reexamined biofilm formation in a 96well assay in which the biofilms were more tightly adherent to the well surface. careful examination of biofilm formation by the two strains revealed that the two strains formed biofilms at different rates (fig. because of this observation, we analyzed biofilm formation at various time points to ensure that we detected peak biofilm formation. specifically, we measured biofilm formation by pa01 at 24 and 36 hours (fig. 3b and 3c) and pa14 at 48 and 72 hours (fig. in contrast to ciprofloxacin, damino acids did not prevent biofilm formation by pa01 or pa14. in fact, the presence of certain damino acids appeared to enhance biofilm formation by pa01. (a) graphic depiction of pao1 and pa14 biofilm development over the course of 96 hours. pao1 biofilm growth was assayed in the presence of ciprofloxacin versus various amino acids at 24 hours (b) and 36 hours (c). pa14 biofilm growth was assayed in the presence of ciprofloxacin versus various amino acids at 48 hours (d) and 72 hours (e). in the absence of amino acids, there was increased biofilm formation with increased incubation time as seen in both the dark field and crystal violet stains (fig. addition of l versus damino acids (alanine, leucine, tyrosine, methionine, or tryptophan) did not have an inhibitory effect on formation of biofilms at 24, 48, and 72 hours (fig. mixes of lamino acids also had no effect, whereas mixes of damino acids delayed biofilm formation at 24, 48, and 72 hours. however, this effect dissipated by 168 hours, indicating that damino acids do not prevent biofilm formation (fig. sidebyside comparison of biofilms grown in the absence or presence of d and lamino acids at 24, 48, 72, and 168 hours. mixture 1 contains 10 mm concentration of alanine, leucine, methionine, tryptophan, and tyrosine at 10 um. biofilm grown in the presence of amino acids at 24, 48, and 72 hours. we then examined biofilm formation by a second p. aeruginosa strain : pa14. in the absence of amino acid supplementation, pa14 formed biofilms similarly to pa01 (fig. in contrast to pa01, individual damino acids delayed biofilm formation up to 72 hours (fig. 2b) but did not ultimately prevent biofilm formation (fig. 2c). thus, for both pa01 and pa14, damino acids do not prevent biofilm formation. sidebyside comparison of biofilms grown in the absence or presence of d and lamino acids at 24, 48, 72, and 168 hours. mixture 1 contains 10 mm concentration of alanine, leucine, methionine, tryptophan, and tyrosine at 10 um. (a) biofilm growth in the absence of amino acid. (b) biofilm grown in the presence of amino acids at 24, 48, and 72 hours. although it was clear that damino acids do not prevent biofilm formation, there was some inconsistency in the crystal violet staining in the 24well plates (fig. 1 and fig. this may have been due to the loose adherence of the biofilms to the surface of the plate, resulting in loss of material during the wash prior to staining with crystal violet (compare dark field photographs and crystal violet stains in fig. 1 and fig. 2). because of this inconsistency, we quantitatively reexamined biofilm formation in a 96well assay in which the biofilms were more tightly adherent to the well surface. careful examination of biofilm formation by the two strains revealed that the two strains formed biofilms at different rates (fig. because of this observation, we analyzed biofilm formation at various time points to ensure that we detected peak biofilm formation. specifically, we measured biofilm formation by pa01 at 24 and 36 hours (fig. 3b and 3c) and pa14 at 48 and 72 hours (fig. in contrast to ciprofloxacin, damino acids did not prevent biofilm formation by pa01 or pa14. in fact, the presence of certain damino acids appeared to enhance biofilm formation by pa01. (a) graphic depiction of pao1 and pa14 biofilm development over the course of 96 hours. pao1 biofilm growth was assayed in the presence of ciprofloxacin versus various amino acids at 24 hours (b) and 36 hours (c). pa14 biofilm growth was assayed in the presence of ciprofloxacin versus various amino acids at 48 hours (d) and 72 hours (e). because the use of damino acids might be a welltolerated antibiofilm factor in the treatment of biofilm infections, we chose to study a variety of these molecules in their ability to inhibit two common laboratory strains of p. aeruginosa. in this study, we looked at the formation of biofilms by pa01 and pa14 in the presence and absence of d and lamino acids using both a 24well and 96well assay. although we observed a modest delay in biofilm formation by both strains in the presence of damino acids, this effect dissipated in all conditions by 72 to 168 hours. thus, we were unable to reproduce a previously published result, suggesting that damino acids inhibit biofilm formation by p. aeruginosa. the effect of damino acids on biofilm formation has recently come under scrutiny because the original result showing biofilm dissolution in b. subtilis was shown to be due to a mutation in the strain used in that study.21 although kolodkingal. also reported that 10 m dtyrosine or 5 nm mixtures of several damino acids were able to inhibit biofilm formation in p. aeruginosa in their original study,20 they did not repeat their experiments on p. aeruginosa biofilm in their subsequent publication.21 several other studies have examined the effect of damino acids on biofilm formation by p. aeruginosa. for example, brandenburg. showed that both d and ltryptophan inhibited biofilm formation in p. aeruginosa.22 sanchez.23, 24, 25 investigated the effect of dmethionine, dphenylalanine, and dtryptophan on biofilm dispersal in six strains of p. aeruginosa and found that biofilm dispersal was straindependent. study,20 sanchez.23 clearly demonstrated that dtyrosine was ineffective in biofilm dispersal of p. aeruginosa. finally, sanchez.25 also reported toxic effects of damino acids on p. aeruginosa viability, thus confounding the interpretation of their results. considering that the inhibitory effects of damino acids on biofilm formation were first observed using a b. subtilis mutant strain that is sensitive to the presence of damino acids, this has raised questions about the validity of this finding. in a careful review of the literature, damino acids occasionally have an effect on biofilm formation that is not replicable between studies. in our study, we systematically examined the effect of damino acids on biofilm formation by p. aeruginosa and were unable to reproduce earlier reports. this was true for two p. aeruginosa strains, pao1 and pa14, in the presence of dalanine, dmethionine, dleucine, dtryptophan, dtyrosine, and damino acid mixtures. thus, we conclude that damino acids do not represent a viable therapeutic option for treatment of p. aeruginosa biofilmrelated infections. | objective pseudomonas aeruginosa, a known biofilmforming organism, is an opportunistic pathogen that plays an important role in chronic otitis media, tracheitis, cholesteatoma, chronic wounds, and implant infections. eradication of biofilm infections has been a challenge because the biofilm phenotype provides bacteria with a protective environment from the immune system and antibiotics ; thus, there has been great interest in adjunctive molecules that may inhibit biofilm formation or cause biofilm dispersal. there are reports that damino acids may inhibit biofilms. in this study, we test the ability of various damino acids to inhibit p. aeruginosa biofilm formation in vitro.study designwe evaluated the effect of dalanine (10 mm), dleucine (10 mm), dmethionine (10 mm), dtryptophan (10 mm), and dtyrosine (10 um and 1 mm) on biofilm formation in two commonly studied laboratory strains of p. aeruginosa : pao1 and pa14.methodsbiofilms were grown in 24well and 96well tissue culture plates, documented photographically and stained with 0.1% crystal violet and solubilized in 33% glacial acetic acid for quantification.resultsin strains pao1 and pa14, the addition of damino acids did not result in an inhibitory effect on biofilm growth in 24well plates. repeating the study in 96well plates confirmed our findings that damino acids do not inhibit biofilm formation of p. aeruginosa.conclusionwe conclude that damino acids only slow the production of biofilms rather than completely prevent biofilm formation ; therefore, damino acids represent a poor option for potential clinically therapeutic interventions.level of evidencen / a. |
plasticity of gabaergic synapses onto excitatory neurons, in the form of long - term potentiation (ltpi) and/or depression (ltdi) of inhibitory postsynaptic potentials (ipsps), was initially reported in layer 5 of the rodent primary visual cortex. following these pioneering studies, neonatal hippocampus, deep cerebellar nuclei [3, 4 ], lateral superior olive, brain stem, and onto dopaminergic neurons in the ventral tegmental area (vta) [7, 8 ]. although there are significant differences in the induction and expression mechanisms of high - frequency long - term inhibitory plasticity (hf - ltpi and hf - ltdi, figure 1), some common features have been identified across several brain circuits. the source of ca is specific to the inhibitory synapse : voltage - gated calcium channels (vgcc) in neonatal hippocampus (figure 1(b), left panel) ; astrocytes in juvenile hippocampus ; postsynaptic intracellular stores in cortex (figure 1(a), left panel) [11, 12 ] ; activation of postsynaptic nmda receptors in the vta (figure 1(c), left panel). in several systems, the induction of hf - ltpi and hf - ltdi depends on high - frequency activation of glutamatergic and gabaergic axons. postsynaptic activation of glutamatergic receptors is often required for the induction of hf - ltpi, while gabaa receptor activity is involved in maintaining the plasticity [1, 8 ]. by sampling and integrating gabaergic and glutamatergic inputs, heterosynaptic forms of inhibitory plasticity the intracellular mechanisms involved in the induction and expression of hf - ltpi differ significantly between brain circuits. in visual cortex, ca release from intracellular stores is triggered by the activation of gabab receptors, facilitated by the activation of serotoninergic (5-ht) and/or -adrenoreceptors and mediated by the activation of ip3 [12, 13 ] (figure 1(a) left panel). in both developing visual cortex and hippocampus, intracellular ca release initiates a bdnf / trkb signaling cascade that modulates gaba release [14, 15 ] (figures 1(a) and 1(b) left panels). while in the hippocampus, hf - ltpi is induced and maintained after the hfs, in visual cortex the maintenance of hf - ltpi requires constant low - frequency stimulation. the specific mechanisms for this requirement remain to be elucidated. although expressed presynaptically as in visual cortex and hippocampus, hf - ltpi in the vta requires retrograde signaling via a nitric - oxide-(no-) guanylate cyclase (gc-) protein - kinase - g-(pkg-) dependent pathway (figure 1(c), left panel). different mechanisms of induction and expression for hf - ltpi suggest that the specificity of the connection and the patterns of activity may be important for the function of heterosynaptic inhibitory plasticity in different circuits. for example, bdnf retrograde signaling allows for a local action of gabaergic plasticity at specific synapses [14, 18 ], suggesting a prominent function of hf - ltpi on the local integration of excitatory and inhibitory synaptic events. differently, at synapses in which production of no is involved in inhibitory plasticity, the widespread diffusion typical of no may influence several presynaptic terminals simultaneously, possibly promoting changes in the state of excitability of a large portion of a microcircuit. the mechanisms for induction and expression of heterosynaptic ltdi show significant differences in different circuits (figures 1(a), 1(b), and 1(c), right panels). in l5 of primary visual cortex, hf - ltdi is induced by activation of glutamatergic and gabaergic axons and is dependent on ca inflow in the postsynaptic excitatory neuron either through nmda receptors [1, 13 ] or through l - type ca channels (figure 1(a), right panel). it has been speculated that nmda - dependent hf - ltdi and l - type cachannels - dependent hf - ltdi may differ in that the former produces a focal, spatially restricted depression of inhibition, and the latter contributes to depressing many inhibitory synapses onto the same postsynaptic neuron. hf - ltdi (or hf - i - ltd) was also induced in l2/3 of primary visual cortex and in the hippocampus. the mechanisms for these forms of plasticity have been investigated and are known to involve the production of endocannabinoids (ecb) in both l2/3 of visual cortex and hippocampus. in the hippocampus, the production of ecb is dependent on the activation of postsynaptic type i metabotropic receptors (mglur - i), while in visual cortex the mechanism of activation has not been identified. a second widely investigated form of heterosynaptic ltdi is induced by low - frequency (lf) activation of glutamatergic axons, which can heterosynaptically depress gabaergic inputs converging onto the activated postsynaptic neuron (lf - ltdi or i - ltd, figure 1(b)). this form of plasticity has been reported in several areas of the brain including vta, basolateral amygdala (bla), dorsal striatum, prefrontal cortex, and corticotectal cocultures. i - ltd is induced by activation of metabotropic glutamate receptors (mglur1) and is maintained by postsynaptic gabaa receptors activity. intracellular pathways activated by the induction of i - ltd lead to the production and the release of endocannabinoids (ecb) from the excitatory neuron [22, 23, 27 ], which in turn promote changes in strength at inhibitory synapses onto the same postsynaptic target neuron (figures 1(b) and 1(c), right panels). in the hippocampus, i - ltd requires the activation of inhibitory afferents in the presence of ecb, suggesting that a raise in presynaptic ca in the presynaptic interneuron terminal is required for the induction of this form of inhibitory plasticity. in the vta, i - ltd is expressed presynaptically and involves a protein - kinase - a-(pka) dependent modulation of gaba release [22, 30 ]. similarly to bdnf - dependent hf - ltpi, ecb - i - ltd signaling is more localized to the area of induction [31, 32 ]. given the induction requirements, ecb - i - ltd may contribute to the integration of local associative inputs elicited by long - lasting presynaptic activity in the low - frequency range. homosynaptic forms of inhibitory plasticity have recently been reported in cortex and hippocampus (figure 2) [15, 18, 3335 ]. the patterns of activity reported for homosynaptic inhibitory plasticity differ between brain areas and with the developmental stage of the circuit. furthermore, homosynaptic monosynaptic forms of inhibitory plasticity have been reported : gabaergic synapses from a single inhibitory neuron of a specific subtype onto a postsynaptic excitatory neuron can change their strength in response to patterned stimulation [34, 35 ]. gabaergic synapses may modulate their strength not only to regulate the integration of excitatory and inhibitory inputs, but also in response to a variety of input patterns, possibly increasing the range of functions that inhibitory plasticity may perform in different circuits. in immature hippocampus, when gaba is excitatory, stimulations eliciting action potential firing in afferent axons 15 ms before postsynaptic firing potentiate gaba postsynaptic currents onto ca3 pyramidal neurons (figure 2(a), left panel), while the opposite timing relationship induces a consistent depression of gaba postsynaptic current amplitudes. the mechanisms leading to spike - timing - dependent long - term potentiation of inhibition (std - ltpi) have been further investigated, showing that this form of gabaergic plasticity is induced postsynaptically and expressed presynaptically. the signaling pathways involved in std - ltpi in neonatal hippocampus depend on the increase in postsynaptic ca levels and on the activation of a camp - pka intracellular pathway. std - ltpi required retrograde bdnf signaling (figure 2(a), left panel), consistent with reports that postsynaptic backpropagating action potentials trigger the release of bdnf in the postsynaptic neurons. the site of action of bdnf is at the moment unclear, as postsynaptic and/or presynaptic actions of the bdnf / trkb signaling pathway have been reported. std - ltpi in the immature hippocampus increases the activity of excitatory neurons and is thought to contribute significantly to the development and refinement of hippocampal circuits [37, 38 ]. in adult hippocampal slices, when gaba is hyperpolarizing and exerts an inhibitory action, homosynaptic plasticity of gabaergic synapses is successfully induced when presynaptic action potentials are elicited at inhibitory axons coincidentally with the generation of postsynaptic action potentials in ca1 pyramidal neurons (std - long - term depression of inhibition, std - ltdi : figure 2(a), right panel) [33, 3941 ]. std - ltdi induction depends on postsynaptic ca influx through voltage - gated ca channels and on the depolarization of the equilibrium potential for chloride by the neuron - specific chloride extruding transporter kcc2 (figure 2(a), right panel). interestingly, activation of the bdnf - trkb pathway regulates the levels of expression of kcc2 in the adult hippocampus, suggesting possible complementary mechanisms of expression for hf - ltpi in cortex and std - ltdi in the adult hippocampus (figure 2(a)). in hf - ltpi the high - frequency stimulation may promote a bdnf - dependent increase in inhibitory synaptic conductance through modulation of presynaptic release probability (figure 1(b), left panel), while the lower frequency of stimulation required by std - ltdi induction may favor bndf - dependent downregulation of the kcc2 transporter. timing is a fundamental feature of homosynaptic inhibitory plasticity ; however, there are substantial differences in the requirements for induction and expression of gabaergic homosynaptic plasticity in hippocampus and sensory neocortex. in visual cortex in particular, homosynaptic inhibitory plasticity has been studied at monosynaptic connections from identified inhibitory neurons onto pyramidal neurons. therefore, the plasticity i will describe in this paragraph is both homosynaptic it depends on the activation of inhibitory axons alone and monosynaptic it is induced by activation of connections mediated by a single axons from an indentified inhibitory neuron subtype onto a pyramidal neuron. in layer 2/3 of visual cortex, the sign of plasticity at inhibitory synapses from fast spiking (fs) onto pyramidal neurons depends on the timing between presynaptic fs neuron firing and postsynaptic pyramidal neuron bursting. burst timing ltd (bt - ltdi) is induced if fs action potentials are elicited 100 ms after pyramidal neuron bursting, while bt - ltpi is induced when fs firing was elicited 200 to 300 ms after postsynaptic bursting (figure 2(b), left panel). both bt - ltpi and bt - ltdi depend on postsynaptic calcium influx, although the source of such increase has not been determined. differently from the forms of inhibitory plasticity reported in l5 of primary visual cortex or in the hippocampus, bt inhibitory plasticity does not appear to require gabab receptor activation, activation of nmda receptors, or changes in kcc2 activity (figure 2(b), left panel), suggesting that this form of inhibitory plasticity may rely on a set of mechanisms yet to be identified. although timing of pre- and postsynaptic activity is indeed a general feature of fs to pyramidal neuron inhibitory plasticity, the requirements for timing and patterns of activity differ significantly, even between layers of primary visual cortex. in layer 4, potentiation of fs to pyramidal neurons synapses (ltpi) requires coincident activation of fs and postsynaptic pyramidal neurons, but fs interneuron bursting needs to be paired with pyramidal neuron subthreshold depolarization (figure 2(b), right panel). despite the differences, both bt - ltpi and ltpi are expressed as changes in the conductance of inhibitory synapses and appear to have a postsynaptic site of expression, possibly depending on an increase in gabaa r number [34, 35, 43 ]. both bt - ltpi and ltpi are induced using paired recording experiments, indicating that these forms of plasticity are specific to the interneuron type [34, 35 ]. several intracellular pathways have been reported to regulate the number of gabaa receptors at inhibitory synapses [4447 ]. an intriguing possibility is that some of these mechanisms may be also involved in the fast transport required for plasticity. a different subset of inhibitory synapses in neocortex, the ones from low - threshold spiking interneurons (lts) onto spiny stellate neurons in the barrel cortex and from regular spiking non - pyramidal neurons (rsnp) onto pyramidal neurons in visual cortex, show modulation of synaptic efficacy in response to changes in circuit excitability [48, 49 ]. in visual cortex, reduction of visual drive right at eye opening strengthens their synapses onto pyramidal neurons, decreases their connection probability, and leaves their short - term dynamics unaffected. in barrel cortex, lts neurons also classified as som neurons change their short - term dynamic and intrinsic properties in response to activity blockade. the induction and expression requirements for plasticity at these inhibitory synapses have yet to be identified. overall there is richness in the forms of inhibitory plasticity and in the variety of mechanisms involved in their induction and expression in different areas and at different developmental stages. this evidence suggests that inhibitory synapses may have important and highly specific functions that contribute to the control of the excitability of neural circuits in complex ways. a number of studies have shown that inhibitory synaptic transmission is crucial for the development [5153 ] and stability of neural circuits [54, 55 ], sharpens tuning of principal excitatory neurons [5659 ], and contributes to the formation of receptive fields [60, 61 ]. all of these functions are based on the assumption that inhibitory synapses are not plastic and exert what is thought to be their principal task : suppress excessive excitability and possibly increase the signal - to - noise ratio. the ability to directly and dynamically control pyramidal neuron input integration, excitability, and output in an activity - dependent manner, suggests that inhibitory synaptic plasticity may be crucial to preserve the dynamic range of excitatory neurons even when the excitability of the circuit is perturbed by changes in environmental inputs. the maintenance of circuit stability is indeed a dynamic process that requires plasticity of many cellular and synaptic components of a neural circuit [65, 66 ]. the richness in plasticity and the specificity of inhibitory circuits suggest that the dynamic regulation of circuit homeostasis is not the sole function of inhibitory plasticity. in primary sensory areas, incoming inputs regulate the maturation of gabaergic transmission, promote the refinement of the connectivity of local microcircuits, and regulate the overall excitability of the circuit [21, 34, 49, 6770 ]. the sharpening of cortical receptive fields during development is also temporally correlated with the maturation of inhibitory synapses, and the modulation of excitability by dynamic adjustment of the balance between excitation and inhibition favors the refinement of neuronal receptive fields [72, 73 ]. at the network level, the regulation of inhibitory synaptic efficacy through plastic changes may contribute to the formation and/or rearrangement of cortical maps. specific inhibitory circuits may contribute differently to the refinement process, as suggested by the effects that paradigms of sensory deprivation produce on the two major populations of inhibitory neurons [4850 ]. in rodent neocortex, both the barrel field of somatosensory cortex and primary visual cortex show depression of fs synaptic inhibition onto pyramidal neurons in response to sensory deprivation during early postnatal development [48, 49 ]. fs to pyramidal neuron synapses receive direct thalamocortical projections [74, 75 ] and, thus, are in a particularly favorable anatomical position to convey information about changes in sensory inputs. a possible function of reduced fs inhibition at this stage in development is to preserve the overall state of excitability of the circuit in the face of a reduced driving input. in primary visual cortex, evidence in favor of this hypothesis comes from the lack of ocular dominance shifts following visual deprivation between eye opening and the beginning of the classical critical period for amblyopia. in the barrel cortex, the same inhibitory synaptic connection may play different functions during different stages in postnatal development. after the third postnatal week, instead of weakening fs to pyramidal neurons synapses, visual deprivation induces ltpi of these synapses in monocular cortex and a general potentiation of inhibitory drive in binocular visual cortex. the switch in sign of md - induced inhibitory plasticity correlates with the time of initiation of the critical period for ocular dominance plasticity [76, 77 ]. a possible interpretation of these results is that as the visual cortex matures, the role of inhibitory plasticity changes, going from regulator of global circuit homeostasis to driver of activity - dependent circuit refinement. ltpi may contribute to the silencing of neurons driven by the deprived eye, possibly favoring the shift in ocular dominance to the eye that remained open. to perform these functions, ltpi should be connection - specific, regulating the excitability of excitatory neurons only within local microcircuits. in addition, it should have an effect on the sign of plasticity at excitatory synapses. while the role of inhibition in controlling excitatory neurons excitability is widely accepted besides modulating fs to pyramidal neurons synapses, visual deprivation significantly affects another inhibitory connection in neocortex : the one from rsnp and lts interneurons onto pyramidal neurons. similar paradigms of sensory deprivation modulate the strength of rsnp and lts inhibitory synapses onto excitatory neurons. it is tempting to speculate that environmental stimuli or behaviors may modulate the strength of inhibitory synapses in ways that are specific for the type of inhibitory neuron and favor different circuit - rewiring patterns. lts and rsnp inhibitory neurons contact the apical dendrites of pyramidal neurons while fs synapses are found at the soma and proximal dendritic shafts. plasticity at lts and rsnp synapses may be involved in the modulation of the local integration of distal inputs, while fs synapses regulate the integration of all inputs reaching the soma. the integration of all inhibitory and excitatory inputs shapes the state of excitability of a circuit throughout life ; thus, the contribution of inhibitory plasticity to neural circuit function is likely to extend beyond circuit refinement. the dynamic regulation of the balance between excitation and inhibition that is induced by changes in inhibitory and excitatory synaptic strength may affect the coding of specific sensory stimuli and serve as an important mechanism for cortical sensory processing throughout life. beyond sensory cortices, inhibitory plasticity is induced in circuits involved in learning and addictive behaviors [7, 8, 15, 22, 33, 79, 80 ]. indeed, in the vta, hf - ltpi is impaired by morphine exposure, and i - ltd is favored by repeated cocaine exposure. these forms of plasticity alter the activity of dopaminergic neurons in the vta following drug abuse, possibly facilitating the development of addictive behaviors. in the vta, other forms of inhibitory plasticity have been reported that do not require patterned activation of afferent fibers but are dependent on the administration of drug of abuse [80, 82, 83 ] and appear to be facilitated by coactivation of serotoninergic receptors. it is currently unknown whether the administration of a drug of abuse and the patterned activation of neurons may activate convergent of cellular targets or whether the electrical and chemical inhibitory plasticity are distinct processes. inhibitory plasticity in the vta was also proposed as mechanism for metaplasticity, a way to constrain or change the state of a postsynaptic neuron to limit or direct the plasticity at other synapses converging onto it. while there is no clear experimental evidence for a metaplastic role of inhibitory plasticity, it would certainly add to the already complex set of functions this plasticity appears to perform. in neonatal hippocampus, when gaba is excitatory, the role of gabaergic plasticity may be to promote circuit wiring and maturation by activating pyramidal neurons [18, 86 ]. in the adult hippocampus, std - ltdi modulates the output and the dynamic range of pyramidal neurons in ca1 globally by shifting the reversal potential of chloride of the postsynaptic neuron [39, 40 ]. in addition, it was recently suggested that std - ltdi regulates the effectiveness of backpropagating action potentials, suggesting that inhibitory plasticity may be a modulating mechanism for the induction of ltp or ltd at synapses between excitatory neurons. a more focal regulation of specific inputs onto excitatory neurons may be performed locally, at specific synapses, by heterosynaptic hf - ltpi and bdnf - trkb signaling. both global and synapse - specific inhibitory synaptic plasticity likely regulate the local integration of incoming inputs onto pyramidal neurons in a complementary fashion, possibly favoring or impairing the induction of other forms of plasticity. although the study of inhibitory plasticity started a couple of decades ago, it has had a fast and steady growth only recently. besides regulating the activity and computation of local microcircuits in many areas of the brain, inhibitory plasticity has been implicated in sensory processing, in the learning of sound localization, in the regulation of neuropathic pain, in the regulation of neural activity following brain injury [89, 90 ], as well as in changes in neuronal excitability induced by pregnancy. much work is needed to identify mechanisms as well as targets for the selective manipulation of gabaergic synaptic plasticity in different brain circuits. the findings that specific inhibitory neuron subtypes contact excitatory neurons at different locations [92, 93 ] and that the postsynaptic membranes opposite to the different subtypes of interneurons contain gabaa receptors with specific subunit composition [9497 ] offer remarkable tools to jump start this investigation. the compelling data about locations, range of induction and expression mechanisms, specificity, and associativity, together with the functional implications of inhibitory synaptic plasticity, strongly support the idea that plasticity at gabaergic synapses is a fundamental regulator of the physiology of neural circuits. advancements in our understanding of the different forms of inhibitory plasticity are crucial to address more directly their many roles in healthy brain function and disease. | on february 12th 1973, bliss and lomo submitted their findings on activity - dependent plasticity of glutamatergic synapses. after this groundbreaking discovery, long - term potentiation (ltp) and depression (ltd) gained center stage in the study of learning, memory, and experience - dependent refinement of neural circuits. while ltp and ltd are extensively studied and their relevance to brain function is widely accepted, new experimental and theoretical work recently demonstrates that brain development and function relies on additional forms of plasticity, some of which occur at nonglutamatergic synapses. the strength of gabaergic synapses is modulated by activity, and new functions for inhibitory synaptic plasticity are emerging. together with excitatory neurons, inhibitory neurons shape the excitability and dynamic range of neural circuits. thus, the understanding of inhibitory synaptic plasticity is crucial to fully comprehend the physiology of brain circuits. here, i will review recent findings about plasticity at gabaergic synapses and discuss how it may contribute to circuit function. |
studies of human origins have witnessed a radical transition from studies based on morphological comparisons to a reliance on molecular genetics. one of the first molecular comparisons estimated human divergence from the african apes at a now generally accepted timescale of 5 million years ago, when most paleontologists then placed it between 15 and 30 million years ago. later, measurement of genetic diversity revealed the relatively recent origin in africa of modern humans, who then spread over the entire globe (see and citations therein). much current research in anthropology centers on understanding the timing and migration routes of modern humans. an analysis of y - chromosome genetic diversity published by shi. in bmc biology has now clarified migration routes and times of settlement for east asia, with wide - ranging implications. previously, it seemed equally possible that the modern humans who settled east asia came either from southeast asia or, alternatively, migrated southward from northern asia. researchers have long noted a significant genetic difference between northern and southern east asian populations that could be interpreted to support either scenario or some mix of the two. both archaeological and genetic evidence for settlement times were also ambiguous. timings for southern east asia ranged from an earliest date of 30,000 years ago to 50,000 years ago at the latest, while dates for the settlement of siberia ranged from 40,000 to 45,000 years ago, or even earlier. however, the new data from shi. suggest that our species reached southern east asia 60,000 years ago, twice as long ago as most previous estimates, and then spread rapidly northward. the amount of genetic diversity in present - day populations is a useful variable for inferring geographic origins and migration routes. africa was pinpointed as the homeland of homo sapiens because of the higher genetic diversity among africans compared with populations elsewhere in the world, while the last geographic regions to be settled, south america and the pacific islands, show the lowest genetic diversity. greater variation has been noted among africans not only in their genes but in variables such as craniometrics, dental traits and even skin color. the original ' out of africa ' hypothesis of modern human origins and subsequent pattern of global migration was based on mitochondrial dna (mtdna) evidence, which revealed a series of population bottlenecks and a progressive loss of diversity moving away from east africa. mtdna, because it is located outside of the cell nucleus, is inherited only through the female line. in contrast, the y chromosome is passed only from father to son, and so can be similarly used to follow the male line. both mtdna and most y - chromosomal dna are non - recombinant and analysis of their inheritance is therefore more straightforward than for other parts of the genome, which are mostly scrambled samples of dna from both parents. human populations on the branches of the mtdna or y - chromosome ' tree ' can be distinguished by sets of accumulated mutational differences or haplotypes in stretches of dna. because these mutations accumulate at a fairly regular rate over time, they can be used as a ' clock ' to estimate the time of human population splits. reconstruction of human origins and migration now mainly relies on the y chromosome because it is much larger than the mitochondrial genome, consisting of tens of millions of nucleotides compared to the 16,000 of human mtdna. thousands of differences can be found in y - chromosomal dna from different human populations. the different y - chromosome haplotypes function as signatures for different human lineages and are often highly associated with different geographic regions, making them extremely useful for tracing human origins and migration. shi. thoroughly tested settlement hypotheses by collecting the largest east asian sample to date, more than 5,000 males from 73 populations. many new samples came from underrepresented south and southwestern china and they also incorporated data from many published reports. the d - m174 lineage was already known from previous studies, and, even if thought to be up to 50,000 years old, the prevalent hypothesis viewed it just as one of the various lineages moving northward with the predominant o - m175 lineage around 25,000 to 30,000 years ago. recently, it was found that d - m174 has a high frequency in andamanese (considered the earliest settlers of southeast asia). then cogently argued that the disjunct distribution of lineage d - m174 today found primarily in the andaman islands, tibet and japan indicated that it is the oldest lineage in east asia. its sporadic presence in other populations, they concluded, was due to recent gene flow that previously went unappreciated. a more detailed subtyping within the d - m174 lineage also allowed shi. to identify the deep, hidden structure of various male lineages. dates in excess of 60,000 years were needed to account for the differences and distributions of these subtypes. this date is older than those previously reported based on both y - chromosomal dna and mtdna. it seems an inescapable conclusion that the lineage is indeed ancient, southern in origin and preceded others found in contemporary east asian populations. recent archaeological evidence of tibetan settlement between 30,000 to 40,000 years ago also supported this conclusion. this genetic signature of early migration has been masked in much of east asia by a later, overriding migration due to the population explosion in neolithic times of the han ethnic group, which is characterized by a different y - chromosome lineage. today, both d - m174 and the han y - chromosome lineages are found in tibet and japan, showing that both these populations are the result of two distinct migrations. shi. also find from their new data that previous impressions that northern asian genetic diversity is greater than southern diversity are probably incorrect and due to incomplete sampling, thus removing one important motive for proposing a northern asian origin. the authors also estimate the age of the unique japanese haplotype d2-m57 as 37,000 years, which suggests that this first wave of migration brought people to japan before the date of the earliest archeological evidence at 30,000 years ago. the origin of the tibetan d - m174 sublineage is older (52,000 years ago) and the tibetan population also has a higher genetic diversity, indicative of earlier settlement. pushing back the date of migration into east asia to 60,000 years ago has wide implications for global scenarios of human dispersion. it makes it necessary to opt for the earliest possible dates for the initial migration out of africa and for settlement in the indian subcontinent. estimates of the time of origin of modern humans in africa fall between 150,000 and 200,000 years ago (figure 1), as supported by paleontological finds in ethiopia. most dates for the coastal migration out of africa range from 50,000 to 77,000 years ago, but these now seem to be too late. there is paleontological evidence for modern humans in the middle east around 92,000 years ago and, if populations were as small as predicted, then we should seriously consider an even earlier out - of - africa date, perhaps even before 100,000 years ago. better sampling of african populations maybe needed to answer the question of whether there was more than one migration and route out of africa. however, the limited worldwide genetic variability among present - day humans has led many researchers to exclude such a possibility and suggests that only a handful of people were involved, perhaps as few as a total of 600 founding females. a simplified scenario of early human migration routes and dates. modern humans originated in africa, probably around 200,000 years ago (200 kya). one or more routes out of africa are possible, but the number of individuals involved was very limited, with perhaps only 600 females. migration probably followed a coastal route, with humans arriving in the indian subcontinent about 70,000 years ago. suggests that humans arrived in southern east asia around 60,000 years ago and then proceeded north to occupy northern east asia and japan. genetic diversity in present - day india is second only to that in africa and implies settlement soon after humans left africa. dispersal across eurasia from africa to india was often previously estimated to have occurred between 45,000 and 59,000 years ago, but a recent proposal of 66,000 to 70,000 years ago would be more congruent with the estimate of shi. genetic markers and an early date for east asian settlement also support the hypothesis that independent migrations populated east asia and australia, as current estimates for human settlement in australia and new guinea are around 55,000 years ago. asia earlier than 30,000 years ago could also have implications for the time of entry of humans into north america ; this is currently put at 16,000 years ago, but could be an underestimate. we should caution, however, that dates inferred from present - day genetic diversity can vary greatly, as a result of unknown differences, in variables such as population size, rates of genetic drift, gene flow, and the presence of selection. for example, y chromosome and mtdna reconstructions of human origins can differ, in part because y chromosomes reflect the activities of males whereas mtdna reflects those of females. it is often the case that dates from mtdna are up to twice as old as those for the y chromosome. the widespread practice of polygyny means that the number of males contributing to the next generation is always smaller than that of females. migration patterns of men and women may also differ due to patterns of marriage exchange and post - marital residence. in brief, it is clear that cultural differences can strongly affect levels of genetic diversity and a correct interpretation of human diversity requires that the biases introduced by migration and admixture patterns must be disentangled from the effects of selection, drift and demography on the human genome. the work of shi. also indicates that human founder populations were small and isolated, and that the initial migration signal may be difficult to detect, especially when it is hidden beneath layers of subsequent migrations. in part analysis of ancient dna can also cut through the layers of time and is an important test of conclusions based on dna from living populations. ancient dna served as an important test of the out - of - africa theory, which predicted that modern humans replaced regional archaic hominids. indeed, analysis of mtdna extracted from neanderthal specimens and from early modern human remains strongly suggests that neanderthals did not contribute to our genome. although accurate characterization of geographic ancestry is possible using a small number of markers, a detailed understanding of human diversity will require more extensive sampling. together, mtdna and the y chromosome constitute only a small fraction of the human genome and we must take more of the genome into consideration for a full understanding of human evolution. a more complete survey of populations and sophisticated statistical analysis of thousands of additional markers is needed. the results will not be trivial and will permit a better understanding of human adaptation, susceptibility to diseases, and even success in pharmacological therapy. high - throughput genotyping and massively parallel sequencing technologies hold great promise that we will soon achieve much more than the tracking of human global migration and settlement. the golden age of human evolutionary genetics is just beginning to dawn. | a recent report in bmc biology indicates that modern humans first arrived in southern east asia 60,000 years ago and settled the rest of east asia from there. this early date and migration route has significant implications for our understanding of the origins of present - day human populations. |
multicentric reticulohistiocytosis (mrh) is a rare disease in which papulonodular skin lesions containing a proliferation of true histiocytes (macrophages) are associated with severe and rapidly destructive arthritis. the disease can involve the bones, tendons, muscles, joints, and nearly any other organ (e.g., eyes, larynx, thyroid, salivary glands, bone marrow, heart, lung, kidney, liver, and gastrointestinal tract). it has been associated with an underlying internal malignancy in about one - fourth of the cases. females are affected more than men, with the ratio of woman to man being 3 : 1. goltz and laymon proposed the name multicentric reticulohistiocytosis in 1954 because of the multifocal origin and systemic nature of the disease. the etiology has not been fully elucidated and no consistently effective treatment has been identified. a 55-year - old female presented with severe debilitating joint pain, involving elbows, wrists, and fingers, and multiple cutaneous lesions, since six months. this was associated with swelling and deformity of the joints, recurrent episodes of high grade fever, and loss of appetite. there was no history of morning stiffness in the joints or a past major medical or surgical illness. on examination, the patient was febrile and pale with restricted mobility of the metacarpophalangeal, proximal and distal interphalangeal, elbow, and shoulder joints, with flexion deformity of the bilateral distal interphalangeal joints [figure 1 ]. nodular lesions over fingers with flexion deformitites of distal interphalangeal joints a dermatological examination revealed discrete, but grouped, firm, reddish brown, non - scaly, non - tender papules and nodules ranging from 2 mm to 2 cm over the elbows, back, and ears [figure 2a, b ]. a hemogram revealed anemia (7.2 gm%) and a raised erythrocyte sedimentation rate (48mm / hour). routine hematological investigations, lipid profile, thyroid profile, rheumatoid factor, and c reactive protein were normal. (b) multiple skin colored papules over pinna, neck and pre - auricular lesion the enzyme - linked immunosorbent assay (elisa) for human immunodeficiency virus (hiv) was non - reactive. an electrocardiogram, chest x - ray, and ultrasound of the abdomen / pelvis were unremarkable. histopathological examination of the skin nodule revealed multiple multinucleated giant cells and the histiocytes showed eosinophilic cytoplasm with a ground glass appearance, confirming the diagnosis of multicentric reticulohistiocytosis [figure 3 ]. multinucleated giant cells and histiocytes with eosinophilic cytoplasm (h and e, 40) the patient was started on tab prednisolone 1 mg / kg per day and simultaneously methotrexate was started at a dose of 15 mg / week. multicentric reticulohistiocytosis, also known as lipoid dermatoarthritis, has a worldwide distribution, with a female preponderance (60 75%). it usually begins during the fourth decade of life with isolated polyarthritis (50%), cutaneous lesions (25%) or both concurrently (25%). the polyarthritis is usually diffuse, symmetric, progressive, and destructive, with a predilection for the distal interphalangeal joint. the appearance of cutaneous manifestations and joint involvement in our patient was simultaneous in the fifth decade of her life. more than 100 cases of multicentric reticulohistiocytosis have been reported in literature, since it was first described by weber and freudenthal. our patient had no mucosal involvement and balcandran. and mittal. cutaneous manifestations include pink, red or brown papules, and nodules ranging from one millimeter to several centimeters in diameter, occurring most frequently on the dorsal aspect of the hands and on the face. other sites of involvement include the arms, scalp, pinnae, and neck, which are also the sites of involvement in our patient. mucosal involvement, which includes the tongue and buccal or nasal mucosa, occurs in roughly one - third of the cases. active disease often remits after approximately eight years, and although it is not considered a paraneoplastic syndrome, it occurs with an associated malignancy in about 20% of the cases. other sites of involvement can include the heart, lungs, thyroid, and bone marrow. constitutional symptoms such as fever, malaise, and weight loss have been described, as have an elevated erythrocyte sedimentation rate (esr), anemia, and hypercholesterolemia. our patient had raised esr and low hb without any serum lipid abnormality or malignancy. diagnosis is based on the histological and immunological features of the proliferating histiocytes. on histopathology, immunohistologically they are positive for trap (tartrate resistant acid phospatase), cd68, lysozyme, and human alveolar macrophage-56 (ham-56), whereas negative for s100 protein, cd1a, and factor xiiia. due to financial constraints systemic steroids, cytotoxic drugs such as cyclophosphamide, chlorambucil, methotrexate, etanercept, and infliximab have been reported to be effective. bisphosphonates such as alendroante and zolidronate have been reported to improve both arthritis and skin lesions.[79 ] we have reported this case because of its rarity and the characteristic histopathological findings. the proliferation of histiocytes can be a paraneoplastic response and there may be significant morbidity and mortality because of joint involvement and malignancy. | multicentric reticulohistiocytosis is a rare systemic granulomatous disease of an unknown cause, characterized by distinct histopathology. the skin, mucosa, synovial, bone, and internal organs may be involved. cutaneous nodules and distinctive arthritis are the most prominent clinical features. a 55-year - old female was referred from orthopedic outpatient department, with multiple, painful and tender nodules on the dorsum of her hands, forearms, elbows, back, and neck. the lesions were present predominantly around the joints with associated arthropathies. smaller nodules were seen on the ear helices. there was no other clinically evident or investigative abnormality. a histopathological study confirmed the diagnosis of multicentric reticulohistiocytosis. |
for a long time history and physical examination have been the most important part of patient 's assessment.1 however, in recent years, the care of patients has become increasingly dependent on the results of laboratory investigations, and clinical laboratories have become a major component in the delivery of health care. laboratory investigation in primary health care (phc) centers should not be considered an isolated activity of the general practitioner. it has always been and will continue to be an integral part of clinical practice in primary health care.2 the factors which motivate physicians to request laboratory services are (i) confirmation of clinical impression ; (ii) reassurance of patients or colleagues that something was being done, even if the results will not affect the diagnosis or therapy and (iii) occasionally requests are based on a desire to do a complete work - up.3 the study of ferrier et a14 showed that women physicians tended to order more laboratory tests than male physicians, and had a higher mean monthly cost of laboratory service per patient. the physicians who are more knowledgeable and clinically skilled used fewer and more appropriate investigations and medical services57 many educational programs such as weekly seminars, lectures, senior clinical staff reviews, computer - based systems (to detect inappropriate test usage) or educational material circulated in the form of summaries of some actual patient cases were brought to the attention of the physicians to correct improper usage. all these programs elicited a satisfactory response during the study time, or shortly after that. however, clinicians reverted to their previous patterns of test utilization after a few months. to be effective, educational programs must be continued indefinitely.811 a more promising approach, which appeals not to the economic but to scientific and clinical sense, is the introduction of agreed request protocols for the investigation of specified types of patients. guidelines consisting of short advisory statements for the ten commonest medical emergencies (myocardial infarction, overdose, hematemesis, pneumonia, etc.) were developed by consultant staff.9 junior staff were given copies of these guidelines and encouraged to use them. there was an immediate reduction in the average number of hematological tests (g4%) and biochemical test (64%). wong and lincoln12 found that clinicians ordered laboratory tests according to an informal protocol which was a product of accumulated experience sanctioned by general use, and thus appeared to have the weight of authority. the problem of laboratory test misuse was therefore not a defect in cognitive knowledge, but primarily protocol. thus, clinicians focus their attention on the broader issue of protocols as a form of information management in medicine.69101321 a cross sectional study was conducted in al - khobar area, eastern province of saudi arabia in 1995 using a questionnaire. for this purpose, all 9 phc centers in the al - khobar area were taken as population under study. a random sample of 5 phc centers, vas selected to study the utilization pattern of laboratory investigations. there were 20 physicians, in 5 phc centers, amounting to 33% of the total physicians in all phc centers in ai - khobar area. the physicians in the selected phc centers were given the questionnaires to survey their knowledge, attitude and practice (kap) towards laboratory investigations and factors which influenced their pattern of utilization and also to correlate their answers with their real life practice, which was explored by studying the medical records. the variables of interest were the physicians age, gender, nationality, place of graduation, postgraduate qualification, years of experience, duration of work in kingdom of saudi arabia, their duration of stay in phc centers and personal data including their professional status. all the physicians involved were informed about the study, and their role in it was fully explained. a scoring system was used to evaluate physicians answers of questionnaire, where 45% was allocated to knowledge, 34% to attitude and 21% to practice. the idea of using a scoring system and grading was based on work done by marion et a110 and pick et a1.22 after computing the scores of every part of kap, they were graded (table 1). for a pilot study, two phc centers were selected in dammam and all the doctors from these two centers were given questionnaires, relevant changes in the questionnaire were made in the light of the pilot study. physician 's grading according to kap scoring of the 20 physicians, 10 were selected at random for re - interviewing to check the reliability of answers given on the kap questionnaire. kappa23 test of reliability was used as test - retest method and it was found that there was 73% agreement between first and second sets of answers. this indicated a high agreement between both sets of answers, hence, a high reliability. the method of multiple regression was applied to the study to see which factor(s) had an effect on their knowledge, attitude and practice. there were 9 (45%/o) saudi and 11 (55%) non - saudi physicians. more than half (55%) of the sample were females. only 3 (15%) physicians were specialists with diplomas or master 's degree. the master degree was in infectious diseases, the two diploma holders were in pediatrics and ophthalmology and the remaining 17 (85%) physicians had only mbbs. the mean duration of experience in years was 9.0 + 7.2 and mean duration of stay in primary health centers was 4.4 + 3.9 years. of the 20 physicians, 15% indicated that laboratory services were always important, 55% said that they were important most of the time, and 30% of them mentioned that laboratory services did not always contribute to the management of their patients. forty percent of the physicians indicated that the quality of laboratory services was good to excellent, 30% indicated that quality of laboratory service was satisfactory, whereas 30% indicated that it was deficient. in the opinion of 65% of the physicians, the laboratory services available in phc was inadequate, whereas 35% indicated that laboratory services were satisfactory. more than half (55"/ ") of the physicians indicated that their practice would be adversely affected if there were no laboratory facilities in the primary health care centers, and 45% indicated that their practice would be moderately to slightly affected. no one mentioned that his practice would remain unaffected if there were no laboratory facilities. more than half (55%) of the physicians indicated that they would order laboratory tests for a patient who asked for a periodic check - up, 40% indicated that they would only take a history and carry out a physical examination and only 5% physicians, would do both (take history and examine, and order laboratory tests). no one would refuse to do a periodic check - tip for a patient, and none would send a patient to a hospital for these procedures. eight out of 20 (40%) physicians practiced medicine outside saudi arabia. the remaining 12 (60%) physicians were either saudi (45%) or non - saudi (15%) but did not practice medicine outside saudi arabia. of the eight (40%) physicians who practiced medicine outside saudi arabia, six (75%) indicated that they carried out laboratory investigations more in saudi arabia. no one mentioned that he / she carried out more in his own country than saudi arabia. the other two (25%) physicians indicated that their laboratory use in saudi arabia was the same as in their own country. the reasons for the different rates of utilization, as indicated by the six (75%) physicians who used laboratory services more in saudi arabia are illustrated in table 2. the fact that laboratory services were free and easily available and that patients demanded them were the most important factors in utilization differences between saudi arabia and other countries. factors causing more utilization in ksa than original countries (6 physicians) when crucial investigations needed for a patient were not available in the phc centers, 85% of physicians would send the patient to king fahd university hospital for them, 10% would ask the patient to have them done in a private hospital and 5% would not do the test but would depend on his / her clinical sense to diagnose and manage the patient. other factors such as medico - legal reasons and the need to reassure patients were less important. most physicians (60%) believed that laboratory investigations were properly utilized, 25% believed they were over - utilized, and the remaining 15% believed that they were under - utilized (table 3). physicians who thought that laboratory tests were over utilized were asked to indicate why they held that view. the important factors mentioned were free laboratory services, physicians lack of knowledge, routine ministry of health requirements and patients demand, early detection and management, medico - legal factors and availability of laboratory services (table 4). opinion about utilization of lab investigations physicians opinion about factors causing over utilization (5 physicians) the kap questionnaire for every physician was evaluated and scored. there was a variety of ranges between the lowest and the highest scores in knowledge, attitude and practice. the range was much wider in attitude and practice, both of which had a lower mean score. the attitude of most of the physicians (80%) toward laboratory services was negative, 10% were neutral, and the remaining 10% had a good attitude. almost 60% of physicians who scored negative in attitude questions had worked for more than ten years. the overall kap grading was fair for 50% of the physicians, whereas 20% had good grade and 30% had a poor grade (table 5). grading of physicians kap scores the physicians characteristics, personal and professional, were studied to measure the influence of these factors on the knowledge, attitude and practice scores. it was found that the length of experience played a role in practice score : as the physician acquired more years of experience, his / her practice scoring regarding laboratory test utilization improved (p >.0296) with r = 0.237. this implies that around 24% of the variation in the physicians practice score, could be explained by the differences in the variables employed. none of these independent variables was significant in affecting the score in knowledge or attitude. the proportion of saudi physicians working in phc centers was 45% in contrast to the basuliman's24 study in dammam city (1992), where 9.5% of all physicians working in phc centers were saudis. this may be due to increased awareness of the importance of phc centers for the provision of health. another important reason could be the increase in the number of saudi graduates from medical colleges and the requirement by ministry of health for all new saudi graduates to work in phc centers for at least two years before joining the hospital service or applying for postgraduate study. the vast majority of physicians (25%) who practiced medicine outside saudi arabia indicated that laboratory service was utilized more in kingdom of saudi arabia. this was unlike the result obtained by willimas et a125 who surveyed a group of residents, hospital consultants and community internists in usa to determine physicians perception of the reasons for overuse of diagnostic tests. community physicians cited routine screening, habitual ordering of groups of tests, and were concerned about possible malpractice suit as being an important reason. nearly all (25%) physicians had practiced medicine in developing counties with a lower economic status than in saudi arabia. this was reflected by the finding that the availability of laboratory facilities was the most important factor for the higher utilization in saudi arabia as indicated by physicians themselves. the attitude of most of the physicians (80%) towards laboratory service was negative. the reason may be that, 60% of them had worked for more than ten years, and had been trained in an era of medical practice not as dependent upon technology as the present time, and they were still unaccustomed to use diagnostic technology. patients demand was cited by physicians in this study as one of the important factors promoting over utilization. unfortunately, it seems to be easier and often safer medico - legally to comply with such demands than not, even when they are totally unjustified. the wide variation among physicians in the use of ancillary services suggests that both over - utilization and under - utilization may occur. this variation in utilization was attributable to the physicians age, specialty, type of practice and experience. the physicians knowledge of cost and efficacy of the diagnostic tests is also likely to influence the use of tests.26 physicians practice scoring indicated that the physicians years of experience (practice since graduation) was the only important factor in determining their utilization of laboratory tests. several other investigators have studied the role of different physicians characteristics on the use of diagnostic services. salloum and franssen27 performed a covariant analysis to identify any trend in the use of lab tests by family physicians in canada. independent variables were physicians gender, years of practice, laboratory facilities in the building, number of patients seen per day, and certification. no significant factors were found to be responsible for variations in the use of laboratory tests. eisenberg and nicklin28 studied the effect of physicians personal characteristics on the use of laboratory tests. these characteristics were the physician 's age, specifically, years since graduation, graduation from private or public school, postgraduate degree, foreign or domestic graduate and urban or rural practice. they came out with the following results : (i) the use of laboratory tests was inversely related to the number of years since graduation from medical school. (iii) there was no statistically significant difference in the use of laboratory tests between foreign and domestic graduates. lave and leinhardt29 reported that older physicians order fewer tests for hospitalized patients than do younger physicians. eisenberg and nicklin28 quoted rose and abel - smith who found that the period of time since graduation was inversely related to a physicians use of lab services, but was not statistically significant. pineault30 studied 34 internists practicing in prepaid group practice and suggested that physicians order fewer diagnostic tests with an increasing number of years since residency. schroeder et a131 were unable to demonstrate a statistically significant relationship between age and lab use. freeborn showed a tendency for younger physicians to order more tests, but were also unable to obtain am statistically significant difference. epstein et a133 in boston correlated the use of outpatient diagnostic tests with personal characteristics of the prescribing physician, by studying the records of 351 hypertensive patients cared for by 30 physicians. they found that the pattern of test utilization was not strongly related to the number of years since graduation from medical school. ferrier et a14 showed that women physician tended to order more laboratory tests than men physicians, and had a higher mean monthly cost of laboratory service per patient. these differences were correlated with practice features related to physicians in their study such as the proportion of total work done in the office, the proportion of female patients aged i s to 49, and the proportion of the physicians practice that is psycho - therapeutic. year of graduation, medical school, practice location and board certification had no significant contribution towards explaining the difference in laboratory ordering. there had been an increase in the number of saudi physicians in phc centers.not a single physician had any post - graduate degree in family medicine or in general practice.laboratory investigations are essential services for phc center physicians.the quality of current laboratory services is deficient in the opinion of 30% physicians.the utilization of laboratory tests in saudi arabia is most probably higher than many other developing countries with less economic welfare, but this does not necessarily mean better utilization. not a single physician had any post - graduate degree in family medicine or in general practice. the utilization of laboratory tests in saudi arabia is most probably higher than many other developing countries with less economic welfare, but this does not necessarily mean better utilization. undergraduate curricula of medical colleges should include courses on health economics and cost effective decision making.test selection and interpretation of results should be adequately taught in the medical colleges.a regular audit should be established to make it evident to all in the primary health care centers and laboratory clinics.continuing medical education should be directed towards the indications, rational use and cost of investigations.it is essential to change the practice behavior of physicians.half-day group discussions of all primary health care centers physicians and a clinical chemists should be held on a regular basis.to improve the services provided by phc center physicians, and for better cooperation between physicians and laboratory specialists, a diagnostic center should be established to serve all phc centers.as a part of health education, efforts should be directed at raising public awareness of the cost and benefits of laboratory investigations.another study is required to measure the utilization vis - a - vis clinical outcome of care, and to examine the reliability of the results in phc center laboratories. undergraduate curricula of medical colleges should include courses on health economics and cost effective decision making. a regular audit should be established to make it evident to all in the primary health care centers and laboratory clinics. continuing medical education should be directed towards the indications, rational use and cost of investigations. half - day group discussions of all primary health care centers physicians and a clinical chemists should be held on a regular basis. to improve the services provided by phc center physicians, and for better cooperation between physicians and laboratory specialists, a diagnostic center should be established to serve all phc centers. as a part of health education, efforts should be directed at raising public awareness of the cost and benefits of laboratory investigations. another study is required to measure the utilization vis - a - vis clinical outcome of care, and to examine the reliability of the results in phc center laboratories. | objectives : the aim of the study was to assess the physicians knowledge, attitude and practice towards laboratory services in the primary health care (phc) centers at al - khobar area.methodology:five primary health care centers were selected out of 9 (56%) from the al - khobar area. twenty physicians (33%), in these primary health care centers were included. a questionnaire was given to all physicians to explore the knowledge, attitude and practice.results:all physicians considered laboratory investigations an essential service that contributed to the management of their patients most of the time. knowledge and practice of most (80% of them) regarding laboratory investigations was between fair to good but the attitude of 80% of them was poor since postgraduation experience was the only factor which influenced their practice.conclusions:there has been an increase in number of saudi physicians working in the primary health care centers. the quality of the current laboratory services was deficient according to 30% of physicians. they considered that investigations were essential for primary health care centers. and finally the utilization of laboratory tests in the kingdom of saudi arabia was higher than many developing countries. |
thymus - parathyroid unit (tpu) (parathymus) occurring in adults is rare, reported in the perithyroid / thyroid and cervical location when extrathoracic and extrathymic. rare cases of parathyroid adenomas, in the context of parathyroid hyperfunction or thymic cyst, have been reported as developing in such units. in the present study, we discuss the features observed in 2 cases where tpu was noted in the perithyroid site (diagnosed during thyroid surgery for basedow graves disease), as well as 2 cases wherein tpu was located in the neck. two patients (a woman and a man, aged 28 and 27 years, respectively) diagnosed with basedow graves disease presented with perithyroid tpu. the basedow graves disease was diagnosed at the age of 25 and 24 years, respectively ; both patients showed tachycardia, loss of body weight, and bilateral exophtalmia. for the first patient (woman), the serum anti - tsh antibodies were 32.1 (positive, > 1.5 ui / l), the antithyroperoxidase antibodies 123 ui / ml (normal, < 34), and thyroid function tests showed < 0.01 mu / l ; 9.93 and 59 pmol / l for tsh, t3 and t4, respectively (normal ranges, 0.274.20 mu / l, 3.106.80 and 1222 pmol / l, respectively). this patient also showed persistent hypoleucocytemia with neutropenia (without positivity for hiv, viral hepatitis virus or tuberculosis tests) as well as a history of hyperprolactinemia in relation with a hypophysal microadenoma diagnosed on magnetic - resonance - imaging at the age of 25 and treated by cabergoline. furthermore, ultrasound examination showed a normally sized thyroid with heterogeneous echogenicity and with several hypoechogenic nodules (2-mm). for the second case, the serum levels of anti - tsh antibodies was 4.7 both patients were treated by neomercazole (and propranolol for the female patient), but the anti - tsh antibodies remained high, although fluctuating. macroscopic and microscopic features were consistent with basedow graves disease ; the thyroid parenchyma showed dystrophic vesicles, multifocal inflammation, fibrosis, and rare calcifications. the parathyroid and the thymus were focally separated from the thyroid capsule by sparse adipocytes (figure 1). the parathyroid tissue appeared normal and showed some adipocytes and chief cells (some with water - clear cytoplasm). the parathyroid, which was surrounded almost entirely by a capsule was focally in contact with the thymic tissue (figure 1). in addition, the thymic tissue, which was also almost completely surrounded by a capsule appeared normal (with both cortical and medulla). several hassal corpuscles were noted, some of which were calcified. in one of the cases, one other parathyroid, (normal), was detected in the perithyroid adipose tissue at distance from the tpu. postoperatively, both patients showed hypocalcemia (1.72 and 1.64 mmol / l, respectively) thereby requiring medication. in the 2 cases of perithyroid tpu (cases 1 and 2) (a, b, c, and d, e, f, respectively), the thymic tissue was situated in the perithyroid tissue, focally separated either by parathyroid tissue or by fibroadipose tissue (a and d, respectively). thymic and parathyroid tissues were focally separated by an inconspicuous fibrous layer or a calcified hassal corpuscle (b) or adipose tissue (d and e). the thymic tissue contains several hassal corpuscles, some of them calcified (a, b, c, and f). the white arrows indicate hassal corpuscles (calcified), the black arrows uncalcified hassal corpuscles. hematoxylin - and - eosin stain, original magnification2.5 (a and d) and 20 (b, c, e, and f). the tpu was observed on the central neck dissection specimen, which was performed in the third case during thyroidectomy for thyroid papillary carcinoma (woman, 42-years) and in the fourth, for thyroid medullary carcinoma extension. for the third case, the ultrasound - examination showed a homogeneous right thyroid lobe and 2 adjacent hypoechogenic nodules (3 and 1.6-cm) with microcysts, microcalcifications, and hypervascularisation. further, in the fourth case, the follow - up positron - emission - computed - tomography showed 3 hypermetabolic foci of paratracheal and right hilar, subcarina location. the parathyroid tissue, which was situated between the thymic lobules (figure 2), showed an incomplete capsule and comprised chief cells and focally oncocytic cells. in addition, an epithelial microcyst was found in the fourth case and another parathyroid. in the other case, parathyroid cells, some of which had clarified cytoplasm, were focally found in direct contact with or intermingled within the thymus (figure 2). in the 2 cases of cervical tpu (cases 3 and 4) (a, b, c and d, e, f, respectively), the parathyroid tissue was situated between thymic lobules (a f), separated either by a thin fibrous capsule, or at contact or intermingled with thymic tissue (b, c, e, and f). the white arrows indicate hassal corpuscles (uncalcified), hematoxylin - and - eosin stain, original magnification2.5 (a), 5 (d), 20 (b, c, and e), and 40 (f). here we report the features observed for 2 cases of tpu at a perithyroid location, both of which were incidentally diagnosed upon the inspection of thyroidectomy specimens obtained for basedow graves disease. features were similar to those previously observed in the cases reported in 1928 and in 2001, occurring in the context of basedow disease and lymphocytic thyroiditis with adenoma. however, in one of the cases presented here, both parathyroid and thymic tissues were observed in direct proximity to the thyroid capsule. the 2 cases showing perithyroid thymus were both young adults and had been diagnosed with basedow graves thyroiditis. in the report of a large series of patients with basedow graves disease, ectopic thymus was observed in 1.8% of patients but at a cervical location. one hypothesis suggests that the incomplete migration of the thymus during embryogenesis results in its ectopic presence within the perithyroid tissue, along with the parathyroid. furthermore, the fact that the tpu may occur in adults in the neck at a distance from the thyroid (as observed in 2 cases), in addition to the presence of intermingled parathyroid cells in the thymic lobule, also favors the hypothesis of developmental abnormalities of the third and/or fourth pharyngeal pouches. a direct fusion of thymus and parathyroid has been reported in 65-mm - fetus and older fetus, being known as the parathymus. water - clear cell buds, similar to the clear cells we observed in the cervical thymus of patient 3, were already mentioned in 1937 as rudiments present in the embryofetal life. moreover, the coexistence of thymic and parathyroid tissues has been reported in the subangulomaxillary region, incidentally, or in the context of hyperparathyroidism in esophageal and jaw sites, in association or not with the adenomatous parathyroid. however, one may question whether metabolic factors related to iodine intake could also be involved in the development of abnormal perithyroid thymus because increased iodine has been reported to result in perithyroid thymic tissue occurrence as demonstrated in rat models wherein the perithyroid / thyroid lymphoid tissue with thymus - like cd4/cd8/tdt immunophenotype and without hassal corpuscles appeared more frequently after consuming enriched - iodine than after normal - iodine or low - iodine diets. the presence of a normal ioduria in one patient, with basedow graves disease, in which this analysis was performed, does not favor the hypothesis of iodine - induced thymic tissue. however, the finding of high levels of anti - tsh antibodies in both patients could be representative of an increased tolerance toward thyroid autoantigens, including during neomercazole treatment. moreover, the presence of persistent neuropenia may suggest a specific immunological profile with regard to the evolution of the disease and response type to treatments. the clinical significance of tpu diagnosed on thyroidectomy specimens performed for a benign inflammatory, autoimmune disease is limited except the postsurgical hypocalcemia, which may occur similarly to patients having the cervical unit and which may require treatment. however, the differential diagnosis with perithyroid lymph node metastases of thyroid tumors, such as papillary or medullary carcinomas may rise as thymic hassal corpuscles may also show calcifications on imaging procedures. although rare, the presence of parathyroid cells intermingled with thymic tissue (as we have observed in one of the cervical units) warrants further interest because of eventual abnormalities related to an parathyroid hormone secretion or, to a lesser extent, immunological disturbances, since in adults. | abstractthe thymus - parathyroid unit (tpu) occurring in adults is rare.the main symptoms and important clinical findings are as follows : 2 patients presented with neomercazole - resistant basedow graves disease. a third patient presented with thyroid nodules and a fourth patient with a neck mass after thyroid resection for medullary thyroid carcinoma.the main diagnoses were those of thyroid nodules (either in the context of goiter, or not). in the fourth case the diagnosis was of thyroid medullary carcinoma recurrence in the neck.thyroidectomy was performed in the 2 cases of basedow graves disease and in the third case (wherein selective neck dissection was also performed). in the fourth case, a neck dissection was performed for a possible medullary carcinoma recurrence.a tpu was microscopically detected in 2 cases with perithyroid location, on thyroidectomies for basedow graves disease and in the 2 other cases with neck soft tissue location (associated with thyroid papillary carcinoma and thyroid medullary carcinoma extension). postsurgical hypocalcemia requiring treatment occurred in both patients with basedow graves disease and in the fourth patient.the presence of tpu should be acknowledged because such lesions can be misdiagnosed as suspect lymph nodes during thyroid surgery for malignant tumors. |
cellular systems maintain homeostatic equilibrium through a constant balance between biosynthetic (anabolic) processes and catabolism. macroautophagy, herein referred to as autophagy, is an evolutionarily conserved, catabolic metabolic program that is a key pathway for cellular adaptation to metabolic stresses such as nutrient withdrawal (amino acids and glucose) or hypoxia. during autophagy, internal cellular components, including bulk cytoplasm and organelles, are sequestered into double - membrane structures known as autophagic vesicles (avs). following fusion of avs to lysosomes, the internal contents are degraded, and the degradation products are used to fuel catabolic metabolic processes for energy generation. recycle existing contents for fuel to promote cell viability, while basal levels of autophagy play a critical role in protein and organelle quality control. autophagy is induced through a stepwise process culminating in the assembly of the autophagosome by core autophagy machinery. a distinct family of autophagy - related genes that mediate the assembly and processing of the autophagosome have been identified. at the molecular level, the induction of autophagy is linked to signal transduction pathways involved in nutrient sensing (figure 1). signalling by the phosphatidylinositol 3-kinase (pi3k)/akt / mammalian target of rapamycin (mtor) pathway downstream of growth receptors engages cellular programs of growth and proliferation and inhibits catabolic metabolic pathways, including autophagy. inhibition of mtor, which integrates growth factor signals and amino acid availability to regulate cap - dependent protein translation, is associated with the induction of autophagy. low cellular energy levels can stimulate autophagy by inhibiting mtor, a process regulated in part by an lkb1/ampk (amp - activated protein kinase)-mediated energy checkpoint [6 - 8 ]. (a) under normal growth conditions, signal transduction downstream of growth factor receptors activates mtorc1 via the pi3k / akt pathway. activation of mtorc1 promotes cell growth through the regulation of cap - dependent protein translation and the simultaneous inhibition of autophagy. glutamine (gln) is transported into cells via the slc1a5 glutamine transporter and is subsequently used to import leucine (leu) via the slc7a5-slc3a2 complex. basal autophagy is maintained under these conditions by the activity of beclin-1, which is inhibited by anti - apoptotic bcl-2 and bcl - xl. bcl-2 and bcl - xl also function to maintain viability by antagonizing bax / bak - dependent mitochondrial apoptosis. (b) under conditions of metabolic stress, including nutrient depletion (glucose and amino acid), growth factor withdrawal, or energy deficit, the mtor pathway is inhibited, resulting in autophagy induction. under conditions of glutamine depletion, activation of the lkb1-ampk pathway by energetic imbalance results in mtorc1 inhibition through activation of the tsc1-tsc2 complex and inhibition of the mtor - binding partner raptor. extended periods of nutrient withdrawal can induce mitochondrial - dependent apoptosis through activation of caspases downstream of bax / bak - dependent cytochrome c (cyt c) release. antagonism of bcl-2 family members by bh3-only proteins may trigger either autophagy or apoptosis, depending on the context. ampk, amp - activated protein kinase ; mito, mitochondrion ; mtor, mammalian target of rapamycin ; mtorc1, mtor complex 1 ; pi3k, phosphatidylinositol 3-kinase ; tsc, tuberous sclerosis protein. several recent breakthroughs have advanced our understanding of the molecular mechanisms governing the regulation of autophagy. mtor activity has long been known to be responsive to nutrient levels ; amino acid depletion is a potent stimulator of autophagy. however, the mechanisms linking amino acid levels to mtor activation and autophagy inhibition have remained one of the key outstanding questions in the field. three independent studies by the groups of guan, sabatini, and murphy have provided new mechanistic insight into how nutrients direct autophagy. the first insight was the identification of the rag family of small gtpases as key stimulators of mtor complex 1 (mtorc1) activity in response to amino acids. amino acids stimulate the association of rag gtpases with the mtor - binding partner raptor, resulting in mtor activation (figure 1). constitutively active rag mutants mimic a nutrient replete state, conferring resistance to starvation - induced autophagy triggered by amino acid withdrawal. provided evidence for a coupled glutamine - leucine amino acid shuttle system involved in mtor regulation and autophagy induction. they demonstrated that glutamine import by the glutamine transporter slc1a5 is coupled to the import of leucine via the slc7a5-slc3a2 antiporter ; intracellular leucine is then sensed by intracellular mediators (possibly rag gtpases) to stimulate mtor activity (figure 1). knockdown of expression of either slc1a5 or slc7a5-slc3a2 results in the induction of autophagy and a reduction in cell size. thus, amino acid transporters upstream of mtor play a key role in autophagy regulation by dictating amino acid availability. autophagy has been ascribed both cytoprotective and pro - apoptotic functions, and as such the role of autophagy in cell death has remained controversial. autophagic cell death is loosely defined by the presence of autophagosomes in dying cells. the use of this classification is poor as autophagy, like apoptosis, is a cellular morphology, and many, if not most, cells increase their rate of autophagy under conditions of stress that promote cell death. autophagic based on the presence of avs may not be accurate. confounding this classification, autophagy can induce cell death directly through both conventional apoptotic machinery and caspase - independent processes, depending on the context. moreover, when metabolic stress is induced in cells lacking the function of conventional apoptotic pathways, autophagy ultimately results in energetic crisis, leading to necrosis. thus, under pathophysiological conditions of nutrient or oxygen limitation (that is, a growing tumour lacking vasculature), autophagy may promote necrosis instead of apoptosis. it remains unclear whether autophagic cell death functions as a central mediator of programmed cell death or is simply a mechanism of recent findings in lower organisms have suggested a physiological role for autophagy in cell death control during normal development. autophagy is specifically induced in drosophila melanogaster at two developmental stages - germarium and mid - oogenesis - and induction of autophagy at these stages promotes starvation - induced cell death. interestingly, these developmental stages are highly influenced by nutritional status, which may suggest that autophagy as executioner is primarily linked to cellular bioenergetics rather than other apoptotic pathways such as those triggered by dna damage. to date, the demonstration of a required role for autophagy in cell death control during development in vivo has been limited to experimental systems in the fruit fly. whether autophagy plays a similar role in mammals remains to be determined. while its role in cell death control remains unclear, deregulation of autophagy has been implicated in various pathological conditions, including neurodegeneration and tumourigenesis (discussed in the following section). the involvement of autophagy in cancer development and progression has been an important recent advance in the field of cancer biology. the upregulation of autophagy has been correlated with differing stages of cancer progression. in particular, autophagy is believed to be upregulated in cancerous lesions marked by environments of decreased oxygen or nutrient stress or both. multiple lines of evidence suggest that oncogene and tumour suppressor networks exert opposing effects on autophagy. when activated, several oncogenes, including pi3k / akt, mtor, and bcl-2, function largely as inhibitors of autophagy, while tumour suppressors [that is, pten (phosphatase and tensin homologue), beclin-1, tuberous sclerosis protein 2 (tsc2), lkb1, and p53 ] stimulate autophagy. this dichotomy has remained controversial though, as autophagy can promote cell survival in response to cellular stress and thus autophagy could potentially contribute to oncogenesis. however, the involvement of autophagy in tumour suppression may actually stem from its role in the degradation of damaged proteins and organelles, including mitochondria, rather than its role in stress responses. haploinsufficiency of beclin-1 promotes tumourigenesis in mouse models and is associated with breast and ovarian tumours in humans. this may be due in part to a still poorly defined role for beclin-1 in the maintenance of chromosome integrity. modifiers of beclin-1 activity can alter tumourigenic potential ; positive regulators of beclin-1, including uvrag (uv radiation - associated gene) and bif, display tumour suppressor properties, while beclin-1 function is inhibited by bcl-2, a known oncogene. together, these data suggest that regulation of basal levels of autophagy through beclin-1 is an important gateway to tumourigenesis. another regulator of autophagy is the tumour suppressor p53, although its role in autophagy induction remains controversial. the ability of p53 to induce autophagy appears to depend on its cellular localization ; nuclear localized p53 triggers stress - induced autophagy through transcriptional control of autophagy mediators, including dram (damage - regulated autophagy modulator), while cytoplasmic p53 appears to function as a negative regulator. growing tumours require p53-dependent autophagy to survive metabolic stress in vivo and may represent an important avenue for therapeutic intervention, particularly in p53-deficient tumours. several recent breakthroughs have advanced our understanding of the molecular mechanisms governing the regulation of autophagy. mtor activity has long been known to be responsive to nutrient levels ; amino acid depletion is a potent stimulator of autophagy. however, the mechanisms linking amino acid levels to mtor activation and autophagy inhibition have remained one of the key outstanding questions in the field. three independent studies by the groups of guan, sabatini, and murphy have provided new mechanistic insight into how nutrients direct autophagy. the first insight was the identification of the rag family of small gtpases as key stimulators of mtor complex 1 (mtorc1) activity in response to amino acids. amino acids stimulate the association of rag gtpases with the mtor - binding partner raptor, resulting in mtor activation (figure 1). constitutively active rag mutants mimic a nutrient replete state, conferring resistance to starvation - induced autophagy triggered by amino acid withdrawal. provided evidence for a coupled glutamine - leucine amino acid shuttle system involved in mtor regulation and autophagy induction. they demonstrated that glutamine import by the glutamine transporter slc1a5 is coupled to the import of leucine via the slc7a5-slc3a2 antiporter ; intracellular leucine is then sensed by intracellular mediators (possibly rag gtpases) to stimulate mtor activity (figure 1). knockdown of expression of either slc1a5 or slc7a5-slc3a2 results in the induction of autophagy and a reduction in cell size. thus, amino acid transporters upstream of mtor play a key role in autophagy regulation by dictating amino acid availability. autophagy has been ascribed both cytoprotective and pro - apoptotic functions, and as such the role of autophagy in cell death has remained controversial. autophagic cell death is loosely defined by the presence of autophagosomes in dying cells. the use of this classification is poor as autophagy, like apoptosis, is a cellular morphology, and many, if not most, cells increase their rate of autophagy under conditions of stress that promote cell death. autophagic based on the presence of avs may not be accurate. confounding this classification, autophagy can induce cell death directly through both conventional apoptotic machinery and caspase - independent processes, depending on the context. moreover, when metabolic stress is induced in cells lacking the function of conventional apoptotic pathways, autophagy ultimately results in energetic crisis, leading to necrosis. thus, under pathophysiological conditions of nutrient or oxygen limitation (that is, a growing tumour lacking vasculature), autophagy may promote necrosis instead of apoptosis. it remains unclear whether autophagic cell death functions as a central mediator of programmed cell death or is simply a mechanism of recent findings in lower organisms have suggested a physiological role for autophagy in cell death control during normal development. autophagy is specifically induced in drosophila melanogaster at two developmental stages - germarium and mid - oogenesis - and induction of autophagy at these stages promotes starvation - induced cell death. interestingly, these developmental stages are highly influenced by nutritional status, which may suggest that autophagy as executioner is primarily linked to cellular bioenergetics rather than other apoptotic pathways such as those triggered by dna damage. to date, the demonstration of a required role for autophagy in cell death control during development in vivo has been limited to experimental systems in the fruit fly. while its role in cell death control remains unclear, autophagy has been implicated in coordinating the clearance of dying cells and cellular debris. deregulation of autophagy has been implicated in various pathological conditions, including neurodegeneration and tumourigenesis (discussed in the following section). the involvement of autophagy in cancer development and progression has been an important recent advance in the field of cancer biology. the upregulation of autophagy has been correlated with differing stages of cancer progression. in particular, autophagy is believed to be upregulated in cancerous lesions marked by environments of decreased oxygen or nutrient stress or both. multiple lines of evidence suggest that oncogene and tumour suppressor networks exert opposing effects on autophagy. when activated, several oncogenes, including pi3k / akt, mtor, and bcl-2, function largely as inhibitors of autophagy, while tumour suppressors [that is, pten (phosphatase and tensin homologue), beclin-1, tuberous sclerosis protein 2 (tsc2), lkb1, and p53 ] stimulate autophagy. this dichotomy has remained controversial though, as autophagy can promote cell survival in response to cellular stress and thus autophagy could potentially contribute to oncogenesis. however, the involvement of autophagy in tumour suppression may actually stem from its role in the degradation of damaged proteins and organelles, including mitochondria, rather than its role in stress responses. haploinsufficiency of beclin-1 promotes tumourigenesis in mouse models and is associated with breast and ovarian tumours in humans. this may be due in part to a still poorly defined role for beclin-1 in the maintenance of chromosome integrity. modifiers of beclin-1 activity can alter tumourigenic potential ; positive regulators of beclin-1, including uvrag (uv radiation - associated gene) and bif, display tumour suppressor properties, while beclin-1 function is inhibited by bcl-2, a known oncogene. together, these data suggest that regulation of basal levels of autophagy through beclin-1 is an important gateway to tumourigenesis. another regulator of autophagy is the tumour suppressor p53, although its role in autophagy induction remains controversial. the ability of p53 to induce autophagy appears to depend on its cellular localization ; nuclear localized p53 triggers stress - induced autophagy through transcriptional control of autophagy mediators, including dram (damage - regulated autophagy modulator), while cytoplasmic p53 appears to function as a negative regulator. growing tumours require p53-dependent autophagy to survive metabolic stress in vivo and may represent an important avenue for therapeutic intervention, particularly in p53-deficient tumours. much insight has been gained into the mechanisms and biology of autophagy, but many questions remain. although we have focussed solely on macroautophagy in this report, autophagy exists in several distinct forms (that is, microautophagy and chaperone - mediated autophagy) and can target specific cellular organelles (that is, pexophagy and mitophagy). in addition, despite recent advances, our knowledge of the signalling networks and layers of regulation that govern autophagy is limited. for example, what are the mechanisms by which amino acids signal to rag gtpases (or other mediators) to limit autophagy ? how do other non - metabolic stressors such as dna - damaging agents signal to the autophagy machinery ? finally, recent studies have implicated autophagy as an integral biological process involved in a number of pathophysiological conditions, including cancer, neurodegeneration, aging, and infectious disease. the challenge will be to identify the exact role - positive or negative - that autophagy plays in these conditions, to determine the underlying mechanisms that regulate autophagy in each case, and to translate this knowledge into autophagy - based therapeutics to treat disease. | autophagy is a universally conserved metabolic program of catabolism that plays important roles in energy homeostasis and impacts both normal physiology and multiple disease processes, including cancer. autophagy has been documented as a pro - survival mechanism used to maintain viability under starvation conditions ; however, conflicting findings have also implicated autophagy in the control of cell death. adding to the controversy, central mediators of autophagy have been implicated in both pro - survival and pro - death processes. this report highlights recent insights into our understanding of how autophagy is regulated and newly discovered physiological roles for autophagy in normal biology and disease. |
internal root resorption is the progressive destruction of intraradicular dentin and dentinal tubules along the middle and apical thirds of the canal walls as a result of clastic activities. although the exact cause is unknown, chronic pulpal inflammation from bacterial invasion, trauma, and orthodontic treatment have been suggested as a cause in most cases. pain or discomfort may be the chief complaint if the granulation tissue has been exposed to oral fluids. radiographic examination usually reveals a fairly uniform radiolucent area with disrupted outline of the root canal. diagnostic accuracy based on conventional and digital radiographic examination is limited by the fact that the images produced by these techniques only provide a two - dimensional (2d) representation of three - dimensional (3d) objects. this might lead to misdiagnosis and incorrect treatment in the management of internal root resorption. the advent of cone beam computed tomography (cbct) has enhanced radiographic diagnosis providing greater 3d appreciation of the tooth, the resorption lesion, and the adjacent anatomy. the true nature of the lesion might be assessed, including root perforations and whether the lesion is amendable to surgical or non - surgical treatment. andearson has stated that an untreated internal resorption leads to premature loss of the tooth. therefore, nonsurgical root canal therapy is the treatment of choice to arrest the destructive process in the absence of concomitant external resorption. however, the irregular confines of the resorptive cavity pose technical difficulties for thorough debridement and obturation of the pulp space. and if the internal resorption has extended to the point that it reaches the external root surface, root integrity is lost and there may be destruction of the adjacent periodontal tissues which also has to be treated. mineral trioxide aggregate (mta) has several clinical applications due to its superior sealing properties, bacteriostatic effects, biocompatibility, and ability to set in presence of blood. mta has suggested indications i.e., root - end filling, pulp capping, apical filling of teeth with open apices, apexification therapy, and repair of root perforations. mta is a suitable material for the treatment of root perforations with the goal of regenerating a periodontal attachment and inducing osteogenesis and cementogenesis. this paper reports a case of mta repair of mandibular right premolar with an inflammatory, perforating resorptive defect in the coronal third of the root canal with use of cbct to enhance radiographic diagnosis of internal resorption. a 54-year - old woman reported to the dental office with pain and swelling in the right mandibular posterior region. the tooth was slightly tender to percussion and served as an abutment to a long span bridge extending upto 48. intraoral periapical radiographic examination revealed a well - circumscribed, fairly round radiolucency in the cervical third of the root with diffuse periapical rarefaction [figure 1 ]. the bridge was cut and removed to preserve the integrity of tooth with resorption and endodontic treatment was scheduled. it was decided not to include 44 as the patient was reluctant to sacrifice a healthy tooth and was ready to wear a partial denture in case of future loss of tooth. implant was not considered due to financial constraint of the patient. as the conventional intraoral radiographs do not indicate the true size and spread of the lesion, the patient was sent for cbct to evaluate the true size and nature of the lesion. informed consent was obtained from the patient and a cbct imaging of the mandible was performed by using the cbct scanner (cs 9300 3d digital imaging system, hatfield, pa). the involved tooth was focused, and the morphology was obtained in axial sections of 90 m thickness with 300 m spacing. the cbct images revealed well - circumscribed radiolucent area with relation to 45 below the level of cemento - enamel junction, involving the distal wall of the root and extend apically upto the middle of the root length suggestive of perforating internal resorption [figures 2 ]. preoperative periapical radiograph showing well defined internal resorption with prosthesis preoperative 3d images showing axial plane based on above finding, root canal therapy was initiated. the tooth was isolated under rubber dam (hygienic dental dam, coltene whaledent, germany) and accessed without anesthesia. presence of hemorrhagic fluid in the canal suggested of a small communication between the resorptive defect and the lateral periodontium. working length determination was established using apex locator (root zx ii ; morita, tokyo, japan) and radiography [figure 3 ]. the canal was prepared using crown down technique with protaper files (dentsply, tulsa dental specialities, ballaigues, swizterland) accompanied by copious irrigation with 2.5% sodium hypochlorite (dentpro, chandigarh, india). a calcium hydroxide paste dressing (metapex ; meta biomed ltd, choenju city, chungbuk, korea) was placed to alkalinize the environment and control bleeding at the perforation. master cone radiograph was taken [figure 4 ] and the canal below the resorptive defect was obturated with gutta percha (dentsply, maillefer) and ah plus sealer (dentsply, maillefer, konstanz, germany) using a combination of cold lateral condensation and vertical compaction with heated pluggers and condensers. the resorptive defect and the remaining canal were then condensed with mta (proroot mta, maillefer, dentsply, swizterland), using mta carrier and root canal pluggers [figure 5 ] (dentsply, maillefer). a wet cotton pellet was placed on this and the access was temporarily sealed with md temp (meta biomed ltd, choenju city, chungbuk, korea). an immediate postoperative radiograph was taken to confirm a satisfactory filling of the resorptive defect [figure 6 ]. fourty - eight hrs later after checking the set of mta the access cavity was finally sealed with light cure composite resin (tetric ceram, ivoclare, and vivadent). finally, porcelain fused to metal fixed partial denture was placed after 4 weeks. intraoral periapical radiograph working length intraoral periapical radiograph master cone intraoral periapical radiograph immediately after mta filling in the resorptive defect follow up intraoral periapical radiograph after 1 month the patient was recalled after 3 months and 6 months and the tooth was found to be symptom - free with healthy gingiva. the periapical radiographs also showed satisfactory healing of the apical rarefaction and also formation of a calcific barrier between the perforation and lateral periodontium [figure 7 ]. this was further confirmed with 6 month follow up cbct scan [figure 8 ]. follow up intraoral periapical radiograph after 6 months follow - up 3d images showing axial plane internal resorption is undoubtedly an endodontic challenge, especially, if the resorptive area is extensive and perforating. when diagnosed, immediate removal of the causative agent must be considered, aiming to arrest the cellular activity responsible for the resorptive activity. however, the complex irregularities of the root canal system and the inaccessibility of internal resorption defect provide technical difficulties for thorough cleaning and obturation of the root canal. the persistence of organic debris and bacteria in these areas may jeopardize the long - term success of the endodontic treatment. therefore, a detailed exploration of the interior of the root canal is necessary for a successful treatment outcome. although periapical and panoramic radiography produce acceptable details in the mesio - distal direction, the observation of details in the bucco - lingual direction is inadequate. the severity of internal root resorption can not be accurately judged from 2d radiographs alone. recently, cbct systems (high - resolution 3d images) have attracted considerable attention as a new diagnostic imaging technique in endodontics. the advantage is that cbct offers a noninvasive reproducible technique for 3d assessment of root canal systems and aids the clinician to visualize the internal anatomy precisely. several case reports and studies have confirmed the usefulness of cbct in diagnosing and managing resorptive lesions. computed tomography is a technique of image acquisition that combines the use of x - rays and computer technology, directing the x - ray beams from different angles to achieve axial section images of the object analysed. the cs 9300 3d digital imaging system used in this case gave series of cross sectional views to clearly observe the size and location of the defect. with the axial view the perforation site was detected at the distal wall of the root canal. a combined approach involving both hand instrumentation and antibacterial irrigation was performed on the involved tooth. sodium hypochlorite is the most commonly use irrigant during root canal treatment due to its tissue dissolving and broad antimicrobial properties. ultrasonic activation of irrigants should be viewed as an essential step in the disinfection of the internal resorption defect. however, even with the use of ultrasonic instruments, bacteria might still remain in confined areas. chemo mechanical debridement of the root canal space fails to consistently render the root canal system bacteria - free. thus, an intracanal, antibacterial medicament should be used to improve disinfection of the inaccessible root resorption defects. calcium hydroxide is antibacterial and has been shown to effectively eradicate bacteria that persist after chemo mechanical instrumentation. calcium hydroxide has also been shown to have a synergistic effect when used in conjunction with sodium hypochlorite to remove organic debris from the root canal. root canal therapy combined with surgical correction may be the only option in some cases. remineralization therapy with calcium hydroxide to form a hard tissue matrix, against which the root - filling material is condensed, has been advocated by others. application of mta at the perforation site precluded, in this case, the need for surgical intervention or prolonged treatment with calcium hydroxide. more importantly, the biologic response to this material was excellent, and complete resolution of the alveolar bone lesion had occurred by the time of a follow - up visit 3 months after the procedure. indeed, it has been shown that mta stimulates the propagation of human osteoblasts by offering a biologically active substrate for the cells. furthermore, the longevity of an endodontic treatment is significantly increased by selecting an appropriate restoration that considers saving the tooth structure and preventing reinfection. porcelain fused to metal bridge was then given extending from 45 to 48 to restore function and esthetics. this case report emphasizes on how conventional intraoral radiography provides clinicians with an accessible, cost - effective, high - resolution imaging modality that continues to be of value in endodontic therapy. there are, however, specific situations, both pre- and postoperatively, where the understanding of spatial relationships afforded by cbct facilitates diagnosis and influences treatment. the usefulness of cbct imaging can no longer be disputed - cbct is a useful task specific imaging modality and an important technology in comprehensive endodontic evaluation. | this case report demonstrates the benefits of utilizing cone beam computed tomography (cbct) in the assessment and mineral trioxide aggregate (mta) in the management of perforating internal resorption in a 54-year - old woman. the advent of cbct has enhanced the clinician 's ability to make a confirmatory diagnosis and determining the treatment plan before undertaking the actual treatment. thorough cleaning and shaping of the root canal space and the resorptive defect was achieved by mechanical instrumentation, irrigation, and interim calcium hydroxide dressing. following this obturation of the canal below, the resorptive defect was done with gutta percha using lateral and warm vertical condensation. the resorptive defect was filled with mineral trioxide aggregate. follow - up intraoral periapical radiographs and cbct scans at 6 months showed adequate repair of the resorption and periapical rarefaction and the tooth remained asymptomatic. |
studies have shown that the majority of children are overweight due to the intake of low - density foods instead of fruits and vegetables and also due to high - fat and high - carbohydrate foods and a sedentary life style ; in addition, some socioeconomic factors are involved. obesity consists of an exceedingly complex group of diseases and probably should be characterized as a syndrome. the etiologies that contribute to obesity and can be modified by physicians include dietary and exercise patterns, endocrine and metabolic diseases, genetic factors, environmental factors, familial and ethnic factors, stress, drugs, and abnormal regulation of body weight or body fat [14 ]. during the past 15 years, there has been an ever - increasing trend in weight gain in iranian children. it appears that common risk factors such as intake of foods rich in carbohydrates influence the incidence of tooth decay and obesity [35 ]. it is clear that some dietary habits or malnutrition affects caries rate due to the irrefutable effects on the secretion and composition of saliva [57 ]. presence of confounding variables such as diet or the individual 's socioeconomic status or background variables, including age, personal hygiene, and the amount of fluoride in the diet, has resulted in an inability to express a firm opinion in the regard. several studies to date have evaluated the relationship between tooth decay and obesity ; however, the results have always been different and sometimes contradictory [814 ]. to our knowledge no studies have so far been carried out in this respect in iranian preschool children and also since there are contradictions in the results of studies carried out elsewhere in the world, the present study was undertaken to evaluate the relationship between the deciduous tooth decay and bmi in a population of 36-year - old iranian children. the present cross - sectional descriptive / analytical study was carried out by clinical examination and the questionnaires were completed by an examiner. the subjects consisted of 36-year - old children in 23 kindergartens and preschool centers in kerman, south east of iran, in 2009 - 2010. data were collected through clinical oral examinations, anthropometric measures, and structured questionnaires in order to assess socioeconomic conditions (level of education and job). a total of 1482 children were examined and data associated with different socioeconomic parameters were determined in 763 of the subjects. the sample size was estimated according to prior studies (calculated as p = 0.05, confidence coefficient 99%). the inclusion criteria were 3 years of age and full eruption of deciduous teeth (20 teeth). children with conditions affecting body weight or tooth decay, including diabetes, hyperthyroidism, hypothyroidism, malignancies, and renal diseases, and those who had permanent teeth other than the first molars in the oral cavity were excluded from the study. the list of the centers eligible to be included in the study was provided by the department of general education and the organization of welfare and public health in kerman. cluster sampling method was used to select kindergartens and preschool centers from the list available. before any clinical examinations the examination was carried out one hour after breakfast, which included a piece of cheese, a slice of bread, and a cup of milk. children 's heights were determined in meter using a laika (taiwan) wall measuring tape. a microlife digital weighting machine (japan) was used to determine children 's weight while they were wearing light cloths without any shoes. body max index (bmi) was calculated using this formula : (1) bmi = mass(kg)[heightm]2. a special form was completed for each child, which included personal data, age, gender, height, weight, and phone number. in subjects in whom the socioeconomic status was evaluated, the educational status of parents, parents ' occupations, and the number of family members were also recorded in the form. bmi was classified as follows [5, 7, 15]:bmi percentile of 95 : obese. bmi percentile of 95 : obese. in the next stage, the teeth were examined for caries, restorations, and extractions based on dmft using a disposable dental mirror under 100 w light based on criteria proposed by the who. a postgraduate student of pediatric dentistry, who had been trained for one week in the department of pediatric dentistry in the faculty by a trained assistant before examination, carried out all the procedural steps and calculations. based on the study protocol, the who definition was used to diagnose caries and in determining decayed teeth (dt) the following clinical forms were considered carious lesion : white spots, a discolored area or an area with a rough surface ; teeth with temporary dressings ; and teeth with any form of restorative material with signs of recurrent carries [15, 16 ]. in addition, the mean sd for dmft index and its components according to our variables were reported. poisson 's regression model was used to evaluate the effect of bmi and various socioeconomic variables on dmft. the final model contained only factors that were significant at the level p 0.05. the rate ratios (rr) and 95% confidence intervals were reported in relation to poisson 's model corresponding to dmft index. this study was a cross - sectional study. of 1482 children evaluated, 257 (17.4%) were in the 3 - 4 age range, 451 (30.4%) were in the 4 - 5 age range, and 774 (52.2%) were in the 5 - 6 age range (table 1). table 2 presents dmft and bmi values along with the three d, m, and f components of dmft separately based on the age groups. poisson 's regression model showed that after adjustment for age, bmi had a significant effect on children 's dmft (p = 0.0001), with an inverse relationship between these two variables. on the whole, the mean of dmft for children with normal bmi was 1.5-fold higher than that in children who were overweight. in addition, age had a significant effect on dmft (p = 0.0001). in this context, with a one - year increase in age, the mean of dmft was 1.3-fold higher (table 2). various socioeconomic variables were evaluated in only 763 subjects because the parents had not provided answers to some questions. in the classification of bmi of the subjects in three groups, there were no subjects in the group with weight under normal ; 83.8% subjects were in the group with normal weight and 12.1% subjects were in the overweight group (bmi percentile more than 85) and 4.1% had bmi percentile more than 95. poisson 's regression model did not reveal any significant effect of birth rank (p = 0.7), mothers ' educational status (p = 0.26), and fathers ' occupation (p = 0.5) on children 's dmft. however, some other variables, including fathers ' educational status and mothers ' occupation, had a significant effect on children 's dmft (table 3). in addition, the effect of gender on dmft was significant (p = 0.0001). in this context, the mean of dmft in boys was 1.19-fold that in girls (table 3). the prevalence of caries - free children in the present study was 38.3% and there were more caries - free children in the overweight children (41.2%). reported a prevalence of 20.8% for obesity in 39-year - old children in iran based on bmi. in addition, the prevalence of dmft in 39-year - old iranian children was reported to be 1.93.6. it is probable that intake of sugary foods might simultaneously result in weight gain and an increase in caries risk. of course, when a low - sugar diet is consumed, bmi returns to normal but tooth decay continues, which is a factor that has resulted in identification of a significant relationship between dmft and bmi in some studies. no doubt dmt / dmt score (carious and lost teeth) is calculated throughout life by considering all the teeth, but bmi is determined for each individual in a cross - sectional manner at a specific time interval. however a potential limitation of this cross - sectional study is that no cause - effect relationship can be deduced. in addition, it should be pointed out that the majority of studies have been retrospective studies carried out simultaneously in different communities with different cultures. considering the detrimental effects of obesity during adulthood and its importance, the present study was undertaken to evaluate the relationship between bmi and tooth decay in 36-year - old children in the kindergartens and preschool centers in kerman during 2009 - 2010. the results showed a significant effect of bmi on children 's dmft, with an inverse relationship between these two variables, consistent with the results of different studies carried out [7, 12, 1820 ]. in contrast, some researchers have reported a direct relationship between bmi and dmft [11, 13, 2125 ]. in other words, they reported that children 's dmft increases with an increase in bmi. the reasons for the discrepancies between the results of various studies might be evaluated from different aspects as follows. the results of the present study showed that age has a significant effect on dmft ; in other words, the mean dmft of children increases 1.5 times with a one - year increase in age : the mean of dmft increases by 32% with one - year increase in age. the majority of these studies have been carried out on subjects over 6 years of age [6, 1114, 16, 22, 25, 26 ]. growth and weight changes in children occur rapidly at this age and obesity can rapidly take place after changes in the diet in children this age. therefore, if a child has tooth decay, the diet is one of the etiological factors. the recent international growth reference, the who child growth standards reference, is used to evaluate nutritional status. this growth reference is recommended for young children from birth to 5 years and provides a scientifically reliable yardstick of children 's growth achieved under desirable health and nutritional conditions and establishes the breastfed infant as the normative model against which all alternative feeding methods must be measured in terms of growth, health, and development ; however it was not recommended for the present study due to children 's age. tooth decay and obesity. tooth decay and obesity are both considered multifactorial entities with predisposing genetic and environmental conditions and it is not possible to designate them as separate diseased conditions. in addition to the diet, some of the confounding factors involved in tooth decay are oral microflora, age, use of fluoride, and socioeconomic status. the effects of all these factors have not been evaluated in any study to date. children who are underweight are more susceptible to infectious diseases such as tooth decay due to compromised immune system. in addition, families with children who are prone to obesity take greater care of their children 's diet and provide them with less sweets and desserts, resulting in a low - caries prevalence in these children. the results of a study in turkey (2011) showed that bmi increases in underweight children after receiving dental services. although obesity has been associated with factors such as the intake of fast - cooking foods and tooth decay has been associated with excessive intake of sweets, the real etiologies have not been definitely identified because the etiologies are complex and multifactorial. a large number of factors involved in obesity and tooth decay have originated from changes in life styles and environmental factors such as changes in the patterns of physical activities and the nutritional services offered in schools [1013, 29, 30 ]. the results of the present study in subjects whose socioeconomic conditions were available showed that variables such as father 's educational status and mother 's occupation had a significant effect on children 's dmft. mother 's occupation significantly altered caries status and children of mothers with better occupations experienced less caries ; comparable results have been observed in previous studies [3133 ]. a review of risk factors for dental caries in young children associated low father 's and mother 's education with high caries prevalence, similar to a study by luepker. (2007) reported that low socioeconomic status results in an increase in the incidence of caries and obesity. in addition, the results of a study by cinar and murtomaa (2011) in turkey showed that higher dmft rates in children are associated with the parents ' low socioeconomic status [13, 2835 ]. researchers believe that there is no significant relationship between bmi and the family 's socioeconomic status because in higher socioeconomic status a high bmi might be due to a high intake of proteins and in lower socioeconomic status high bmi might be the result of high carbohydrate intake. in the present study the mean of dmft in boys was higher than that in girls. in this context the prevalence of caries - free children in the present study was 37.1% and more caries - free children were found in the obese group, consistent with the results of a study by sadeghi and alizade in 2007. the present study showed that caries rate in the deciduous teeth of 36-year - old children decreases with an increase in body weight. on the whole, the mean of dmft for children with normal bmi was 1.5-fold that of children who were overweight. since tooth decay and obesity are multifactorial conditions and their relationship with individuals ' nutritional habits has been established, the triad of sugar, tooth decay, and obesity should be evaluated in various studies. therefore, despite an inverse relationship between tooth decay and bmi in the present study, prevention of tooth decay and obesity should be considered more than ever ; in addition, the 3-day analysis of the diet in children should be considered in future studies. and also, no cause effect relationship can be deduced from a cross - sectional design study such as this one. | the aim of the present study was to evaluate the relationship between bmi and tooth decay in a population of iranian children. in this cross - sectional descriptive / analytical study, 1482 children were selected from kindergartens and preschool centers in kerman, iran. the children underwent examination of deciduous teeth (using the dmft index) after determination of height and weight for calculation of bmi. the relationship between bmi (after adjustment for age) and dmft was determined using poisson 's regression model. the mean of dmft in children with normal bmi was 1.5-fold that in subjects with extra body weight. age had a significant effect on dmft. in addition, dmft was higher in boys compared to girls. the results of the present study showed that caries rate in the deciduous teeth of 36-year - old children decreases with an increase in body weight. |
approximately one in every five hospitalized patients is readmitted within 30 days. currently, two - thirds of us hospitals have reimbursement penalties for higher than expected 30-day readmission rates from the center for medicaid and medicare services [2, 3 ]. it is expected that similar penalties will be extended to other procedures and diagnoses including cardiac surgery. in preparation for the expansion of the penalty system in the usa and to improve prediction of patients at high risk of postdischarge complications leading to readmissions or premature death, risk factors must be identified early in the hospital course to align the best possible quality and continuity of care. currently, a validated risk model for predicting readmissions after cardiac surgery is not available and few risk factors for readmission are known. recent evidence from california reported an association between infection and higher rates of 30-day readmission after cardiac surgery. however, identification of infection after discharge without routine monitoring of a postcardiac surgical patient is problematic. what is needed is for clinical care teams to identify patients at high risk of infection before cardiac surgery to determine readiness and safety for the patient to undergo surgery. a common marker of inflammation is white blood cell (wbc) count, routinely measured prior to cardiac surgery. wbc count provides a broad measure of inflammation status, whether as a result of infection or proinflammatory disease states such as diabetes, copd, or hemodialysis [58 ]. elevated wbc count is reported as a component of the systemic inflammatory response syndrome (sirs) to sepsis and is endorsed as a marker for reporting the systemic inflammatory response to cardiopulmonary bypass [9, 10 ]. in addition, current evidence has shown that preoperative wbc count is predictive of in - hospital mortality and stroke and major bleeding after coronary artery bypass graft surgery and associated with complications in other endovascular and thoracic procedures [13, 14 ], suggesting that preoperative wbc count may aid clinical care teams in risk - stratifying patients prior to surgery. however, it is not known if a patient 's baseline inflammatory state measured by crude wbc count could predict 30-day readmission. therefore, we sought to evaluate whether preoperative wbc count was associated with 30-day readmissions after cardiac surgery. patients undergoing coronary artery bypass graft (cabg) surgery and/or valve surgery within the northern new england cardiovascular disease study group (nne) between july 2008 and december 2010 were enrolled in the cohort. a total of 2,209 consecutive patients were included along with 268 readmissions to the hospital performing the index cardiac surgery. twelve patients were excluded due to missing white blood cell counts and twenty - one for incomplete data, leaving a total of 2,176 patients and 259 readmissions occurring within 30 days of discharge from the index cardiac surgery admission. all institutional review boards for each center reviewed and approved the data collection for the nne registry and supplementary data collection for readmissions. the nne is a voluntary regional consortium of physicians, allied health professionals, research scientists, and hospital administrators from institutions in maine, new hampshire, and vermont that support coronary revascularization and open - heart surgery. the goal of the consortium is to foster continuous improvement in the quality, safety, and effectiveness of care for patients with cardiovascular disease through the analysis of process and outcomes data with timely feedback to the health care professionals providing these services. all the hospitals providing open - heart surgery in this region contribute data on consecutive cases with validation of procedure numbers and mortality performed every two years. the registry collects data on patient characteristics, procedural indication, priority, and process, and in - hospital outcomes (see http://www.nnecdsg.org/ for the data forms and publically available data). wbc count was defined as the last preoperative measurement of wbc taken prior to procedure, was collected by data abstractor at each center. categories of wbc counts were divided into predefined categories (12.0 thousands per cubic millimeter, mm). baseline, operative, and postoperative outcomes were compared using chi - square tests and continuous data using student 's t - test or wilcoxon rank sum tests where appropriate. we conducted both univariate and backwards stepwise logistic regression removing risk factors that did not reach an alpha 12,000 mm) had higher 30-day readmission than those with lower wbc counts prior to surgery (15% and 18% compared to 10%12%, p = 0.037, figure 1). after backwards stepwise regression, wbc count and other risk factors remained significantly associated with 30-day readmission including number of diseased vessels, on - pump surgery, nadir hematocrit 12,000 (mm) (table 2). the calculated c - statistic was 0.66 with a hosmer - lemeshow goodness of fit chi - square of 10.94 and p value of 0.2. patient and procedural characteristics stratified by white blood cell categories are summarized in table 4. we explored the predictive ability of wbc counts prior to cardiac surgery on 30-day readmission. with and without adjustment of other risk factors for readmission, patients with preoperative wbc counts > 12,000 (mm) were significantly more likely to be readmitted to the hospital within 30 days from discharge. we are the first to demonstrate that a marker of inflammation prior to the start of surgery demonstrates increased risk of 30-day readmission and should be incorporated into risk models to predict readmission prior to discharge from cardiac surgery. wbc count has enjoyed a resurgence in recent years as a valid marker of inflammation and as a strong independent predictor of future coronary heart disease and stroke [15, 16 ]. after an acute event, patient outcomes remain influenced by wbc count at the time of hospital admission. in several studies, peak wbc count or elevated monocyte count has been linked to death or major adverse cardiac events (maces) outcomes, including readmission [1719 ]. other strong evidence has linked high wbc count at admission with adverse outcomes (mortality and bleeding) in patients undergoing coronary revascularization with cardiopulmonary bypass [11, 12 ]. however, in the case of cardiopulmonary bypass it is unclear whether high wbc count contributes to preexisting risk or to development of the systemic inflammatory response postoperatively or both. the systemic inflammatory response is a complication in cardiopulmonary bypass patients that is caused by a combination of surgical stress and contact activation of blood component in the extracorporeal circuit [20, 21 ]. it is poorly defined and the only formal definition is the systemic inflammatory response syndrome (sirs), borrowed from the sepsis field. according to the definition, sirs exists when any two out of four criteria relating to abnormal temperature, heart rate, respiratory rate, or white cell counts exist. an evidence - based review of the inflammatory response indicated that all four sirs criteria were rarely monitored in the setting of cardiopulmonary bypass as they were felt to be too nonspecific and if taken literally would apply to approximately 40% of all patients [2426 ]. a more recent update on minimal reporting criteria by the outcomes consensus panel singled out wbc count as the only criterion measured on its own as being relevant to the inflammatory status. this recommendation was supported by other fields in which wbc count is recognized as a valid marker of inflammation [5, 6, 8, 27 ]. an alternative theory for the development of the systemic inflammatory response is that this is determined less by the extracorporeal circuit itself but rather by preexisting activation of white cells and endothelium or by preoperative transfusion. consistent with this theory is that high wbc count prior to coronary surgery utilizing cardiopulmonary bypass is linked with adverse outcomes including mortality and bleeding [11, 12 ]. our present findings that high wbc count before - surgery is linked to an increased risk of 30-day readmission after discharge add further weight to this idea. we therefore conclude that wbc count measured prior to cardiac surgery may serve as a measure of the patient 's inflammatory status and could aid in identifying and managing patients at heightened risk of readmission after discharge from cardiac surgery. this becomes especially relevant in an era when higher than expected readmission rates may attract financial penalties to hospitals. | approximately 1 in 5 patients undergoing cardiac surgery are readmitted within 30 days of discharge. among the primary causes of readmission are infection and disease states susceptible to the inflammatory cascade, such as diabetes, chronic obstructive pulmonary disease, and gastrointestinal complications. currently, it is not known if a patient 's baseline inflammatory state measured by crude white blood cell (wbc) counts could predict 30-day readmission. we collected data from 2,176 consecutive patients who underwent cardiac surgery at seven hospitals. patient readmission data was abstracted from each hospital. the independent association with preoperative wbc count was determined using logistic regression. there were 259 patients readmitted within 30 days, with a median time of readmission of 9 days (iqr 416). patients with elevated wbc count at baseline (10,00012,000 and > 12,000 mm3) had higher 30-day readmission than those with lower levels of wbc count prior to surgery (15% and 18% compared to 10%12%, p = 0.037). adjusted odds ratios were 1.42 (0.86, 2.34) for wbc counts 10,00012,000 and 1.81 (1.03, 3.17) for wbc count > 12,000. we conclude that wbc count measured prior to cardiac surgery as a measure of the patient 's inflammatory state could aid clinicians and continuity of care management teams in identifying patients at heightened risk of 30-day readmission after discharge from cardiac surgery. |
traumatic brain injury (tbi) is a heterogeneous phenomenon with a variety of external force causes, severities, and anatomical injuries. the most common causes of tbi include falls, sports - related injuries, and motor vehicle accidents. tbi severity ranges from mild to severe with brain functional deficits for survivors manifesting across motor, sensory, cognitive, psychological, and socioemotional domains. the temporal lobes are the most vulnerable areas to acute injury in tbi, in part related to their location near the base of the skull and the free edge of the tentorium [13 ]. hippocampal atrophy in tbi, which has been demonstrated to be related to injury severity, is likely to reflect an aggregated effects of trauma - induced cellular loss that develops over time. protracted neuronal loss of the hippocampus has been well documented in human postmortem studies and in an extensive experimental animal literature that records cell loss [68 ]. longitudinal studies showed that hippocampal volumes will decline over a prolonged period from 1 week (largest decline) to 2.5 years [9, 10 ]. the hippocampus is critical for episodic memory [11, 12 ], which contains personally experienced events situated in subjective time and space. it has been proposed that both remembering past and imagining novel scenarios rely on an intact hippocampus as the physiologic basis for memory formation and consolidation in a coherent scene. recent evidence suggests that amnesic patients with hippocampal damage have difficulty not only projecting back in time to mentally simulate the past (retrospection), but also projecting forward in time to mentally simulate novel and specific future scenarios (prospection) [15, 16 ]. it has also been documented that some tbi survivors seem to live in a timeless world, in a sort of perpetual present experiencing difficulties in traveling back and forward into subjective time. for example, tbi survivors may fail to recall specific events from the personal past [18, 19 ], may be incompetent in conscious recollection of personal events [19, 20 ], and may present disturbances in the ability to imagine future (episodic future thinking). it was putative that the prolonged hippocampal damage may impair the episodic memory system in tbi survivors. however, there was no quantitative mri study examining how deteriorating structural abnormality of the hippocampus affected episodic memory network in tbi survivors. it prompted us to examine brain connectivity changes of the hippocampus - dependent network in tbi. brain connectivity is now being explored by depicting neuronal coupling between brain regions through various techniques [2224 ], among which resting state fmri (rsfmri) analysis not only has the noninvasive advantage but also possesses additional gains : resting state networks (rsns) are highly organized in space, reproducible from subject to subject, and allow the search for significant baseline fluctuations to obtain task - free functional network information. to examine cognitive changes specifically, we highlighted how responses in individual brain regions can be effectively combined through functional connectivity. effective connectivity quantifies directed relationships between brain regions and controls for confounds that limit functional connectivity features that facilitate insight into functional integration. it overcomes important pitfalls of functional connectivity that limit our understanding of neuronal coupling, such as involvement of functional connection of other cognitive processes, observational noise, or neuronal fluctuations. one popular approach to make effective connectivity analysis is granger causality analysis (gca). it uses multivariate autoregressive models of time series data to illustrate the amount of variance in one region explained by the signal history in another region and quantifies the magnitude and direction of influence of one region time series on another. by examining altered effective interaction in tbi survivors ' episodic memory network, we sought to enrich its neural connectivity pathology which is of high importance for accurate diagnosis and early intervention. the current study planned to focus on mild traumatic brain injury (mtbi) survivors. mtbi is most popular, as it accounts for the overwhelming majority of the head - injured population treated in emergency departments. a recent who systematic review suggests that the annual incidence of mild tbi is probably over 600/100,000. moreover, moderate and severe tbi consumes most resources per individual, yet mtbi 's magnitude and societal ramifications are often underestimated. with the label of mild, patients do not seek medical attention, are not systematically assessed, or are lost to medical follow - up. it has been estimated that 4080% of mtbi survivors experience postconcussive syndrome, a constellation of physical, cognitive, and behavioral difficulties that may persist up to 2 years after tbi. along with the significant number of mtbi, these negative outcomes underline the value of accurate identification and adequate management. further, according to a cohort study with standardized tests of language skills, severe tbi group displayed greater improvement in scores from the acute period to 12 months after tbi. however, scores for the mild - moderate tbi groups remained quite stable over time. to keep subjects more homogeneous and reduce intersubject variabilities, we only focused on patients over 3 months to 14 months after tbi. the tbi group was composed of 19 patients (16 males, mean age 38.21 13.42 years) with mild tbi. screening for mild tbi was based on the world health organization 's collaborating center for neurotrauma task force. inclusion criteria were (i) conscious survivors at the time of testing ; (ii) a favorable outcome (glasgow coma score of 1315) according to glasgow outcome scale (gos) ; (iii) loss of consciousness (if present) 10 voxels. the statistics were color - coded and mapped in mni space, while brain regions were estimated from talairach and tournoux after adjustments for differences between mni and talariach coordinates with a nonlinear transform. rois for further multivariate granger causality analysis (mgca) were defined as regions that showed significant functional connectivity with the left hippocampus in the episodic memory network in healthy controls. the granger causality is an optimal candidate for its data - driven nature and is widely used in fmri studies. the entire time series of bold signal intensities from rois, averaged across voxels within each roi among subjects of the same group, were normalized to form a single vector per roi. the mgca uses directed transfer function (dtf), computed from a multivariate autoregressive model of the time series in the selected rois. in this study, we also adopted the weighted dtf with partial coherence to emphasize direct connections and inhibit mediated influences [43, 44 ]. to assess the significance of path weights, a null distribution was obtained by generating 2500 sets of surrogate data and calculating the dtf [43, 44 ]. for instance, fourier transform was applied to each regional time series, and the phase of the transformed signal was randomized. in addition, a difference of influence (doi) term was used to assess links that showed a dominant direction of influence to limit potentially spurious links caused by hemodynamic blurring. the doi was compared with the null distribution for a one - tailed test of significance with a p value of 0.01 (fdr corrected for multiple comparisons). the stringent threshold was chosen to avoid potentially spurious causal links introduced by low temporal resolution and hemodynamic blurring in the fmri signal. (number of granger causal afferent connections to a node) to find the central targets of network, out - degree (number of granger causal efferent connections from a node) to find the central sources [46, 47 ], and in + out degree to find network hubs. further, three kinds of hubs of the network were defined if the sum of in - degree, out - degree, or in + out degree of a node was at least 1 standard deviation (sd) greater than the average degree of all nodes in the network respectively [48, 49 ]. between - group degree difference was carried out by the two - sample t - test analysis of the in - degree, out - degree, and in + out degree of each roi in each individual, respectively. connection density difference was determined by calculating between - group difference about the numbers of significantly causal interactions in each individual. between - group differences in the causal connectivity graphs were determined using permutation tests to get a data - driven nonparametric approach. permutation tests constructed a distribution of a test statistic by freely resampling the ddtf values without replacement. the key and only assumption for permutation tests is data exchangeability which means the distributions of two group data are identical under the null hypothesis. here, we randomly permuted the ddtf values to two new groups. as a result, an empirical distribution of the data sets was constructed using test statistic values for all possible permutations. the true doi obtained with the correct pairs of subjects was then compared with the obtained distribution. thus, p value was calculated by dividing the frequency of permutations presenting more extreme test statistic value by the number of all permutations (10000 times). whole brain functional connectivity maps for the mtbi group, healthy controls, and their contrast results are illustrated in figure 1 and listed in table 1. these maps illustrate significant functional connectivity between the left hippocampus and a widespread set of brain regions belonging to the episodic memory system. in common, the hippocampal network of both groups covered bilateral inferior frontal gyrus (ifg, ba47), bilateral medial frontal gyrus (mefg, ba10/11), left inferior temporal gyrus (itg, ba21/20), bilateral middle temporal gyrus (mtg, ba 21/22), left fusiform gyrus (ba20/37), bilateral parahippocampal gyrus (phg), left anterior part of the superior temporal gyrus (stg, ba38), the middle portion of the left stg (ba 41/22), bilateral anterior cingulate cortex (acc), and left posterior cingulate cortex (pcc). the hippocampus - dependent fc network of the mtbi group additionally involved the bilateral posterior parts of stg (ba39) and left supramarginal gyrus (ba40), as well as the right itg, right fusiform gyrus, right anterior and middle parts of right stg, and right pcc. by contrast, functional connectivity between the left hippocampus and the right middle / posterior stg (ba22/41 and ba39) were significantly stronger in mtbi survivors than in normal controls. however, functional connectivity between the left hippocampus and other components in the hippocampal network was not significantly stronger in normal controls than that in mtbi survivors. we then determined regions in the hippocampus - dependent functionally connected network in healthy controls as rois to form a canonical network and evaluated effective connectivity of the episodic memory network by means of mgca. a causal connectivity graph was constructed using the thickness of connecting lines to indicate strengths of causal influences (see figures 2(a) and 2(c)). for both mtbi survivors and healthy controls, causal influences within the episodic memory network presented strongly covarying relations (figures 2(a) and 2(b)). results from mgca analysis showed that causal interactions became denser for the mtbi group than that of the nc group. however, connection density between the two groups was not significantly different from each other (p = 0.84). for example, strong causal outflow originated from the left hippocampus to left anterior stg (astg), from the left phg to left astg, and from the left pcc to left phg. besides, bidirectional interactions in the left hemisphere between the left hippocampus and left phg and between the left ifg and left mefg were significant too. however, we also observed two cross - hemisphere causal connectivity, such as from the left mefg to right mefg, from the left astg to the right ifg, and from the right phg to left hippocampus (see figure 2(b)). for the mtbi group, in the contrary, significant causal interaction mainly existed across hemispheres, mainly flowing out from the right hemisphere. for example, strong causal outflow originated from the right mefg to left mefg and from the right hippocampus to left astg, as well as bidirectional interactions between the left ifg and right mefg and between the left astg and right mtg. also significant interhemisphere causal connectivity originating from the left hippocampus to left phg and left pcc and from the left ifg to left astg was detected (see figure 2(d)). the only overlapped causal interaction in both the mtbi and nc groups was bidirectional connectivity between the right hippocampus and right phg. node degree analysis showed that, in healthy controls, there were three hubs in the hippocampus - dependent episodic memory network, such as the left hippocampus, left phg, and left astg. specifically, central target hubs (flow - in hubs) were the left mefg, left hippocampus, and left phg, while the left phg and left astg were the central source hubs (flow - out hubs). in the mtbi survivors, all three kinds of network hubs shifted from the left to the right hemisphere. specifically, network hubs were the right mefg and right hippocampus, the central target hub was the right hippocampus, and central source hubs were the left phg, left astg, and right mefg. out - degree, and in + out degree values of each roi were not significantly different (p value ranging from 0.09 to 0.96). between - group analysis also showed increased driving effect between nodes in bilateral structures (see figure 3). in detail, increased interhemisphere causal effects were found in the interactions from the left fusiform gyrus to right ifg (nc mean sd versus mtbi mean sd : 0.0052 0.0061 versus 0.0169 0.0205, p = 0.02), from the right mtg to left fusiform gyrus (0.0014 0.0016 versus 0.0044 0.0054, p = 0.01), and from the right hippocampus to left mtg (0.0049 0.0110 versus 0.0169 0.0230, p = 0.04) and bilateral interaction between the right mefg and left mtg (0.0030 0.0050 versus 0.0096 0.0114, p = 0.02 ; 0.0031 0.0045 versus 0.0099 0.0205, p = 0.01). increased intrahemisphere causal connectivity was also identified from the left fusiform to the left stg (0.0035 0.0059 versus 0.0113 0.0171, p = 0.04) and from the left mtg to left mefg (0.0036 0.0049 versus 0.0148 0.0263, p = 0.03). no decreased driving effect was detected by comparing the mtbi group with that the nc group. resting state fmri maps have been shown to reveal the full distribution of memory - related regions, as they coincide with regions showing activation across a variety of task - based memory studies. the current study sought for covarying areas with the left hippocampus and determined the hippocampus - dependent neural network of episodic memory in mtbi survivors with reference to previous findings. further effective connectivity analysis of this hippocampal network by gca revealed significant findings, such as contralateral shift of source, target and network hubs, weakened ipsilateral causal interactions, and increased causal interactions across the two hemispheres, which reflected the neural compensatory mechanism of mtbi survivors. the hippocampus - dependent neural network identified in the current study was consistent with previous findings. specifically, functional connectivity was observed between the hippocampus and many regions of the brain, including the ifg, the mefg (anterior prefrontal gyrus), the anterior part of stg, the mtg, the itg, the parahippocampus, and the pcc / retrosplenial cortex [14, 5255 ]. these structures are also known to be involved in the default mode network (dmn). as part of episodic memory network, the dmn is an interconnected and anatomically defined brain system that preferentially activates in states of relative rest but deactivate during tasks. as expected, mtbi survivors compensated for hippocampal deficits by relying on an intensified and extended functionally connected network. along with the heavier manipulation of the hippocampal neural network, both the anterior and posterior parts of right stg were overrecruited by mtbi survivors. it was consistent with many neuroimaging studies concerning episodic memory, which have reported that functional brain activity in elders increases in the right hemisphere. recruitment of the right hemisphere reflected that mtbi patients compensated for decreased functional connectivity of one brain region through recruiting additional brain resources in the contralateral hemisphere to perform a cognitive task. by means of gca, directed relationships with different linking weights within the episodic memory network provided more information. causal interaction analysis between rois determined by functional connectivity analysis found that hubs of patients ' episodic memory network displayed a contralateral shift. in nc, network hubs were the left hippocampus and the phg (also flow - in hubs, with a dominant role of receiving information) as well as the left astg (as a flow - out hub, with a dominant role of sending information). these areas constituted an interactive model of episodic memory in which the anterior stg and hippocampus / phg were information integration centers for semantic and episodic memory in the declarative memory system. as a macromodel based on neuropsychological data which presents an interactive construction of memory systems, the mnesis model (short for memory neostructural inter - systemic model) specifies the dynamic and reconstructive nature of memory by highlighting a hierarchical order of three long - term representation systems of perceptual memory (the lowest level), semantic memory, and episodic memory (the highest level) and adds two retroactive processes of memory semanticization and perceptual memory transfer during experience reliving. the perceptual representation system receives, stores, and makes the basic unit of information about perceptual features of physical objects available to other systems. semantic memory refers to one 's noetic awareness of the existence of the world and objects, events and other regularities in it, independent of self, autonoetic awareness and time. when semantic information of the memory, which has no contextual richness but present a schematic version of the memory, is established, retrieval processes are required to reactivate its mental representations and return the individual to his or her conscious experience of the event. these personal experiences covered detailed contextual information, scene construction, and a sense of reliving or autonoetic consciousness. semantic memory encompasses a rich fund of general knowledge about the world, represented in visual, olfactory, gustatory, tactile, and auditory cortices [65, 66 ], conducive to the identity of perceptual events. it is proposed that these multiple sensory inputs are converged in the left anterior temporal lobe (atl), a transmodal representational hub, to form a concept. the atl as a concept formation center in the semantic memory network has been demonstrated by magnetoencephalography, distortion - corrected functional mri, pet, or repetitive transcranial magnetic stimulation techniques. meanwhile, the hippocampus is always involved whenever detailed ; contextual information is recalled according to the transformation hypothesis of memory consolidation and the binding of items and contexts model. personal experience is represented as a pattern of features that correspond to different facets processed during the encoding of the episode. the hippocampus is more adept at associating multiple attributes of differential forms of memory than other structures. inputs from regions of the recognition (or context) memory network are not directly involved in memory strength, but converge at the hippocampus to become cohesive memories of individual events via the formation (binding) of episodic memory [72, 73 ]. after binding, the outputs of the hippocampus return to cortical regions from which the inputs arose. thus, the hippocampus performs complex high - resolution binding of the different qualitative aspects of an event, both at encoding and at retrieval. the parahippocampal area is also an important hub as it enables the communication between the hippocampus and neocortical areas. it has been proposed that the phg provides contextual information about the where and when of a target item for memory encoding to the hippocampus to bind new memories and link the memory of that particular episode within a larger network [11, 59, 69 ], underlining its integrative and maintenance functions. in mtbi survivors, these network hubs in normal controls were replaced by the right hippocampus and right mefg. it was compatible with the pathological observation that reduced volume of the temporal lobe is commonly found following moderate to severe tbi, due to abrasive contact with bony plates of the skull during the acute injury. the observed compensation mechanism in mtbi was also consistent with previous findings. a latest research, which performs a direct test of the relationship between episodic memory 's perceptual richness and hippocampal function, claims that the right hippocampus plays an important role in the recruitment of posterior cortical regions that support the representation of perceptual memory content. in the current study, it reflected that mtbi survivors compensated for episodic memory dysfunction by means of overenrollment of the perceptual memory system. meanwhile, greater activation of the right pfc also indicated an optimized compensation mechanism in the mtbi survivors. the pfc is generally thought to be the primary site of scaffolds to compensate for declines in functioning in other regions, due to its versatile and flexible nature. it was suggested that at least some of the age - related overrecruitment in prefrontal cortex may reflect attempted compensation for reduced activation in the hippocampus. a recent review also highlighted that the pfc was heavily activated during memory encoding and retrieval in elderly participants. with reference to neurocognitive models, it was postulated that the strategic component of episodic memory depends primarily on the pfc to constrain memory search and monitor the appropriateness of recovered memories. the intensified interconnection between the right mefg and other regions in the current study showed that mtbi survivors manipulated the right mefg to a greater degree to make up for the left hippocampus deficit. however, overactivation of the pfc is not always related to better performance but a poorer memory, because recruitment of additional brain regions might come with additional cost. we postulated that overrecruitment of the right mefg may cause dysfunction of its primary role due to overload. the affected executive control and working memory thus led to failed episodic memory retrieval. a study analyzing story narratives from 10 participants with tbi reported a normal microlinguistic processing (lexical and syntactic) but impaired macrolinguistic abilities (pragmatic, cohesive, and coherent) in tbi survivors. the authors defined these patients as nonaphasic and suggested that their confused and impoverished language was caused by reduced ability to organize information at the macrolinguistic level (unable to guide comprehension and production of logical relationships, both temporal and causal, between agents and events). our primary finding of a disrupted causal connectivity between the left astg (the concept formation center in the semantic memory network) and the hippocampus may provide their research a possible explanation. we demonstrated that it was due to the broken - down interaction of the concept forming center and the context binding center that caused the abnormal macrolinguistic processing but preserved lexical and syntactic competence in tbi survivors. hence, tbi survivors always presented a confused and impoverished language but no obvious symptoms of other language deficits, such as anomia. brain injury of the hippocampus was consistently reported to cause a chronic post - tbi episodic memory impairment. by examining altered effective connectivity of the episodic memory network in mtbi survivors, we found that the pattern of brain network changes detected in tbi survivors has similar functional consequences to normal aging. even though functionally connected regions with the hippocampus were extended, dysfunction of neural network hubs of the astg, hippocampus, and phg in the dominant hemisphere and overrecruitment of the right mefg lead to an abnormal episodic memory network. our findings also demonstrated that effective connectivity analysis was more suitable to represent the working mechanism of an episodic memory network than functional connectivity analysis. to the best of our knowledge, this is the first in vivo demonstration of the dynamic relationships between nodes in the hippocampal episodic memory network in mtbi survivors. | traumatic brain injuries (tbis) are generally recognized to affect episodic memory. however, less is known regarding how external force altered the way functionally connected brain structures of the episodic memory system interact. to address this issue, we adopted an effective connectivity based analysis, namely, multivariate granger causality approach, to explore causal interactions within the brain network of interest. results presented that tbi induced increased bilateral and decreased ipsilateral effective connectivity in the episodic memory network in comparison with that of normal controls. moreover, the left anterior superior temporal gyrus (astg, the concept forming hub), left hippocampus (the personal experience binding hub), and left parahippocampal gyrus (the contextual association hub) were no longer network hubs in tbi survivors, who compensated for hippocampal deficits by relying more on the right hippocampus (underlying perceptual memory) and the right medial frontal gyrus (mefg) in the anterior prefrontal cortex (pfc). we postulated that the overrecruitment of the right anterior pfc caused dysfunction of the strategic component of episodic memory, which caused deteriorating episodic memory in mtbi survivors. our findings also suggested that the pattern of brain network changes in tbi survivors presented similar functional consequences to normal aging. |
human brucellosis remains as one of the most common zoonotic diseases being reported worldwide, with approximation of more than 500,000 new cases annually. disease is acquired upon direct contact with infected animals, consumption of infected dairy products, or inhalation of aerosols generated by any activity during handling of the isolate in the laboratory. the prevalence of brucellosis varies between countries with high endemicity being associated with the mediterranean countries, middle east, southwest asia, and some areas in latin america [1, 3 ]. implementation of the national surveillance programme and a massive vaccination programme of the livestock has resulted in eradication of the disease in europe, australia, new zealand, and canada. the incidence of brucellosis was first reported in malaysia following the isolation of brucella abortus in 1950 and brucella suis in 1963 among the livestock [4, 5 ]. subsequently, cases of human brucellosis surged, urging the initiation of national programme for the area - wise eradication of bovine brucellosis which was implemented nationwide in 1979. this effort had markedly reduced the prevalence of bovine brucellosis in malaysia from 3.3% in 1979 to 0.23% in 1988. limited adequate biosafety level 3 (bsl3) facility required for the handling of brucella isolates impinged on the proper identification of the organisms. as a consequence, the sensitivity pattern of the brucella isolates encountered in this region is also unknown as in vitro susceptibility testing is not done routinely due to above reason. therefore, clinical cases are treated based on previous literature reports as well as who recommendations. currently, tetracycline, rifampicin, trimethoprim - sulfamethoxazole / cotrimoxazole (sxt), streptomycin, and other aminoglycosides, separately or in combinations, are the most commonly used antimicrobial agents for brucellosis treatment. however, reduced susceptibility of the organisms to streptomycin, rifampicin [8, 9 ], and sxt [10, 11 ] has been reported recently warranting a proper investigation to assess the susceptibility pattern of the local isolates. the main aim of this study is to ascertain the most common species causing brucella infection in malaysia and to determine the susceptibility patterns of these clinical isolates against the commonly used antibiotic agents. all isolates were obtained from blood culture except for one, which was derived from tissue biopsy of lumbar area. the isolates come from various parts of malaysia, mainly penang 33 isolates (80.6%), selangor and kuala lumpur 2 isolates (4.9%), respectively, and 1 isolate (2.4%) each from negeri sembilan, melaka, perlis and kedah. the identification of the organisms was done based on conventional method by taking into consideration the gram stain characteristic, colonial morphology, the fastidious requisite of the organism, and requirement of co2 for growth. the production of urease, oxidase, and hydrogen sulphide, ability to reduce nitrate to nitrite, motility test using semisolid motility medium, and sensitivity to the dyes, basic fuchsin and thionine (at final concentrations of 2040 g / ml), were also observed. agglutination with monospecific antisera for a and m antigens (animal health veterinary laboratories agency, uk) is also taken into consideration in identification of the organisms to the genus and species level and to identify the biotype of the organism. the minimal inhibitory concentration (mic) values of tetracycline (tc), streptomycin (sm), doxycycline (dc), trimethoprim - sulfamethoxazole (sxt), rifampicin (rf), and gentamycin (gm) were determined using the e - test (biomerieux, sweden) method. organism inoculum equivalent to 0.5mcfarland turbidity was prepared and lawned on mueller - hinton agar (bd bbl) supplemented with 5% sheep blood. each antibiotic strip was individually placed on the inoculated agar and incubated at ambient air, 35c for 48 hours. the mic was interpreted as the value at which the inhibition zone intercepted the scale on the e - test strip. mic50 and mic90 levels were defined as the lowest concentration of the antibiotic at which 50% and 90% of the isolates were inhibited, respectively. three brucella reference strains (b. abortus 544, b. melitensis 16 m, and b. suis 1330) were used as controls for identification, biotyping, and antimicrobial susceptibility testing. in addition to those brucella reference strains, 2 other organisms, escherichia coli atcc 25922 and staphylococcus aureus atcc 29213, were also used as the quality control strain for susceptibility testing. all the isolates included in this study were identified as brucella melitensis except for one isolate which was identified as b. suis. serotyping using specific monoclonal antisera revealed that 31 (76%) isolates of b. melitensis were serotype 3, seven (17%) were serotype 2, and two (5%) were from serotype 1. interpretation of the mic value for all antibiotics tested was based on breakpoints for brucella spp. this drug is available, breakpoint for slow - growing bacteria (haemophilus influenza) is referred to. only 11 isolates (26%) exhibit mic 1 g / ml for rifampicin. the mic50 and mic90 values of each antibiotic are displayed in table 1. based on the mic90, sxt was noted to be the most potent agent against brucella spp. (0.125 g / ml), followed by tetracycline and gentamicin (0.19 g / ml), doxycycline (0.25 g / ml), streptomycin (0.75 g / ml), and rifampicin (1 g / ml). rifampicin showed the least activity against brucella, with elevated mic level of above 1 g / ml observed in 30 isolates (70%). therefore, all the drugs were found to be effective to combat brucellosis except for rifampicin. brucellosis occurs as sporadic cases with occasional outbreak mainly among people who work with farm animals, especially cattle, sheep, and goats in malaysia. cases were also noted among those who consumed raw milk due to traditional belief that it carries some hidden health benefits. the diagnosis of brucellosis is made either by serological detection of the antibody or isolation of the organism. nonetheless, the gold standard method for the diagnosis of the disease is by isolation of causative agent. as this organism is highly infectious with infective dose of 10100 bacteria sufficient to cause infection, manipulation of these organisms warrants bsl 3 laboratory facilities. therefore, attempts at isolation and identification of brucella spp. from clinical specimen are not extensively performed. misidentification of these organisms with other slow - growing gram - negative, nonoxidizer bacteria using commercial test kits, further accentuates the low number of detectable cases. to overcome this devoid of information, we conducted this study, which revealed that human brucellosis in malaysia is exclusively caused by b. melitensis. our finding is in concordance with the previous reports from different regions of turkey, mediteranean and south america basin [1719 ]. there have been many reports on the susceptibility testing for brucella, based on many different methods [2022 ]. although there was noted to be no significant variation between the methods, susceptibility testing using microbroth dilution is the most recommended method for in vitro efficacy testing of antibiotics against brucella sp. however, we opted for e - test method for the present study as it was reported to be reliable, reproducible, less labour - intensive, and less time - consuming, as well as imposing less risk of laboratory acquired infection due to lesser manipulation of the organism during the process. the world health organization has released recommendations on the use of doxycycline in treating adults with acute infection, combined with either rifampicin or streptomycin for six - week duration. however there were reports that brucellosis with osteoarticular and visceral complications is associated with lesser risk of relapse with triple therapy with streptomycin, rifampicin, and doxycycline. a review and meta - analysis of 30 randomised controlled trials also concluded that preferred treatment should be with combination of doxycycline and gentamicin or triple (e.g., doxycycline with rifampicin and gentamicin) regimens.. however these relapse cases were associated with inadequate treatment due to improper dosing or poor patient compliance rather than to antimicrobial resistance [8, 27 ] and therefore antimicrobial susceptibility testing is still not crucial in management of brucellosis cases. doxycycline has become the most prescribed tetracycline derivative in the treatment of brucella infection because of its superior pharmacokinetic nature. in the present study, amongst the tested antimicrobial agents, the mic breakpoint for both tetracycline and doxycycline was found to be within the susceptible category and among the lowest recorded. this is in concordance with the previous reports [11, 22, 29 ]. our mic50 and mic90 values were lower than previously being reported [21, 30 ]. similar findings were noted with its other derivative agent, tetracycline which displayed even a more potent activity compared to doxycycline. however, the most potent agent with lowest mic50 and mic90 values recorded compared to other agents tested in this study was sxt. this proved that the agent can serve as a good alternative agent for oral treatment. the drug is furthermore cheap, associated with lesser side - effects and is the preferred agent for the treatment of brucellosis among children and pregnant woman. similar findings which were reported by few other studies proved that the in vitro activity for this drug was consistent. the current mic value was also much lower than other studies conducted in area endemic of brucella [22, 30, 31 ]. aminoglycosides such as gentamicin, streptomycin, and its derivative were also reported to be effective drugs against brucellosis. a multicentre study reported that combination of gentamicin and doxycycline is considered as of equal efficacy to the recommended regime of doxycycline and streptomycin. therefore, this may serve as alternative if patients were not tolerating well the first line treatment. the main factor that preludes its wider use is probably the side effect and the parenteral mode of administration. in this study gentamicin showed a good in vitro activity against all the tested brucella isolates as reported previously in other countries [18, 28, 33 ]. similar finding as well as lower and higher mic values were reported in turkey for gentamicin. the breakpoint for rifampicin has not been established, and therefore these organisms can not be confidently characterised as susceptible, intermediate or resistant. however, based on the breakpoint used, a worrying pattern was observed with our isolates. elevated mic level of > 1.0 g / ml were detected among as high as 70% of the isolates. our mic50 and mic90 values were similar to some reports [28, 34, 35 ] however was lower than others [20, 29 ]. this elevated pattern is of clinical concern as rifampicin is widely used for the treatment of brucellosis in this country. total eradication of brucella infection has proven to be difficult to achieve as sporadic cases and occasional outbreak were still observed in malaysia despite the measures previously taken. in this study, we are reporting a reduced susceptibility towards rifampicin in majority of our tested brucella isolates. as routine antimicrobial testing is not feasible in many of the laboratory settings, the use of rifampicin is best avoided as the treatment for brucellosis. a regular screening to monitor for the presence of resistance phenotype if possible, is advisable to ensure early detection of any resistance that may develop and may lead to treatment failure. | brucellosis is a world - wide zoonotic disease with a major impact on the public health. due to the high risk of laboratory acquired infection, limited laboratory investigations were performed on this organism, including detailed identification and susceptibility study. brucella melitensis is the commonest aetiological agent for human brucellosis in this region. the in vitro susceptibility pattern against selected antimicrobial agents was assessed using e - test. all isolates were noted to be sensitive to all the antimicrobial agents tested except for rifampicin where elevated mic > 1 g / ml was noted in 30 out of 41 isolates tested. |
herein we report an immunocompetent person who presented with fever, headache, atypical rash, and hazy vision. ophthalmoscopy of both eyes revealed perivascular sheathing with frosted branch angiitis pattern in veins, patchy retinal hemorrhages. this case illustrates that vzv should be considered in the differential diagnosis of retinal perivasculitis. since a rapid and accurate diagnosis is crucial for prompt administration of antiviral therapy, pcr - based analysis of aqueous humor is a valuable tool for detecting viruses. frosted branch angiitis (fba) is a panuveitis associated with sheating of all retinal vesssels. the first comprises patients affected by lymphoma (e.g. large cell lymphoma) and leukemia (e.g. acute lymphoblastic leukemia). the second group includes patients with associated autoimmune disorders including systemic lupus erythematosus, crohn 's disease, behcet disease which can have fba as a clinical sign of the underlying disease.the third group includes patients with viral infections such as cytomegalovirus (cmv), herpes simplex (hsv), varicella zoster (vzv) or non - viral infections like toxoplasma retinochoroiditis. finally the last group comprises patients with acute idiopathic disorder (1, 2). arn is caused mainly by vzv and hsv, and patients present by decrease in vision, photophobia, and eye pain (3). fba secondary to viral infection can progress to acute retinal necrosis with poor and permanent damage to vision (4, 5). we describe a 33-year - old woman who was diagnosed with a severe case of meningitis and eye infection by vzv. a 33-year - old woman was seen by a clinician because of fever, chills, myalgia and arthralgia that started 2 weeks prior to the visit. after 5 days, she developed maculopapular rash on her abdomen and right flank. after several days, she reported pulsatile headache, nausea, vomiting and progressive blurring of her vision and was admitted to rasoul - e - akram hospital in tehran, iran. she had no history of rheumatic disease but had recurrent oral ulcerations whose frequency was less than 3 times per year. she was married, living with his husband and had a technical office job with no relevant exposure. on examination, her temperature was 39c, blood pressure was 110/70 mm hg, the weight 101 kg. her body - mass index was 34.6 (bmi : the weight in kilograms divided by the square of the height in meters. on eye examination the visual acuity was 20/2000 in both eyes). anterior segment examination showed mild ciliary injection, fine granulomatous keratic precipitates and no posterior synechiae. both eyes had mild inflammatory reactions in anterior chamber (1 + cell) and vitreous (1 + cell). in funduscopy there was diffuse and extensive retinal perivascular sheathing (mostly phlebitis) and scattered intraretinal hemorrhage along with severe macular edema in both eyes. retinal examination showed perivascular sheathing with frosted branch angiitis pattern in veins and patchy retinal hemorrhages based on an inconclusive initial work up, an anterior chamber paracenthesis was performed to investigate the presence of viral footsteps. aqueous specimen was subjected to hsv, vzv and cmv pcrs and intravitreal (2000 microgram in 0.004ml) and systemic ganciclovir was given to patient because of high suspicion about cmv infection. aqueous pcr analysis (qualitative) turned positive for vzv (done in a private laboratory by conventional procedure) therefore, ganciclovir switched to acyclovir 10 mg / kg every 8hr along with oral prednisolone 60 mg daily for 7 days that followed by tapering doses. laboratory data in table 1 show increased alt and ldh that is usual in viral infection. tests for rheumatologic diseases (anti - smooth muscle ab, anti - ssa, anti - ssb, anti - cardiolipin igm, igg, anti - b2 glycoprotein igm, igg, ana, anti - ds dna, anti - ccp, ace, rf) were negative. tests for infectious diseases (wright s test, anti - brucella igg ab, vdrl, r pr, anti - toxoplasma ab igm, igg, anti - cmv ab igm, igg, anti- vca ab igm, igg, anti - hsv ab 1,2 igm, igg, anti - hiv ab, hbsag, anti - hcv ab, anti vzv igm) were negative. serum anti - vzv ab igg and vzv dna on aqueous sample by pcr method were positive. laboratory data for patient with frosted branch angiitis inr : international normalized ratio one week later, the patient s systemic condition improved, however, no significant improvement in visual acuity was noticed. repeated ocular coherence tomography (oct) revealed the persistence of macular edema in both eyes. therefore, the second intravitreal ganciclovir injection (2000 microgram in 0.004 ml) was combined with intravitreal bevacizumab injection (1.25 mg in 0.05 ml). two weeks after second injection, the visual acuity improved to 20/1000 and 20/1000, bilaterally. parenteral acyclovir was replaced by oral valacyclovir (12 weeks) and systemic corticosteroid was tapered off gradually over a month. herein we report a case of an immunocompetent person who presented with vzv meningitis and fba. frosted branch angiitis is a rare disease characterized by visual disturbance associated with anterior chamber and vitreous inflammation and severe sheathing of retinal vessels resembles the appearance of frosted branches of a tree. it is postulated that the clinical picture of fba can be caused by direct invasion of infective agent or secondary to immunologic response. shenoy. reported a 26-year - old lady who presented with fever, headache and sudden loss of vision in her right eye. fundus examination showed a yellow infiltrate above the fovea with minimal surrounding retinal edema in the right eye. she was diagnosed as acute hsv infection by positive igg and igm antibodies to hsv (6). in our case, however, the haze media prevents us to examine the peripheral retina completely to detect any probable area of retinal necrosis. together with systemic antiviral therapy, an intravitreal injection of ganciclovir was administered at same session after anterior chamber paracenthesis to further combat any undetected area of retinal necrosis. our case proves that fba is only a descriptive fundus finding and not a disease entity by itself and physicians must try to identify a possible etiology. vzv, hiv, ebv, cmv, rubella, toxoplasma and streptococci group a can be the triggering antigen source, so only after effective and proper antiviral coverage, systemic steroid would result in controlling inflammation (7). the reactivation of vzv infection in immunocompetent persons that presented by aseptic meningitis or other neurological disease with or without rash, is reported previously (8). given the atypical rash in our patient, we did nt think of vzv reactivation at the outset which resulted in delayed institution of antiviral agent. therefore, awareness of the protean vzv manifestations is of utmost importance to prevent permanent damage. diagnosis of vzv - induced neurological and ocular disease may be facilitated by using pcr technology to detect vzv dna in csf and aqueous humor of patients with uveitis. unfortunately, we did nt look for anti - vzv antibody in the csf that is thought to be superior to the detection of vzv dna in csf in the diagnosis of vzv meningoencephalitis (11). in our case, the initiation of the systemic steroid together with anti - viral agent improved her systemic condition as well as intraoculr inflammation. the anterior chamber and vitreous inflammation subsided, vascular sheathing resolved and retinal hemorrhages gradually disappeared. however, the visual acuity did not improved because of the presence of a recalcitrant macular edema. intravitreal injection of anti - vascular endothelial growth factor (anti - vegf) has been widely used in recent years to treat macular edema secondary to diabetic retinopathy, central and branch retinal vein occlusion and uveitis. because of inheriting immunosuppressive potential of anti - vegf agent, we combined bevacizumab intravitreal injection, with ganciclovir intravitreal injection from a different site, to prevent potential chance of viral proliferation and invasion. most patients with fba have been treated with systemic steroids and acyclovir has been used with unknown effect because herpes virus group may cause frosted branch retinal angiitis(1, 12). in our patient with blurred vision, aseptic meningitis, positive pcr for vzv in aqueous humor and positive serum varicella igg, the diagnosis of reactivation of vzv is confirmed. this case illustrates that vzv should be considered in the differential diagnosis of retinal perivasculitis. ziyaeyan showed that a significant proportion of children and adults are at risk of vzv infection (13) so, vzv vaccine should be incorporated in childhood vaccination program in iran. | introduction : frosted branch angiitis(fba) is a panuveitis with sheating of all retinal vesssels.case presentation : herein we report an immunocompetent person who presented with fever, headache, atypical rash, and hazy vision. ophthalmoscopy of both eyes revealed perivascular sheathing with frosted branch angiitis pattern in veins, patchy retinal hemorrhages. aqueous pcr analysis turned positive for vzv.discussion:this case illustrates that vzv should be considered in the differential diagnosis of retinal perivasculitis. since a rapid and accurate diagnosis is crucial for prompt administration of antiviral therapy, pcr - based analysis of aqueous humor is a valuable tool for detecting viruses. |
however, it may cause unexpected complications during or after surgery, the most common of which include temporary dysphagia, hematoma, and hoarseness due to the injury of recurrent laryngeal nerve7). in rare cases the authors experienced a case of a large perforation in the hypopharynx that occurred after an anterior cervical discectomy in a patient transferred to our institute with major complaints of slight fever and subcutaneous emphysema. to avoid a catastrophic complication,, we report a case of large perforation of hypopharynx with a review of relevant literature, then we discuss the imaging and clinical features of this rare complicated case. a 58-year - old male transferred to our emergency room (er) complaining of painful swelling in the anterior cervical region. the patient underwent c3 - 4 discectomy and total disc replacement 3 days ago with the diagnosis of c3 - 4 disc herniation in local spine clinic. a physical examination identified painful edema in the surgical site of the anterior cervical region and necrotic skin tissue. a hematological analysis demonstrated that his leukocyte count was 9670/ml, with an erythrocyte sedimentation tare of 70 mm / hr and a c - reactive protein of 19.9 mg / dl. an esophagogram with gastrografin showed that a large amount of contrast medium had leaked into the right lateral cervical region (fig., the patient was diagnosed with a perforation in the hypopharynx following the anterior cervical discectomy. as a result the operative findings included extensive soft tissue necrosis and abscess formation, both of which were removed and cultured. the perforated site was approximately 2 cm long and was found outside the pyriform sinus in the right hypopharynx (fig. the mucous membrane was sutured with vicryl 3 - 0 in two layers from the outside. there was an attempt to reinforce the perforated part with the surrounding muscle, but severe inflammation prevented its use. consequently, tissel was spread on the surgical site before the tube was inserted for drainage. on the 10th day after the operation, an esophagogram conducted on the 13th day after the operation showed no contrast leakage (fig. thereafter, the patient was transferred to the plastic surgery department of our institute to receive skin graft. a full - thickness skin graft was performed before the patient was discharged from the hospital. the patient was monitored for six months after the operation and showed no sign of complications. although anterior approaches to the cervical spine are popular and safe, they are not free of complications7). other complications include hematoma, vocal cord paralysis due to damage of the recurrent laryngeal nerve, leakage of cerebrospinal fluid, and perforation of the esophagus or hypopharynx. among these complications, the incidence of esophageal perforation is low and it has been reported to be approximately 0.3% ; the rate of hypopharynx perforation is much lower than that of esophageal perforation3). the symptoms of hypopharynx perforation include hemorrhage, pain, dysphagia, and subcutaneous emphysema. among these symptoms, as the symptom develops, it can lead to cellulitis of the soft tissue due to inflammation, edema of the cervical region, fever, and hematosepsis5,6). in this case, the patient did not have a conspicuous fever but had painful swelling in the anterior cervical region as well as cellulitis and necrosis in the surrounding soft tissue. in addition, other findings included subcutaneous emphysema and pneumomediastinum in the cervical region. if hypopharynx perforation is diagnosed late, it is likely to cause fatal complications, such as retropharyngeal abscess, mediastinitis, hematosepsis, and meningitis, which may lead to death4,8). to diagnose perforation of hypopharynx at an early stage, it is critical to examine the medical history and conduct a careful physical examination. cervical and chest radiographs enable the detection of subcutaneous emphysema, pneumomediastinum and pneumothorax, and cervical or chest ct scans enable the detection of a cervical abscess. the esophagogram which uses the water soluble contrast medium, gastrografin and ct may also determine the accurate location and degree of damage, including whether it has spread to the mediastinum, based on an air - biliarygram. therefore, such methods are essential for determining the appropriate surgical treatment and access route2,11). if clinical suspicion remains, the use of barium, which is more sensitive, is suggested instead of a water - soluble contrast medium1). this case also showed that it was possible to make a precise diagnosis using ct and an esophagogram with gastrografin after examining the medical history and conducting a physical examination. because the perforation was large, it was conspicuous enough to avoid the use of barium. conservative treatment may be effective when the perforation less than 2 cm, the damage is limited to the pharynx without spreading to the cervical esophagus, the cervical abscess is not found, or there are no general symptoms of mediastinitis or hematosepsis. the conservative treatment for perforation requires the extensive administration of intravenous antibiotics and strict restriction of oral diet. when a perforation is diagnosed early and its size is considered large, emergent repair is essential. in addition, surgical intervention is also necessary when contrast medium has leaked into the mediastinum or the pleural cavity, leading to the symptoms of pneumothorax and hematosepsis5). as soon as the patient, in this case, transferred the er, cervical ct and esophagogram with gastrografin were performed to confirm the perforation of hypopharynx. due to the suspicion of inflammation and abscess formation at the surgical site, the patient underwent exploration and drainage, and the primary suture as shown in this study, primary suture can be sufficient for healing the damage to the hypopharynx. however, if the damage is more large, pedicled sternocleidomastoid muscle flap interposition may be required to reinforce the sutured site10). in this case, oral food intake after the cervical discectomy resulted in a leakage of the ingested food into the surrounding tissues. this leakage aggravated inflammation of the tissue and abscess formation, which led to severe necrosis of the surrounding region. as a result, it was impossible to use the muscle for reinforcement. however, the irrigation and complete removal of the necrotic tissues were performed without muscle reinforcement. after anterior cervical discectomy, a diagnosis of perforation of hypopharynx must be precise and prompt if conservative treatments are unlikely to suffice or if serious complications, such as mediastinitis or hematosepsis, are likely to arise. | perforation of the hypopharynx, which can occur after anterior cervical approach, is a very rare type of complication. if diagnosed late, it can lead to very fatal course, such as mediastinitis and hematosepsis. therefore, a precise and prompt diagnosis is crucial. when conservative treatment alone is not expected to heal the perforated site or is likely to lead to serious complications, surgical treatment becomes necessary. this report demonstrates that surgical intervention performed immediately after an early diagnosis can lead to the successful treatment of a large perforation in the hypopharynx on a 58-year - old male patient. |
the opportunistic yeasts belonging to the genus candida have been associated with a wide range of human infections and significant mortality and morbidity. the clinical manifestations range from infections of the superficial skin and its appendages to deep - seated or disseminated candidiasis. have been the third most common nosocomial pathogens associated with blood stream infections (bsis). among noninvasive infections, vulvovaginal candidiasis (vvc) affects approximately 75% of the women with at least one episode during their lifetime. though c. albicans has been associated mostly with human infections, there has been an increase in the prevalence of infections due to non - albicans candida in the recent past [3, 4 ]. of the many pathogenic non - albicans species known, c. tropicalis, c. parapsilosis, c. kefyr, c. krusei, and c. guilliermondii are mostly associated with human infections. candida spp. have grown from successful commensals to pathogens in various body sites with the help of many virulence determinants. though non - albicans candida have proven to be common pathogens in most invasive infections, little attention is given to their virulence attributes. adherence to host tissue, response to environmental changes, secretion of hydrolases, and biofilm production are a few of the most important virulence mechanisms of these fungi. the exact mechanism of candidal pathogenesis is of prime interest for many researchers globally. with the increasing rate of infections due to non - albicans candida and varying susceptibilities to commonly used empirical antifungal agents like fluconazole however, from a microbiologist 's perspective, conventional methods like sugar assimilation, fermentation reactions, and identification based on morphological characters are tedious and time - consuming. this emphasizes the need for testing various work flow algorithms using commercially available diagnostic modalities to generate a rapid and accurate identification of candida species. the aim of this study was to evaluate the diagnostic utilities of commercial chromogenic medium and vitek 2 compact system against polymerase chain reaction (pcr) in species identification of clinical isolates of candida. it was also aimed at detection and comparison of in vitro production of virulence factors among invasive and noninvasive (mucosal) clinical isolates of candida and at studying their antifungal susceptibility patterns. the present study was carried out on 110 clinical isolates of candida spp. obtained from patients diagnosed either with blood stream infection or with vulvovaginal candidiasis during a period of one year (march 2012february 2013) in the laboratory attached to a tertiary care teaching hospital in southern india. two commercially available methods for the species identification of candida, namely, hicrome candida differential agar (himedia, mumbai, india) and vitek 2 compact system (biomerieux, marcy l ' etoile, france), were compared with species - specific pcr as the gold standard. candida isolates were streaked on hicrome agar plates as per manufacturer 's instructions and incubated at 37c for 48 hrs. the plates were examined at 24 hrs and 48 hrs for the colony morphotypes. at the end of 48 hrs, the final reading of all the plates was taken by two independent observers to determine the interobserver measure of agreement (). fresh subcultures of all the candida isolates were obtained on sabouraud 's dextrose agar (sda) plates for identification and antifungal susceptibility testing using the vitek 2 compact, i d yst, and aft (ys06) cards as per manufacturer 's instructions. in ys06 cards, antifungal susceptibility results based on interpretative guidelines recommended by clinical laboratory standards institute (clsi) 2012 were obtained for flucytosine, fluconazole, voriconazole, amphotericin - b, and caspofungin. fresh subcultures of the isolates were used for dna extraction by the heat lysis method with slight modifications. briefly, 2 - 3 colonies of candida spp. were suspended in 350 l of molecular grade water and vortexed to make a uniform suspension followed by series of steps including ultrasonication for 2 mins and heating at 80c for 5 mins followed by centrifugation for 3 mins at 10,000 rpm. the supernatant containing dna was then subjected to multiplex pcr, targeting the species - specific genes of candida from the internal transcribed spacer (its) region. the pcr conditions and reaction composition were modified and optimized at our laboratory from previously described protocol. briefly, the pcr reaction mix (25 l) is composed of 12 l of pcr ready mix (genei, bangalore, india), 8 l of molecular grade water, 0.16 m each of primers, and 5 l of dna. the amplicons c. albicans (218,110 bp), c. glabrata (482 bp), c. parapsilosis (229 bp), c. tropicalis (218 bp), and c. krusei (182 bp) were illuminated under uv light in 3% agarose incorporated with ethidium bromide and analyzed using the gel image analysis software of bio - print mega system (vilber lourmat sas, marne - la - valle, france). the proteolytic activity (prz) was calculated as the ratio of diameter of the colony to the total diameter of the colony and the precipitation zone. phospholipase production of the isolates was determined using egg yolk agar medium as previously described. the phospholipase activity (pz) was measured in a similar manner as the proteinase activity. esterase production was determined using a medium containing tween 80 according to earlier reported method. presence of a halo around the colony when observed against transmitted light was considered as positive for esterase production. the plates were incubated for a maximum of 5 days before terming them nonesterase producers. microtitre plate assay was used to determine the biofilm producing ability of the isolates. sabouraud 's dextrose broth (6% glucose ; ph 6) was used to induce the biofilm production in microtitre plates inoculated with 10 cfu / ml freshly subcultured candida isolates. the plates were incubated at 30c for 48 hrs, washed with 0.15 m phosphate buffer saline to remove the planktonic cells, and then stained with 0.1% crystal violet, followed by estimation of optical density at 540 nm. for each isolate the biofilm production was tested in triplicate and c. albicans atcc 24433 was used as a standard strain. the strength of biofilm production (weak, moderate, and strong) was calculated using a method previously reported. 16, il, chicago) was used for data analysis. to estimate the frequencies, measure of agreement () between the diagnostic tests was estimated using cross tabulations and kappa () value > 0.6 was considered as a test with good agreement with the gold standard, and test was used for determining the association of virulence factors with site of isolation. of the total 110 isolates studied, 54 (49%) were obtained from the blood cultures of patients with candidaemia comprising c. tropicalis (25, 46%), c. parapsilosis (16, 30%), c. albicans (7, 13%), c. glabrata (2, 4%), c. krusei (1, 2%), and other candida spp. another 56 (51%) candida isolates were from women of reproductive age group diagnosed with vvc involving c. albicans (34, 61%), c. glabrata (11, 20%), c. tropicalis (5, 9%), c. parapsilosis (2, 4%), c. krusei (2, 4%), and other candida spp. all the isolates were primarily identified using species - specific pcr (figure 1). among the candida spp. isolates, c. albicans (61%) and c. glabrata (20%) were most common in vvc, whereas c. tropicalis (46%) and c. parapsilosis (30%) were most common in bsis. as pcr did not identify the species of 5 candida isolates, only 105 candida were included in the analysis of diagnostic utility of hicrome candida differential agar and vitek 2 compact system. the sensitivity and specificity of hicrome candida differential agar and vitek 2 compact for the species identification of candida are shown in table 1 and the comparison between the three methods is depicted in table 2. the antifungal susceptibility testing was done using the vitek 2 compact system for all the 105 candida isolates. isolates were classified as sensitive, intermediately sensitive, and resistant to each antifungal agent based on their minimum inhibitory concentrations (mics). all the isolates were uniformly susceptible to amphotericin - b and flucytosine in our study. c. parapsilosis demonstrated the least susceptibility (64%) to voriconazole followed by c. tropicalis (79%). isolates from bsis showed intermediate sensitivity or resistance to various antifungal agents, while fluconazole intermediate susceptibility was seen in two c. albicans (2/34, 6%) isolated from vvc. of the 105 isolates, 11 had lost viability and hence were not available for further virulence studies. we could study virulence characters of 94 isolates comprising 48 candida from blood and 46 from the high - vaginal specimens. varied production of virulence factors among the two infection groups and various species of candida were observed. proteinase production was more common among invasive isolates (17, 35%, p = 0.017) in comparison with high - vaginal isolates (6, 13%) when tested using test. phospholipase production was more common among high - vaginal isolates (13, 28%, p = 0.001) than in blood isolates (2, 4%). esterase production and biofilm production were observed in isolates from both infections with no statistically significant difference (p > 0.05) (table 4). among the various species tested, c. albicans (24/39, 62%) and c. tropicalis (18/30, 60%) were the leading producers of biofilm. comparison of production of virulence factors among c. albicans and non - albicans candida did not show a statistically significant difference with respect to the production of biofilm (p = 0.295), esterase (p = 0.478), and proteinase (p = 0.09). production of phospholipase was significantly higher in c. albicans (93%, p < 0.001) than in non - albicans candida (7%). distribution of virulence factors among study isolates based on site of isolation and various species is depicted in table 4. coproduction of proteinase and esterase was observed in 12/48 (25%) of the blood isolates, in comparison with 2/46 (4%) of the high - vaginal isolates (p < 0.001). however, production of more than one virulence factor among c. albicans and non - albicans candida did not show any statistical significance (14/39 (36%) and 15/55 (27%), p = 0.55) on analysis. increasing number of treatment failures, associated mortality, and shift to more resistant isolates advocate the need for species identification in candida. utility of molecular techniques like pcr, rflp, and aflp is very promising but is expensive restricting their utility to research and reference laboratories. in our study, we used pcr for species identification as the standard method and compared the utilities of vitek 2 compact and hicrome candida differential agar in identification of candida. we observed that the sensitivity and specificities of both methods were comparable with each other and pcr. the measure of agreement () for vitek 2 compact and hicrome candida differential agar with pcr was 0.895 and 0.826, respectively, suggesting the applicability of both methods in routine diagnostic laboratories. the performance of hicrome candida differential agar in our study was comparable to previous reports. however, we observed low sensitivity (79%) of hicrome agar in identification of c. parapsilosis, which could probably be due to the same color of colony (cream to white) for c. parapsilosis, c. glabrata, and c. kefyr as instructed by the manufacturer. however, in our study three isolates of c. parapsilosis had white colonies with light green centers mimicking the colonies of c. albicans. vitek 2 compact system was rapid in reporting the identity and antifungal susceptibility results with an average turnaround time of 17 hrs, while hicrome candida differential agar scores better for its general utility as a primary isolation and identification medium from clinical specimen. we also noticed a high interobserver measure of agreement (= 0.892) for interpreting the results of hicrome candida differential agar indicating high reproducibility. the present study demonstrates higher isolation rates of non - albicans candida in bsis possibly due to the involvement of cases from high risk patient care areas. c. tropicalis was the most common pathogen isolated from patients with blood stream infections. we report a higher proportion of c. albicans in vvc, which is in contrast to the observations of few studies indicating the changing trends in etiologies with a shift towards non - albicans candida. geographic differences and increased use of self - medications with antifungal agents might have contributed to upsurge of infections by more resistant candida spp. [16, 17 ]. however various studies worldwide have shown a predominance of c. albicans in mucosal infections and colonization [18, 19 ]. sensitivity rates of c. albicans for fluconazole, flucytosine, voriconazole, amphotericin - b, and caspofungin were 95%, 100%, 97.5%, 100%, and 97.5%, respectively, and these rates are similar to those reported by pahwa. and higher than the sensitivity rates reported by chander.. the latter group reported a far lower sensitivity rate of c. albicans to fluconazole (38%) as the study isolates were obtained from nosocomially acquired candidemia and hence higher resistance to common antifungal agents. however, as was observed in our study, no resistance to amphotericin - b was observed. there are reports of varying rates of resistance to azoles among isolates, primarily non - albicans candida from vvc. numerous virulence attributes of candida have been postulated that can facilitate the transition of a mucosal colonizer to a fatal disseminating pathogen. while adhesions help the yeast in adhering to the host cell surfaces, hydrolytic enzymes like proteinases, phospholipases, and lipases along with hyphal forms promote the penetration through cells. in addition to these virulence attributes of candida, host immunity plays a major role in restricting these infections to either a localized form as seen in immunocompetent hosts or a deep - seated and disseminating form of infections common among immunocompromised hosts. an emphasis on hydrolytic enzymes produced by candida spp. can help in understanding the disease process better as these enzymes have activity on a wide array of host substrates. secreted aspartyl proteinases (sap) in candida are known to enhance the hypha formation, epithelial cell damage, invasion, and inflammatory responses. in vivo experimental models also demonstrated an increase in the invasiveness of yeasts with the production of proteinases [22, 23 ]. an association between production of sap and increased hyphae formation in vivo has been reported previously. considering the coregulation of sap and hyphal production in candida spp., we assume the isolates from bsis to be predominantly proteinase producers. in our study, we observed a statistically significant increase in the production of proteinases among the blood isolates compared to those from vvc. the production of proteinases was significantly high among c. tropicalis, possibly contributing to its increased occurrence in bsis. though c. albicans is the leading producer of proteinases among all the species of candida, recent studies suggest the production of 4 types of sap by c. tropicalis. phospholipases are a group of enzymes produced by candida species that primarily help in digesting the phospholipids of the host cells leading to cell lysis. c. albicans is the major producer of phospholipases, whereas a less proportion of non - albicans candida produce this enzyme. it is postulated that more sensitive methods are needed to detect the lesser amount of phospholipases produced by non - albicans candida. in our study, the majority (93%) of phospholipase producers were c. albicans, while only 7% of non - albicans candida were positive for phospholipases. phospholipase production was predominately seen in vaginal isolates possibly due to higher number of c. albicans in this group. though production of esterase has been documented as virulence character among clinical isolates of candida, little is known about the pathogenesis. recent investigations suggest the mechanism of virulence is due to the cytotoxic effects of lipases and esterase in the host tissues. however, we did not find a significant difference in esterase production among the invasive and noninvasive isolates. the proportion of esterase positive isolates in this study was lower in comparison to few published reports [28, 29 ]. biofilm production is considered as one of the most potent pathogenic traits attributed to treatment failures and recurrent infections. we report similar rates of biofilm production among blood and mucosal isolates (52% and 54%), but higher biofilm production was seen in c. albicans as compared to non - albicans candida. though the exact reason for the higher biofilm production rates by c. albicans is not completely understood, studies using the scanning electron microscopy to understand the complex biofilm architectures have attributed the integrity and strength of these biofilms to the higher number of hyphal elements produced by c. albicans than c. tropicalis and c. parapsilosis. the latter two species form biofilms of lesser strength and the biofilm in these two species is primarily constituted of micro colony aggregates of yeast cells. considering the need of high glucose concentrations for non - albicans candida to produce biofilms in vitro we used sdb with 6% glucose. we anticipated high glucose concentration to simulate in vivo conditions for non - albicans candida thus promoting the production of detectable biofilms in vitro. notably, biofilm production of c. tropicalis (62%) was comparable to c. albicans (60%) in our study. with an increasing trend of blood stream infections due to c. tropicalis, further studies to understand its biofilm architecture and antifungal susceptibility are warranted. we could not compare the production of virulence characters among the same species isolated from different sites due to small number of few candida species. however, current information might be useful for a better understanding of the role of host environmental factors and individual virulence factors in site specific disease development. similarly, interspecies comparison of virulence mechanisms among all the pathogenic species of candida could not be achieved in the current study due to the small numbers of few species of candida. however, our study involved common candida isolates form invasive and mucosal infection sites and analyzed the production of important virulence determinants, especially among less explored non - albicans candida. exploring genetic mechanisms of virulence in non - albicans candida would help in understanding the spectrum of disease and changing trends in disease epidemiology. from this study, we found c. albicans followed by c. glabrata were the leading pathogens causing vvc, while c. tropicalis and c. parapsilosis were predominant in blood stream infections. vitek 2 compact and hicrome differential agar have proven their efficacy in speciation of candida ; however incorporation of either or both techniques in daily practice depends on the throughput and resource settings of the laboratory. we also observed an overall increase in the production of virulence characters among non - albicans candida, especially c. tropicalis. most of c. albicans in our study were susceptible to fluconazole, favoring its use in empiric therapy of vaginal infection. resistance to various commonly used antifungal agents among non - albicans candida suggests the need for species identification in routine laboratories for initiation of appropriate antifungal therapy. | candida spp. have emerged as successful pathogens in both invasive and mucosal infections. varied virulence factors and growing resistance to antifungal agents have contributed to their pathogenicity. we studied diagnostic accuracy of hicrome candida differential agar and vitek 2 compact system for identification of candida spp. in comparison with species - specific pcr on 110 clinical isolates of candida from blood stream infections (54, 49%) and vulvovaginal candidiasis (56, 51%). c. albicans (61%) was the leading pathogen in vvc, while c. tropicalis (46%) was prominent among bsis. hicrome agar and vitek 2 compact had good measures of agreement () 0.826 and 0.895, respectively, in comparison with pcr. we also tested these isolates for in vitro production of proteinase, esterase, phospholipases, and biofilms. proteinase production was more among invasive isolates (p = 0.017), while phospholipase production was more among noninvasive isolates (p = 0.001). there was an overall increase in the production of virulence factors among non - albicans candida. identification of clinical isolates of candida up to species level either by chromogenic agar or by vitek 2 compact system should be routinely done to choose appropriate therapy. |
the main constituents of cannabis are 9-tetrahydrocannabinol (thc) and cannabidiol (cbd) [13 ]. they act on specific cannabinoid receptors, mainly cannabinoid receptors type 1 and 2, cb1, and cb2 [48 ]. although it is still debated whether to consider the g protein - coupled receptor 55 (gpr55) as a cannabinoid receptor, several evidences suggest that it might also be a cannabinoid receptor. beside the effects caused by exogenous cannabinoids, cb1 and cb2 are stimulated by two major endogenous ligands : n - arachidonoylethanolamide (anandamide, aea) and 2-arachidonoylglycerol (2-ag) [10, 11 ]. membrane phospholipids are metabolized by calcium - dependent phospholipases to release aea and 2-ag (for a review, see). briefly, the precursor of aea is n - arachidonoyl phosphatidylethanolamine (nape) which is hydrolyzed by a phospholipase d to release aea and phosphatidic acid. 2-ag synthesis is based on hydrolysis of diacylglycerols (dag) by two dag - lipase isozymes, dagl and dagl. the cellular level of these ligands is regulated by several enzymes, especially fatty acid amide hydrolase (faah), monoacylglycerol lipase (mgl), and cyclooxygenase-2 (cox-2). together, cannabinoid receptors, ligands, and enzymes constitute a functional cannabinoid system involved throughout the body in several physiological mechanisms as well as in pathophysiological conditions. importantly, this system is implicated in the regulation of the central nervous system (cns) neurobiology. interestingly, the cannabinoid system plays a major role in the regulation of cns neurotransmission [14, 15 ]. as cb1 receptors have predominantly a presynaptic localization in the brain neurons, they play a postsynaptic regulatory role by modulating the release of several neurotransmitters such as gamma - aminobutyric acid (gaba), glutamate, and dopamine [1618 ]. for example, the stimulation of a glutamatergic neuron results in a synaptic release of glutamate, which induces a postsynaptic influx of calcium through nmda receptors. consequently, the process of synaptic strengthening is activated by this increase of the calcium concentration. afterward, there is a postsynaptic synthesis of endocannabinoids by the stimulation of postsynaptic metabotropic glutamate receptors, mglu. through cb1 presynaptic receptors, endocannabinoids regulate the presynaptic glutamate release and avoid any excessive postsynaptic release of calcium [13, 14 ]. exogenous cannabinoids, by binding to the cb1 receptors, disrupt the regulation of glutamate release mediated by endocannabinoids. an excess of postsynaptic influx of calcium occurs, thus accelerating the pruning synaptic process and the apoptosis of the cell [13, 14 ]. in the cns for example, through postsynaptic cb1 receptors they regulate the slow self - inhibition of neocortical interneurons and pyramidal neurons, an endocannabinoid dependent persistent change of somatodendritic excitability. the retina is anatomically and developmentally an extension of the cns, and the retina and the brain are connected by the optic nerve, the axons of the ganglion cells, through the lateral geniculate nucleus. they express several neurotransmitters such as dopamine [21, 22 ], serotonin, glutamate, and gaba [24, 25 ]. retinal processing, as measured by electrophysiological measurements (flash electroretinogram (ferg), pattern electroretinogram (perg), and electrooculogram (eog)) is sensitive to pharmacological drugs acting on the cns neurotransmission [2629 ]. finally, cns disorders, such as neurological, psychiatric, and addictive diseases, display manifestations in the retina [15, 3034 ]. briefly, the retina represents the neural portion of the eye and is composed of several neuronal cell layers (figure 1). then, it reaches the outer segment of the retina where the photoreceptor cells are located, namely, rods and cones. at this level, the light is absorbed by the photopigments of the photoreceptors, thus initiating the conversion of light into an electric signal, called the phototransduction process. this signal is transferred to the bipolar cells and then to the ganglion cells, the axons of which form the optic nerve, which transfers the visual information to the visual cortex via the lateral geniculate nucleus. this signal is also under the influence of interneuron cells, namely, amacrine and horizontal cells. the retinal pigment epithelium is also part of the retina and plays a role in several trophic functions such as light absorption, photoreceptor disk renewal, and immune modulation, to name a few [20, 35 ]. in the cns, the manipulation of endocannabinoids has been reported to attenuate brain damages induced by a variety of insults. moreover, the cannabinoid system signaling pathways is considered as a critical process involved in neuron survival. for instance, an upregulation of the endocannabinoid system is known as an adaptive response that attenuates cns damages in the context of trauma and neurodegenerative diseases [36, 37 ] and is considered as a neuroprotective strategy against neuropathological states. in line with these observations, the inhibition of endocannabinoid degradation induces a decrease in pathological tissues in experimental conditions of cns injuries. as the retina is considered as a part of the cns, it is now crucial to assess whether the cannabinoid system is distributed in the retina and is involved in retinal pathological or retinal protective conditions. the cannabinoid system has been detected in ocular tissues and other critical stages of visual information processing. in ocular tissues, cb1 receptors have been localized in the ciliary body of rat, bovine, and human and in the trabecular meshwork in bovine and human [4042 ]. cb1 receptors were also found in the nonpigmented ciliary epithelium in human and bovine and in the conjunctival epithelium in mouse and human [4244 ]. hydrolysis of anandamide was detected in the porcine iris, choroid, lacrimal gland, and optic nerve. aea and 2-ag were expressed in human cornea, ciliary body, iris, and choroid [46, 47 ]. endocannabinoids are also found in later and more integrated stages of visual processing, especially in the lateral geniculate nucleus (lgn) and in the visual brain (for a review see). briefly, cb1 receptors are found in the lgn, superior colliculus, and suprachiasmatic nucleus in rat and mouse and in the lgn and the superior colliculus in human. cb1 and cb2 are expressed in the primary (v1) and secondary (v2) visual cortex of rat and mouse. in nonhuman we first review the distribution of the cannabinoid receptors, ligands, and enzymes in the retina. then, we review studies that have examined the role of the cannabinoid system in retinal neurotransmission, neuroplasticity, and neuroprotection. based on these results, we argue for the development of synthetic cannabinoids as therapeutic agents for the treatment and the prevention of retinal diseases. finally, we support potential effects of cannabis use on human retinal processing and we present several functional measurements allowing the rigorous assessment of the retinal function in cannabis users. in order to thoroughly explore the role of the cannabinoid system in the retina, a search for relevant articles was conducted in the pubmed, sciencedirect, and google scholar databases using the following keywords : (cannabinoid system or endocannabinoids or cannabinoid ligands or cannabinoid receptors or cannabinoid enzymes) and (neurotransmission or neuroplasticity or neuroprotection) and (retina or retinal). all results up to june 1, 2015, were examined for the selection process. relevant publications were chosen through an individual independent selection of titles by the following authors : thomas schwitzer, raymund schwan, anne giersch, and vincent laprevote. the articles selected had to be written in english and be related to the topic of the review. numerous studies have shown the presence of the cannabinoid receptors cb1 and cb2 in the retina of several animal species such as tiger salamander, goldfish, rat, mouse, chick, and monkey [41, 4858 ]. cb1 receptors are detected in inner and outer plexiform layers and two synaptic layers of the retina of all of these species. cb1 receptors are also expressed in the main cells of the neural retina, especially in the cone pedicles and rod spherules of photoreceptors of the same species. furthermore, they are found in horizontal cells, amacrine cells, ganglion cells, and ganglion cell axons of these species except goldfish [53, 56 ]. in monkey, rat, mouse, and chick, cb1 receptors are detected in inner and outer segments of the photoreceptors. in monkey, cb1 receptors are expressed throughout the retina from the fovea to the retinal periphery and in particular in cones of the central retina. in goldfish, cb1 receptors are mainly localized intracellularly and on the plasma membrane of photoreceptors, bipolar cells, and amacrine cells. cb1 receptors are also found in mller cells and in synaptic terminal of on and off bipolar cells in goldfish. although the presynaptic location of cb1 receptors was readily described, especially in gabaergic and glutamatergic neurons, there are several evidences also indicating a postsynaptic localization.. showed that retinal ablation induced an increase in level of cb1 proteins in the optic tectum and other retinorecipient visual areas in the adult chick brain with no change in cb1 mrna levels. this increase in cb1 receptors expression after retinal ablation suggests a postsynaptic location of these receptors in the retinotectal axons., cb2 receptors are expressed in photoreceptors, horizontal cells, amacrine cells, inner nuclear layer, inner plexiform layer, retinal pigment epithelium, and retinal ganglion cell layer, especially in the somas of retinal ganglion cells [54, 55 ]. in mouse, cb2 receptors are localized in cone and rod photoreceptors, horizontal cells, amacrine cells, bipolar cells, and ganglion cells. cb1 receptors are detected in outer segments of photoreceptor cells, inner plexiform layer, outer plexiform layer, two synaptic layers of the retina, inner nuclear layer, ganglion cell layer, and retinal pigment epithelium cells [40, 43, 60 ]. the two main endogenous cannabinoid ligands acting on cannabinoid receptors, namely, anandamide and 2-ag, are found in the retina of tiger salamander, goldfish, rat, mouse, chick, bovine, porcine, and monkey [45, 56, 61, 62 ]. in human, 2-ag is expressed at a high level in the retina [46, 47 ], whereas anandamide is detected at a lower level in the retina [42, 46, 47 ]. the cellular level of retinal endocannabinoids is regulated by several main enzymes : faah, mgl, and cox-2. faah is an integral membrane protein and is expressed throughout the monkey retina, from the fovea to the retinal periphery. most specifically, faah is detected in photoreceptors, outer plexiform layer, inner nuclear, inner plexiform layer, and retinal ganglion cell layer in monkey. concerning photoreceptors in monkey retina, faah is preferentially expressed in cones of the central retina and is mainly located in cone pedicles and rod spherules. faah is also detected in cone and rod bipolar cells and in ganglion cell somas and axons in the monkey retina. in the rat, goldfish, and bovine retina, mouse and rat a faah activity is detected in rods, bipolar cells, horizontal cells, amacrine cells, mller cells, and ganglion cells. in goldfish, faah is most prominent over mller cells and cone inner segments and is observed at a lower level in amacrine cells, cell bodies in the ganglion cell layer, and the inner plexiform layer. faah is also expressed in the human retina, in particular in retinal pigment epithelium cells. preliminary findings are consistent with the detection of mgl in inner plexiform layer, rod bipolar cells, amacrine cells, and ganglion cells in the mouse retina [13, 64 ]. similarly, the presence of cox-2 has been shown in horizontal cells, amacrine cells, ganglion cells, and mller cells in the rat retina whereas cox-2 has been detected in rod and bipolar cells in the mouse retina. enzymes allowing the synthesis of cannabinoid ligands are also expressed in the retina [64, 67 ]. for instance, nape responsible for the synthesis of aea was identified in the bovine retina by means of gas chromatography - electron impact mass spectrometry. the enzyme responsible for the synthesis of 2-ag named dagl has been detected in the two synaptic layers, the outer plexiform layer, and the inner plexiform layer of the mouse retina. importantly, dagl is localized in postsynaptic terminals of type 1 off cone bipolar cells whereas the expression of dagl appears to be restricted to retinal blood vessels in the mouse retina. dagl is detected early in postnatal development in the rat retina in photoreceptors cones and rods cone bipolar cells, horizontal, amacrine, and ganglion cells. the endocannabinoid system is detected in critical stages of retinal information processing such as photoreceptors, bipolar cells, and ganglion cells. these findings support a role of endocannabinoids in the modulation of retinal neurobiology as well as in the regulation of vertical transmission of the retinal information. the modulation of ionic channels or enzymatic activity, to name a few, can affect neurotransmission. previous studies have outlined the involvement of the cannabinoid system in these mechanisms, thus allowing the regulation of the retinal neurotransmission [13, 15 ]. several inward and outward ionic channels are known to play a major role in retinal physiology. for example, sodium, calcium, chloride, and potassium channels are involved in the phototransduction process and especially in the depolarization and the hyperpolarization of photoreceptor and bipolar cells. different studies have shown that cannabinoid agonists induced a dose - dependent reversible modulation of calcium, potassium, and chloride currents in bipolar, rod, cone, and ganglion cells [53, 56, 57, 6874 ]. these findings suggest a regulatory role of the cannabinoid system in the retinal neurotransmission at the level of photoreceptor, bipolar, and ganglion cells, which constitute three critical stages of the neural retina. as a consequence, stimulation of the cannabinoid system may modulate the vertical transmission of the retinal information and consequently may alter visual perception. a direct action of cannabinoids on retinal enzymatic activity or retinal transmitter release has also been described [72, 7579 ]. in the bovine retina, thc has led to a dose - dependent regulation of monoamine oxidase activity thus altering the retinal neurotransmission. similarly, in the isolated bovine retina, cannabinoid cb1 receptor agonists, but not cb2 agonists, inhibited aspartate release, which was blocked by cannabinoid antagonists. in perfused guinea - pig retinal discs, dopamine and noradrenaline transmission release was inhibited by activation of cannabinoid receptors cb1, which was blocked by cannabinoid antagonists [77, 79 ]. of interest, the release of several retinal neurotransmitters such as dopamine, noradrenaline, gaba, and glutamate is therefore modulated by cannabinoids [72, 73, 7679 ]. finally, studies on the goldfish retina argue for a role of the cannabinoid system in the regulation of the phototransduction cascade, the dark and light retinal sensitivity, the dark and light retinal adaptation, and the retinal contrast sensitivity [57, 70, 80 ]. the role of the cannabinoid system in the regulation of short - term and long - term plasticity in the cns has been readily described [8184 ]. the messenger ribonucleic acid (mrna) for cannabinoid receptors has been detected during development, especially in the embryonic and adult rat retina [50, 55 ]. recent findings suggest that exogenous cannabinoids may alter both synaptic transmission and synaptic plasticity in the retina, in particular in retinal ganglion cells. using whole - cell voltage - clamp recordings in retinal ganglion cells in adult and young mice (p14p21), the administration of an exogenous cannabinoid agonist significantly reduced the frequency of spontaneous postsynaptic currents (spscs) in these cells. consequently, it was suggested that the cannabinoid agonist had a presynaptic action and that it could decrease the release of both gaba and glutamate. as the largest effect was found in young mice and was different from adult mice, these results argue for a developmental role of the cannabinoid system in the maturation of synaptic retinal circuits. neuroprotection is a protecting mechanism that consists in preventing the death of damaged neurons and their degeneration, due to a hostile environment created by an initial cellular stress. using several models of retinal diseases, mounting evidence suggests that the retinal cannabinoid system might play a neuroprotective role in the retina. oxidative stress is known to be a key mechanism in the pathological process of age - related macular degeneration (amd) and diabetic retinopathy (dr) [85, 86 ]. in amd, a trend towards an increase in retinal anandamide level was observed. in a cellular model of amd, the dr is characterized by an oxidative stress, a breakdown of the blood - retinal barrier, and a proinflammatory effect to name a few, which are associated with retinal neuronal death. a recent study showed that the retinal concentration of anandamide was increased in dr whereas no change in the retinal level of 2-ag was observed. in a rat model of dr, treatment with cannabidiol significantly reduced both oxidative stress and neurotoxicity and prevented retinal cell death. consequently, exocannabinoids may be a relevant therapeutic strategy decreasing oxidative stress signaling and preventing neurodegeneration of retinal cells in amd and dr. the neuroprotective role of cannabinoids was also shown in an animal model for autosomal dominant retinitis pigmentosa (rp) on the photoreceptor degeneration, synaptic connectivity, and functional activity of the retina. in this rat model, the administration of a synthetic cannabinoid agonist from p24 to p90 induced improvements of visual function compared to vehicle - administered animals. in fact, the enhancement of vision loss was demonstrated by increased electroretinogram signals, especially scotopic a- and b - wave amplitudes. these changes were correlated with a delay in the degeneration of photoreceptors and with the preservation of presynaptic and postsynaptic elements. these crucial findings support the retinal protective role of exocannabinoids on both the structural and functional properties of retinal cells. retinal ganglion cell death may be a consequence of a neurodegenerative retinal disease such as glaucoma. an excessive extracellular glutamate release has been identified as one of the pathophysiological mechanisms inducing excitotoxicity in glaucoma via the excessive formation of peroxynitrite. the neuroprotective effect of thc and cannabidiol was observed through the limitation of peroxynitrite production in a rat model of excitotoxicity consisting in intravitreal injection of n - methyl - d - aspartate (nmda). similarly, weekly injections of thc decreased intraocular pressure and reduced the loss of retinal ganglion cells in the peripheral and central retina. in another model of intraocular pressure, an ischemic - reperfused retina model, systemic administrations of a faah inhibitor decreased enzymatic activity and consequently reduced the retinal damage caused by the ischemic - reperfusion mechanism. additionally, intravitreal injections of an aea agonist reduced retinal ganglion cell loss which was abolished by the systemic administration of a cb1 antagonist. in another example, the administration of an inhibitor of faah increased retinal ganglion cell survival following optic nerve axotomy in young and aged rats. this effect was affected by a cannabinoid antagonist and was associated with both an increase in anandamide and a decrease in anandamide metabolites, with no effect on 2-ag level. all these results are in accordance with the relevance of local or systemic administrations of exocannabinoids in the prevention of retinal ganglion cell loss due to retinal diseases. finally, anandamide and 2-ag were also involved in the modulation of the innate immune response in human retinal mller glia to combat inflammation in the retina during human immunodeficiency virus (hiv) infection. the hiv infection induces a retinal neurodegeneration by increasing the inflammatory response, which consequently causes retinal impairments. in this case, both anandamide and 2-ag induced a decrease in the inflammation and limited the retinal loss. these data argue for the use of cannabinoids in retinal diseases as new therapeutic agents to prevent neurodegeneration and cell death. according to the large distribution of endocannabinoids in the retina and considering the role of cannabinoids in modulating the retinal neurophysiology, it is now crucial to consider potential effects of cannabis, after both acute and regular use, on the structural and functional characteristics of the human retina [15, 31 ]. the retina is organized in cell layers of which functional properties can be assessed by electrophysiological techniques (figure 1). the electroretinogram (erg) measurements, the ferg, and the perg allow the assessment of the functional properties of specific cell types in the neural retina [96, 97 ]. the ferg measures the electric biopotential evoked mainly by photoreceptor cells, namely, rods and cones, and the on - bipolar and mller cells complex, in response to a light stimulation. ferg recordings performed under photopic and scotopic conditions are called light- and dark - adapted ferg, respectively, according to the flash luminance intensity used, which is measured in candelassm (cdssm). two main components are usually observed on a typical ferg trace : an electronegative component called a - wave, followed by an electropositive component named b - wave. the a - wave is generated by the hyperpolarization of the photoreceptors and the b - wave reflects the depolarization of the on - bipolar and mller cells complex. although the a - wave and the b - wave are commonly the most analyzed, other components are detected in the full - field erg such as the photopic negative response (phnr). this response is a negative component that follows the b - wave and reflects the activity of innerretinal cells, especially the ganglion cells. indeed, a brief red flash against a blue background is an optimal configuration for eliciting this response. the perg records the central macular function of the retina, as well as the retinal ganglion cells response using reversal black and white checkerboards viewed with a central fixation. to investigate the transient perg, two main waves are usually described on the transient perg trace : a positive wave named p50, followed by a negative wave called n95. like the ferg, both the amplitude and implicit time of these waves can be measured. p50 is, in part, attributed to the retinal ganglion cells and macular photoreceptors and is used to evaluate the macular function. the eog measures a variation of electrical potentials between skin electrodes located on the external and internal canthi. this variation corresponds to an electrical potential between the front and the back of the eye and is called standing potential. this potential mainly originates from the retinal pigment epithelium and varies with the retinal illumination. it is obtained by asking the subjects to make lateral eye movements in both photopic and scotopic conditions. it is possible to derive two parameters from the eog trace, namely, the dark trough, which represents the trough of the curve in dark condition whose origin remains unclear, and the light peak, which represents the maximal peak in light condition and corresponds to the maximal depolarization of the basal membrane of the retinal pigment epithelium. the neural retina is composed by three critical stages : the photoreceptor cells, called rods and cones, the bipolar cells, and the ganglion cells. the retina also contains interneuron cells : amacrine and horizontal cells and the retinal pigment epithelium. the functional properties of photoreceptors, bipolar cells, ganglion cells, and retinal pigment epithelium cells are evaluated by objective electrophysiological retinal techniques. considering the large distribution of the cannabinoid system in the different retinal cell layers and its broad involvement in the regulation of retinal neurophysiology, these electrophysiological measures may be of benefit to the evaluation of the impact of cannabis on the human retinal function. to date, only one study, to our knowledge, has evaluated the impact of cannabis on the retinal function. in this study, no ferg abnormality was found neither in a man suffering for hallucinogen persisting perception disorder after marijuana consumption nor in four heavy cannabis smokers with no visual disturbance. however, several eog anomalies were observed in the patient with hallucinogen perception showing effects of cannabis on the retinal pigment epithelium functioning. the small number of subjects and the absence of control group probably explain the lack of erg alterations. although cannabis is one of the most prevalent drugs used worldwide, there is to date few studies that have evaluated the impact of cannabis on the human visual function. these studies were already reported in several reviews [13, 15 ]. as discussed in these reviews, there is no certainty that the visual abnormalities found in cannabis users originated from the retina because no studies evaluating the retinal structural and functional properties have yet been performed in cannabis users. cannabis is a neurotoxic and neuromodulator substance that acts on several inhibitory and excitatory neurotransmitters signaling pathways in the cns. the main effects of cannabis concern the glutamatergic, gabaergic, and dopaminergic brain synaptic transmissions. all of these neurotransmitters are detected in the retina and play several key roles in retinal physiology. for instance, dopamine is the main catecholamine expressed in the human retina and is known to be involved in light adaptation. as another example, glutamate and gaba are two amino - acid neurotransmitters expressed in the retina and are involved in numerous regulatory mechanisms in the retina, especially those concerning the synaptic transmission [24, 25 ]. in addition, glutamate plays a major role in the vertical transmission of the retinal signal [24, 25 ]. accordingly, cannabis could disrupt the regulatory role of the cannabinoid system in the retina and consequently alter the transmission of the retinal information. as a consequence, studies evaluating the impact of cannabis on human retinal function are genuinely needed [15, 31 ]. this review outlines the presence of endocannabinoids in critical stages of the neural retina such as photoreceptor cells, bipolar cells, and ganglion cells. endocannabinoids are also detected in interneuron cells, namely, amacrine and horizontal cells, as well as in mller and retinal pigment epithelium cells. the presence of endocannabinoids in the retina supports a regulatory role of the cannabinoid system in the vertical transmission of visual information from photoreceptors to ganglion cells, the ultimate stage before the transmission of the visual information to the brain, mostly the visual cortex. this also supports a potential dysregulation induced by exogenous cannabinoids, such as thc and cannabidiol, the main constituents of cannabis. this review also described the localization of the cannabinoid system in the retinal pigment epithelium suggesting a role of this system in the renewal of the photoreceptor disks as well as in trophic functions. another important finding of this review is the involvement of cannabinoids in retinal neurotransmission, neuroplasticity, and neuroprotection. firstly, cannabinoids regulate the release of retinal neurotransmitters by acting on ionic channels or enzymatic activity and are thus able to alter the retinal signal. these results were recently supported by alterations of electroretinogram recordings in mice lacking cannabinoid receptors cb1 and cb2. unfortunately, only one study has investigated the impact of cannabis use on the human retinal function. secondly, the cannabinoid system seems to play a major regulatory role in retinal synaptic plasticity, in particular during the postnatal development. as an extension of these results, it is legitimate to question the effects of prenatal cannabis exposure on the retinal development in offspring. thirdly, using several animal models of retinal diseases, numerous studies have shown that cannabinoids could play a neuroprotective function by preventing the retinal cell death. these results can support the development of synthetic cannabinoids as new therapeutic strategies to prevent and treat retinal diseases. the retina constitutes a relevant and useful site to investigate neurotransmission signaling pathways as well as cns processes. the retinal organization in mammals is well known and the retina is an accessible part of the cns that can be evaluated with noninvasive, objective, relatively rapid, and costless methods. furthermore, its measure is quite standardized allowing good reproducibility [97, 101, 104, 106 ]. as the cannabinoid system is involved in cns processes, the retina therefore represents a useful tool to evaluate the cns pathophysiology and might eventually also serve to monitor curative and preventive treatment efficiency for cns disorders. this review gives an overview of the distribution of the cannabinoid system in the retina together with its involvement in the regulation of retinal neurotransmission, neuroplasticity, and neuroprotection. these suggest potential alterations of structural and functional retinal properties by exogenous cannabinoids, especially thc and cannabidiol contained in joints. as cannabis is widely spread worldwide, it is now critical to explore the effects of cannabis on the human retina. based on experimental studies in animals, this review also aims to provide several retinal methods to correlate the cellular and molecular changes induced by cannabinoids to potential functional retinal deficits in cannabis users. however, considering the neuroprotective role of the cannabinoid system in the retina, this review also argues for therapeutic uses of synthetic cannabinoids in the treatment and the prevention of retinal diseases. | cannabis is one of the most prevalent drugs used in industrialized countries. the main effects of cannabis are mediated by two major exogenous cannabinoids : 9-tetrahydroxycannabinol and cannabidiol. they act on specific endocannabinoid receptors, especially types 1 and 2. mammals are endowed with a functional cannabinoid system including cannabinoid receptors, ligands, and enzymes. this endocannabinoid signaling pathway is involved in both physiological and pathophysiological conditions with a main role in the biology of the central nervous system. as the retina is a part of the central nervous system due to its embryonic origin, we aim at providing the relevance of studying the endocannabinoid system in the retina. here, we review the distribution of the cannabinoid receptors, ligands, and enzymes in the retina and focus on the role of the cannabinoid system in retinal neurobiology. this review describes the presence of the cannabinoid system in critical stages of retinal processing and its broad involvement in retinal neurotransmission, neuroplasticity, and neuroprotection. accordingly, we support the use of synthetic cannabinoids as new neuroprotective drugs to prevent and treat retinal diseases. finally, we argue for the relevance of functional retinal measures in cannabis users to evaluate the impact of cannabis use on human retinal processing. |
according to the world health organization (who), health education is any combination of learning experiences designed to help individuals and communities improve their health, by increasing their knowledge or influencing their attitudes. thus, health education not only plays a very crucial role but also remains as an elementary instrument in health promotion and disease prevention, especially in developing countries. it is important that health education should not just confine to the transmission of information, but should aim to make the target population understand the given information, execute the recommendations, and adapt to healthier lifestyles. however, in clinical practice, it was discerned that doctors tend to undervalue the patient 's desire for information and misperceive the process of sharing information. with a shift toward patient - centered care, it becomes important to equip patients with appropriate knowledge and to construct scientific beliefs enabling them to make appropriate healthcare decisions and to rationally redesign their lifestyles. oral health education could be comprehended in a similar way, and a shift in the terminology from dental health education to oral health education over the years should be appreciated at this juncture, which emphasizes on the health of the mouth as a whole rather than confining to the teeth. dental health education has also been subjected to many practical refinements together with theoretical and terminological modifications. one such refinement is the increased utilization of inputs from the fields of education, sociology, and psychology. the prevalence of periodontal disease is high universally and is a substantial public health problem for countries. the fact that periodontal disease contributes to the global burden of chronic disease makes it a problem of profound importance that has to be addressed. in the first ever national - level epidemiological survey on oral health conducted by the dental council of india (dci), it was identified that the prevalence of periodontal disease was high among the indian population, which increased with age. the prevalence was highest among those in the age group of 3544 years, with 89.6% of subjects in this age group identified as having periodontal diseases. it has evolved as a significant public health problem reckoning its impact on the quality of life of individuals and communities. hence, it becomes exceedingly important for a dentist or a periodontist to corroborate their patient 's understanding of the etiology of periodontal diseases, the wide range of treatment choices available, and the sequelae of neglect. knowledge is an invaluable asset to the patients both for staying healthy and seeking required care in situations of need. health literacy also plays a role in the verbal communication between the patient and the healthcare professional, which often gets filled with a lot of intricate medical terminology. oral health education in clinical practice largely involves display of static pictures and face to face provision of informal education by the oral healthcare professionals. however, there is sound evidence that three - dimensional (3d) animations help the viewers to acquire better understanding of objects with intricate details compared to static orthographic [two - dimensional (2d) ] depictions. the greatest advantage with 3d animations is that over - reliance on the health professional patient communication and its apparent limitations can be partially negotiated, if not completely. it is inferred that periodontal structures and the causes, presenting features, preventive measures, and treatment modalities for periodontal problems, which often are difficult to comprehend in 2d depictions, could be better understood in 3d projections. nevertheless, there is limited evidence about the distinguished effect of 3d oral health educational videos in improving oral health knowledge of the received. the purpose of this randomized controlled trial was to test the effectiveness of 3d oral health educational videos over conventional 2d projections among the first year dental students in a dental college in south india. this was a parallel, randomized controlled trial with an allocation ratio of 1:1 using computer - generated randomization method. the study protocol was approved by the institutional ethics committee and no alterations to the trial methods were executed. this randomized controlled trial is reported in compliance with the consolidated standards of reporting trials (consort) statement. this study was conducted in a dental college in south india from october 2014 to february 2015. the eligibility criteria for the recruitment of the study sample are as follows : (1)first year dental students who got admission in the 20142015 academic year ; (2) students who did not receive a formal periodontal health education before ; and (3) students without any systemic problems and vision impairment. out of 90 students who were evaluated for eligibility, 80 students met the eligibility criteria. randomization was done to ensure unbiased allocation of study subjects to the test group and control groups. we believe that there could be extraneous variables, other than the type of intervention, which may influence the final outcome, like the ability of the participants to concentrate during the educational session, iq levels, previous exposure to basic oral health education, etc. controlled randomization of study participants was done to ensure equal size of groups and to avail its statistical advantages over simple random allocation. the participants in the test group were given oral health education pertaining to periodontal anatomy, etiology, presenting conditions, preventive measures, and treatment for periodontal problems using a 3d animation video. participants in the control group were given the same education using regular 2d video projections. both the interventions were carried out in a classroom setting and the duration of intervention for both the groups was same (4 min and 15 s). the content of the voice over was repeatedly reviewed by experts for content validity till no further modifications were required. as this was a homotrait (same content), multimethod (different forms) approach, care was taken to ensure complete emphasis to the method of delivery of education (2d or 3d) by matching the voice over and thereby eliminating potential confounding effects. oral health knowledge, in general, and periodontal knowledge, in particular, was the elementary factor of interest. we used a multiple - choice questionnaire for the assessment of outcome measure [table 1 ]. the questionnaire consisted of 10 questions, with each question having four options to choose the answer from. every question had one correct option, two wrong options, and a do nt know option. do nt know was given as an option for every question to ensure complete filling of the questionnaire, as some participants may not want to answer something they do not know, and also to curtail the possibility of correct answers by chance. without this option, the obligation to choose one of the options may yield good results just by chance. we also assessed the oral hygiene status of the participants at baseline and 1 month post intervention using oral hygiene index simplified (ohi - s), plaque index (pi), and the ginigival status of the participants using gingival bleeding index (gbi). these were collectively referred to as oral health status in the context of this article. study questionnaire and the percentage of participants choosing correct options in both the groups at baseline and post intervention the null hypothesis is that there were no differences between the test and control groups with respect to their mean scores in the study questionnaire and the mean scores as recorded by the aforementioned indices post intervention. differences between the knowledge of the participants and the oral health status in the test and control groups at baseline and post intervention were analyzed using unpaired t - test. paired t - test was used for analyzing differences between baseline and post intervention scores in both the test and control groups. descriptive statistics were used and the likelihood of being more knowledgeable post intervention was depicted with odds ratio [95% confidence interval (ci) ]. the videos with voice over were assessed for clarity and quality by administering them to five patients attending the outpatient department of the institution who could read, write, and understand english. the pace at which the voice over was recorded was adjusted based on the suggestions received and few sentences were re - recorded owing to dearth of clarity. the intention behind the study was to test the effectiveness of 3d animation videos over conventional 2d videos and the study was considered as an initial move before translating the content of voice over to the regional language and administering it to a larger group of patients. all the participants were administered the questionnaire before receiving the allotted intervention and were clinically examined to record the baseline oral health status. participants in the test and control groups received the intervention at the same time in different classrooms. the participants had to choose the answers on their own on all three occasions and measures were taken to avoid sharing opinions and views during the time of answering the questionnaire. clinical examination was conducted 1 month after the intervention by the same examiner who had performed the examination at baseline, to eliminate observational bias. this was a parallel, randomized controlled trial with an allocation ratio of 1:1 using computer - generated randomization method. the study protocol was approved by the institutional ethics committee and no alterations to the trial methods were executed. this randomized controlled trial is reported in compliance with the consolidated standards of reporting trials (consort) statement. this study was conducted in a dental college in south india from october 2014 to february 2015. the eligibility criteria for the recruitment of the study sample are as follows : (1)first year dental students who got admission in the 20142015 academic year ; (2) students who did not receive a formal periodontal health education before ; and (3) students without any systemic problems and vision impairment. out of 90 students who were evaluated for eligibility, 80 students met the eligibility criteria. randomization was done to ensure unbiased allocation of study subjects to the test group and control groups. we believe that there could be extraneous variables, other than the type of intervention, which may influence the final outcome, like the ability of the participants to concentrate during the educational session, iq levels, previous exposure to basic oral health education, etc. controlled randomization of study participants was done to ensure equal size of groups and to avail its statistical advantages over simple random allocation. the participants in the test group were given oral health education pertaining to periodontal anatomy, etiology, presenting conditions, preventive measures, and treatment for periodontal problems using a 3d animation video. participants in the control group were given the same education using regular 2d video projections. both the interventions were carried out in a classroom setting and the duration of intervention for both the groups was same (4 min and 15 s). the content of the voice over was repeatedly reviewed by experts for content validity till no further modifications were required. as this was a homotrait (same content), multimethod (different forms) approach, care was taken to ensure complete emphasis to the method of delivery of education (2d or 3d) by matching the voice over and thereby eliminating potential confounding effects. oral health knowledge, in general, and periodontal knowledge, in particular, was the elementary factor of interest. we used a multiple - choice questionnaire for the assessment of outcome measure [table 1 ]. the questionnaire consisted of 10 questions, with each question having four options to choose the answer from. every question had one correct option, two wrong options, and a do nt know option. do nt know was given as an option for every question to ensure complete filling of the questionnaire, as some participants may not want to answer something they do not know, and also to curtail the possibility of correct answers by chance. without this option, the obligation to choose one of the options may yield good results just by chance. we also assessed the oral hygiene status of the participants at baseline and 1 month post intervention using oral hygiene index simplified (ohi - s), plaque index (pi), and the ginigival status of the participants using gingival bleeding index (gbi). these were collectively referred to as oral health status in the context of this article. study questionnaire and the percentage of participants choosing correct options in both the groups at baseline and post intervention the null hypothesis is that there were no differences between the test and control groups with respect to their mean scores in the study questionnaire and the mean scores as recorded by the aforementioned indices post intervention. differences between the knowledge of the participants and the oral health status in the test and control groups at baseline and post intervention were analyzed using unpaired t - test. paired t - test was used for analyzing differences between baseline and post intervention scores in both the test and control groups. descriptive statistics were used and the likelihood of being more knowledgeable post intervention was depicted with odds ratio [95% confidence interval (ci) ]. the videos with voice over were assessed for clarity and quality by administering them to five patients attending the outpatient department of the institution who could read, write, and understand english. the pace at which the voice over was recorded was adjusted based on the suggestions received and few sentences were re - recorded owing to dearth of clarity. the intention behind the study was to test the effectiveness of 3d animation videos over conventional 2d videos and the study was considered as an initial move before translating the content of voice over to the regional language and administering it to a larger group of patients. all the participants were administered the questionnaire before receiving the allotted intervention and were clinically examined to record the baseline oral health status. participants in the test and control groups received the intervention at the same time in different classrooms. the participants had to choose the answers on their own on all three occasions and measures were taken to avoid sharing opinions and views during the time of answering the questionnaire. clinical examination was conducted 1 month after the intervention by the same examiner who had performed the examination at baseline, to eliminate observational bias. ten were excluded from the study, and finally 80 participants were randomized and enrolled in the trial to receive the intended interventions. demographic characteristics on age and gender are not provided here as all the participants belonged to the same age group and a majority of the participants were females (92.5%). so, a separate analysis based on gender was not considered owing to the small number of male participants in the trial. the analysis was limited to comparisons between the test and the control groups with respect to their baseline and post intervention performances in the study questionnaire and oral health status. the descriptive statistics of the oral health knowledge questionnaire at baseline, immediately post intervention, and 1 month post intervention are given in table 2. the test and the control groups were not different with respect to their baseline mean knowledge scores (p = 0.092, unpaired t - test). nevertheless, significant differences in the immediate post intervention and 1 month post intervention scores were found between the groups as assessed by unpaired t - test (p < 0.001). descriptive statistics of oral health knowledge scores at baseline, immediately post intervention, and 1 month post intervention in the test (3d) and control (2d) groups we also analyzed the differences between mean baseline scores and mean post intervention scores in the test and control groups using paired t - tests. there were significant (p < 0.001) differences between the mean scores at baseline and post intervention in both the test and control groups [table 2 ]. we calculated the odds ratio to assess the likelihood of choosing a correct answer by the kind of intervention received. the participants in the test group were found to be 3.91 times more likely to choose a correct answer compared to the participants in the control group immediately post intervention and this was statistically significant (95% ci : 2.645.81). the test and control groups were homogenous with respect to their pi scores at baseline. no significant differences in mean pi scores were found between the groups after 1 month. there was significant reduction in the mean pi scores post intervention in both the groups (p < 0.001) [table 3 ]. similar findings were observed with respect to the bleeding index scores within and between the test and control groups (p < 0.001) [table 3 ]. comparative evaluation of pi, bi, ohi - s scores at baseline and 1 month post intervention in the test and control groups however, there were no differences in the ohi - s scores between the test and control groups both at baseline and post intervention. significant improvement in oral hygiene was found in the both groups from baseline to post intervention (p < 0.001) [table 3 ]. it is clear from the results that both the interventions have a profound impact on the periodontal health knowledge of the participants and their oral health status. participants who had received 3d education showed significantly better improvement compared to those who received 2d interventions, with the exception of ohi - s scores. the baseline scores in the test and control groups were homogenous without significant differences. it was found that the periodontal knowledge scores of the participants had significantly improved with both the interventions. this could be easily understood as transmission of information more likely results in an improvement in knowledge when properly executed. nevertheless, there were significant differences between the periodontal knowledge scores of the test and control groups after the intervention, which highlights the effectiveness of 3d education over 2d projections. the pi scores in the test group were significantly less compared to those of the control group 1 month after the intervention. the 3d video on mechanical plaque removal might have had a better impact on the participants of the test group than the 2d mechanical plaque control education received by the control group. brushing endured through time as the most effective method for plaque control and for maintenance of gingival health. educational videos administered in the study consisted of the brushing technique, and this could have positively influenced a change in the bleeding index of the participants in both the groups from baseline to post intervention. the fact that calculus is so tenacious that it can not be removed by home - based mechanical oral hygiene measures could have played a role in the ohi - s scores being similar between the test and control groups post intervention. it was inferred that the percentage of participants who got oral prophylaxis done between the baseline and post intervention examinations was relatively comparable. the periodontal knowledge scores as assessed by a close - ended questionnaire were found to be improved in both the groups. this improvement was not just limited to the data collected immediately after the intervention, but persisted as observed from the data collected 1 month post intervention. these results highlighted knowledge recall, and the recall of knowledge was considerably high among the participants in the test group compared to those in the control group. this increase in post intervention knowledge in the test group has a consistent explanation from the results of a study conducted using 3d multimedia system for patient education. another finding from the study was improvement in compliance of the participants after the intervention, which is clear from the post intervention oral health status. regardless of the type of intervention received, participants compliance to oral hygiene measures had increased along with the periodontal health knowledge. the reasons for such improvement could be found in the comprehensiveness of the information provided through the interventions, which included the etiology, symptoms, preventive actions, available treatment options, and the sequelae of neglect. these results are in accordance with a study conducted on 30 male patients who were matched for age and education. there was enough evidence suggesting the advantages of computer - based oral health education over personal communication and printed material. though the information provided is same, computer - based education does have an extra edge in terms of enabling the patients to visualize the information and to better accumulate this information to the existing knowledge. such visualization and comprehensive information obviously aid in improvement of knowledge, better retention of the acquired knowledge, realization of the importance of compliance to oral health instructions, and better oral health practices. this study should be looked upon as an initial move to test for the effectiveness of 3d periodontal health education over that of 2d education. the ultimate aim of the investigators is to develop a 3d periodontal health educational video in the regional language, based on the findings from this study. this would involve translation, checking for cross - cultural validity, and a pilot study to realize the scope for improvement. the reason for choosing first year dental students was that they were comparable with the general population in terms of their periodontal health knowledge with only a month of experience as a student of dentistry. also, the interventions may help them to better understand their curriculum and incorporates a sensible view on how to comprehend oral health related problems. since there was no standardized measure for assessment of periodontal health knowledge, we had designed a questionnaire applicable to the area of the study, which was checked for content validity by experts in the field. however, there is a need to develop a comprehensive, standardized measure to assess periodontal health knowledge of people, and such measures should be thoroughly reviewed for validity and reliability. to sum up, 3d videos serve as an invaluable tool in improving patients periodontal health knowledge and understanding of the disease process. they also enable the patients to better comply with instructions, which apparently results in better oral health status. the ultimate objective of oral healthcare professionals is improvement in the health status of their patients, in general, and oral health status, in particular. | background : there is limited evidence about the distinguished effect of 3d oral health education videos over conventional 2 dimensional projections in improving oral health knowledge. this randomized controlled trial was done to test the effect of 3 dimensional oral health educational videos among first year dental students.materials and methods:80 first year dental students were enrolled and divided into two groups (test and control). in the test group, 3d animation and in the control group, regular 2d video projections pertaining to periodontal anatomy, etiology, presenting conditions, preventive measures and treatment of periodontal problems were shown. effect of 3d animation was evaluated by using a questionnaire consisting of 10 multiple choice questions given to all participants at baseline, immediately after and 1month after the intervention. clinical parameters like plaque index (pi), gingival bleeding index (gbi), and oral hygiene index simplified (ohi - s) were measured at baseline and 1 month follow up.results:a significant difference in the post intervention knowledge scores was found between the groups as assessed by unpaired t - test (p<0.001) at baseline, immediate and after 1 month. at baseline, all the clinical parameters in the both the groups were similar and showed a significant reduction (p<0.001)p after 1 month, whereas no significant difference was noticed post intervention between the groups.conclusion:3d animation videos are more effective over 2d videos in periodontal disease education and knowledge recall. the application of 3d animation results also demonstrate a better visual comprehension for students and greater health care outcomes. |
oxidative stress refers to an imbalance between free radical [especially reactive oxygen species (ros) ] generation and antioxidant defense1. the antioxidant system in the human body includes enzymatic [e.g. superoxide dismutase (sod) and glutathione peroxidase (gpx) ] and non - enzymatic antioxidants [e.g. glutathione (gsh) ] and it plays an important role in the prevention of oxidative stress. oxidative stress is involved in the pathogenesis of hypertension, atherosclerosis, diabetes, osteoporosis, and cancer2. ros initiates lipid peroxidation through an attack on polyunsaturated fatty acids, and generates products such as mda (malondialdehyde)4. ros also causes oxidative damage to proteins and dnas4. among these damages, oxidative dna damage is the most detrimental one to human health because of its role in the pathogenesis of the various diseases mentioned above. ros, especially hydroxyl radical (oh), can cause dna base changes, strand breaks, damage to tumor - suppressor genes, and enhanced expression of proto - oncogenes5. in addition to the increase of ros, the decrease of dna repair capacity is the other reason for the accumulation of oxidative dna damage in the human body6. physical exercise is an important factor affecting oxidative stress and oxidative dna damage, since a sharp increase in oxygen consumption during exercise results in an increase in ros generation. however, the effect of exercise - induced oxidative damage is variable depending on several factors such as type, mode, duration, and intensity of exercise7. a single bout of exercise induces oxidative stress and dna damage, whereas regular moderate intensity exercise decreases them8. under the concept of hormesis, low - to - moderate ros generation induced by regular moderate intensity exercise a previous study demonstrated that long - term high - intensity exercise (75%vo2max) increased 8-hydroxy-2-deoxyguanosine (8-ohdg), while regular exercise with moderate - intensity (50%vo2max) tended to decrease 8-ohdg10. the activity of 8-oxoguanine dna glycosylase (ogg1), a crucial enzyme for the repair of 8-oxoguanine lesions, is up - regulated after regular exercise in human and animals11. tai chi (tc), a traditional chinese exercise, is classified as a moderate intensity exercise, and its intensity is basically similar among different age and gender groups12. previous studies have reported the numerous tangible benefits of tc practice, such as improvement of balance and prevention of falls, enhancement of flexibility and strength, as well as amelioration of cardio - respiratory and immune function impairments. studies of the effects of tc exercise on oxidative stress and oxidative dna damage are limited. tc exercise reduced the degree of oxidative stress in middle - aged women13, older adults14, and obese patients with type 2 diabetes15, and long - term tc practice reduced the degree of dna damage, measured by the comet assay, in middle - aged13 and older16 adults. however, the effects of tc intervention on oxidative stress and dna damage have not been investigated in young females who seldom participate in physical exercise. the efficacy of tc exercise on dna base damage and its repair also remains unclear. young females are less conscious about physical exercise compared to age - matched males17. although tc exercise is suitable for any population18, only middle - aged and elderly age groups frequently practice tc for health purposes. tc is becoming popular among chinese college students since it has been designated as a compulsory sport in the physical education curriculum of most colleges. therefore, the objective of this pilot study was to determine the effects of tc intervention on oxidative stress, dna damage and repair in young females who did not participate in regular physical exercise. ten sedentary female students from gannan normal university, china, voluntarily participated in this program. they completed inclusion and lifestyle questionnaires. exclusion criteria were as follows : having experience of tc exercise ; having performed physical exercise for more than 1 hr per week in the past 3 months ; smoker or regular alcohol drinker ; consuming supplements or medical products with antioxidant properties ; having a history of cardiovascular and respiratory diseases ; having knee joint pain ; having a history of lower - extremity fracture within the past 1 year. they were asked to keep their usual lifestyle including dietary behavior and daily activities, and medication (if any) throughout the study period. at the time of recruitment, their demographic characteristics were as follows : age, 20.400.70 yrs old ; height, 155.486.15 cm ; weight, 47.136.11 kg ; body mass index (bmi), 19.421.54 kg / m ; and resting heart rate, 79.1012.65 beats / minute. all of the participants completed the 12-week tc training program. according to this protocol, they practiced the 24-form simplified tc, 5 times (sessions) per week (monday to friday), on campus. the first 2 weeks involved familiarization with tc through teaching and learning. in the following 10 weeks, the participants practiced 5 rounds of tc (1 round refers to practice from the 1st movement to the 24th movement of this tc style) accompanied by a classic music for this tc style in each session. an experienced tc instructor led all participants in practice and taught them necessary movement corrections in the intervals between rounds. thus, each session lasted about 60 min including a 10-minute warm - up for stretching and a 5-minute cool - down for relaxation. heart rate (hr) during tc exercise was monitored in the last session of the 5 rounds of the 24-form simplified tc in the last 4th, 8th, and 12th weeks. the exercise duration of five rounds of tc practice lasted for around 30 minutes. hr parameters such as average hr, maximum hr, and minimum hr were recorded using a polar watch (suunto t6d running pack, finland) and the telemetry hr team system (suunto team manager, finland). blood samples were collected at baseline, 8, and 12 weeks. in each session of blood collection, 3 ml of whole blood was taken after overnight fasting between 7:308:30 a.m. from a forearm vein and placed into a heparinized tube. plasma was separated by centrifugation at 3,000 rpm for 5 min and kept frozen at 80c until assayed for 8-ohdg, ogg1, sod, gpx, mda, gsh, and hydroxyl radical inhibiting capacity (oh-ic). sod was measured by the hydroxylamine assay using a spectrophotometer (unico7200, usa) at 550 nm. gpx was detected by the rate method using a spectrophotometer (unico7200) at 412 nm. mda was assessed by the thiobarbituric acid (tba) assay using a spectrophotometer (unico7200) at 532 nm. gsh was analyzed by colorimetry using a spectrophotometer (unico7200) at 420 nm. hydroxyl radical inhibiting capacity was determined by the griess colorimetric method using a spectrophotometer (unico7200) at 550 nm. the kits for sod, gpx, mda, gsh, and oh-ic measurements were all purchased from nanjing jiancheng bioengineering institute, china. plasma 8-ohdg and ogg1 levels were measured using commercial elisa (enzyme - linked immunosorbent assay) kits according to the manufacturer s instructions (santa cruz biotechnology, inc. then, either 10 l of anti-8-ohdg or anti - ogg1 antibody in combination with 50 l of streptavidin - biotin - horseradish peroxidase were added to the sample wells in turn. to the standard sample wells, 50 l of 5 different concentrations of standard samples and 50 l of streptavidin - biotin - horseradish peroxidase were added. the microtiter plate was then incubated for 1 hour at 37 c. then, the plate was washed with wash buffer 5 times. during the procedure of washing, before discarding the buffer, each well was filled with wash buffer for 30 seconds. color reactions were then conducted for 10 min in the dark at 37 c after 50 l of chromogenic agent a and 50 l of chromogenic agent b were added in turn to each well. finally, 50 l of stop solution was added to terminate the reaction and the blue color changed to yellow. shanghai, china) at 450 nm within 15 min, and the concentrations of 8-ohdg and ogg1 in plasma were read from standard curves. all data were expressed as the mean sd. the differences of plasma and hr monitoring parameters among different time points were determined by repeated measures analysis of variance (anova). post hoc pair - hoc comparisons (lsd) were performed to test the differences when significance was shown. the correlation between plasma 8-ohdg and ogg1 levels at the same time point was determined using pearson s correlation coefficient. the statistical package for social sciences (spss) version 17.0 was used for the analysis and values of p0.05) (table 1). hraverage = mean heart rate, hrmin = minimum heart rate, hrmax = maximum heart rate, bmp = beats per minute the sod level was significantly increased (p0.05). these results suggest that the increase of ogg1 activity after 12 weeks of tc exercise exceeds the requirements for immediately excising all 8-ohdg in young females. based on the results of this pilot study, a 12-week tc exercise intervention is effective at increasing the activity of plasma sod and ogg1 and decreasing plasma mda concentration in young sedentary females. it suggests that tc exercise helps to alleviate the oxidative stress response and dna damage in those who have serious levels of oxidative stress and dna damage, such as older and sick people. third, the authors only told the participants to keep their daily dietary habits and did not ask them to record their dietary details, especially before each blood sampling. thus, dietary variation might have affected the data of blood parameters. nevertheless, this pilot study showed tc intervention can improve the response to oxidative stress and the dna damage / repair process in female students. further studies with a longer intervention duration, larger groups, and a control group are needed to confirm our present results. moreover, the long - term effects of tai chi on oxidative stress response and dna / repair also need to be studied in the future. | [purpose ] this study was to examine the effects of 12 weeks of tai chi (tc) exercise on antioxidant capacity, and dna damage / repair in young females who did not perform regular physical exercise. [subjects and methods ] ten female students from a chinese university voluntarily participated in this program. all of them practiced the 24-form simplified tai chi, 5 times weekly, for 12 weeks. plasma levels of superoxide dismutase (sod), glutathione peroxidase (gpx), malondialdehyde (mda), glutathione (gsh), hydroxyl radical inhibiting capacity (oh-ic), 8-hydroxy-2-deoxyguanosine (8-ohdg), and 8-oxoguanine dna glycosylase (ogg1) were measured at 0, 8, and 12 weeks. heart rate (hr) was monitored during the last set of the training session at 4, 8, and 12 weeks. [results ] plasma sod and oh-ic levels were increased at 8 and 12 weeks compared to the baseline (0 weeks). gpx and gsh levels did not change significantly throughout the study period. the plasma mda level was decreased significantly at 8 weeks but not at 12 weeks compared to the baseline value. while the plasma 8-ohdg level did not change throughout the study period, the plasma ogg1 level was significantly increased at 8 and 12 weeks compared to the baseline value. [conclusion ] tc practice for 12 weeks efficiently improved the oxidative stress response in young females who did not perform regular physical exercise. the tc exercise also increased the dna repairing capacity. |
this study was nested into a larger efficacy study, and a total of 116 pregnant women in the second and third trimesters were enrolled in the pharmacokinetic cohort. in addition, 17 nonpregnant women with uncomplicated p. falciparum malaria in uganda were enrolled in the pharmacokinetic cohort (table 1). the standard oral fixed dose treatment of artemether and lumefantrine (twice daily for 3 days) was well tolerated and no cases of vomiting were reported. no patients presented with recurrent malaria infections during the follow - up (until delivery or day 42). twenty - six pregnant and 17 nonpregnant women contributed with densely sampled plasma, and 90 pregnant women contributed with sparsely sampled capillary plasma (table 1). one individual in the capillary arm was omitted from the data analysis because of an unexplainable high baseline plasma lumefantrine concentration of 7,717 ng / ml. demographic values were generally similar between pregnant and nonpregnant women, except for a higher body weight in pregnant women as compared with that in nonpregnant women (table 1). furthermore, pregnant women in the group with venous plasma sampling had lower admission parasitemias as compared with both pregnant women with capillary sampling and nonpregnant women (table 1). several absorption, distribution, covariate, and residual error models were fitted to the data to construct the best - performing model. the final nonlinear mixed - effects model consisted of a flexible transit absorption model (five transit compartments) followed by a two - compartment disposition model and described the lumefantrine data well (figure 1 supplementary data). residual variability was best described using an additive error model on the log - transformed data. implementation of between - patient variability, between - dose occasion variability, and a box cox transformation of the relative bioavailability of lumefantrine resulted in a significant improvement of the model (objective function value differences (ofv) of 261, 539, and 21.5, respectively). venous and capillary data were successfully modeled simultaneously using a proportional correction factor for the capillary samples on a population level because no patient contributed with both capillary and venous samples. capillary concentration samples were estimated to be 11.9% (relative standard error of 9.32%) lower compared with venous concentration samples. however the following covariates were all selected in the forward covariate search (p 250,000 parasites/l), severe anemia (hb 8 weeks) if a match (with respect to history of fever ; axillary temperature > 37.5 c ; smoking ; and parasitemia : 6.63) was considered as significant in the backward elimination. the impact of bodyweight, age, estimated age of gestation, parasitemia, body temperature, and pregnancy (categorical) were evaluated on all parameters (cl / f, vc / f, q / f, vp / f, mean transit time, and f). systolic blood pressure was evaluated on elimination clearance and intercompartmental clearance and liver status results (alanine transaminase) on elimination clearance. a linear (equation 1), exponential (equation 2), and power relationship (equation 3) were tested sequentially for continuous covariates. all covariates were centered on the median value of the population except estimated gestational age that was centered on nonpregnant woman. categorical covariates were implemented as a relative difference (%) between groups. where pi is the individual parameter estimate, ptv is the typical parameter estimate for the study population, n is a fixed effect, and n is a random effect. body weight was also evaluated as an allometric function on all clearance and volume parameters. one model with pregnancy as a categorical covariate on all clearance parameters, volume parameters, and mean transit time was also developed and bootstrapped for a full - covariate model approach. parameter - covariate relations were considered potentially significant in the full - covariate model if the 95% ci did not include zero effect. to assess the reliability of individual parameter estimates, - and - shrinkages were calculated. a stratified (for pregnancy, trimester, and biological matrix) nonparametric bootstrap of 1,000 data sets was performed to calculate reliable standard errors of parameter estimates and the nonparametric cis around these estimates. using 2,000 simulations of each individual plasma sample, a prediction - corrected visual predictive check and a numerical predictive check the 95% cis of the simulated 5th, 50th, and 95th percentiles were overlaid with the 5th, 50th, and 95th percentiles of the observed data to visualize the predictive check. monte carlo simulations were performed using the population pharmacokinetic parameter estimates of the final model to assess potential differences in exposure between pregnant and nonpregnant women. the wellcome trust is a uk - based medical research charity and is independent of all drug companies. it has no financial links with the manufacturers of either the diagnostic tests or the drugs used in this study. | pregnancy alters the pharmacokinetic properties of many antimalarial compounds. the objective of this study was to evaluate the pharmacokinetic properties of lumefantrine in pregnant and nonpregnant women with uncomplicated plasmodium falciparum malaria in uganda after a standard fixed oral artemether lumefantrine treatment. dense venous (n = 26) and sparse capillary (n = 90) lumefantrine samples were drawn from pregnant patients. a total of 17 nonpregnant women contributed with dense venous lumefantrine samples. lumefantrine pharmacokinetics was best described by a flexible absorption model with multiphasic disposition. pregnancy and body temperature had a significant impact on the pharmacokinetic properties of lumefantrine. simulations from the final model indicated 27% lower day 7 concentrations in pregnant women compared with nonpregnant women and a decreased median time of 0.92 and 0.42 days above previously defined critical concentration cutoff values (280 and 175 ng / ml, respectively). the standard artemether lumefantrine dose regimen in p. falciparum malaria may need reevaluation in nonimmune pregnant women. |
the spleen is a reticuloendothelial organ that constitutes a crucial part of the hematologic and immunologic system. the standard management of splenic injury has shifted from surgical splenectomy to spleen - preserving nonoperative procedures. however, approximately 22,000 all - cause splenectomies are still performed per year in the united states. the most common indications for splenectomy are hematologic disorders, such as hemolytic anemia, autoimmune hemolytic anemia, sickle cell disease, -thalassemia, and immune thrombocytopenic purpura. although splenectomy in trauma patients has decreased in recent years, this procedure is still recommended for severe splenic injury (grade iii moreover, emergent splenectomy remains a life - saving technique for patients who exhibit life - threatening hemorrhage caused by splenic injury. several complications of splenectomy, including bacterial infections, thrombosis, thromboembolism, pulmonary arterial hypertension, and cancer, have been described. thrombosis and thromboembolism may complicate ischemic heart disease, ischemic stroke, hemorrhagic stroke, pulmonary embolism, deep vein thrombosis, and portal vein thrombosis. however, the association of thrombosis and thromboembolism with splenectomy has been inconsistently reported in previous studies. splenic injury can impair the function of the spleen by causing the loss of functional splenic tissue. however, no study has evaluated the increased risk of stroke in patients with splenic injury. in addition, most studies have investigated vascular complications of splenectomy that was indicated for hematologic disorders. therefore, we conducted a nationwide retrospective cohort study to determine the risk of ischemic and hemorrhagic strokes in patients with splenic injury and splenectomy by analyzing the national health insurance research database (nhird) of taiwan. the taiwan national health insurance (nhi) program is a single - payer universal insurance program that was initiated in 1995 and covers approximately 99% of the taiwanese population (> 25 million people ; http://www.nhi.gov.tw/english/index.aspx). the government of taiwan has authorized the national health research institutes (nhri) to establish the nhird and release decoded and scrambled data for research purposes. in this study, we analyzed the hospitalization claims data, which constitute a subdataset of the nhird, containing information on sex, age, dates of admission and discharge, diagnoses, surgical procedures, discharge status, and medical expenditures. diagnoses and management were classified according to the international classification of diseases, ninth revision, clinical modification (icd-9-cm) codes. this study was approved by the ethics review board of china medical university (cmu - rec-101012). the study patients were selected from the inpatients claims of patients who were diagnosed with splenic injury (icd-9-cm code 865) between 1998 and 2010. patients aged 20 years or older with splenic injury who underwent splenectomy (icd-9-cm procedure codes 41.2, 41.3, 41.5) were classified as the splenectomized cohort, and patients with splenic injury who did not undergo splenectomy were classified as the nonsplenectomized cohort. the dates of the first hospitalization for splenic injury were defined as the index dates. to create a control cohort, patients without splenic injury and splenectomy were randomly selected and matched with splenic injury patients in a 4:1 ratio according to age group with a 5-year interval, sex, and the year of the index date. the exclusion criteria were age 25 million people ; http://www.nhi.gov.tw/english/index.aspx). the government of taiwan has authorized the national health research institutes (nhri) to establish the nhird and release decoded and scrambled data for research purposes. in this study, we analyzed the hospitalization claims data, which constitute a subdataset of the nhird, containing information on sex, age, dates of admission and discharge, diagnoses, surgical procedures, discharge status, and medical expenditures. diagnoses and management were classified according to the international classification of diseases, ninth revision, clinical modification (icd-9-cm) codes. this study was approved by the ethics review board of china medical university (cmu - rec-101012). the study patients were selected from the inpatients claims of patients who were diagnosed with splenic injury (icd-9-cm code 865) between 1998 and 2010. patients aged 20 years or older with splenic injury who underwent splenectomy (icd-9-cm procedure codes 41.2, 41.3, 41.5) were classified as the splenectomized cohort, and patients with splenic injury who did not undergo splenectomy were classified as the nonsplenectomized cohort. the dates of the first hospitalization for splenic injury were defined as the index dates. to create a control cohort, patients without splenic injury and splenectomy were randomly selected and matched with splenic injury patients in a 4:1 ratio according to age group with a 5-year interval, sex, and the year of the index date. the exclusion criteria were age 1 year. table 3 shows a comparison of the risk of stroke between splenectomized and nonsplenectomized patients who had splenic trauma, revealing a 1.21-fold higher risk of stroke for patients who received splenectomy (95% ci 1.011.44). females exhibited a significantly higher risk of stroke after splenectomy (adjusted hr 1.5 ; 95% ci 1.052.16). however, the increased risk of stroke in male patients receiving splenectomy compared with nonsplenectomy was nonsignificant (adjusted hr 1.13 ; 95% ci 0.921.39). hrs of hemorrhagic and ischemic strokes among splenic injury patients with and without splenectomy stratified by demographics and comorbidity this large nationwide cohort study confirmed the increased risk of hemorrhagic and ischemic strokes among patients with splenic injury. we observed that splenectomy increased the risk of stroke compared with nonsplenectomy in splenic injury patients. the spleen is a reticuloendothelial organ that constitutes a vital part of the hematologic and immunologic systems. multiple factors are likely to contribute to vascular events in patients with splenic injury and splenectomy, including platelet activation, hypercoagulability, activation of the endothelium, and altered lipid profiles. several studies have reported that splenectomy resulted in thrombocytosis, leukocytosis, concentrated hemoglobin, hyperlipidemia, and increased c - reactive protein levels. all these alterations are independently associated with an increased risk of vascular complications in splenectomized patients. in addition to the aforementioned factors, bidirectional interaction between brain injury and spleen is proposed. brain injury, including trauma, hemorrhage, and ischemia, induces an immunoinflammatory response and release of inflammatory cytokines by macrophages in the spleen, the so - called brain - spleen inflammatory coupling. for example, tumor necrosis factor (tnf)- has been shown to result in exacerbation of brain ischemia. immune alterations, including increased ratio of interferon (ifn)- to interleukin-10 and significant elevation of absolute lymphocyte, cd4 + t cell, and cd8 + t cell counts, have been described after splenectomy. t lymphocytes are associated with the production of ifn- and tnf-, which have shown a direct pathogenetic role in neuronal ischemic damage. tuttolomondo reported a significant elevation in peripheral frequency of cd4 + cells and cd28 null cells (cd4+cd28- t cells) in patients with acute ischemic stroke, particularly in those with cardioembolic stroke. these complex multifactorial interactions could possibly play a critical role in the pathogenesis of stroke in splenectomized patients. our study revealed that the cumulative incidence of stroke was the highest in splenic trauma patients who underwent splenectomy, followed by those who did not receive splenectomy. cox proportional hazard modeling showed that splenectomy and splenic injury were independently associated with an increased risk of stroke. patients with splenic injury are typically young (approximately 70% were younger than 50 years in this study) and have few medical comorbidities. recently, surgery and invasive procedures were shown to be associated with an increased risk of stroke within 30 days after the procedures and operations. however, the risk of stroke returned to the baseline if the surgery and invasive procedures were performed > 30 days previously. the increased risk of stroke after splenic injury and splenectomy may be a postprocedural or postoperative phenomenon. but our study showed that splenic trauma patients, both those who underwent splenectomy and those who did not, exhibited a higher hr of stroke during a follow - up period of > 1 year than those who were observed during a follow - up duration of 1 year. therefore, merely the postprocedural or postoperative attribution of increased risk of stroke can not explain the results of our study. in other words, the increased risk is likely attributable to the splenic injury and splenectomy. kristinsson reported the long - term complications of 8149 veterans who underwent splenectomy irrespective of indications. during a 27-year follow - up, they determined that splenectomized patients exhibited an increased risk of pulmonary embolism and deep vein thrombosis, but not ischemic stroke, coronary artery disease, or acute myocardial infarction. by contrast, our study revealed that splenic trauma patients exhibited a 1.74-fold greater risk of stroke if they did not receive splenectomy and a 2.05-fold increased risk of stroke if they received splenectomy. the conditions under which splenectomy is indicated and the presence or absence of ongoing intravascular hemolysis are likely to determine the risk of thromboembolic events after splenectomy. patients with thalassemia intermedia, hgb e/-thalassemia, sickle cell anemia, and hereditary stomatocytosis exhibit a greatly increased risk of vascular complications after splenectomy. trauma patients with splenectomy are thought to have a baseline or increased risk. in our study the differences in indications for splenectomy may explain the conflicting results regarding the risk of thromboembolic events after splenectomy. our study revealed that both patients with splenic injury who underwent splenectomy and those who did not exhibited an increased risk of hemorrhagic and ischemic strokes. the increased hrs of hemorrhagic stroke were higher than those of ischemic stroke after adjustment for age, sex, and comorbidities. based on our research, there has been no comprehensive study that evaluated the risk of hemorrhagic stroke after splenic injury and splenectomy. ischemic stroke and hemorrhagic stroke also share several similar risk factors, including hypertension, atherosclerosis, coronary artery disease, heart failure, atrial fibrillation, hematologic disorders, hyperlipidemia, diabetes mellitus, obesity, and alcohol use. some factors, such as diabetic foot, increased cd4+cd28- t cells (cd28 null cells), arterial stiffness, and elevation of immuno - inflammatory markers, are found commonly in patients with stroke and coronary artery disease. the mechanism of ischemic stroke in splenectomized patients could be also responsible for the increased incidence of hemorrhagic stroke in patients after splenectomy. although this study was a large nationwide study, first, the lack of data on risk factors for stroke, such as smoking, obesity, and socioeconomic status, represents a major limitation. therefore, we could not evaluate the association of the severity of stroke with splenic injury and splenectomy. fourth, information on the etiology and subtypes of ischemic stroke was not available in the nhird. we can not discriminate the subtypes of ischemic stroke according to the trial of org 10172 in acute stroke treatment (toast) classification. finally, the diagnoses of stroke, splenic injury, and splenectomy could not be validated using the nhird. misclassification of diseases may cause bias in our study, although several studies have proven the accuracy of the nhird. in conclusion, our study results show that splenic injury and splenectomy are significantly associated with an increased risk of hemorrhagic and ischemic strokes. higher hrs of hemorrhagic stroke than of ischemic stroke were observed among patients with splenic injury and splenectomy. the results of this study may alert physicians and patients to the severe complications of splenic injury and splenectomy. | abstractthe spleen is a crucial organ in humans. little is known about the association between stroke and splenic injury or splenectomy. the aim of this study was to determine the risk of stroke in patients with splenic injury and splenectomy.a nationwide cohort study was conducted by analyzing the national health insurance research database in taiwan. for comparison, control patients were selected and matched with splenic injury patients in a ratio of 4:1 according to age, sex, and the year of hospitalization. we analyzed the risks of stroke using a cox proportional - hazards regression analysis.a total of 11,273 splenic injury patients, including 5294 splenectomized and 5979 nonsplenectomized patients, and 45,092 control patients were included in this study. the incidence rates of stroke were 8.05, 6.53, and 4.25 per 1000 person - years in splenic injury patients with splenectomy, those without splenectomy, and the control cohort, respectively. compared with the control cohort, splenic injury patients with splenectomy exhibited a 2.05-fold increased risk of stroke (95% confidence interval [ci ] 1.82.34), whereas those without splenectomy exhibited a 1.74-fold increased risk (95% ci 1.512). splenectomy entailed an additional 1.21-fold increased risk of stroke compared with nonsplenectomy in patients with splenic injury.this study revealed that splenic injury and splenectomy were significantly associated with an increased risk of hemorrhagic and ischemic strokes. the results of this study may alert physicians and patients to the complications of splenic injury and splenectomy. |
the identification of sclerostin as a therapeutic target and the optimisation of anti - sclerostin antibodies (scl - mab) have led to a vast array of preclinical studies documenting its ability to enhance bone formation, strength, and density [1, 2 ]. developments have even attracted the attention of nasa with treatment potentially capable of reversing bone density deterioration experienced by astronauts during prolonged space flight. the first human clinical trial continued the success story with a single injection of scl - mab surpassing gains in bone mineral density beyond levels expected after six months of daily teriparatide injections. amgen (romosozumab), eli lilly (blosozumab), and novartis (bps804) represent the main industrial backers of scl - mab therapy. in light of recent clinical trial developments, industry commentators fully expect market approval for a humanized anti - sclerostin antibody by 2017. there is little doubt that there is a serious need for effective treatment. the world health organization considers osteoporosis second only to cardiovascular disease as a threat to global health with over 200 million sufferers globally facing an increased risk of fracture and related complications. current approaches encompass largely anti - resorptive treatments such as bisphosphonates, selective oestrogen receptor modulators, oestrogen, and denosumab antibody therapy. teriparatide available as either full - length (recombinant human pth 1 - 84) or the active fragment (pth 1 - 34) is currently the only clinically available anabolic treatment for osteoporosis. for instance, the use of teriparatide and recombinant human pth has been limited to 1824 months in the usa and eu, respectively. also, while initially associated with increases in bone formation eventually their use leads to a rise in markers of bone resorption. consequently, while many existing therapies are available, new treatment options are continuously sought. in this review, we provide a perspective of the field of scl - mab therapy. specifically, we examine the development of scl - mab through preclinical and clinical studies while assessing the strengths and potential short - comings of treatments. a commentary on areas for further research and novel future applications osteoporosis is a metabolic bone disease characterised by a significant decrease in bone mineral density (bmd) and structural changes to the bone that greatly increase the risk of fracture. however, it is particularly prevalent in postmenopausal women with one in three women aged over 50 likely to experience an osteoporotic fracture compared to one in five men. the increased prevalence after menopause is due to decreased oestrogen levels which accelerate bone loss. these result in reduced mobility, chronic pain, and a greatly increased level of dependence, with 1020% of previously independent sufferers being admitted to nursing homes. it is estimated that 1.6 million osteoporotic hip fractures occur annually and are set to increase to 6.3 million globally by 2050. within the eu in 2010, approximately the cost of treatment for osteoporotic fractures and pharmaceutical intervention of the disease are projected at 37 billion per annum. osteoporotic fractures are further complicated by the inherent difficulties in ensuring adequate fixation of pins and screws due to diminished quality of the surrounding bone [10, 11 ]. the occurrence of osteoporosis is set to increase drastically with the number of sufferers in the eu alone forecasted to rise by 23% to 33.9 million by 2025. this will create a serious medical and economic challenge and highlights the need for effective strategies to deal with the issue on a global scale. in order to be successful, the development of these international strategies must be built on a solid foundation of safe and effective therapies that can be applied at an individual level. the role of sclerostin in the modulation of the wnt/-catenin dependent pathway came to light specifically through the study of sclerosteosis and van buchem disease, two rare genetic disorders associated with high levels of bmd and an associated low risk of fracture. both diseases were traced to mutations impacting a single gene, sost, which is mainly expressed from osteocytes. nevertheless, its expression in chondrocytes, osteoblasts, the bone marrow, heart, pancreas, liver, and some foetal tissue has been reported [1517 ]. the amino acid sequence of sclerostin distantly resembles that of the cerebus / dan (differential screening - selected gene aberrative in neuroblastoma) family of glycoproteins, defined by 190 residues with a cysteine knot - like domain. however, while dan proteins act as classical antagonists of bmp signalling, sclerostin - bmp interactions are weak and appear to be specific to bmp-7 in osteocytes alone [18, 19 ]. others suggest that sclerostin exhibits a catabolic effect on bone by increasing osteoclast production via increased osteocytic expression of receptor activator of nuclear factor kappa b (rankl). the decline in rankl signalling may at least in part explain the sustained decreases in bone resorption markers observed with antisclerostin therapy [20, 21 ]. several sources of evidence implicate a prominent role for sclerostin as an antagonist of the wnt signalling pathway [22, 23 ]. the wnt gene family are largely characterised by their highly conserved glycosylated secreted proteins and can be broadly defined by canonical and noncanonical mediators. the latter typically involve cgmp - related signalling, jun - kinase activation, and/or activation of protein kinase a. primarily the noncanonical pathway is involved in processes such as tissue formation during development, maintenance of adult stem cells, and tumour repression. canonical wnts are defined by their ability to stabilise -catenin via wnt ligand binding to the frizzled (fzd) receptor and low - density lipoprotein receptor - related proteins 5 and 6 (lrp5/6). this results in the phosphorylation of lrp5/6 thus permitting axin to bind to the receptor complex. this results in the inhibition of glycogen synthase kinase-3 's (gsk-3) activity, which ordinarily targets -catenin for degradation. however, the creation of this complex stabilises cytosolic -catenin resulting in its nuclear translocation. within the nucleus, it induces downstream transcription of bone - related genes such as runx2 and osteocalcin via t - cell factor / lymphoid enhancer binding factor (tef / lef) cofactors [2527 ] (figure 1(a)). activation of the wnt/-catenin dependent pathway also modulates osteogenic and chondrogenic differentiation of mesenchymal stem cells as well as regulating bone mass by increasing osteoprotegerin expression thereby reducing osteoclastogenesis [28, 29 ]. it has been shown that sclerostin exerts its negative regulation of bone formation by binding via its central core to the extracellular domains lpr5/6 at the first -propeller via an nxi motif. fundamentally, the interference of sclerostin in the wnt / lrp / fzd complex results in uninhibited gsk-3 activity and the phosphorylation of -catenin leading to its subsequent degradation. consequently, translocation into the nucleus is not actively facilitated which renders wnt pathway gene promoters inactivated. this effectively inhibits the anabolic function of wnt signalling for bone and as a consequence decreases bone formation (figure 1(b)). mutations in the lrp receptors initially helped establish a link between wnt signalling and disease and their role as positive regulators of bone formation. the lrp5 gene was initially identified as a determinant of bone mass in the 1990s. specifically, linkage analyses studies demonstrated that the human chromosome 11q13 was associated with two extremes of low and high bone mass [33, 34 ]. osteoporosis pseudoglioma syndrome, an autosomal recessive hereditary disorder (characterised by low bone mass and abnormal eye vasculature), was traced to an inactivation mutation of lrp5. in contrast, gain - of - function lrp5 mutations are linked to the autosomal dominant high bone mass (hbm) which are defined by excessive bone formation, bone thickening, and reduced risk of fracture [34, 35 ]. briefly, lrp5-deficient mice manifested an osteoporotic phenotype arising from the impaired bone formation while bone resorption remained unaffected. in contrast, mice carrying an hbm mutation within the lrp5 gene display osteosclerosis [36, 37 ]. several studies using cre - loxp - technology focused on the inactivation of -catenin specifically in osteoblasts and osteoclasts in an effort to understand the molecular signature of the skeletal phenotype displayed by these diseases. essentially, these and other similar studies demonstrated that -catenin deletion in cells within the osteoclast lineage resulted in increased osteoclastogenesis. in contrast, -catenin deletion in mesenchymal osteoprogenitor cells resulted in the prevention of osteoblast differentiation with an associated preference for chondrogenesis [38, 39 ]. importantly, -catenin inactivation in fully differentiated osteoblasts and osteocytes resulted in increased osteoclastogenesis arising from the decreased osteoprotegerin while bone formation remained unaffected [29, 40, 41 ]. more recently it has been demonstrated that frizzled-8 and -catenin negatively regulate osteoclast differentiation independent of osteoblasts and that canonical wnt signalling controls bone resorption by two different mechanisms. the authors report that mice deficient in frizzled-8 manifest osteopenia associated with the unhindered bone formation and increased osteoclastogenesis. nevertheless, this phenotype was not associated with impaired osteoprotegerin production or wnt signalling by osteoblasts. further, -catenin deletion in the osteoclast lineage confirmed the negative influence of canonical wnt signalling on osteoclastogenesis. here increased bone resorption was evident despite the apparently normal production of osteoprotegerin by osteoblasts being observed. the reader is directed to a recent review for a more comprehensive insight into the molecular associations of lrp5 and bone formation. the potential of sclerostin as a therapeutic target was soon recognised and gained momentum when animal studies with sost knock - out mice were also found to have increased bmd over wild - type counterparts, whereas transgenic mice overexpressing sost displayed an osteoporotic bone phenotype [18, 44 ]. given the similarities to the changes observed in lrp5 mutations, the notion sclerostin functioned as an lrp5 antagonist was pursued and later confirmed nonetheless, it is now recognised that other interactions are also involved in the antiosteoanabolic role of sclerostin [22, 23 ]. more recently, yorgan and colleagues attempted to clarify if sclerostin truly acts as a wnt signalling antagonist by interacting with lrp5. the authors generated col1a1-sost mice with transgenic overexpression of sclerostin under the control of a 2.3-kb col1a1 promoter, resulting in a low bone mass phenotype. the two mouse lines used carried different high bone mass mutations of lrp5 (lrp5 (a170v) and lrp5 (g213v)). subsequently, the authors found that the inhibitory function of sclerostin overexpression on bone formation was not observed in lrp5 (g213v / g213v) mice and was strongly reduced in lrp5 (a170v / a170v) mice. the authors then adopted a similar approach whereby the transmembrane wnt signalling antagonist krm2 was overexpressed in mice. interestingly, the antiosteoanabolic influence of the col1a1-krm2 transgene was not affected by either lrp5 mutation investigated. due the heterogeneous cellular make - up of bone, gaining detailed insight into the transcriptional response to scl - mab and the associated mechanistic nature of its modulation of wnt signalling pathway has proved challenging. in the context of the mesenchymal stem cell osteogenic differentiation pathway, sclerostin activity has the most effect on the late osteoblast cells, inhibiting the terminal differentiation of osteoblasts and the associated mineral deposition. it has been postulated that sclerostin may function in the upregulation of small integrin - binding ligand n - glycoprotein (sibling) family formation. sibling proteins bind to newly mineralised surfaces preventing further mineralisation through acidic serine aspartate - rich motifs (asarm peptides). it also appears that the formation of phex, a metalloprotease capable of inactivating asarm peptides disruption of mineralisation, is inhibited by sclerostin too. more recently, nioi and colleagues used laser capture microdissection to assess changes in mrna expression of specific canonical wnt - related genes in osteoblasts, osteocytes, and bone lining cells in ovx - rat vertebrae that had undergone a single administration (100 mg / kg) of 1scl - mab. samples were obtained 6, 24, 72, and 168 hours after scl - mab administration. microarray analyses revealed that five canonical wnt - related genes in particular, namely, gja1, bglap, twist1, mmp2, and wisp1, were markedly upregulated subsequent to scl - mab exposure, suggesting a targeted activation of canonical wnt signalling associated genes. notably, there was a significant upregulation of extracellular matrix - associated genes in osteoblasts, osteocytes, and bone lining cells in response to scl - mab treatment. this would suggest that bone anabolism is facilitated via the activation of matrix - producing osteoblasts and transition of bone lining cells to active matrix - producing osteoblasts. interestingly, while scl - mab treatment has been observed in several animal and human studies to enhance bone formation and reduce bone resorption, here gene modulation of osteoclastogenesis remained unaffected. increased osteoprotegerin is a likely candidate modulator given it is recognised as a canonical wnt target. the complexity of the mechanisms by which scl - mab exert their effect is an ongoing area of research and vital to a full understanding of its potential as a therapeutic target. meanwhile, the neutralisation of sclerostin using monoclonal antibodies has been subject to numerous preclinical studies and a range of clinical trials which are addressed below. these reviews include its discovery and development as a therapeutic target, its mode of action, and its suitability as a biochemical marker, along with more general overviews [2, 4749 ]. the initial development and screening of sclerostin neutralising antibodies involved a murine igg1 produced in a hybridoma cell line which was validated in vitro. due to the subsequent commercial development of the antibody, information relating to its characteristics has been limited. nevertheless, the eli lilly anti - sclerostin antibody blosozumab is described as an igg4 antibody while the novartis humanized scl - mab is described as an igg2 antibody. the first in vivo study to test anti - sclerostin antibodies for increasing bone mass involved a rodent model of postmenopausal osteoporosis (figure 2). ovariectomised (ovx) rats were left untreated for one year in order to develop significant levels of bone loss. treatment of 19-month - old ovx - rats with scl - mab (25 mg / kg, biweekly for 5 weeks) appeared to completely reverse the bone loss exhibited over the previous year - long period. in fact, bone strength and mass were both reportedly higher in scl - mab treated ovx - rats compared to non - ovx control animals. more recently, it has been determined that the increases in bone formation observed due to scl - mab treatment in rats and primates do not negatively influence bone matrix quality even where increases in bone volume as high as 54% are noted. the transition from preclinical models to human clinical trials was facilitated by a major primate study that used dosage levels and a delivery schedule intended to replicate those of early - stage human trials. this study confirmed a strong correlation between serum antibody levels and serum levels of the bone formation markers osteocalcin and p1np and was considered to provide strong evidence that the effect of treatment was robust and reproducible. a complete chronological summary of all clinical trials investigating scl - mab is presented in table 1. data from two phase i trials of the humanized scl - mab romosozumab (amgen, formally known as amg785) have been published. the initial first - in - human single - dose randomized study included healthy men and postmenopausal women (aged 4559 ; nct01059435). this was followed by a multidose study (nct01825785) [4, 53 ]. describe a randomized, double - blind, placebo - controlled, ascending - single - dose study in healthy men and postmenopausal women which received romosozumab or placebo (3 : 1) subcutaneously (0.1, 0.3, 1, 3, 5, or 10 mg / kg) or intravenously (1 or 10 mg / kg). the primary objective of the study was to establish safety and tolerability of romosozumab. secondary objectives included the evaluation of pharmacokinetics, bone turnover markers, and bone mineral density. a total of 72 subjects were enrolled with 56 (14 placeboes, 42 romosozumab) receiving investigational product administered subcutaneously while 16 subjects (4 placeboes, 12 romosozumab) received the investigational product intravenously. both subcutaneous and intravenous dosing resulted in a greater - than - dose - proportional increase in romosozumab serum concentrations, with apparent clearance decreasing as dose increased. increases in p1np, bone - specific alkaline phosphatase (bap) and osteocalcin (markers of bone formation) were dose - related with maximum increases from baseline of 184%, 126%, and 176%, respectively. the serum concentration of c - terminal telopeptide of type 1 collagen (ctx ; bone resorption marker) concomitantly decreased in an approximate dose - dependent manner with a maximum decrease of 54% below baseline reported. as expected, bmd was also significantly enhanced with maximum increases of 5.3% in the lumbar spine and 2.8% in the hip observed. overall, treatment was generally well - tolerated with one serious adverse event being reported for a subject that received 10 mg / kg dose. the subject experienced nonspecific hepatitis which commenced 1 day after dosing but resolved after approximately 1 week. of the 54 subjects receiving romosozumab 11% (6/54) tested positive for binding anti two of these subjects tested positive for neutralising antibodies (1 subject 10 mg / kg subcutaneous ; 1 subject 5 mg / kg intravenous). nonetheless, when placed in context with existing therapies, a single 10 mg / kg dose of scl - mab was found to produce bmd increases that were either equivalent or greater than that observed after 6 months of daily teriparatide treatment. a subsequent double - blind, placebo - controlled, randomized, ascending - multiple - dose phase i trial (nct01825785) was completed to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of romosozumab treatment for 12 weeks in 16 healthy men and 32 postmenopausal women with low bone mass. the first cohort received 6 doses of 1 or 2 mg / kg every 2 weeks (q2w). the second group of participants received 3 doses of 2 or 3 mg / kg every four weeks (q4w). the healthy male subjects received 1 mg / kg q2w or 3 mg / kg q4w or placebo. romosozumab treatment in all groups was observed to increase pinp by 66147% and lumbar bone mineral density by 47%, associated with a concomitant 1550% decrease in serum ctx. bone formation marker levels (p1np, osteocalcin, and bsap) returned to baseline 48 weeks subsequent to the last romosozumab dose. with the exception of increased mild injection site reactions with romosozumab, adverse events appeared balanced between treatment and placebo groups. similarly to the single - dose study, two subjects tested positive for neutralising antibodies which reportedly had no apparent effects on the primary study objectives. mcclung and colleagues have recently published results from a phase ii, multicentre, international, randomized, placebo - controlled, parallel - group, eight - group study, where the efficacy and safety of romosozumab over a 12-month period was evaluated in 419 postmenopausal women (5585 years of age ; nct00896532). these comprised either subcutaneous romosozumab monthly injections (dose of 70 mg, 140 mg, or 210 mg) or every 3 months (140 mg or 210 mg), subcutaneous placebo, or an open - label active comparator - oral alendronate (70 mg weekly) or subcutaneous teriparatide (20 g daily). the primary endpoint was the percentage change in bmd in the lumbar spine at 12 months compared to baseline. secondary endpoints included the percentage change in bmd at other sites and changes in markers of bone turnover. after 12 months, the pooled romosozumab group had significantly higher bmd compared to the pooled placebo group irrespective of the frequency of dose (monthly / every 3 months) and dose concentration (140 mg/210 mg)., mean increases (compared to baseline) of 11.3%, 4.1%, and 3.7% in the lumbar spine, 4.1% total hip, and femoral neck were observed, respectively. furthermore, the increases in bmd observed were significantly greater than the alendronate (4.1% increase in bmd in the lumbar spine) and teriparatide groups (7.1% increase in bmd in the lumbar spine). interestingly, serum levels of bone formation markers in this trial were reported to increase transiently. this contrasts the robust and reproducible increases in serum bone formation markers reported subsequent to romosozumab treatment in the iconic primate study. mcclung and colleagues observed that increases in serum bone formation markers occurred as soon as 1-week after administration with peak levels reported after 1 month. however, levels returned or fell below baseline values within 29 months (dependent on dose and marker evaluated). all romosozumab groups demonstrated a decrease from baseline in the level of ctx initially, with the largest median decrease evident after one week. furthermore, subjects receiving monthly doses of romosozumab maintained levels of ctx below baseline after 12 months. the incidence of serious adverse events in the pooled romosozumab group was 7% which was comparable to the placebo (14%), alendronate (8%), and teriparatide (9%) groups. the 210 mg (administered every 4 weeks) romosozumab group had 10% (5 subjects of 51) occurrence of serious adverse events. these included breast cancer, chronic obstructive pulmonary disease, noncardiac chest pain, wrist fracture, and begin renal oncocytoma. however, no serious adverse event was reported by more than 1 participant in any group and none were considered by the investigator to be treatment - related. as observed in the previous phase i studies, binding antibodies were identified in 20% of subjects receiving romosozumab, with 3% of these demonstrating in vitro neutralising activities. despite their occurrence, there was no apparent effect on the occurrence of adverse events, pharmacokinetics, or pharmacodynamics. overall, given the significant and prompt increases in bmd subsequent to romosozumab treatment compared to alendronate and teriparatide, this study further supported the application of romosozumab in the treatment of osteoporosis. more recently, this trial was extended to include an additional 12-month treatment period under similar study conditions as outlined above. although only currently available in the abstract form, the increases in bmd in the lumbar spine and total hip observed during the first 12 months of treatment were further increased with the additional 12-month romosozumab. the largest gains in bmd were again observed with the 210 mg monthly dose (15.7% lumbar spine bmd ; 6.0% total hip bmd). this study was extended to include a one - year double - blind extension phase where eligible subjects (women receiving 210 mg romosozumab dose) were rerandomized 1 : 1 within their original treatment group to placebo or denosumab 60 mg once every six months (n = 260). women receiving romosozumab 210 mg monthly who then transitioned to treatment with denosumab (targets rankl) after 12 months continued to accrue bmd at a rate similar to that observed during the second year of treatment with romosozumab. however, subjects that transitioned to placebo demonstrated a return in bmd towards pretreatment levels. furthermore, in subjects where romosozumab treatment was discontinued after 2 years, a decrease in bmd towards baseline levels and the return of bone formation marker p1np to pretreatment levels were observed. another ongoing study is assessing the impact of romosozumab treatment compared to teriparatide treatment on vertebral mass, thickness, and density. although not published, initial results conveyed by amgen suggest that treatment with romosozumab produced an increase in cortical thickness of 11.2% compared to 5.6% with teriparatide. furthermore, romosozumab induced an increase of 22.2% in trabecular bmd compared to a 17.4% increase with teriparatide. in contrast, a reduction of 4.3% was reported in the placebo group (nct01796301). a phase i trial assessing changes in baseline lumbar spine bmd after the transition from alendronate to romosozumab for 3 months has recently been completed with results pending (nct01588509). it would certainly appear that romosozumab has great promise in the treatment of osteoporosis and based on these results it would appear that market approval may be foreseeable. less clear at the moment, however, are efficacy issues regarding long - term treatment (i.e., in excess of 2 years as a decrease to baseline is observed after discontinuation) and safety concerns relating to potential long - term treatment schedules. for the moment, at least, it would appear that romosozumab would be highly beneficial in the short - term and is compatible with antiresorptive therapies. the first study included a single - dose, dose escalation study (7.5 to 750 mg i.v. ; this was followed by a multicentre safety and tolerability study of multiple - dose administration (q2w, q4w ; nct01742091). participants were otherwise healthy postmenopausal women ranging in age within 4570 years of age (single - dose) and 4580 years of age (multidose). however, antibodies to the drug were discovered upon screening (23% of patients in the single - dose study ; 36% in the multidose study). nonetheless, titres were low and their occurrence did not appear to impact pharmacodynamics. furthermore dose - dependent increases in drug serum levels and bone formation markers p1np and bap and osteocalcin along with a decrease in ctx were recorded. treatment also resulted in increased bmd in the lumbar spine with a maximum increase above baseline of 3.41% after a single dose and 7.71% after multiple doses after 85 days (compared to 5.3% and 7.2%, resp. these positive clinical results were followed in a more recently randomized, double - blind, placebo - controlled multicentre phase 2 clinical trial of blosozumab comprising 120 postmenopausal women (4585 years) with low bone mineral density (lumbar spine bmd t - score of 2.0 to 3.5 ; nct01144377). the study encompassed a 1-year treatment period with a 3-month follow - up period once treatment ended. the primary objective was to evaluate the dose - response of 180 mg (every 2 or 4 weeks) to 210 mg (q2w) (compared to 70 mg210 mg in the phase two trial of romosozumab) of blosozumab on lumbar spine bmd. in terms of efficacy, mean increases in bmd in the lumbar spine were statistically significant for all treatment groups. a maximum increase in the lumbar bmd of 17.7% above baseline in the group receiving 270 mg every two weeks was reported. in the 3-month follow - up study, levels of p1np peaked at week 4 and remained significantly above baseline for 24 weeks and returning towards baseline levels by the end of the study. levels of osteocalcin and bone alkaline phosphatase also increased with blosozumab treatment and returned towards baseline by the end of the study. the bone resorption marker ctx levels decreased to less than placebo within two weeks and were similar to placebo at 12 weeks and reduced compared to placebo at 52 weeks. mild injection site reactions were reported by up to 40% of patients receiving blosozumab ; otherwise, adverse events were similar between treatment and placebo groups. four women taking blosozumab were diagnosed with breast cancer within 3 months to 1 year of the beginning of the trial ; however, examination suggested these were likely to be preexisting tumours. anti - blosozumab antibodies were found in 35% of those treated with one patient developing neutralising antibodies that resulted in greatly reduced efficacy from treatment. a one - year follow - up study assessing the effects of discontinuing blosozumab treatment in the phase ii patient cohort has recently been published. no serious adverse effects after discontinuation of the treatment were observed. with the discontinuation of treatment, a decline in bmd in both the femoral neck and the lumbar spine was observed in all blosozumab treatment groups, which continued through the 1-year follow - up period. moreover, serum biochemical formation and resorption markers did not differ significantly between previously treated blosozumab and placebo groups. while in the initial phase ii trial approximately 35% of blosozumab treated patients presented with antitreatment antibodies, these were seen to decline with discontinuation of the treatment. while no serious safety issues were raised, john lechleiter the chairman of the board, president, and chief executive officer of eli lilly explained that phase iii trials proposed for 2014 were delayed due to higher than desirable levels of injection site reactions. this led to the reassessment of the formulation used in their phase ii trials before moving forward. the occurrence of injection site reactions appears to be a common occurrence subsequent to antisclerostin treatment for all humanized scl - mab tested and is currently under investigation by both amgen and eli lilly. three bps804 phase ii trials to treat postmenopausal women with low bmd (nct01406548), osteogenesis imperfecta (nct01417091), and hypophosphatasia (nct01406977) have been completed. to date, no data has been published and there is no information available in relation to plans for phase iii trials. an additional phase ii trial to investigate the safety and tolerability of bps804 in patients with late - stage chronic kidney disease (nct01806610) has since been withdrawn prior to initiation of patient enrolment. not surprisingly, therefore, bps804 does not currently feature in the clinical pipeline reports for 20142018 published by novartis. nevertheless, the company 's interest in fundamental research pertaining to the mechanistic actions of sclerostin in bone repair persists [6062 ]. the combined therapy approach of scl - mab antibodies followed by ongoing use of antiresorptive drugs to maintain enhanced bone formation is another emerging area of investigation. the use of alendronate in relation to pretreatment and cotreatment with antisclerostin therapy has been assessed in animal studies with encouraging results. this stands in contrast to the currently approved anabolic treatment with human parathyroid hormone (pth). in this case, it was hypothesised that cotreatment with alendronate would enhance the anabolic qualities of pth. however, the combined treatment was actually found to have a reduced anabolic effect in clinical studies [64, 65 ]. the cotreatment of anti - tumour necrosis factor (anti - tnf) along with anti - sclerostin antibodies in human tnf transgenic mice has recently been shown to be more effective than either treatment administered alone. specifically, cotreatment of mice with anti - tnf and scl - mab was found to be effective in repairing cortical lesions, cartilage destruction, and preventing proteoglycan loss. these results were an improvement compared to anti - tnf or scl - mab alone which appeared to prevent further disease progression but did not support tissue repair. for instance, in this model, the combination of anti - tnf and scl - mab treatment was observed to prevent cortical and trabecular bone loss (an increase of 34% compared to baseline) and restored vertebral bone to levels observed in nonarthritic wild - type mice. the combination of anti - tnf and scl - mab significantly decreased arthritic bone erosion compared to baseline as well as reducing osteoclast number. interestingly, compared to baseline levels, scl - mab alone or in combination with anti - tnf significantly increased cartilage thickness, area, and proteoglycan content. this combination of anti - inflammatory treatment with bone enhancing antibodies could also have potential to aid the treatment of decreased bmd due to colitis. the option of alternating anabolic and antiresorptive therapies has also been the focus of several recent clinical trials relating to romosozumab treatment and is highlighted in section 4.1. the efficacy of anti - sclerostin antibody therapy in the treatment of fragility fractures has been tested in rodent and nonhuman primate preclinical models [6669 ]. more recently, two phase ii clinical trials (nct00907296) (nct01081678) have also investigated this application. in all rodent experimental models (rat diaphyseal defect, rat closed mid - diaphyseal femoral fracture, rat femoral osteotomy, and murine femoral osteotomy), subcutaneous administration of 25 mg / kg of scl - mab twice weekly for varying time courses confirmed enhanced bone healing with significant increases in bone formation, mass, and strength [6669 ]. furthermore, cui and colleagues report that the size of the repair callus in scl - mab treated mice with osteotomies was increased as early as 2 weeks after treatment compared to controls. the scl - mab treated group was associated with a faster fracture union by week 6 and significantly higher maximal loading capacity. similarly, suen and colleagues report an increase in fracture callus size of 2330% at 3, 6, and 9 weeks after scl - mab administration compared to vehicle controls. histologically more bony tissue and less cartilage tissue were observed in fracture calluses across all time points in the scl - mab treated groups. moreover, the proportion of mature callus tissue was significantly greater with the scl - mab treatment at weeks 6 and 9. this was reflected by the significant increases in total bone volume (2633%) and high - density bone volume (3842%) compared to vehicle groups. the scl - mab treatment also resulted in faster mineral deposition compared with vehicle controls. furthermore, scl - mab increased the rate of new bone formation in both the total callus (41%) and the periosteal callus subregion (42%) at 9 weeks. specifically, a significantly higher load in scl - mab treated groups was reported at weeks 6 (98%) and 9 (53%) after fracture compared to controls. similarly, scl - mab treatment resulted in significant increases in callus stiffness and energy to failure. the positive impact of scl - mab treatment in fracture repair setting is also reflected elsewhere. more recently, yee and colleagues have demonstrated enhanced bone formation using scl - mab in an early on - set type i diabetic mouse fracture model. here the authors report that scl - mab treatment rescued impaired osteogenesis and marrow adiposity that is associated with the diabetic phenotype. moreover, in uninjured bone, the positive effect of scl - mab on bone formation persisted for up to 3 weeks after discontinuation of the biweekly (25 mg / kg) treatment. despite the promise of scl - mab treatment in fracture repair, the reparative effect does not appear to reproducibly extend to nonunion fractures [72, 73 ]. have recently demonstrated that treatment with scl - mab can enhance bone repair in the surrounding bone of a rat femoral critical - sized bone defect but does not possess osteoinductive activity to heal it. thus, it seems that scl - mab is effective in enhancing bone formation of preexisting and regenerating bone tissue but does not appear to be an osteoinductive agent. this appears to be supported by a recent study that has shown that endochondral bone formation persists in fractures lacking sclerostin (sost / mice) while fibrocartilage callus removal was enhanced. examined scl - mab treatment for the repair of bilateral fibular osteotomies in cynomolgus monkeys. treatment consisted of 30 mg / kg of anti - sclerostin antibody twice weekly for 10 weeks. the therapy led to an increase in serum levels of the bone formation markers osteocalcin and procollagen 1 n - terminal propeptide (p1np) and an associated increase in bone formation. after just 7 weeks, fractures in the vehicle and scl - mab groups healed to 27% and 48% of the mean peak load of the intact contralateral femurs in the vehicle group. in addition, scl - mab administration significantly improved the rate by which the majority of intact strength was achieved compared to vehicle - treated groups. the rate to union was also markedly improved with scl - mab treatment. specifically, 9/10 in the latter group achieved union compared to 4/9 fractures in the vehicle - treated group. histologically, there was a significant increase in bone formation within the fracture area in scl - mab treated groups. this was associated with reductions in the persistence of a cartilaginous callus within this group. the smaller fracture gaps observed within the scl - mab groups also displayed less fibrovascular tissue compared to vehicle - treated groups, although this difference was not statistically significant. at the fracture site, scl - mab treatment resulted in a 27% increase in mature bone callus formation associated with a 30% increase in bone mineral content. furthermore, scl - mab produced a 48% greater mean torsional stiffness and 32% greater peak torque compared to vehicle controls. both phase ii trials assessing scl - mab in fracture repair (nct00907296 and nct01081678) directed by amgen have submitted requests in may 2013 and january 2014, respectively, to delay publication of the trial results. at the time of writing, neither blosozumab (eli lilly) nor bps-804 (novartis) appears to be undergoing investigation at clinical trial. therefore, to date, the efficacy of scl - mab therapy in the application of fracture repair has not been established in human trials. furthermore, a press release early in 2013 confirmed that amgen would not be pursuing scl - mab treatment for fracture healing into phase iii trials. sclerostin levels are greatly increased in the early stages of fracture healing which may add to the complexity of providing effective neutralisation to enhance fracture repair. fracture repair and related aftercare currently account for a staggering 95% of the cost of all osteoporosis treatments. the lack of scl - mab therapy in this area will see patients remain restricted to the prophylactic treatment options to reduce fracture risk for the foreseeable future. high failure rates are reported for total hip replacement due to the weakness of the surrounding bone. the anchorage of screws and pins used in fracture repair can also be compromised due to poor bone quality [10, 11 ]. preclinical studies have demonstrated efficacy in significantly enhancing bone fraction volume adjacent to the implant subsequent to scl - mab administration. treatment has also been shown to increase peak pull - out forces [76, 77 ]. moreover, screws inserted postmortem also showed increased pull - out force in the treated group. this would appear to imply a general rather than injury specific response to antibody treatment. moreover, scl - mab administration has demonstrated promise in preventing periprosthetic osteolysis and aseptic loosening in a preclinical study. more recently, in a severe model of osteoporosis (approximately 78% trabecular bone loss at the time of implantation) systemic scl - mab administration has also proven effective in stimulating osseointegration via enhanced bone formation. here, the authors report improved trabecular bone volume and architecture as well as decreased bone resorption subsequent to scl - mab treatment. in addition to the treatment of osteoporosis, antisclerostin therapy has been explored as a treatment for other conditions causing reduced bone mineral density. preclinical animal studies encompassing periodontitis, colitis, rheumatoid arthritis, osteoarthritis, bone health complications of diabetes mellitus, chronic kidney disease, and osteogenesis imperfecta (oi) have all been encouraging [8085 ]. however, there appears to be some contradiction between studies possibly resulting from the different animal models used or the disease stage in which scl - mab were administered. for instance, treatment of severe oi with scl - mab had limited success in enhancing trabecular bone volume and cortical thickness in growing mice (4 weeks). in contrast, treatment failed to have any significant impact in adult oi mice (20 weeks). furthermore, neither growing nor adult oi mice displayed treatment - associated alterations in bone remodelling serum markers compared to wild - type controls. in contrast, moderately severe oi in the brtl/+ mouse model of oi responded positively to scl - mab treatment. here, treatment resulted in stimulated osteoblast - mediated bone formation leading to increases in bone mass and reduced long - bone fragility [83, 86 ]. bisphosphonates are currently used in the treatment of oi and have been shown to lower the risk of fracture in some cases [87, 88 ]. while generally well - tolerated, there are, however, unresolved issues relating to the use of bisphosphonates in oi. these include issues relating to long - term retention, clinical reproducibility of lowering fracture risk, functional benefit, and potential childhood bisphosphonate toxicity [8992 ]. moreover, other anabolic treatments such as growth hormone and teriparatide have provided mixed outcomes. for instance, prolonged use of teriparatide may be potentially associated with the development of osteosarcoma [93, 94 ]. given these issues and the observed anabolic effect of scl - mab in osteoporosis and fracture repair, specifically, it is thought that scl - mab treatment may benefit paediatric oi patients in particular by increasing bone mass and mechanical strength and reducing the risk of fracture. a 2-week treatment course of scl - mab was found to produce increases in bone mass and mechanical strength in the well - established brtl/+ mouse model of moderately severe type iv oi. however, more extensive studies are required to fully establish the potential of scl - mab in oi, moreover, given that it is likely that scl - mab therapy will need to be administered as an adjunctive therapy (see section 7), the long - term benefits in oi need to be addressed. recently, chen. combined anti - tnf t with scl - mab treatment in a mouse model of rheumatoid arthritis. the combination therapy was found to contribute to repair of damaged articular cartilage and eroded bone, whereas either treatment alone only prevented progression of disease symptoms. however, a recent study has suggested that pharmacological inhibition of sclerostin does not impact articular cartilage remodelling ; therefore, an explanation for the positive outcomes described above is yet to be fully elucidated [81, 95 ]. periodontitis, a destructive disease of the tooth - supporting structures ultimately resulting in tooth loss, is another area which may potentially benefit from scl - mab therapy to repair large osseous defects. recent evidence suggests that sclerostin (as well as dkk-1) is significantly increased in the gingival tissue and the serum of chronic periodontitis patients. these findings suggest a possible molecular link between sclerostin and periodontal disease. using a variety of methods including lineage tracing and knock - out models, it has recently been shown that activation of the wnt - canonical pathway via scl - mab treatment results in cementum and alveolar bone regeneration [85, 97, 98 ]. taut and colleagues recently investigated the effect of administering 25 mg / kg scl - mab administration delivered twice weekly for a therapeutic duration of 3 and 6 weeks in an experimental periodontal rat model. notably, after 6 weeks, the scl - mab treated group displayed reversed ligature - induced bone loss. this outcome was also associated with a significant increase in bone volume and tissue mineral density compared to vehicle - treated controls. interestingly, local administration of scl - mab displayed limited effect on volumetric alveolar bone healing. alveolar bone loss was, however, significantly improved after 6 but not 3 weeks subsequent to systemic scl - mab treatment compared to vehicle - treated groups. bone densitometry scanning was also performed on femora to assess off - site skeletal responses to scl - mab administration. the femora of all treatment groups demonstrated significant increases in bone mineral density at both 3- and 6-week time points. bone formation markers were also increased after scl - mab administration which translated to the restoration of lost bone microarchitecture, volume, and density to levels comparable to intact control after 6 weeks. interestingly, the application of scl - mab as a preventative measure to alveolar bone loss (administered when sutures were initially placed and while at the site of defect) only resulted in a slight and nonsignificant increase in bone mass after 2 and 4 weeks. these findings highlight the suitability of scl - mab as an adjunctive therapy in this context. chen and coworkers also studied the effect of administering 25 mg / kg vehicle or scl - mab subcutaneously twice weekly for 6 weeks in ovariectomised rats with experimental periodontitis (ligature). the authors noted that the administration of scl - mab leads to significant increases in the bone mineral apposition rate in ovx - rats compared to controls. the authors attribute this finding to increased bone formation and decreased bone resorption evident from significant increases in osteocalcin and osteoprotegerin and serum tartrate - resistant acid phosphatase and ctx, respectively. it is worth considering that current osteoconductive agents, such as bone substitutes used in this application, are clinically unpredictable in their consistency to form bone. consequently, in an effort to recapitulate the complex biological events involved in wound healing and repair, a multiphasic approach is becoming necessary. as noted previously, current evidence would suggest that scl - mab possesses osteoconductive rather than osteoinductive properties [62, 73 ]. therefore, it remains to be seen if (a) scl - mab is superior to current available osteoconductive agents such as commercial bone substitutes / grafts at producing more clinically consistent bone regeneration and (b) if scl - mab treatment, either systemically applied or locally administered via a multiphase scaffold or current commercial grafts, could provide a more robust outcome of targeted bone repair. growing evidence suggests that circulating wnt signalling inhibitors such as dkk-1 and sclerostin may crucially contribute to the pathogenesis of chronic kidney disease - associated bone mineral disorder (ckd - mbd). serum sclerostin levels have been shown to increase with ckd - mbd progression and may be potentially linked with cardiovascular events observed in this patient population [103, 104 ]. in addition to its prominent role in bone remodelling, the wnt pathway and several of its inhibitors including sclerostin are increasingly associated with the occurrence of extraosseous mineralisation. this occurrence is similar to that observed in cardiovascular calcification and calciphylaxis, a rare life - threatening condition which manifests predominately in patients with ckd or end - stage renal disease [105, 106 ]. the biological similarities of vascular calcification and bone formation are gaining increased attention and it is believed that bmp-2, in particular, may be a vital link. specifically, bmp-2 function can be inhibited by active matrix gla protein (mgp), the activator of which is vitamin k dependent. moreover, vitamin k antagonist usage has been implicated as an independent risk factor for the development of calciphylaxis. sclerostin also antagonizes the effect of bmp-2 indirectly via inhibition of the wnt/-catenin signalling pathway. this may help explain the biological link between circulating serum sclerostin levels and its link to cardiovascular events observed in recent studies. nevertheless, the potential of sclerostin as a biomarker in this context requires additional confirmation. preliminary evidence in a cross - sectional multislice computed tomography scanning study of 67 chronic haemodialysis patients suggests that increased sclerostin expression was colocalised at the sites of calcifying aortic heart valve disease. others have implicated increased serum sclerostin levels in ckd patients to be statistically correlated with inflammation, vascular lesions, uremia, and potentially mortality. in contrast, others have found a contradictory correlation whereby higher levels of serum sclerostin were associated with improved survival in prevalent haemodialysis patients. moreover, in a recent prospective study of 673 incident dialysis patients, increased serum levels of sclerostin were associated with lower short - term (up to 18 months) cardiovascular (hazard ratio 0.29 ; 95% ci 0.130.62) and all - cause mortality (hazard ration 0.39 ; 95% ci 0.220.68). at this point, there is no clinical data to underline the therapeutic efficacy of scl - mab in ckd - mbd. however, recent preclinical animal studies have reported enhanced bone volume and mineralisation with scl - mab treatment specifically when pth levels are low [113, 114 ]. good tolerability has generally been reported subsequent to scl - mab administration. however, the development of neutralising antibodies to antisclerostin treatment has been reported including one case which seriously reduced the efficacy of treatment [4, 21, 53, 55 ]. moreover, the potential immunogenicity of full - size monoclonal antibodies has been raised as a general safety concern. ultimately, ongoing phase iii trials of full - size sclerostin neutralising antibodies will determine if immunogenicity is sufficient to cause concern. nevertheless, while isolated, these events have contributed to the ongoing development of smaller antibody fragments [95, 115, 116 ]. have shown the sclerostin - neutralising activity of sclerostin antibody fragment (scl - fab ; mw 48 kd) to be as effective as that of the parent scl - mab igg in the rat medial meniscus tear model of osteoarthritis. moreover, the sclerostin single chain fragments (scl - scfv ; 35 kd) were observed to increase bone density, enhance bone formation, and improve bone microstructure in a rat preclinical model of osteoporosis. however, while scl - scfv demonstrated high specificity and affinity, the authors note it had lower stability than the full - sized antibody. the route to market approval for any novel therapeutic agent is never easy and in the case of antisclerostin treatment the stakes are incredibly high. years of research stand in the balance along with an estimated $ 99 million in global sales annually if market approval is secured. while encouraging results from both preclinical and clinical studies have grabbed attention, a relatively small number of clinical safety and efficacy considerations have emerged. one serious adverse event has been reported in the first clinical trial of amg785 (nct01059435). here, one subject developed severe nonspecific hepatitis which began one day after receiving 10 mg / kg of amg785 (romosozumab). the hepatitis was resolved after 26 days, and additional cases of hepatitis have not been reported in subsequent trials to date. as previously mentioned, the study of sclerosteosis and van buchem disease uncovered the role of sclerostin in the modulation of the wnt/-catenin dependent pathway. as a precaution, therefore, prominent health issues relating to both conditions were investigated. while heterozygous carriers of sclerosteosis have higher than normal bmd, they are otherwise considered clinically normal. however, bone overgrowth in the skull that constrains the cranial nerves and induces facial palsy can occur. there was no indication of a disproportionate increase in the bone mineral content of the skull. results from brainstem auditory evoked potential tests to determine any negative impact on nerve function and hearing were also considered unremarkable by a blinded clinician. the wnt/-catenin signalling is involved in a wide variety of developmental and adult tissue processes. unsurprisingly, therefore, dysregulation of this integral pathway is associated with a multitude of diseases including cancer, fibrosis, and neurodegeneration [119, 120 ]. a commercial therapeutic specifically targeting the wnt - pathway is not currently available. with market approval of scl - mab fast approaching, jia and colleagues constitutively activated -catenin using catnb+/lox(exon 3) mice which were crossed with mice expressing a tamoxifen - inducible procollagen i cre - er promoter. they demonstrate that constitutively activated -catenin resulted in an excessive bone volume within the vertebral column in both early postnatal (3 days) and mature tissue (up to 7 months postnatally). however, the authors noted that early stabilisation of -catenin essentially slowed linear bone growth within the vertebrae by retarding growth plate maturation. moreover, histologically the excessive newly formed bone appeared immature and occurred primarily adjacent to the growth plate. in contrast, late - stage -catenin stabilisation appeared to not affect bone maturity or distribution. more recently, it has also been shown that timely suppression of the wnt/-catenin pathway is required for osteocytic differentiation to occur adequately. in this study, -catenin was constitutively activated in osteocytes by crossing catnb+/lox(exon 3) mice with dentin matrix protein 1-cre transgenic mice. interestingly, stabilisation of -catenin in osteocytes was observed to substantially increase cancellous bone mass. however, the activation was noted to have severe adverse effects on bone strength and bone growth. mice were shorter and presented with impaired linear growth of the long bones. the integral role of wnt/-catenin signalling in development and disease, as well as the effects described in recent studies, should certainly be seriously considered prior to the adoption of an application involving its therapeutic interference. direct comparisons between humanized scl - mab are currently unavailable. however, a curious difference between a recent phase ii blosozumab study and a prominent primate study assessing romosozumab is of potential interest [21, 52 ]. clearly, comparison of a year - long human clinical trial and a 2-month long primate study assessing structurally different humanized forms of scl - mab is anecdotal. nevertheless, in the absence of an explanation, it is worth briefly discussing. in the human clinical trial using blosozumab, p1np levels increased promptly during the first 4 weeks of treatment. concurrently, the serum concentration of the bone resorption marker ctx rapidly decreased to a concentration below that noted for the placebo group within the first 2 weeks of treatment. by the end of the study, p1np levels were comparable to pretreatment levels while ctx remained reduced. the authors suggest that the return of p1np towards baseline levels in the later stage of treatment (after week 25) may be a result of the initial increase in bone formation reducing stresses and strains on the skeletal system. moreover, signalling molecules such as dkk-1 may be actively involved by reducing bone formation via negative counterregulation. importantly, the observations that blosozumab increases bone formation, decreases bone resorption, and increases spine and total hip bmd are consistent with the recently published romosozumab phase ii trial comprising over 400 subjects. the clinical implications, if any, in the discrepancy of serum p1np levels in human and nonhuman primate studies remain unclear. similarly, the causes relating to the transient changes in biochemical markers of bone formation during blosozumab treatment have yet to be elucidated. some clarification may be sought in a recent study which assessed scl - mab (25 mg / kg) treatment administered once weekly for 6 or 26 weeks in ovx - rats. nevertheless, the study remains relevant given its investigation of the mechanistic effect on longer - term responses of osteoclasts, osteoblasts, and osteocytes subsequent to scl - mab treatment. specifically, the authors observed that changes observed in bone resorption and formation at 6 weeks in ovx - scl - mab treated rats largely did not persist after 26 weeks. for instance, at 6-week scl - mab treatment induced a net reduction in bone resorption as evident from decreases in serum tracp-5b and a reduction in the ex vivo capacity of marrow cells to differentiate into osteoclasts. moreover, an 80% reduction in surface erosion within the vertebrae and trabecular and tibial endocortical surfaces compared to ovx - vehicle samples was noted. at 26 weeks, however, the serum tracp-5b and reduced ex vivo osteoclastic differentiation capacity of marrow cells were no longer evident. interestingly, at this time, the authors did not observe changes in rankl or osteoprotegerin expression. as expected, scl - mab treatment induced significant increases in bone formation reflected by augmentations in serum p1np and osteocalcin. additionally, increases in the bone formation rate within trabecular, endocortical, and periosteal regions were observed. by week 26, however, only changes within the endocortical regions persevered while increases in skeletal mrna of osteocytic genes were evident. in particular one can cautiously speculate that the net increases in bone volume caused by the constitutive activation of wnt - canonical signalling via scl - mab treatment may be synergistically counterregulated via increased osteocytic mediated sclerostin expression. although these findings can not be directly inferred to human outcomes, they may provide some insight as to the interstudy differences noted above. the potential for counterregulatory signalling to reduce bone formation will require further study to address its impact on longer - term treatments. to date, however, all published studies have demonstrated efficacy despite these observations. taken together for now, the combination of scl - mab in conjunction with antiresorptive agents has demonstrated the most efficacy at trial. therefore, it is likely that this approach will be the regimen that receives regulatory approval. generally, the safety profile of scl - mab treatment is positive, especially when compared to competitor osteoporotic therapeutics in development. for instance, the cathepsin k inhibitor mk 0822 (merck, odanacatib) appears to increase, albeit not to statistically significant levels, the occurrence of atrial fibrillation and stroke in treated versus placebo groups. these safety concerns with mk 0822 were only brought to light with the release of data from a phase iii trial. similarly, the full extent of the safety profiles of romosozumab and blosozumab can not be fully determined from the comparatively small phase ii studies. compared to current anabolic market competitors such as teriparatide which is limited to 2 years ' lifetime use, the substantial improvements in bone formation and microarchitecture subsequent to scl - mab treatment have been unrivalled. consequently, the results of clinical studies demonstrating the therapeutic potential of romosozumab in phase iii trials are eagerly awaited. while the clinical data relating to efficacy appears clear, there remain significant questions relating to optimal treatment interval. for instance, given its apparent superiority to increase bmd, should scl - mab be the first line of treatment for osteoporotic patients ? however, it remains to be established if scl - mab can be used for a long term in a cyclical manner either alone or in a complementary manner with antiresorptive options. many studies have already been performed following scl - mab therapy with an antiremodelling agent. this approach may help address questions attesting to the tolerability of scl - mab and whether the emergence of neutralising antibodies may become more of an issue with increased exposure. the emergence and clinical success of scl - mab have reinvigorated the therapeutic market for osteoporosis. despite the many questions that remain, preclinical studies and published clinical trial results would imply that scl - mab will emerge as a dominant first - line treatment in the management of osteoporosis. moreover, scl - mab is set to become a valuable tool in the development of international strategies to address the rise of osteoporosis and osteoporotic fracture management on a global scale. | it is estimated that over 200 million adults worldwide have osteoporosis, a disease that has increasing socioeconomic impact reflected by unsustainable costs associated with disability, fracture management, hospital stays, and treatment. existing therapeutic treatments for osteoporosis are associated with a variety of issues relating to use, clinical predictability, and health risks. consequently, additional novel therapeutic targets are increasingly sought. a promising therapeutic candidate is sclerostin, a wnt pathway antagonist and, as such, a negative regulator of bone formation. sclerostin antibody treatment has demonstrated efficacy and superiority compared to other anabolic treatments for increasing bone formation in both preclinical and clinical settings. accordingly, it has been suggested that sclerostin antibody treatment is set to achieve market approval by 2017 and aggressively compete as the gold standard for osteoporotic treatment by 2021. in anticipation of phase iii trial results which may potentially signify a significant step in achieving market approval here, we review the preclinical and clinical emergence of sclerostin antibody therapies for both osteoporosis and alternative applications. potential clinical challenges are also explored as well as ongoing developments that may impact on the eventual clinical application of sclerostin antibodies as an effective treatment of osteoporosis. |
advances in oncological and supportive care have led to improved prognoses and extension of survival time in cancer patients. consequently, increasing numbers of patients with cancer require admission to intensive care units (icus). in the last issue of critical care, dr taccone and coworkers reported that patients with cancer represent a large proportion of icu patients. in their substudy from the sepsis occurrence in acutely ill patients (soap) study conducted in 198 european icus, 15% of patients had a malignancy, mostly solid tumors but also hematological malignancies. these findings are in accordance with results from the saps-3 study, performed in 2002 in an international population comprising almost 20,000 icu patients ; these results showed that 3% of these patients had metastatic cancer, 6% had non - metastatic cancer and 2% had hematological cancer. less than 10 years ago, in guidelines for icu admission, a taskforce of the american college of critical care medicine concluded that patients with hematological or metastasized solid malignancies were poor candidates for icu admission. these patients were considered to have a very high risk (up to 90%) of mortality. at that time, in contrast with the very poor prognosis reported in the literature, taccone and coauthors reported much lower hospital mortality of 58% in icu patients with hematological cancer and 27% in patients with solid malignancies, compared with 23% in icu patients without cancer. others have also reported the improvement in prognosis after icu admission for patients with hematological cancer. in hematopoietic stem cell transplant recipients who received invasive mechanical ventilation, mortality was uniformly higher than 90% in studies before 1993, but gradually decreased to 52% in 2000. in addition to advances in stem cell transplantations, improvements in critical care may have contributed to this improvement in prognosis for these patients. clearly, patients should no longer be refused admission to icus only because they have hematological cancer. a relapsed / refractory state of leukemia and a poor sequential organ failure assessment (sofa) score were found to be the independent risk factors associated with mortality in patients with acute leukemia and should be considered when decisions regarding icu admission are made about patients with hematological cancer. in the study by dr taccone and colleagues, it should be noted that patients with solid cancers form a very heterogeneous population, with many different forms of cancer, different oncological treatments and different reasons for admission to the icu. most icu patients with cancer are admitted after surgery, often as primary treatment for their cancers, and the short - term prognosis of these patients is mostly good. in patients after transhiatal esophageal resection for esophageal cancer, hospital mortality may be as low as 3.5%. likewise, mortality after pancreatico - duodenectomy in patients with pancreatic cancer may be less than 5% in experienced centers. the outcome after major oncological surgery may be mostly related to the surgical procedure, more than to the critical care on the icu. only limited data are available about patients with cancer admitted to icus for other reasons than post - operative care after oncological surgery. azoulay and coauthors reported 30-day mortality of 58% in patients admitted for medical reasons. in a brazilian study involving 1,090 patients with cancer requiring icu admission for reasons other than routine postoperative care, most of these patients had non - metastasized solid cancer, and most patients required mechanical ventilation. in patients with a prolonged icu length of stay, mortality was independently associated with the number of failing organs, age and performance scale score. clearly, icu treatment is not futile for all patients with cancer. despite these recent data, rates of refusal of icu admission in cancer patients remain high and the criteria on which triage decisions are based differ between oncologists and intensivists. decisions to withhold life - sustaining treatments are more often made for patients with cancer than patients with other terminal diseases, even when these other diseases have at least the same poor prognosis. this has been demonstrated clearly for patients dying from chronic heart failure compared to patients with metastatic cancer. over the past years the prognosis of cancer patients has improved significantly, even when icu admission is necessary. refusal of icu admission should not be based on the diagnosis of cancer as the underlying condition. in contrast, these decisions should be based on the availability of treatment options, and on patients ' own preferences. unfortunately, current prognostic models for icu patients, all based on data from the first 24 hours after icu admission, such as apache (acute physiology and chronic health evaluation) ii and saps (simplified acute physiology score) ii, can not reliably predict whether cancer patients will survive icu admission. when in doubt, it may be a very good option to start full unlimited treatment for a few days. discontinuation of treatment should be considered if progressive organ failure is seen after 3 to 5 days. | a recent paper by taccone and coworkers showed that 15% of patients from 198 european intensive care units (icus) had a malignancy, mostly solid tumors but also hematological malignancies. over the past years, the prognosis of cancer patients has improved significantly, even when icu admission is necessary. refusal of icu admission should not be based on a diagnosis of cancer as the underlying condition. in contrast, these decisions should be based on the availability of treatment options, and on patients ' own preferences. |
epidermal growth factor receptor - tyrosine kinase inhibitors (egfr - tkis) have become the standard of care in the management of egfr mutant lung cancers. compared to chemotherapeutic agents, egfr - tkis have proven their superiority in terms of survival and toxicity profile when treating nonsmall cell lung cancer (nsclc) patients positive for egfr mutation. with the exception of leptomeningeal metastasis where erlotinib has shown better response than gefitinib, both of these tkis are equally efficient when treating egfr mutant nsclc. hence, safer toxicity profile becomes one of the most important factors when choosing these tkis. in this study, we compared the different toxicity profiles of erlotinib and gefitinib among indian population. eighty - five patients of south indian origin were screened for egfr mutation, at cancer institute, chennai, india. egfr mutation test was performed by scorpion probe - based amplified refractory mutation system - reverse transcription - polymerase chain reaction. patients were started on either erlotinib 150 mg or gefitinib 250 mg in the first - line setting based on physician discretion. detailed history and physical examination with special emphasis on drug toxicity was performed at every visit. toxicity of tkis was graded according to the national cancer institute common terminology criteria for adverse events v 4.0. of the 85 patients tested for egfr mutation, 34 (40%) patients were positive for the same. twenty - three patients were started on gefitinib and 11 patients were started on erlotinib. demograpphic profile of patients on tki 's therapy skin toxicity was the major side effect of tkis. nine of the 11 patients treated with erlotinib had skin toxicity compared to 7 of the 23 patients treated with gefitinib. grade 34 skin toxicity was observed in five patients among erlotinib arm compared to only one patient among gefitinib arm. of the nine patients who developed skin rash with erlotinib, four required dose reduction from 150 mg to 100 mg. in four patients, erlotinib was changed to gefitinib, as reducing the dose did not result in decrease in skin toxicities [figure 1 ]. gefitinib - induced drug rash was managed conservatively with antihistamines and clindamycin skin ointment without treatment interruption. erlotinib - induced pustular skin lesions affecting face, leg, and hand other side effects such as diarrhea and deranged liver function were comparable in both the groups as shown in table 2. randomized studies have clearly shown survival benefit of tkis compared to chemotherapy when treating egfr mutated lung cancer. tkis such as erlotinib and gefitinib used in the treatment of egfr mutated lung cancer have similar toxicity profiles, but the grades and severity of the toxicities have not been studied extensively. in this prospective study, we compared toxicity profile of erlotinib and gefitinib in indian patients. in our study, nine of the 11 patients treated with erlotinib had skin toxicity with five of them having grade 34 skin rash (45.45%) whereas only 7 of the 23 patients treated with gefitinib had skin toxicity and only one among them had grade 34 skin rash. grade 34 toxicity due to erlotinib in our study was much higher than that found in optimal and eurtac studies (2% and 13%, respectively). this probably is because the steady - state plasma trough concentration by erlotinib at its maximal tolerated dose of 150 mg was 3.5 times higher than that produced by gefitinib at its approved dose of 250 mg once daily which was approximately one - third of the maximum tolerated dose. because of the skin toxicity, erlotinib dose was reduced to 100 mg in four patients which they could tolerate, and for the other four patients, erlotinib was changed to gefitinib as their skin toxicity recurred with the same severity even following dose reduction. all these four patients tolerated gefitinib well and three of them had grade 1 rash. in only one patient, erlotinib was continued at 150 mg after treating skin rash with antihistamines and clindamycin topical ointment. there was a significant delay in the treatment in erlotinib arm due to skin toxicity. treatment had to be stopped for at least 20 days in six of the patients until the rash subsided. in the gefitinib arm, there were no treatment delays and grade 23 rash was managed with antihistamines and clindamycin topical ointments. other side effects such as diarrhea, deranged liver function test, and hand - foot syndrome were comparable in both arms. the limitation of our study is small sample size. skin toxicity is a major side effect with erlotinib among indian patients which results in significant treatment delay, which in turn may adversely affect the survival of patients with egfr mutant lung cancer. dose reduction and changing the drug were helpful in patients who could not tolerate 150 mg of erlotinib. gefitinib had a much more friendly toxicity profile and was well tolerated among indian patients. 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. | introduction : erlotinib and gefitinib are the most commonly used epidermal growth factor receptor - tyrosine kinase inhibitors (egfr - tkis) in the treatment of egfr mutant nonsmall cell lung cancer (nsclc). both erlotinib and gefitinib have shown equal efficacy in terms of response rates and overall survival. hence, their toxicity profile becomes the most important determining factor in choosing these agents when treating egfr mutant nsclc. in this study, we compared the toxicity profile of erlotinib and gefitinib among an indian subset of lung cancer patients.materials and methods : in this prospective nonrandomized study, 85 patients of south indian origin with nsclc were tested for egfr mutation status, and egfr mutant patients were started on either erlotinib or gefitinib. they were periodically monitored for drug toxicities.results:out of the 85 patients tested, 34 patients were positive for egfr mutation. eleven of them were started on erlotinib and 23 were started on gefitinib. the most common side effect of tkis was skin rash. nine out of the 11 patients started on erlotinib and 7 of the 23 patients started on gefitinib had skin rash. grade 3 and 4 skin rash was significantly more among patients treated with erlotinib which resulted in treatment delays. other side effects of tkis such as diarrhea and deranged liver functions were similar among the both subsets of patients.conclusion:skin toxicity is the major and serious side effect with erlotinib among indian patients with egfr mutant lung cancer. this resulted in significant treatment delay, which might adversely affect the overall survival of patients. gefitinib was better tolerated and had a safer toxicity profile compared to erlotinib in indian patients. |
endoscopic ultrasound - guided fine - needle aspiration (eus - fna) has become a mainstay diagnostic technique for the evaluation of lesions arising from the pancreas and upper gastrointestinal tract as well as adjacent structures, including lymph nodes and the liver. eus - fna is safe and has a diagnostic accuracy of 60 % to 90 % 1 2 3. the diagnostic yield of eus - fna is influenced by several factors : the nature of the target lesion, experience of the endoscopist, presence of an on - site cytopathologist, and various technical aspects, such as needle size and number of passes. in order to limit the influencing parameters, 2011 that features a hollowed - out reverse bevel to trap core samples (echotip procore ; cook medical, bloomington, indiana, usa). a potential advantage of this type of needle is the acquisition of larger amounts of tissue with preserved architecture. preliminary results on the performance of the 19-gauge (g), 22 g, and 25 g procore needles were promising, with reported high diagnostic accuracy rates of 89.6 % 4, 88.5 % 5, and 86 % 6, respectively. however, comparative studies of the 22 g procore needle versus the 22 g standard cytology needle showed similar results for the cytology parameters, amount of blood contamination, and diagnostic adequacy and accuracy of the cell block material 7 8 9. preliminary data demonstrated an 83 % first - pass sensitivity for pancreatic malignancy, with a cumulative sensitivity of 96 % after three passes 6. histologic diagnosis was possible in 63 % after a single pass and in 80 % after multiple passes 6. taking into account the results of a recent meta - analysis 10 that shows a potential advantage of the 25 g needle over the 22 g needle for the diagnosis of pancreatic malignancy, the idea of a 25 g needle that can obtain histologic specimens sounds attractive. therefore, we designed a prospective study comparing the most commonly used 22 g standard cytology needle versus the 25 g echotip procore needle in the same group of lesions in terms of differences in accuracy, technical performance, and quality and quantity of the cytology and cell block specimen obtained. this pilot study included patients with pancreatic mass lesions or lymphadenopathy who were referred for eus - fna sampling at our hospital (grand hpital de charleroi, charleroi, belgium) between december 2012 and october 2013. exclusion criteria included the following : cystic lesion, coagulation disorder (international normalized ratio > 1.5, platelet count < 50 000/mm), pregnancy, age < 18 years, and refusal or inability to provide informed consent. the study was approved by the grand hpital de charleroi review board, and written informed consent was obtained from all patients for participation in the study. a linear array echoendoscope (uct160 ; olympus medical systems, tokyo, japan) connected to a processor featuring color doppler function (prosound alpha 7 ; hitachi medical systems europe [aloka ], zug, switzerland) was used for all procedures, which were carried out by four experienced endosonographers (a.s., p.w., h.h., and c.g.) and one gastroenterology fellow (g.m. the gastroenterology fellow was present during all procedures in order to ensure adherence to the study protocol. after the lesion had been carefully inspected and vessel interposition along the puncture route excluded by color doppler, eus - fna was performed with both a 22 g (echotip) needle and a 25 g echotip procore needle (fig. 1). the order in which the needles were used was according to computer - generated randomization. during each puncture, the needle went through the lesion, and 15 to 20 to - and - fro movements were made with minimal negative pressure simultaneously applied by pulling the needle stylet slowly and continuously (slow - pull technique) 6. after the second puncture, if the acquired specimen was not adequate, eus - fna was repeated with one of the two needles until an adequate specimen had been obtained. fna material was recovered in a vial containing 15 ml of thinprep preservcyt solution (hologic, marlborough, massachusetts, usa) by flushing with 5 ml of this solution and, if needed, by pushing the stylet through the needle. the specimens obtained were labeled according to the aspiration sequence and sent to a single experienced cytopathologist (b.w.), who was blinded to the types of needles used. one monolayer was obtained with thinprep 2000 and stained with papanicolaou stain (cytologic specimen) ; the leftover material was centrifuged and the clot retrieved in agar, fixed in formalin for at least 2 hours, and embedded in paraffin to obtain a cell block (histologic specimen). one 5-m - thick slide stained with hematoxylin and eosin was available for every cell block. only the results of the first and second punctures were compared. the 25 g echotip procore histology needle (left), which has a hollowed - out reverse bevel, and the 22 g echotip standard cytology needle (right). the quality of the obtained cytologic and histologic specimens was reported by the cytopathologist according to the following grading system : insufficient material or contaminated material (score of 0), rare diagnostic cells (score of 1), diagnostic cells at every field with 10 times magnification (score of 2), or diagnostic cells at every field with 20 times magnification (score of 3). the primary outcome parameter was the percentage of cases in which the pathologist classified the quality of the sample as sufficient for histocytologic evaluation (score 1). this grading system, although not published as such, is very easy to apply to a cytologic slide, especially when the specimen has been obtained with a monolayer technique. it is also easily reproducible because one needs only to record the magnification at which diagnostic cells are seen filling the microscopic field. the visibility of each needle was qualitatively scored as optimal or suboptimal and the ease of puncture as easy or difficult 6 11. the amount of blood contamination was scored qualitatively as no blood (score of 1), small quantity of blood (score of 2), or large quantity of blood (score of 3) 12. the quantity of the specimens was graded as small (score of 1) in the presence of fragments with no visible core, medium when one or two cores were visible (score of 2), or excellent when at least three cores were visible (score of 3). a final diagnosis of malignancy or benignancy was made according to one of the following reference methods : (1) definite benign or malignant histologic diagnosis based on surgical resection specimens from patients who had undergone surgery, (2) cytology or histology findings with definite proof of malignancy in patients with unresectable tumors according to imaging findings and compatible clinical follow - up, and (3) cytology or histology findings without proof of malignancy and a minimum clinical and radiologic follow - up of 7 months. for comparison of continuous data, a paired t test was performed if a normal distribution was shown, and the wilcoxon rank sum test was carried out if normality could not be demonstrated. a p value of less than 0.05 was regarded as statistically significant with spss 17.0 for windows (spss inc., a total of 28 eus - fna procedures targeting lesions of the pancreas (n = 19) and lymph nodes (n = 9) were performed in 27 patients (18 women, 9 men) with a median age of 69 years (range, 38 the final diagnoses were pancreatic adenocarcinoma (n = 18), pancreatic neuroendocrine tumor (n = 1), malignant lymphadenopathy (n = 6), and benign lymphadenopathy (n = 3). no benign pancreatic lesions were encountered. in the subgroup of patients with pancreatic lesions, 10 were punctured through the duodenum and 9 were punctured through the stomach. in the subgroup of patients with lymphadenopathy, the median lymph node size was 24 mm (range, 15 final diagnoses were made on the basis of surgery in 3 cases, positive fna for malignancy with a compatible clinical course in 23 cases, and negative fna for malignancy with at least 7 months of follow - up in 2 cases. visualization was suboptimal in 16 % of punctures with the 25 g needle versus 0 % of punctures with the 22 g needle ; however, this difference was not statistically significant (p = 0.125) (videos 1, 2). no relevant differences were found regarding the ease of puncture (p = 0.688), amount of blood contamination (p = 0.705), macroscopic quantity of the material (p = 0.858), and quality of the cytologic (p = 0.438) and histologic (p = 0.220) specimens (table 1). subgroup characteristics regarding the adequacy of histocytologic material for the two needle types and different types of lesions are shown in table 2. video 1 endoscopic ultrasound - guided fine - needle aspiration of a pancreatic mass with a 25 g echotip procore needle. video 2 endoscopic ultrasound - guided fine - needle aspiration of a pancreatic mass with a 22 g echotip needle. wilcoxon rank sum test. in the subgroup of patients with pancreatic cancer, each needle missed two cases. transduodenal puncture with the 25 g needle showed rare benign cells (cytology score = 1, cell block score = 1), while puncture with the 22 g needle was noncontributive (cytology score = 0, cell block score = 0). because of the strong suspicion of malignancy, a follow - up eus - fna a few weeks later was performed with a standard 22 g needle and confirmed the diagnosis of pancreatic adenocarcinoma. each needle missed one other case of pancreatic adenocarcinoma of the head of the pancreas punctured through the duodenum because of insufficient histocytologic material (total histocytologic score = 0). therefore, the single - pass sensitivity for pancreatic neoplasia for both needles was 89.5 % (95 % ci 66.82 98.39). in the subgroup of patients with lymphadenopathy, a total of two false - negative results for malignancy were obtained with both needles that concerned the same patients. the first patient had gallbladder cholangiocarcinoma and perihepatic lymph nodes suspicious for malignancy that were 2 cm in size. transduodenal puncture with both needles resulted into noncontributive histocytologic material (total histocytologic score = 0). the second patient had lung cancer and mediastinal lymph nodes suspicious for malignancy (4 cm in size). eus - fna with both needles showed rare benign cells (cytology score = 1, histology score = 1). however, clinical and radiologic follow - up was compatible with metastatic lymph nodes. therefore, the single - pass sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for malignancy were equal for the needle types : 66 % (95 % ci 24.1 94), 100 % (95 % ci 30.9 100), 44.4 % (95 % ci 39.5 100), 60 % (95 % ci 17 92.7), and 84.8 % (95 % ci 67.3 94.2), respectively. overall, eus - fna is highly effective for most pancreatic tumors and solid malignancies adjacent to the upper gastrointestinal tract, with reliable sensitivity, specificity, and overall diagnostic accuracy of 60 % to 90 % 1 2 3. however, fna cytology specimens may not be adequate in cases in which the diagnosis relies on tissue architecture, such as autoimmune pancreatitis, lymphomas, gastrointestinal stromal tumors, and well - differentiated adenocarcinomas 1 13. in order to procure larger amounts of tissue with preserved architecture that would enable histologic analysis, a novel needle assembly with a reverse - bevel technology (echotip procore) was introduced in 2010 2011. according to the designer, the side notch (not tru - cut) it had the ability to obtain full histology in 89.5 % of cases, with an overall diagnostic accuracy of 93 % 4. however, because technical difficulties were encountered during transduodenal passes, the same needle was later introduced in a 22 g platform. a pubmed search revealed only three studies of procore needles comparing the diagnostic yield of the most commonly used 22 g fna needle with that of the 22 g procore needle (table 3) 7 8 9. no advantage of the procore design was reported in terms of cytology and histology. however, in the study by witt. 8, fewer passes were needed to achieve diagnosis with the 22 g procore needle (2.11 with the 22 g procore needle vs 2.94 with the 22 g standard needle). nevertheless, this result should be interpreted with caution because of the small number of different groups of lesions (n = 18). interestingly, in the study by strand. 9, technical failure was encountered in 5 of 32 patients because of significant resistance to advancement of the needle, with deflection of the echoendoscope (gf - uct140, olympus) while in the transduodenal position. potential advantages of the smaller caliber are greater flexibility, less friction in the needle sheath, easier penetration of hard pancreatic tumors, and fewer bloody aspirates. preliminary data on the performance of the 25 g procore needle suggest an 83 % first - pass sensitivity for pancreatic malignancy, with a cumulative sensitivity of 96 % after three passes 6. histologic diagnosis was possible in 63 % after a single pass and in 80 % after multiple passes. in our study, both the 22 g standard needle and the 25 g procore needle had a slightly better single - pass performance. cytologic and histologic diagnoses were possible in 84.2 % and 78.9 % of cases, respectively, with an 89.5 % overall sensitivity for pancreatic cancer.to the best of our knowledge, this is the first randomized, single - group, prospective study to assess the diagnostic yield of the 25 g procore versus the 22 g standard cytology needle in pancreatic mass lesions and lymphadenopathy adjacent to the upper gastrointestinal tract. although the 25 g needle was not visualized optimally in 16 % of punctures no effect on the quality of cytology or histology, amount of blood contamination, and technical difficulty was observed. we found no significant differences between the two needles in terms of diagnostic yield, with similar diagnostic rates and similar numbers of successful procedures. our prospective, single - group, paired design has the intrinsic strength that performance comparisons were made on the same group of lesions, eliminating in this way the bias of differences in lesion type, size, and location. this is in contrast with most eus - fna studies, which compare different needles in different populations. a larger number of cases may be needed to detect subtle differences between the two needles. furthermore, no benign pancreatic lesion was encountered during the study period, such as focal autoimmune pancreatitis, in which an eus - fna diagnosis is challenging. in conclusion, this pilot study demonstrates that the diagnostic yield of the new 25 g echotip procore needle is comparable with that of a 22 g standard fna assembly. both needles performed equally in terms of the quality of cytologic and histologic specimens, blood contamination, and ease of puncture. macroscopic (top), cytologic (center), and histologic (bottom) images of endoscopic ultrasound - guided fine - needle aspiration specimens of pancreatic adenocarcinoma punctured with a 25 g echotip procore (left) and a 22 g echotip cytology needle (right). | background and study aims : a new 25-gauge (g) endoscopic ultrasound - guided fine - needle aspiration (eus - fna) device (echotip procore ; cook medical, bloomington, indiana, usa) has been developed, which features a hollowed - out reverse bevel to trap core samples. however, data on the differences between the diagnostic yield of the 25 g echotip procore and that of a 22 g standard needle are limited. patients and methods : this pilot study included 27 patients referred during an 11-month period for eus - fna of pancreatic masses and enlarged lymph nodes adjacent to the upper gastrointestinal tract. each lesion was punctured once by both a 25 g echotip procore needle and a 22 g standard needle (echotip ; cook medical) with capillary sampling. blinded histocytologic analyses were conducted. the final diagnosis was based on fna findings of malignant cells, pathologic analysis of the surgical specimen, and/or radiologic and clinical follow - up of at least 7 months. results : a total of 28 eus - fna procedures targeting masses of the pancreas (n = 19) and lymph nodes (n = 9) were performed. no complications were encountered. single - pass sensitivity rates for pancreatic and lymph node malignancy were equal for the needle types : 89.5 % (95 % ci 66.82 98.39) and 66 % (95 % ci 24.1 94), respectively. there were no significant differences between the needles in terms of eus visualization (p = 0.125), amount of blood contamination (p = 0.705), macroscopic quantity of the material (p = 0.858), quality of the cytology (p = 0.438), and adequacy and accuracy of the cell block material (p = 0.220). conclusions : both needles were safe and successful in terms of a high diagnostic yield, with similar histocytologic results.the results of this study were presented at digestive disease week (ddw) 2014, chicago, illinois.this trial was registered at clinicaltrials.gov (b027201316271). |
a 73 year old female patient presented in may 2005 with lower abdominal pain, abdominal bloating and increased frequency of bowel habits. subsequent colonoscopy detected a neoplastic lesion at the splenic flexure confirmed at biopsy to be adenocarcinoma. however, at laparotomy, the primary arising from the splenic flexure was found to be advanced with diffuse intra abdominal metastases. post - operatively, the patient received palliative chemotherapy with good response indicated by tumour shrinkage in serial ct scans and falling carcinoembryonic antigen (cea). she later presented to the emergency department in august 2007 with abdominal pain and marked abdominal distension. abdominal x - ray showing colonic dilatation at laparotomy, the ascending and transverse colon was dilated to 12 cm with features of a giant mucocele which yielded 3.5 litres of clear mucus. this was found to be due to closed loop obstruction between the terminal ileum adherent to the pelvis as a result of peritoneal disease and local relapse at the splenic flexure resulting in accumulation of mucus and formation of a mucocele. a loop ileostomy was fashioned and the transverse colon was drained with a 24 french 2 way catheter brought out at the top end of the wound. post operatively, about 1600 ml of mucus was drained from the catheter in the initial few days and this subsequently reduced to 125 ml daily. appendiceal mucocele is a well recognized entity and occurs due to appendiceal obstruction followed by luminal accumulation of mucus and dilatation of the obstructed appendix (1). it may be associated with concomitant colon cancer and hence thorough investigation of the colorectal tract is recommended after diagnosis of an appendiceal mucocele (2). rarely an appendiceal mucocele can become very large and can present as an abdominal mass (3,4). it has been reported as an unusual complication after diversion transverse loop colostomy in a patient with long - standing ulcerative colitis resulting from distal stomal and rectal stenosis and accumulation of mucus in the closed loop over many years 5). mucocele following sequestration of a segment of bowel wall at the site of colo - colic anastomosis after resection of sigmoid carcinoma has been reported. in this case, the patient had rising carcino - embryonic antigen levels (cea) with increased uptake on positron emission tomography (pet) scan at the site of original anastomosis raising the suspicion of a recurrence. following excision of the mucocele perforation of a colonic mucocele has been reported 22 years after formation of ileostomy in a patient with crohn s disease. this occurred due to stenosis of the distal colonic segment and subsequent development of a mucocele (7). in this case, a closed loop of the right colon and transverse colon was formed by distal obstruction at the splenic flexure by tumour relapse and proximal obstruction at the ileo - caecal valve by peritoneal metastasis. the continued secretion and accumulation of mucus led to distension of the closed colonic loop. a similar presentation has been reported in a patient with carcinomatosis peritonei from recurrent squamous cell carcinoma of the cervix, eight months after palliative bypass of obstructed ascending colon (8). colonic mucoceles mostly present with abdominal distension which usually evolve slowly and insidiously over a period of 2 - 6 years (5). it may cause obstructive symptoms if it is large enough to compress adjacent bowel segments. in patients with loop colostomies or mucous fistulas, the cessation of mucous drainage from either the rectum or the mucous fistula should alert the surgeon to the possibility of a closed loop obstruction (9). imaging modalities such as ultrasound and ct can be used to aid diagnosis and this requires a high index of suspicion. in a majority of cases, the diagnosis is confirmed at exploratory laparotomy. in this case, a ct scan done five months before the acute presentation revealed colonic dilatation and local relapse at the splenic flexure along with liver metastasis and peritoneal disease (figure 2). a diagnosis of colonic mucocele was not made and the air fluid level in the closed loop was thought to be due to competent ileocaecal valve. on hindsight, the absence of intestinal obstruction in spite of impressive colonic dilatation in the ct scan should have raised the suspicion of an evolving colonic mucocele. this reinforces the need for a high index of suspicion so that the diagnosis can be made pre - operatively and the patient managed conservatively. ct abdomen showing dilatation of ascending and transverse colon upto splenic flexure with air fluid levels. in a case where diagnosis has been confirmed on imaging, treatment can be undertaken by ct guided drainage (8). other treatment options include excision of the mucocele and this should usually be undertaken in low - risk surgical patients who have a good prognosis (5). in cases where there is a high chance of mucocele recurrence, a permanent catheter or a sump drainage tube can be placed and the patient can be taught self irrigation (5). colonic mucocele is an extremely rare entity and requires a high index of suspicion for diagnosis. the treatment depends on the underlying disease process and the overall prognosis of the patient. we believe this to be the first reported case in literature of a colonic mucocele following palliative bypass surgery for advanced colonic cancer. | we report an unusual case of a giant colonic mucocele following ileo - sigmoid bypass surgery in a patient with advanced adenocarcinoma of the splenic flexure. the formation of a giant colonic mucocele resulted from distal splenic flexure obstruction due to tumour relapse and proximal caecal obstruction due to peritoneal disease with subsequent accumulation of mucus in the closed loop. |
in this single - center, double - blind, placebo - controlled phase 2 pilot trial, eligible women were randomized 1:1 to either 10 g tx-004hr vaginal e2 softgel capsules or matching placebo vaginal softgel capsules (therapeuticsmd) administered intravaginally once - daily for 14 days. a reproducible, computer - generated block randomization schedule (produced in sas v. 9.1.3, plan procedure) was used to randomize the women to the study groups. biostudy solutions, llc generated the assignment codes, which were locked in a room with restricted access. the packaging, labeling, appearance, and route of administration of tx-004hr and placebo were identical, and all persons participating in the study were blinded to the treatment each woman received. the blind was maintained until study completion. on day 1, participants completed a questionnaire that assessed vva symptoms and specified their most bothersome symptom (mbs), and the investigator assessed the appearance of the vaginal mucosa (secretions, color, epithelial integrity, and epithelial surface thickness ; see table 1). women self - administered their first dose of study medication at the clinical site under supervision, and capsule disintegration was assessed after 6 hours. during screening, vaginal smears were collected to evaluate vaginal ph and cytology (vaginal maturation index [vmi ]). participants were provided with a diary and instructed to record dosing dates and times for the 2-week study period. on day 15, vva symptoms (participant assessment via questionnaire), vaginal mucosa, cytology, ph, and capsule disintegration (investigator assessment) were evaluated. diaries were collected and adverse events (aes) were assessed on days 8 and 15. e2 levels were tested during screening at a local laboratory using a lower limit of quantification of less than 10 pg / ml. an independent institutional review board approved the protocol, and participants provided written informed consent before any study - related activities. the trial was conducted in accordance with good clinical practices, us federal regulations, and the declaration of helsinki regarding treatment of human participants in a study. postmenopausal women (40 - 75 y of age) were enrolled if they had a body mass index less than or equal to 34 kg / m ; less than or equal to 5% superficial cells on vaginal smear cytology ; a vaginal ph more than 5.0 ; an e2 level less than or equal to 50 pg / ml ; and at least one of the following symptoms of vva with a moderate - to - severe severity score : vaginal dryness, vaginal pain associated with sexual activity, vaginal and/or vulvar irritation / itching, dysuria, or vaginal bleeding associated with sexual activity. women were excluded if they had a current or prior medical condition considered clinically significant, undiagnosed vaginal bleeding, or a current vaginal infection requiring treatment ; had a contraindication to estrogen therapy (et) or an allergy to e2 ; smoked at least 15 cigarettes per day ; or had substance abuse in the past year. participants could not have taken any prescription or over - the - counter drug that might interact with e2 therapy within 28 days of screening, or used progestogen implants, vaginal or injectable et, or vaginal or injectable estrogen - progestin therapy (ept) within the past 3 months ; transdermal et or ept or et lotions or gels within the past 8 weeks ; or estrogen pellets or injectable progestogen therapy within the past 6 months. efficacy endpoints were based on the fda 's guidance, and included change from baseline (screening) to day 15 in the percentage of parabasal vaginal cells, percentage of superficial vaginal cells, and percentage of intermediate vaginal cells ; in vaginal ph ; and change from baseline (randomization) to day 15 in self - assessed severity of most bothersome vva symptom (mbs). the range of mbs severity scores was 0 (none) to 3 (severe). endpoints also included change from baseline to day 15 in investigator assessment of the vaginal mucosa (secretions, epithelial integrity, epithelial surface thickness, and color). the severity scale ranged from 0 to 3, with 0 indicating no atrophy and 3 indicating severe atrophy. safety endpoints included changes in vital signs, weight, results of a physical examination (including breast and pelvic examination), and aes. a sample size of 50 (25 women per treatment arm) was considered adequate to estimate the magnitude and variability of efficacy measures needed to determine sample size for a future phase 3 trial. a p value of 0.05 was considered statistically significant, with the exception that confidence intervals were based on an alpha level of 0.10 (ie, 90% ci). for all comparisons except severity of the mbs and investigator assessments of the vaginal mucosa, the change from baseline values was evaluated using ancova with baseline as the covariate, to analyze the differences between the tx-004hr vaginal e2 softgel capsules and placebo groups. in this single - center, double - blind, placebo - controlled phase 2 pilot trial, eligible women were randomized 1:1 to either 10 g tx-004hr vaginal e2 softgel capsules or matching placebo vaginal softgel capsules (therapeuticsmd) administered intravaginally once - daily for 14 days. a reproducible, computer - generated block randomization schedule (produced in sas v. 9.1.3, plan procedure) was used to randomize the women to the study groups. biostudy solutions, llc generated the assignment codes, which were locked in a room with restricted access. the packaging, labeling, appearance, and route of administration of tx-004hr and placebo were identical, and all persons participating in the study were blinded to the treatment each woman received. the blind was maintained until study completion. on day 1, participants completed a questionnaire that assessed vva symptoms and specified their most bothersome symptom (mbs), and the investigator assessed the appearance of the vaginal mucosa (secretions, color, epithelial integrity, and epithelial surface thickness ; see table 1). women self - administered their first dose of study medication at the clinical site under supervision, and capsule disintegration was assessed after 6 hours. during screening, vaginal smears were collected to evaluate vaginal ph and cytology (vaginal maturation index [vmi ]). participants were provided with a diary and instructed to record dosing dates and times for the 2-week study period. on day 15, vva symptoms (participant assessment via questionnaire), vaginal mucosa, cytology, ph, and capsule disintegration (investigator assessment) were evaluated. diaries were collected and adverse events (aes) were assessed on days 8 and 15. e2 levels were tested during screening at a local laboratory using a lower limit of quantification of less than 10 pg / ml. an independent institutional review board approved the protocol, and participants provided written informed consent before any study - related activities. the trial was conducted in accordance with good clinical practices, us federal regulations, and the declaration of helsinki regarding treatment of human participants in a study. postmenopausal women (40 - 75 y of age) were enrolled if they had a body mass index less than or equal to 34 kg / m ; less than or equal to 5% superficial cells on vaginal smear cytology ; a vaginal ph more than 5.0 ; an e2 level less than or equal to 50 pg / ml ; and at least one of the following symptoms of vva with a moderate - to - severe severity score : vaginal dryness, vaginal pain associated with sexual activity, vaginal and/or vulvar irritation / itching, dysuria, or vaginal bleeding associated with sexual activity. women were excluded if they had a current or prior medical condition considered clinically significant, undiagnosed vaginal bleeding, or a current vaginal infection requiring treatment ; had a contraindication to estrogen therapy (et) or an allergy to e2 ; smoked at least 15 cigarettes per day ; or had substance abuse in the past year. participants could not have taken any prescription or over - the - counter drug that might interact with e2 therapy within 28 days of screening, or used progestogen implants, vaginal or injectable et, or vaginal or injectable estrogen - progestin therapy (ept) within the past 3 months ; transdermal et or ept or et lotions or gels within the past 8 weeks ; or estrogen pellets or injectable progestogen therapy within the past 6 months. efficacy endpoints were based on the fda 's guidance, and included change from baseline (screening) to day 15 in the percentage of parabasal vaginal cells, percentage of superficial vaginal cells, and percentage of intermediate vaginal cells ; in vaginal ph ; and change from baseline (randomization) to day 15 in self - assessed severity of most bothersome vva symptom (mbs). the range of mbs severity scores was 0 (none) to 3 (severe). endpoints also included change from baseline to day 15 in investigator assessment of the vaginal mucosa (secretions, epithelial integrity, epithelial surface thickness, and color). the severity scale ranged from 0 to 3, with 0 indicating no atrophy and 3 indicating severe atrophy. safety endpoints included changes in vital signs, weight, results of a physical examination (including breast and pelvic examination), and aes. a sample size of 50 (25 women per treatment arm) was considered adequate to estimate the magnitude and variability of efficacy measures needed to determine sample size for a future phase 3 trial. a p value of 0.05 was considered statistically significant, with the exception that confidence intervals were based on an alpha level of 0.10 (ie, 90% ci). for all comparisons except severity of the mbs and investigator assessments of the vaginal mucosa, the change from baseline values was evaluated using ancova with baseline as the covariate, to analyze the differences between the tx-004hr vaginal e2 softgel capsules and placebo groups. fifty postmenopausal women were enrolled in the study and randomized to either tx-004hr vaginal e2 softgel capsule (n = 24) or placebo (n = 26). two women in the placebo group discontinued treatment ; one was due to an ae (vulvovaginal discomfort) and the other withdrew consent. overall, participant demographics and baseline characteristics were not statistically different between the treatment groups (table 1). fifty postmenopausal women were enrolled in the study and randomized to either tx-004hr vaginal e2 softgel capsule (n = 24) or placebo (n = 26). two women in the placebo group discontinued treatment ; one was due to an ae (vulvovaginal discomfort) and the other withdrew consent. overall, participant demographics and baseline characteristics were not statistically different between the treatment groups (table 1). the prevalence of superficial cells increased from baseline by 35.2 percentage points with tx-004hr at day 15. this increase was significantly greater than the increase seen with placebo of 8.75 percentage points (p = 0.0002 ; fig. the change from baseline to day 15 in the percentage of intermediate cells also increased more with tx-004hr vaginal softgel capsules (18.7 percentage points) compared with placebo (3.54 percentage points ; p = 0.0017 ; fig., the prevalence of parabasal cells showed a significantly greater decrease from baseline to day 15 with tx-004hr than with placebo (54.4 percentage points vs 4.80 percentage points ; p < 0.0001 ; fig. changes from baseline in the maturation index of the vaginal smear (least squares [ls ] means and standard errors [se ]). p values represent significant differences in ls mean change from baseline to day 15 between the vaginal e2 softgel capsule and placebo using ancova with treatment as a fixed effect and baseline as a covariate. vaginal ph decreased significantly more from baseline to day 15 with tx-004hr (0.974) than with placebo (0.339 ; p = 0.0002). overall, no significant difference in the decrease from baseline in severity of the mbs between treatments was found (1.043 vs 1.042 for tx-004hr vs placebo, respectively ; p = 0.9951). although no statistical difference between groups was found, the individual symptoms of dryness and dyspareunia decreased in severity by approximately 1.0 and 0.8 points versus approximately 0.7 points and 0.5 points for the e2 softgel capsules and placebo, respectively. greater mean decreases from baseline were observed with vaginal epithelial integrity (0.342 vs 0.176 ; p = 0.0001) and vaginal secretions (0.643 vs 0.274 ; p = 0.0401) in the tx-004hr vaginal e2 softgel capsules group compared with the placebo group (fig. no significant differences from baseline were observed for vaginal color or epithelial surface thickness between treatments. investigator 's assessment of the mucosal epithelium (least squares [ls ] means and standard errors [se ]). p values represent significant differences in ls mean change from baseline to day 15 between the vaginal e2 softgel capsule and placebo using ancova with treatment as a fixed effect and baseline as a covariate. atrophy subjectively assessed as 0 = none, 1 = mild, 2 = moderate, and 3 = severe. the prevalence of superficial cells increased from baseline by 35.2 percentage points with tx-004hr at day 15. this increase was significantly greater than the increase seen with placebo of 8.75 percentage points (p = 0.0002 ; fig. the change from baseline to day 15 in the percentage of intermediate cells also increased more with tx-004hr vaginal softgel capsules (18.7 percentage points) compared with placebo (3.54 percentage points ; p = 0.0017 ; fig., the prevalence of parabasal cells showed a significantly greater decrease from baseline to day 15 with tx-004hr than with placebo (54.4 percentage points vs 4.80 percentage points ; p < 0.0001 ; fig. changes from baseline in the maturation index of the vaginal smear (least squares [ls ] means and standard errors [se ]). p values represent significant differences in ls mean change from baseline to day 15 between the vaginal e2 softgel capsule and placebo using ancova with treatment as a fixed effect and baseline as a covariate. vaginal ph decreased significantly more from baseline to day 15 with tx-004hr (0.974) than with placebo (0.339 ; p = 0.0002). overall, no significant difference in the decrease from baseline in severity of the mbs between treatments was found (1.043 vs 1.042 for tx-004hr vs placebo, respectively ; p = 0.9951). although no statistical difference between groups was found, the individual symptoms of dryness and dyspareunia decreased in severity by approximately 1.0 and 0.8 points versus approximately 0.7 points and 0.5 points for the e2 softgel capsules and placebo, respectively. greater mean decreases from baseline were observed with vaginal epithelial integrity (0.342 vs 0.176 ; p = 0.0001) and vaginal secretions (0.643 vs 0.274 ; p = 0.0401) in the tx-004hr vaginal e2 softgel capsules group compared with the placebo group (fig. no significant differences from baseline were observed for vaginal color or epithelial surface thickness between treatments. investigator 's assessment of the mucosal epithelium (least squares [ls ] means and standard errors [se ]). p values represent significant differences in ls mean change from baseline to day 15 between the vaginal e2 softgel capsule and placebo using ancova with treatment as a fixed effect and baseline as a covariate. atrophy subjectively assessed as 0 = none, 1 = mild, 2 = moderate, and 3 = severe. over the 14-day study period, a total of 14 (28%) women experienced 17 aes (13 aes in the tx-004hr vaginal e2 softgel group and 4 aes in the placebo group). all aes were mild in severity, no serious aes were reported, and no deaths occurred during the study. this phase 2 study showed that tx-004hr, a novel, solubilized e2 vaginal softgel capsule, was better than placebo in improving the vmi, reducing vaginal ph, and reducing atrophic effects on epithelial integrity and vaginal secretions. in addition, treatment with tx-004hr was safe and well tolerated when administered intravaginally once daily for 14 days. also, as demonstrated in phase 1 pharmacokinetic studies, systemic estrogen levels with tx-004hr were two to three times lower than with an approved vaginal e2 tablet at equivalent doses. thus, this new vaginal e2 softgel capsule is effective at treating the signs of vva with low systemic estrogen levels. furthermore, the results of a survey of the women participating in this study indicate that the majority of women thought the capsule was easy to use, were satisfied with the product, and would consider using the treatment again. the significant improvements in the signs of vva that this study found with the tx-004hr vaginal e2 softgel capsule were clinically meaningful. the efficacy observed with tx-004hr at week 2 with objective markers (ie, changes in vmi) in our study seems to be superior to historical data reported with the vaginal e2 tablet at the same dose (10 g e2). in a 52-week, multicenter, double - blind study (n = 309), improvements were observed from baseline after 2 weeks of daily administration of a 10-g e2 tablet that were significantly better than with placebo. the increase in the percentage of superficial cells at 2 weeks was significantly greater than placebo, but was less than the improvement seen in this study in which superficial cells increased to 37% after 2 weeks of treatment. the significant improvements seen in our pilot phase 2 study in all components of the vmi, in ph, and in observations of vaginal epithelial integrity and secretions are an important indication of potential improvements in symptoms of vva. the vmi and ph are the most useful objective markers of vaginal atrophy due to estrogen decline. visual assessments of the vaginal mucosa have been shown to correlate with the objective measures and, thus, are an additional validation of the diagnosis of vva and of the monitoring of effects of treatment on vva. significant improvements in vmi, ph, and the vaginal examination (vaginal epithelial integrity and vaginal secretions) were observed within 2 weeks. with a longer duration of treatment and follow - up, and a larger sample size no major or unexpected safety concerns were observed with tx-004hr vaginal e2 softgel capsule treatment during the study period. this is consistent with the favorable safety profiles of other low - dose vaginal estrogen therapies that have been previously reported. the testing of the safety and efficacy of tx-004hr vaginal e2 softgel capsules is currently being expanded in the phase 3 rejoice trial. three doses are being tested (25 g, 10 g, and an ultra - low - dose of 4 g) with additional outcome measures. primary outcome measures include change from baseline in vmi, vaginal ph, and the severity of dyspareunia as mbs after 12 weeks. secondary outcome measures include change from baseline in the severity of vaginal dryness and vaginal burning ; appearance of the vaginal mucosa at 2, 6, 8, and 12 weeks ; change from baseline in female sexual function index at 12 weeks ; and hormone concentration assessments at pretreatment, day 1, and weeks 2 and 12. the results of this study suggest that tx-004hr vaginal e2 softgel capsules are a safe, effective, local, novel treatment option for postmenopausal women with moderate - to - severe vva. improvements in vaginal cytology and ph from baseline at week 2 were significantly higher with tx-004hr than with placebo. these improvements on vaginal cell physiology seem to be superior to other vaginal therapies based on historical data. the clinical evidence presented here, coupled with favorable pharmacokinetic results showing two to three times lower systemic exposure of e2 than an approved vaginal e2 tablet at the same dose, along with positive satisfaction survey results, is promising and has helped to frame the design of a larger phase 3 trial to further evaluate the safety and clinical efficacy of tx-004hr. if approved, the tx-004hr vaginal e2 softgel capsule would provide a new alternative for women with vva, with better efficacy, earlier onset of action, easier usage, and lower systemic estrogen exposure than currently available options. | abstractobjective : the aim of the study was to evaluate the safety and efficacy of vaginal estradiol (e2) softgel capsules for moderate - to - severe symptoms of vulvar and vaginal atrophy (vva). previous phase 1 studies showed lower systemic estrogen concentrations with this softgel capsule compared with an approved low - dose vaginal e2 tablet.methods:in this randomized, double - blind, placebo - controlled phase 2 study, 50 postmenopausal women (aged 40 - 75 y) with at least1 moderate - to - severe vva symptom received 10 g vaginal e2 softgel capsules or placebo daily for 14 days. changes from baseline in vaginal maturation index, investigator 's assessment of vaginal mucosa (secretions, epithelial integrity, epithelial surface thickness, color), vaginal ph, and most bothersome symptom were assessed. adverse events were evaluated.results:compared with placebo, the percentage of superficial (35.2 percentage points [pp ] vs 8.75 pp ; p = 0.0002) and intermediate (18.7 pp vs 3.54 pp ; p = 0.0017) cells increased from baseline significantly more with vaginal e2 capsules, and parabasal cells decreased significantly more (54.4 pp vs 4.80 pp ; p < 0.0001). vaginal ph decreased significantly more with vaginal e2 capsules (0.974 vs 0.339 ; p = 0.0002). decreases in severity of atrophic effects on vaginal epithelial integrity (0.342 vs 0.176 ; p = 0.0001) and secretions (0.643 vs 0.274 ; p = 0.0401) were significantly greater with vaginal e2 capsules vs placebo. there was no statistical difference in most bothersome symptom severity change from baseline. no serious adverse events were reported.conclusions:vaginal e2 softgel capsules are a safe, effective, local treatment option for postmenopausal women with moderate - to - severe vva, with lower systemic estrogen absorption than currently available intravaginal treatments. |
pyrrole(py)-imidazole(im) (pi) polyamides are small synthetic molecules composed of aromatic rings of n - methylpyrrole and n - methylimidazole amino acids. synthetic polyamides recognize and bind to specific nucleotide sequences in the minor groove of double - helical dna with high affinity. various sequence - specific dna - binding pi polyamides have been developed to regulate gene expression by targeting the promoter regions of enhancer and transcription factor - binding elements in vitro. pi polyamide targeting rat transforming growth factor (tgf)-1 has been reported to inhibit the expressions of tgf-1 mrna and protein in the renal cortext of dahl - s rats. these observations indicate that pi polyamides will be effective for tgf-1-related diseases, including progressive renal injury [4, 5 ]. pi polyamides targeting human aurora kinase a (aurka) and b (aurkb) promoters significantly inhibited the promoter activities, and mrna and protein expression levels of aurka and aurkb. they also demonstrated a marked antiproliferative synergy in human tumor cell lines as a result of induction of apoptosis - mediated severe catastrophe of cell - cycle progression. pi polyamides specifically inhibited lectin - like oxidized low - density lipoprotein receptor-1 mrna expression and apoptosis induced by oxidized low - density lipoprotein and angiotensin ii in human umbilical vein endothelial cells. pi polyamide that targets the activator protein-1-binding site of the matrix metalloproteinase (mmp)-9 promoter significantly inhibited mmp-9 's mrna expression, protein level, and enzymatic activity in human breast adenocarcinoma cells. moreover, the polyamide treatment significantly decreased metastasis in a mouse model of liver metastasis. from these observations, we have already observed that pharmacokinetic profile of pi polyamide is related to their molecular weights [9, 10 ]. we have already reported bioanalytical method for the determination of some pi polyamides in rat plasma by using high - performance liquid chromatography (hplc), and in this paper, we show more simple and sensitive method using an internal standard (is) that was developed for different sequence and longer pi polyamide. an is is useful to compensate for the errors from sample preparation. if an is is added at the beginning of sample preparation, it is possible to quantitate analytes of interest in more simple ways with robustness. we have developed and validated a new method using an is for the quantitation of some pi polyamides in rat plasma at the lower limit quantitation of 0.25 g / ml, while it was 1 g / ml in our previous method. pi polyamides (hn.49, tgf-1f, tgf-1 t, hn.50f, hn.50 t, and lox-1) were purchased from gentier biosystems co., ltd. hplc - grade methanol, hplc - grade acetonitrile, and lc - ms - grade distilled water were purchased from kanto chemical co., inc. all the other reagents used were of the highest quality and purchased from wako pure chemical industries, ltd. stock solutions of pi polyamides were prepared by dissolving the compounds in water to produce 2 mg / ml stock solutions. the solutions were stored at 20c. calibration standard and quality control (qc) samples were prepared mixed with 10 l of diluted stock solutions and 40 l of blank plasma. the samples were mixed with 100 l of methanol contained 1 mm of phenacetin as an is and vortexed. after centrifugation at 10,000 xg for 5 min at 4c twice, the supernatant was injected in hplc system. pi polyamides and phenacetin were analyzed on a luna c18 (2) (5 m, 4.6 150 mm, phenomenex) column, maintained at 40c. the mobile phase consisted of 0.1% acetic acid (solvent a) and acetonitrile (solvent b) at a flow rate of 1.0 ml / min. the gradient started at the mobile phase a - b (100 : 0, v : v), changed linearly to a - b (20 : 80, v : v) until 10 min, washed with a - b (20 : 80, v : v) until 15 min. the autosampler was maintained at 4c. the injection volume was 30 l. the detection wavelength was set at 310 nm. selectivity was evaluated by confirming that there were no interference peaks at the retention time of pi polyamides in rat blank plasma and that there were sufficient separation of pi polyamides and phenacetin. calibration curve linearity for pi polyamide was evaluated on three different days by preparing the samples each day. calibration curves for pi polyamide were fitted by the least squares method (weighting of 1/x) using the relationship between the ratio of the pi polyamide peak area to the is peak area (peak area ratio) and the nominal analyte concentration. the criteria for the linearity of the calibration curves were defined as follows : the accuracy of the back - calculated values for the nonzero calibration standards should be within 15% (20% at the lower limit of quantitation (lloq)) and at least 75% of the nonzero calibration standards should meet the criteria, including at lloq and the upper limit of quantitation. intraday accuracy and precision were evaluated by analyzing five replicates at four different concentrations of qc samples. lloq, ql, qm, and qh, which relate to the lowest, lower, middle, and higher concentrations ranges for the linear region of the calibration curve, are used as qc samples. interday accuracy and precision were evaluated by analyzing the qc samples on three different days. accuracy, expressed as the relative error (re), should be within 15% (20% for the lloq). precision, expressed as the coefficient of variation (cv), should be less than 15% (20% for the lloq). recovery was determined by comparing the absolute peak areas of the extracted qc samples with those of the neat solutions. five replicates per sample were analyzed and the averages of the responses were used for the calculation. freezer storage stability and extracted sample stability were evaluated by analyzing qc samples stored under the relevant storage conditions in n = 5. three to four male wistar rats (280320 g) were obtained from sankyo labo service corporation (tokyo, japan). they were maintained in a temperature - controlled room in a 12-h light - dark cycle and allowed free access to food and tap water. they were acclimatized for at least one week before the experiments. a polyethylene tube (sp45 ; 0.58 mm i.d., 0.96 mm o.d., natsume seisakusho co., ltd.) was inserted into the right jugular vein and left femoral artery of the rats under anesthesia with pentobarbital sodium (kyoritsu seiyaku corporation). after cannulation, the rats were left alone for at least one night before the experiments. pi polyamide dissolved in water was administered intravenously into the jugular vein of the rats at a dose of 3.0 mg / kg. blood samples (0.5 ml) were collected into heparinized 1.5 ml microtubes at 0 and 300 min after administration. plasma was separated by centrifugation at 10000 xg and 4c for 10 min, then stored at 20c until use. the animal study was approved by the animal ethics committee of the college of pharmacy, nihon university. the peaks of pi polyamides and phenacetin were observed at the retention times of 8.1 min and 10.3 min with sufficient separation. the mean of the back - calculated concentrations over three days, the accuracy and precision are presented in table 1. calibration curve linearity was good over the range of 0.5 to 200 g / ml for hn.49, 0.25 to 200 g / ml for tgf-1f, tgf-1 t, hn.50 t, and lox-1, 1 to 200 g / ml for hn.50f in rat plasma. accuracy and precision of the assay for the determination of pi polyamides in rat plasma were presented in table 2. intraday and interday accuracy were within 14.4% to 9.4% and 14.4% to 7.5%, respectively. all the accuracy values were within 15%, and all the precision values were less than 15%. the study was conducted using low- and high - qc samples (ql and qh) to evaluate the stability of the extracted samples in the autosampler tray at 4c for 24 hours and their freezer storage stability at 20c for 14 days. all results were within 15% of the nominal values, showing sufficient stability for sample analysis and storage. the mean plasma concentration - time profiles after the intravenous administration of pi polyamides at a dose of 3.0 mg / kg are shown in figure 3. although hn.49 was below the lloq at most of the time points of blood collection, the sensitivity of the quantitation of other pi polyamides was sufficient to evaluate the plasma concentration - time profiles of the compounds. as we have already observed, molecular weights of the pi polyamide are thought to be related to their pharmacokinetic profiles [9, 10 ]. additionally, it was suggested that combination of the py and i m were also related to the unique pharmacokinetic profiles judging from the results of hn.50 t and hn.50f, and that of tgf-1 t and tgf-1f, which are almost the same molecular weights. a simple and robust hplc method was developed and validated for the determination of pi polyamides in rat plasma, and applied to the sample analysis after intravenous administration of pi polyamides. the observed pharmacokinetic profiles were unique and they are thought to be related to their molecular weights and the combination of py and i m as well. pi polyamides have been identified as novel candidates for gene therapy, and evaluation of the pharmacokinetic profile of the compounds is quite important for the development. | a simple and robust method using high - performance liquid chromatography with uv detection was developed and validated for the determination of six pyrrole - imidazole (pi) polyamides (hn.49, tgf-1f, tgf-1 t, hn.50f, hn.50 t, and lox-1) in rat plasma. after the plasma proteins were precipitated with methanol containing phenacetin as an internal standard, the analytes were separated on a luna c18 (2) (5 m, 4.6 150 mm). calibration curves were linear over the range of 0.5 to 200 g / ml for hn.49, 0.25 to 200 g / ml for tgf-1f, tgf-1 t, hn.50 t, and lox-1, 1 to 200 g / ml for hn.50f in rat plasma. the inter- and intraday precision were below 15%, and the accuracy was within 15% at the quality controls. the validated method was successfully applied to sample analysis for the pharmacokinetic study. |
gingival recession is defined as the location of the gingival margin apical to the cemento - enamel junction.1 most important factors causing gingival recession are periodontal disease and improper oral hygiene measures along with other predisposing factors such as thin gingiva, a prominent root surface, labially positioned teeth, frenum pull, and bone dehiscence.2 recession of the gingival margin results in an impaired esthetic appearance and sometimes dental hypersensitivity. gingival recessions are treated to increase the attached gingival width and in some cases for esthetics. various techniques are described to achieve root coverage. the procedures to treat the recession are classified into pedicle soft tissue grafts or free soft tissue grafts.3 pedicle graft procedures, depending on their direction of transfer may be grouped as : (i) rotational flaps (such as lateral sliding flaps or papilla flaps) or (ii) advanced flaps with or without rotation or lateral movement.4 two important factors which influence the root coverage outcome include (i) height of the interdental bone and (ii) interdental soft tissue adjacent to the defect. the depth and width of the defect and the amount of avascular tooth surface in contact with a graft during initial healing period will also affect the outcome. the double papilla technique is opted to treat miller s class 1 recession in this case report. a male patient aged 31 years came to the dental office with miller s class i recession in relation to buccal aspect of 24. patient was in good health and had not received any periodontal therapy previously. on intraoral examination, the recession was 2 mm in length and 2 mm in width (figure 1). the attached gingival width adjacent to the recession was 3 mm and probing depth of the adjacent tooth was 2 mm on mesial side, 1 mm on buccal side, 3 mm on distal side, and 2 mm on palatal side. pre - operative incisions given. local anesthesia with 2% lidocaine (2% lignox, 1:80,000) was administered, and two horizontal incisions were made on the interdental papilla, parallel to the cemento - enamel junction of the tooth to be treated with no. 15 blade. two releasing incisions were made obliquely at the line angles of the adjacent teeth, and these incisions were extended beyond the mucogingival junction (figure 2). partial thickness flap on the mesial and distal portions was elevated (figure 3), and the root surface which was exposed was planned thoroughly using a curette. root conditioning was performed using tetracycline hydrochloride (250 mg, ph 1.8)for 5 min (figure 4). de - epithelialization of the flap was done and rotated to cover tetracycline treated root surface. the other pedicle flap which was un - deepithelialized was kept in position to cover the previous flap. interrupted suturing (5 - 0 vicryl) was done across the medial area of the two papilla flaps (figure 5). surgical site was protected with tin foil (figure 6), and periodontal dressing was given with coe - pack. the patient was advised to refrain from brushing at the grafted site for 3 weeks. the patient was instructed to rinse with 0.2% of chlorhexidine mouthwash twice daily for 3 weeks. antibiotic (amoxicillin 500 mg, 3 times a day for 5 days) and analgesic (acecloren - p [aceclofenac 100 mg and paracetamol 500 mg ] 3 times a day for 5 days) was prescribed. the patient was reviewed after a week, 2 weeks, and after a month. complete root coverage was observed at 3 months follow - up (figure 7). local anesthesia with 2% lidocaine (2% lignox, 1:80,000) was administered, and two horizontal incisions were made on the interdental papilla, parallel to the cemento - enamel junction of the tooth to be treated with no. 15 blade. two releasing incisions were made obliquely at the line angles of the adjacent teeth, and these incisions were extended beyond the mucogingival junction (figure 2). partial thickness flap on the mesial and distal portions was elevated (figure 3), and the root surface which was exposed was planned thoroughly using a curette. root conditioning was performed using tetracycline hydrochloride (250 mg, ph 1.8)for 5 min (figure 4). de - epithelialization of the flap was done and rotated to cover tetracycline treated root surface. the other pedicle flap which was un - deepithelialized was kept in position to cover the previous flap. interrupted suturing (5 - 0 vicryl) was done across the medial area of the two papilla flaps (figure 5). surgical site was protected with tin foil (figure 6), and periodontal dressing was given with coe - pack. the patient was advised to refrain from brushing at the grafted site for 3 weeks. the patient was instructed to rinse with 0.2% of chlorhexidine mouthwash twice daily for 3 weeks. antibiotic (amoxicillin 500 mg, 3 times a day for 5 days) and analgesic (acecloren - p [aceclofenac 100 mg and paracetamol 500 mg ] 3 times a day for 5 days) was prescribed. the patient was reviewed after a week, 2 weeks, and after a month. complete root coverage was observed at 3 months follow - up (figure 7). complete coverage of the root is the main objective to be achieved when treating gingival recession in patients who have esthetics as their priority. different surgical techniques are practiced for root coverage which include lateral pedicle flaps, coronally advanced flaps, free gingival grafts, free connective tissue grafts, etc. the double papilla procedure is technique sensitive but has good results in treating isolated recessions. the partial thickness double papilla pedicle graft technique was first proposed by cohen and ross.5 hall stated that double pedicle graft had very low predictability in most practitioners hands.6 the 1989 world workshop in clinical periodontics concluded the double papilla pedicle has very limited usefulness.7 its weaknesses are its poor predictability and the technical skills required to perform the procedure. nelson proposed a technique that combines a free connective tissue graft with a full thickness double papilla graft.8 harris further proposed the use of a partial thickness double pedicle flap rather than a full thickness one overlying a free connective tissue graft as partial thickness flap allows the connective tissue graft to receive vascular supply both from the recipient bed and from the flap overlying it.9 the advantages of this technique include excellent color matching, good vascular supply, root coverage, and decrease in hypersensitivity.10 the greatest advantage of this procedure is that there is no need for an additional donor site.11 few factors have to be considered when opting for this technique. the interdental papilla should be thick next to recessionthere should be an absolutely healthy periodontium adjacent to the recession to be treatedthis technique can not be practiced to treat multiple adjacent recessions. the interdental papilla should be thick next to recession there should be an absolutely healthy periodontium adjacent to the recession to be treated this technique can not be practiced to treat multiple adjacent recessions. in the past decades, several surgical procedures have been proposed for treating gingival recession. however, the choice of mucogingival surgical technique to treat a recession defect depends on the clinician s skill and the type of recession. | gingival recession can result in an unesthetic appearance, hypersensitivity, and root caries. the physiological well - being of the patient is a significant factor associated with the success of dental therapy. esthetics is one of the main concerns of the patient if recession is present especially in anteriors. hence, the treatment of choice for root coverage for the anterior teeth should address the biological as well as the patient s aesthetic demands. numerous surgical procedures have been implicated for root coverage. aesthetic results from using pedicle grafting procedure are superior to the use of free gingival grafts. the double papilla technique evolved from treating defects where tissues adjacent or apical to the defect alone may be inadequate for grafting purpose. this technique can be used in areas with shallow vestibule and palatal areas. the double papilla technique combines the esthetic results of pedicle graft with the predictability and usefulness of free gingival graft and is an effective and predictable method of obtaining esthetic root coverage. |
although -lipoic acid has been firstly explored in the light of its natural antioxidant properties and its ability to induce apoptosis in several tumour cell lines [1, 2 ], the therapeutic implications related to its clinical use have been extensively reviewed underlying overall beneficial effects on several pathological conditions such as diabetes mellitus and atherosclerosis [3, 4 ]. among the putative cellular therapeutic targets of -lipoic acid, also the endothelial compartment has been considered and some reports suggested a protective effect of -lipoic acid on endothelial cell damage. in the same fashion, endothelial cell models (such as human umbilical vein endothelial cells, huvec) as vascular protective agents to attenuate oxidative stress damage. endothelial cells derived from different anatomical districts are heterogeneous in terms of the pattern of antigens expression, secreted molecules, and immunological properties and therefore common in vitro endothelial models, such as huvec, can not properly recapitulate the in vivo features of pathological endothelium. in this light, the responses of large vein endothelium are thought to play important roles in clinical diseases, such as chronic venous disease (cvd) and thrombosis [7, 8 ]. the endothelium actively reacts in response to changes of the local environment by expressing and releasing specific cytokines, chemokines, and soluble chemical mediators that, in turn, play a role in the pathophysiology of cvd [912 ]. the therapeutic options for cvd patients range from conservative therapies, minimally invasive approaches, and surgical treatments ; nevertheless cvd remains a disease with a high recurrence rate. in the present study, we have addressed if the natural compounds -lipoic acid and ginkgo biloba might have a role in treating cvd through a direct effect on the pathological endothelium, by using vein endothelial cells (vec) isolated from patients at different stages of cvd. in particular, we have evaluated the anti - inflammatory effect of the natural compounds -lipoic acid and ginkgoselect phytosome on cytokines / chemokines released by cvd patient - derived vec. for this purpose, we first characterized the systemic levels of a wide panel of cytokines / chemokines (n = 31) in the plasma of patients affected by cvd with respect to normal healthy controls and the release of these cytokines / chemokines by vec purified from the same cvd patients, in unstimulated and tnf--stimulated conditions. finally, we investigated the intracellular signal transduction pathways involved in promoting the vec release of cytokines / chemokines, which are targeted by natural anti - inflammatory compounds. overall, the data reported in the present study provide new insights into the molecular mechanisms of cvd pathogenesis, highlighting new potential therapeutic targets. the main demographic / clinical characteristics of cvd patients enrolled in this study are reported in (see supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2013/423407). forearm blood samples of patients affected by primary cvd with superficial venous reflux (c2 - 4epaspr following the ceap classification) were collected in the presence of sodium citrate before varicose veins surgery. blood samples were immediately centrifuged for plasma isolation that were stored at 80c in single - use aliquots. the surgical specimens were collected during surgery for conservative and hemodynamic treatment of venous insufficiency in ambulatory care. highly purified vec cultures obtained from cvd patients were isolated and characterized for morphological and phenotypic properties, as previously described. the procedures followed were in accordance with the declaration of helsinki, approved by the institutional review board (university hospital of ferrara) and all participant subjects gave written informed consent. cells were grown on egm2 medium (lonza, walkersville, md) with 2% fbs and full supplements (egm2 bullet kit, lonza) in 5 g / cm fibronectin precoated tissue culture plates (bd, becton dickinson, san jos, ca), as described previously. for endothelial cell treatments, the following reagents have been used : recombinant tumor necrosis factor (tnf)- (r&d systems, minneapolis, mn), ginkgoselect phytosome, and -lipoic acid (kindly provided by laborest s.p.a., milano, italy) were dissolved in distilled water and in dimethylsulfoxide (dmso, sigma, st. louis, mo), respectively, and at a final concentration such that the dmso did not exceed 0.1% in the cell culture media. the optimal concentrations of tnf- (5 ng / ml), ginkgoselect phytosome, and -lipoic acid (both used at 100 g / ml) were determined in preliminary dose - response experiments. ginkgoselect phytosome and -lipoic acid were added to the cells 1 hour before treatment with tnf-. in selected experiments, cells were preincubated for 1 hour with pharmacologic inhibitors of erk1/2 (pd98059 ; 50 m ; calbiochem, la jolla, ca), p38/mapk (sb203580 ; 10 m ; calbiochem), nf-b (parthenolide ; 10 m ; enzo life science, exeter, uk), and akt (mk-2206 ; 5 m ; biovision incorporated, milpitas, ca) pathways prior to the addition of tnf-. cell supernatants were collected from endothelial cultures and frozen at 80c as described until the cytokine analyses were performed. cell viability was monitored by light microscopic analysis of the cell monolayers after hematoxylin - eosin staining or by quantitative examination after detachment of the monolayers by means of trypan blue dye exclusion, as described [17, 18 ]. the profiles of expression of the inflammatory markers icam-1 and vcam-1 were analyzed by flow cytometry as previously described. in brief, cells were detached with trypsin - edta and washed and 5 10 cells were resuspended in 200 l of pbs containing 1% bsa (sigma - aldrich) and incubated for 30 minutes at 4c with the following monoclonal antibodies (mab) : fitc - conjugated anti - icam-1 (r&d, clone bbig - i1) or fitc - conjugated anti - vcam-1 (r&d, clone bbig - v3). culture supernatant samples and plasma of patients were frozen and thawed only once before performing the milliplex map human cytokine / chemokine panel (merck millipore, billerica, ma), a bead - based multiplex immunoassay, which allows the simultaneous quantification of the following 29 human cytokines : egf, il-1, il-1 receptor antagonist (ra), il-1, il-2, il-3, il-4, il-5, il-6, il-7, il-8, il-10, il-12(p40), il-12(p70), il-13, il-15, 1l-17a, eotaxin, g - csf, gm - csf, ifn-2, ifn-, cxcl10, mcp-1, mip-1, mip-1, tnf-, tnf-, and vegf. moreover, a custom - made milliplex map human cytokine / chemokine magnetic bead panel (merck millipore) was used to quantify the cytokines pdgf - ab / bb and rantes. culture supernatant samples were processed following the manufacturer 's recommended protocols and read on a magpix instrument equipped with the milliplex - analyst software using a five - parameter nonlinear regression formula to compute sample concentrations from the standard curves. the milliplex map human multi - pathway 9-plex magnetic bead signalling kit phosphoprotein was used to detect changes in phosphorylated erk / map kinase 1/2 (thr185/tyr187), akt (ser473), stat3 (ser727), jnk (thr183/tyr185), p70 s6 kinase (thr412), nf-b (ser536), stat5a / b (tyr694/699), creb (ser133), and p38 (thr180/tyr182) in endothelial cell lysates using the luminex system, according to the manufacturer 's instructions. briefly, endothelial cells were plated in 100 mm dishes, grown at subconfluence, and subjected to partial fcs reduction (to 0.5%) for 18 hours before the addition of treatments. cells were harvested in milliplex map lysis buffer in the presence of protease inhibitor cocktail set iii (calbiochem). each lysate (20 g total protein) was diluted in milliplex map assay buffer 2, incubated at 4c overnight, and analyzed according to the manufacturer 's instructions. the median fluorescence intensity (mfi) was measured with the luminex system. to validate the milliplex map human multipathway data, we have performed western blot analyses as previously described [21, 22 ]. briefly, cells were harvested in lysis buffer containing 1% triton x-100, 150 mm nacl, 50 mm tris - hcl ph 6.8, and protease inhibitors (protease inhibitor cocktail ; roche diagnostics). protein determination was performed by bradford assay (bio - rad, richmond, ca). equal amounts of protein (50 g) for each sample were migrated in acrylamide gels, blotted onto nitrocellulose filters, and probed with the following antibodies for the phosphorylated forms of erk1/2, p38/mapk, and ikb and for the respective total protein kinase content for verifying loading evenness : anti - phospho - p44/42 mapk (erk1/2) and anti - p44/42 mapk (erk1/2) (both from cell signalling technology, danvers, ma) ; anti - phospho - p38/mapkinase and anti - p38/mapkinase (both from cell signaling technology, beverly, ma) ; anti - phospho - akt and anti - akt (both from merck millipore) ; anti - ikb (from santa cruz biotechnology, santa cruz, ca) and antitubulin (sigma - aldrich). after incubation with peroxidase - conjugated anti - mouse and anti - rabbit igg, specific reactions were revealed with the ecl lightning detection kit (perkin elmer, boston, ma). densitometry values were estimated by the imagequant tl software (ge healthcare bio - sciences ab, uppsala, sweden). multiple film exposures were used to verify the linearity of the samples analyzed and avoid saturation of the film. the results were evaluated by using student 's t - test and the mann - whitney rank - sum test, when appropriate. in the first set of experiments, we sought to investigate the pattern of circulating cytokines / chemokines in cvd patients. for this purpose, plasma samples of cvd patients were collected before surgery for conservative and hemodynamic treatment of venous insufficiency in ambulatory care and analyzed for a panel of 31 cytokines / chemokines involved in the inflammatory and/or thrombosis processes. overall, cvd patients (mean age of 52.3 11.5) were characterized by a mean duration of the disease of 19.3 11.6 years ; besides 73% of them showed a family history of cvd with a variable incidence of other relevant comorbidities such as diabetes mellitus, hypertension, hypercholesterolemia, and cardiac disease (supplementary table 1). among the 31 cytokines / chemokines analyzed by multiplex assay, 18 were detectable in plasma samples of both cvd patients and healthy control individuals : mip-1, il-8, il-7, ifn-, il-12(p70), tnf-, gm - csf, ifn-2, g - csf, il-1ra, mip-1, vegf, egf, eotaxin, mcp-1, cxcl10, pdgf, and rantes (table 1). of interest, within the panel of cytokines and chemokines detectable at the plasma level, 12 (mip-1, il-8, tnf-, gm - csf, ifn-2, mip-1, vegf, egf, eotaxin, mcp-1, pdgf, and rantes) were significantly (p < 0.05) increased in cvd patients compared to healthy controls while 2 additional cytokines (g - csf and cxcl10) showed levels close to significance (table 1). these data suggested the potential role of these cytokines / chemokines in the pathogenesis and/or progression of the disease. to evaluate the role of pathological endothelium in the establishment of the increased levels of circulating cytokines / chemokines characterizing cvd, we have isolated pathological vec from surgical specimens obtained from the same cvd patients analysed for the circulating cytokines / chemokines (supplementary table 1 and table 1). vec cultures were characterized and defined as cd31/cd105/cd146/cd144/cd45/cd14 cells, as previously described, and were assessed for the baseline release of the same panel of 31 cytokines / chemokines evaluated in plasma samples of cvd patients. in this respect, it should be noticed that since vegf and egf are essential components of the vec culture medium, it was not possible to distinguish exogenously added vegf and egf from endogenously produced cytokines. of interest, among the cytokines / chemokines detectable at the plasma of cvd patients (table 1), only mip-1 was undetectable in vec culture supernatants (table 2), while il-8, il-7, ifn-, il-12(p70), tnf-, gm - csf, ifn-2, g - csf, il-1ra, mip-1, eotaxin, mcp-1, cxcl10, pdgf, and rantes were released at different levels (table 2). this group of data suggests that vec represent a major cell type contributing to the production of plasmatic cytokines / chemokines in cvd patients. in light of the fact that pathological vec are exposed in vivo to a proinflammatory milieu [711, 14 ] and that we found a significant (p < 0.01) elevation of the plasmatic levels of tnf- in cvd patients, in further experiments vec cultures were exposed in vitro to recombinant tnf- in order to mimic the in vivo inflammatory microenvironment. only those cytokines / chemokines showing a mean increase 2-fold were considered (figure 1). these eight cytokines / chemokines were grouped on the basis of the fold increase : mip-1, pdgf, and ifn-2 (between 2 and 3 mean fold increase), g - csf and il-8 (between 10 and 20 mean fold increase), and gm - csf, cxcl10, and rantes (between 100 and 300 mean fold increase) (figure 1). in the next set of experiments, we have investigated whether the anti - inflammatory natural compounds -lipoic acid [2328 ] and ginkgo biloba derivatives [29, 30 ] affected the release of cytokines / chemokines by pathological vec. indeed, although it has already been reported that both compounds, and in particular -lipoic acid, exhibit anti - inflammatory activities both in vitro and in vivo in the context of endothelial biology of different pathologies [2327 ], it remains to be established whether these compounds are active also on pathological vec isolated from cvd patients. since both compounds have been shown to potentially affect the survival of endothelial cells [3135 ], we started by analysing the morphology and viability of vec exposed to different concentrations (up to 250 g / ml for each compound) of -lipoic acid and ginkgoselect phytosome for 24 hours. as shown in figure 2(a), morphological analysis did not reveal toxic effects at the concentration of 100 g / ml, when compounds were used either alone or together with recombinant tnf-. the lack of toxicity indicated by morphological evidences was confirmed by quantitative cell viability analysis (figure 2(b)). in addition, preexposure for 1 hour to both -lipoic acid and ginkgoselect phytosome (both used at 100 g / ml), before addition of recombinant tnf- for 24 hours, significantly (p < 0.05) inhibited the tnf- mediated induction of the proinflammatory markers vcam-1 and icam-1 (figure 2(c)). therefore, concentration of 100 g / ml for both compounds was selected for further experiments. indeed, having confirmed that both -lipoic acid and ginkgoselect phytosome exerted anti - inflammatory activities also in pathological vec without inducing a specific toxicity (figure 2), we analyzed the effect of both compounds on the specific release of cytokines / chemokines by pathological vec. as shown in figure 3(a), in vitro treatment with -lipoic acid alone was able to significantly decrease the baseline levels of pdgf, rantes, and cxcl10, while ginkgoselect phytosome alone only inhibited basal pdgf. in addition, pretreatment (for 1 hour) with both -lipoic acid and ginkgoselect phytosome before addition of tnf- significantly downregulated the release of tnf--induced pdgf, rantes, and cxcl10 (figure 3(b)), while -lipoic acid, but not ginkgoselect phytosome, significantly inhibited the tnf--induced release of g - csf and gm - csf (figure 3(b)). thus, -lipoic acid exhibited a broader spectrum of inhibitory activity as compared to ginkgoselect phytosome. on the other hand, the release of the other cytokines / chemokines upregulated by tnf- (ifn-2, mip-1, and il-8) was unaffected by either -lipoic acid or ginkgoselect phytosome (data not shown). in the last group of experiments, we have investigated the intracellular signalling pathways mediating the release of the various cytokines and chemokines in the culture supernatants of pathological vec and targeted by -lipoic acid and/or ginkgoselect phytosome. in line with previous reports showing activation of various intracellular pathways by tnf- treatment, we found that exposure of vec cultures to recombinant tnf- resulted in a time - dependent activation / phosphorylation of several signalling pathways such as jnk, p38/mapk, erk1/2, nf-b and akt, stat3, and creb (figure 4(a)). of note, -lipoic acid significantly inhibited both nf-b and p38/mapk pathways at two different time points (figure 4(b)). on the other hand, ginkgoselect phytosome significantly inhibited p38/mapk and akt pathways at two different time points, while it was less efficient than -lipoic acid in inhibiting nf-b. neither of the two compounds was effective in modulating the remaining pathways analysed and shown in figure 4(a). based on the data illustrated above, the evidence that -lipoic acid was more powerful than ginkgoselect phytosome in inhibiting the release of cytokines and chemokines, either spontaneously or in response to tnf-, suggested that nf-b pathway plays a primary role in promoting the tnf- mediated induction of cytokines and chemokines. to confirm this issue, in the last group of experiments, cells were preincubated with pharmacologic inhibitors specific for nf-b, p38/mapk, akt, and erk1/2 pathways 1 hour before tnf- addition. as shown in figure 5, the release of pdgf, g - csf, rantes, gm - csf, and cxcl10 was significantly reduced by parthenolide, an inhibitor of nf-b pathway, which showed an overlapping pattern of inhibition with -lipoic acid (figure 3), thus confirming the prominent role of nf-b in mediating the tnf--induced release of several cytokines / chemokines as well the role of nf-b as the major intracellular pathway targeted by -lipoic acid. interestingly, the selective inhibitor of the p38/mapk pathway sb203580 also significantly inhibited the release of g - csf, gm - csf, and cxcl10, suggesting that this pathway might also contribute to the release of cytokines / chemokines by vec. finally, the inhibitor of the akt pathway induced a selective inhibition of pdgf, rantes, and cxcl10 (figure 5), showing a clear - cut overlapping with the inhibitory activity of ginkgoselect phytosome (figure 3). consistently, akt pathway represented a major target of ginkgoselect phytosome (figure 4(b)). the specificity of the different inhibitors of the pathways activated by tnf- has been validated by western blot, (see supplementary figure 1 in supplementary material available online). by analysing a large panel of cytokines / chemokines (n = 31), we demonstrated for the first time that patients affected by cvd are characterized by increased plasmatic levels of mip-1, il-8, tnf-, gm - csf, ifn-2, g - csf, mip-1, vegf, egf, eotaxin, mcp-1, cxcl10, pdgf, and rantes with respect to healthy control subjects. since the inflammatory properties of vein endothelium in cvd are poorly understood, an important point of our study was that all these cytokines, with the exception of mip-1, were spontaneously released in vitro by vec purified from the same cvd patients analyzed for the plasmatic levels of cytokines / chemokines. an additional interesting finding was that the majority of the cytokines / chemokines found elevated in plasma of cvd patients and released spontaneously in the culture supernatants by vec obtained from cvd patients (with the exception of vegf and egf that were not valuable since they were contained in the endothelial culture medium) were significantly induced by recombinant tnf-, a major proinflammatory cytokine. taken together, the in vivo and in vitro data suggested that a specific pattern of cytokines / chemokines (mip-1, pdgf, ifn-2, g - csf, il-8, rantes, gm - csf, and cxcl10) (i) is produced by pathological vec obtained from cvd patients, (ii) is significantly increased in response to tnf-, and (iii) is elevated in the plasma of cvd patients. these findings suggest that pathological vec significantly contribute to the elevation of the majority of plasmatic cytokines / chemokines in cvd although, obviously, the potential contribution of additional cellular sources to the production of plasmatic cytokines / chemokines is not excluded. in order to evaluate potential anti - inflammatory compounds able to affect the release of this specific set of cytokines / chemokines, we have used -lipoic acid and ginkgo biloba derivatives, taking into account previous studies, which have demonstrated that both compounds show anti - inflammatory activity on a variety of endothelial cell types [2335 ], although they have never been tested before on pathological vec. the anti - inflammatory activity of both -lipoic acid and ginkgo biloba derivatives was confirmed on the basis of their ability to markedly inhibit the surface levels of icam-1 and vcam-1, which are known to promote recruitment of leukocytes, platelets, and erythrocytes to the vein wall [37, 38 ], without inducing toxic effect of vec cultures. we could demonstrate that, among the cytokines / chemokines found elevated in the plasma of cvd patients and whose in vitro release increased in response to recombinant tnf-, -lipoic acid significantly (p < 0.05) decreased the basal release of pdgf, rantes, and cxcl10 and the tnf--induced release of pdgf, rantes, cxcl10, g - csf, and gm - csf. on the other hand, ginkgoselect phytosome decreased the release only of basal pdgf and the tnf--induced pdgf, rantes, and cxcl10. thus, -lipoic acid showed a broader and more potent inhibitory activity on the release of cytokines / chemokines with respect to ginkgoselect phytosome. since recombinant tnf- induced several intracellular pathways in vec, such as the jnk, p38/mapk, erk1/2, nf-b, akt, stat3, and creb pathways, it is noteworthy that -lipoic acid significantly inhibited tnf--induced nf-b and dampened p38/mapk activation, while ginkgoselect phytosome downmodulated tnf--induced p38/mapk and akt activation. consistently with a prominent role of nf-b and to a lesser extent p38/mapk pathway in promoting the release of a specific set of cytokines / chemokines in response to tnf-, parthenolide, a pharmacological inhibitor of the nf-b pathway, inhibited the release of pdgf, rantes, cxcl10, g - csf, and gm - csf, recapitulating the effect of -lipoic acid, while mk-2206, a pharmacological inhibitor of the akt pathway, inhibited the release of pdgf, rantes, and cxcl10, recapitulating the effect of ginkgoselect phytosome. in conclusion, we have demonstrated that pathological vec significantly contribute to the circulating cytokines / chemokines found elevated in plasma of cvd patients and by using both -lipoic acid and ginkgoselect phytosome, we have characterized and targeted the prominent pathways promoting the release of these cytokines and chemokines. | large vein endothelium plays important roles in clinical diseases such as chronic venous disease (cvd) and thrombosis ; thus to characterize cvd vein endothelial cells (vec) has a strategic role in identifying specific therapeutic targets. on these bases we evaluated the effect of the natural anti - inflammatory compounds -lipoic acid and ginkgoselect phytosome on cytokines / chemokines released by cvd patient - derived vec. for this purpose, we characterized the levels of a panel of cytokines / chemokines (n = 31) in cvd patients ' plasma compared to healthy controls and their release by vec purified from the same patients, in unstimulated and tnf- stimulated conditions. among the cytokines / chemokines released by vec, which recapitulated the systemic profile (il-8, tnf-, gm - csf, inf-2, g - csf, mip-1, vegf, egf, eotaxin, mcp-1, cxcl10, pdgf, and rantes), we identified those targeted by ex vivo treatment with -lipoic acid and/or ginkgoselect phytosome (gm - csf, g - csf, cxcl10, pdgf, and rantes). finally, by investigating the intracellular pathways involved in promoting the vec release of cytokines / chemokines, which are targeted by natural anti - inflammatory compounds, we documented that -lipoic acid significantly counteracted tnf--induced nf-b and p38/mapk activation while the effects of ginkgo biloba appeared to be predominantly mediated by akt. our data provide new insights into the molecular mechanisms of cvd pathogenesis, highlighting new potential therapeutic targets. |
conditional gene expression systems such as promoters regulated by tetracycline or doxycycline (dox) have several advantages for testing the effects of genes on aging and lifespan. in the ' tet - on ' system, feeding dox to drosophila melanogaster causes high levels of transgene expression in all tissues. by waiting until the young adult stage to administer dox, all of pre - adult development is identical between control and experimental groups, and any difference in lifespan subtle differences in the genetic background of drosophila strains can have significant effects on lifespan. with the tet - on system, control and experimental animals have identical genetic backgrounds, and therefore any differences in lifespan must be due to dox administration and transgene expression. dox itself, and overexpression of control genes such as escherichia coli lacz, have no detectable effects on lifespan. in contrast, several genes have been identified for which dox - regulated overexpression has negative effects on lifespan, and overexpression of the dgmii gene, encoding -mannosidase ii, was associated with slightly increased lifespan. expression of antisense rna has long been known to be able to inhibit gene expression in drosophila and other organisms. double - strand rna (dsrna) formed by hybridization of sense and antisense sequences is thought to initiate a pathway in which homologous rna sequences are destroyed. this phenomenon has been referred to as post transcriptional gene silencing (ptgs) or rna interference (rnai). expression of inverted - repeat constructs, where the transcript is expected to fold into a dsrna hairpin, has been shown to be an efficient initiator of rnai. in experiments reported here, the tet - on system was used to drive expression of inverted - repeat constructs in drosophila to determine if a conditional system for rnai could be created. in natural populations, lifespan and reproductive period the duration of the reproductive period is subject to natural selection, and lifespan is altered as a consequence. significant experimental support for this model has come from laboratory selection experiments with d. melanogaster. genetically heterogeneous drosophila populations have been selected over hundreds of generations for late - life reproduction. selection is thought to function by altering the frequency of gene alleles present in the starting population. the correlated phenotypes can vary somewhat depending on the starting strains used ; however, increased stress - resistance appears to be common to all or most long - lived strains. in one well studied set of five replicate control (b) and long - lived (o) strains, lifespan was doubled and was correlated with increased stress - resistance and increased glycogen and lipid stores. starch - gel electrophoresis has been used to assay for changes in the frequency of enzyme electrophoretic alleles in the o and b lines. a change in the frequency of cu / zn - superoxide dismutase (cu / znsod) alleles was observed, with the more active allele enriched in the long - lived o strains. consistent with this observation, overexpression of cu / znsod in transgenic drosophila has been shown to be sufficient to cause significant increases in lifespan. the most dramatic change in allele frequency in the o and b lines was observed for the gene for phosphogluconate mutase (pgm). pgm exists in three electrophoretic forms : fast (pgm), medium (pgm) and slow (pgm). pgm predominates in the b strains, whereas pgmallele frequency is on average ten - fold higher in the long - lived o strains relative to the b controls. when o strains were taken off selection for several generations (back - selection), lifespan decreased and pgm allele frequency was reduced to levels more like those in b strains. these results suggest that pgm or a closely linked gene is responding to selection, and make pgm a candidate lifespan regulator. pgm encodes the enzyme phosphoglucomutase, which inter - converts glucose 1-phosphate and glucose 6-phosphate. its activity is therefore important for both glycolysis and glycogen synthesis, and the altered pgm allele frequency might therefore be relevant to the increased glycogen and lipid stores of the o strains. if the increased pgm allele frequency is contributing to the unique phenotypes of the long - lived o strains, this might be because pgm has increased or decreased enzyme activity, or enzyme activity has been altered in some way (perhaps by a change in its regulation). one way to begin to test these models experimentally is to engineer transgenic flies with increased or decreased pgm expression and assay for effects on lifespan. multiple strains were generated that were homozygous for each of the f, m and s electrophoretic alleles of pgm, by appropriate crosses to a third chromosome balancer stock. pgm enzyme activity and lifespan varied greatly across the strains with no correlation with pgm allele (data not shown). this result was expected, as differences in genetic background between such purified chromosome strains has profound effects on lifespan and the activities of various enzymes. the pgm coding - region sequences were cloned and sequenced from the pgm, pgm and pgm homozygous strains and from the canton - s wild - type strain. amino - acid substitutions were identified that predicted pi values for the alleles that correlated with their mobility on starch gels (figure 1). the substitution of t for a in pgm creates the amino - acid sequence ttk (in the single - letter amino - acid code) which is a potential phosphorylation site for protein kinase c (pkc) that is absent in pgm and pgm. if pgm contributes to the increased lifespan of the o lines, this might be due to increased enzyme activity, decreased enzyme activity, or alteration in enzyme activity in some other way such as in its regulation or subcellular localization. the tet - on system was used to test whether simply increasing or decreasing pgm expression would be sufficient to alter lifespan. a p - element transformation vector called usc1.0 was generated, which had unique ecori and psti sites downstream of the tet - on doxycycline - regulated promoter (figure 2a). the pgm cdna sequences were cloned into usc1.0 to create the pgmsense construct (figure 2b), which should allow overexpression of the pgm enzyme. a fragment of pgm coding region of approximately 1 kilobase (kb) (from + 711 to + 1,794) was cloned into usc1.0 in an inverted - repeat orientation to create construct pgminvrpt (figure 2c), which should potentially cause conditional rnai. for each pgm construct five independent transgenic lines were generated using standard methods. as a control, two trangenic lines were generated containing the usc1.0 vector in which no transgene is expressed. expression of the transgenic constructs and the endogenous pgm gene was assayed by northern blot, with or without dox (figure 3a). the pgm inverted - repeat sequences were used as probes and should hybridize to both transgenes and the endogenous pgm transcript. in the sense - construct lines, feeding dox resulted in efficient expression of the transgene at levels five- to ten - fold greater than the endogenous transcript. dox - induced expression of the inverted - repeat construct had two effects detected by northern blot. first, dox produced a smear of hybridization resulting from the inverted - repeat transcript (figure 3a). the smear pattern may have resulted from incomplete denaturation of the hairpin structure and/or instability of the inverted - repeat transcript. second, the level of the endogenous pgm transcript was found to be reduced by dox feeding. the northern blots were also hybridized with a pgm - specific probe corresponding to pgm coding - region sequences (+ 1 to + 700) located outside the region used to create the inverted repeat. this probe is therefore specific for the endogenous pgm transcript. using this probe the smear of hybridization derived from the inverted - repeat construct was no longer detected, and the endogenous pgm rna levels were more readily observed and quantitated (figure 3b). dox induced expression of the inverted - repeat construct was found to cause a reduction of endogenous pgm rna levels. the decrease varied across the five transgenic lines, with reductions ranging from 1.3- to 24-fold. the inverted - repeat construct therefore appears to function as expected to induce rnai and cause decreased expression of the endogenous pgm gene. in the control strains where no transgene is expressed, dox administration was found to have no effect on pgm transcript levels (figure 3c). the indirect pgm activity assay was used to assay for changes in pgm gene expression at the protein level. the sense construct yielded conditional overexpression of pgm activity in each transgenic line, with increases ranging from two- to five - fold (table 1). potential reductions in pgm activity were more difficult to assay because of the inherent limitations of the assay. the pgm activity assay is indirect and is coupled to the conversion of nadp to nadph. the extract will contain other activities capable of converting nadp to nadph, thereby creating a significant background activity. the pgminvrpt construct yielded decreases in pgm activity of as much as 50% ; however, the effect was quite variable from experiment to experiment (table 1). in the control strains where no transgene is expressed, dox administration was found to have no consistent effect on pgm levels (table 1). to determine whether altered pgm gene expression could affect lifespan, mean lifespan was assayed in multiple pgmsense and pgminvrpt transgenic lines with or without dox treatment. the percentage change in mean lifespan caused by dox and transgene expression is presented for each line. lifespan was observed to vary across transgenic lines and replicate experiments with changes ranging from -7% to + 6%, with one outlier at + 16% (table 2). however, no change in lifespan was observed that was consistent across the sense or inverted - repeat lines or in multiple experiments. the lifespan assays were repeated at 29c, and again no consistent changes were observed (table 3). we conclude that under these conditions, altered pgm expression does not have a detectable effect on adult drosophila lifespan. the phenomenon of rnai (or ptgs) is of great interest for two reasons. first, it represents an evolutionarily conserved pathway that probably has important functions in the regulation of gene expression and the control of transposable elements. second, it provides a means for researchers to test gene functions by experimentally downregulating expression of specific genes under well controlled conditions. in drosophila, rnai can be initiated by injection of dsrna into embryos, and this has allowed identification of novel phenotypes for genes during development. however, injection has limited potential application in the adult, as it is unlikely that the rnai could be targeted to all tissues, or to specific tissues, and the trauma of injection is likely to have negative effects on phenotypes such as lifespan. transgenic constructs have been used to cause rnai in drosophila, by using various promoters to drive expression of inverted - repeat fragments of gene coding regions. using the conditional tet - on promoter system to drive expression of an inverted - repeat provided conditional rnai of the pgm gene. this conditional system should have many applications, both in the study of the rnai pathway itself and in the design of experiments where rnai is used a research tool. conditional rnai should be particularly useful to study phenotypes in the adult such as aging and lifespan. conditional gene - expression systems have previously been used to identify positive regulators of lifespan, such as cu / znsod and dgmii. in addition, mutations have been used to identify several genes in drosophila that act as negative regulators of lifespan, such as mth, indy, inr and chico. for mth and inr therefore it is only rare hypomorphic alleles or allele combinations that allow the animals to survive to adult stage where increased lifespan can be observed. conditional rnai should provide a powerful means to study these genes and other negative regulators of aging and lifespan. inactivation or downregulation of the genes specifically in the adult using tet - on rnai may allow increased lifespan without confounding effects on development. lam and thummel have recently reported the use of a heat - shock gene promoter to drive expression of dsrna, and the efficient conditional inhibition of gene expression during larval and pupal stages of drosophila development. genetic selection of drosophila populations for late - life reproduction caused the correlated phenotype of increased lifespan and increased the frequency of the slow electrophoretic allele, pgm, of the pgm gene. if pgm contributes to the increased lifespan phenotype of the selected strains, it might be because pgm has increased activity, decreased activity or activity that is altered in some way such as through its regulation or subcellular localization. the pgm alleles were cloned and sequenced and were found to differ by amino - acid substitutions consistent with the relative electrophoretic mobilities of the encoded enzymes. a recent study reported the sequencing of several fast, medium and slow electrophoretic alleles of pgm from a wild population of d. melanogaster. the sequences reported in that study (verrelli alleles) are comparable to those reported here (ives alleles) as follows : the medium verrelli allele used for alignment is identical in amino - acid sequence to the ives medium allele. the verrelli fast alleles are identical to the ives fast allele in each of the three amino - acid positions at which the ives fast differs from the ives medium (wild - type) allele. there are a number of differences between the verrelli slow alleles and the ives slow allele. the ives slow allele differs from the ives medium (wild - type) amino - acid sequence at two positions (amino - acid positions 6 (e g) and 9 (a t)). the amino - acid change in the ives slow allele at position 6 (e g) is not reflected in any of the verrelli slow alleles. the second amino - acid change in the ives slow allele (at position 9) in addition, the verrelli slow alleles have ammo - acid substitutions that are not shared by the ives slow allele. the data are consistent with the conclusion that pgm is highly polymorphic and subject to selection in natural populations. consistent with this idea, pgm haplotypes and glycogen content have been found to vary among flies at different latitudes. the tet - on promoter system allowed a test of the hypothesis that increasing or decreasing pgm gene expression would affect lifespan. pgm enzyme levels were increased two- to five - fold using a sense transgenic construct, and pgm rna levels were decreased 1.3- to 24-fold by driving expression of an inverted - repeat construct. no changes in lifespan were detected that were consistent across multiple transgenic lines or replicate experiments. the results indicate that simply increasing or decreasing pgm gene expression does not significantly affect the lifespan of adult drosophila, at least under the conditions tested. the pgm allele used in these experiments was cloned from the ives strain, which is the progenitor of the long - lived selected strains and their controls. the high degree of polymorphism of pgm makes it possible that the slow electrophoretic allele in the ives strain could consist of multiple dna sequence forms. the failure to observe effects on lifespan could therefore conceivably have resulted from overexpression of the wrong dna sequence form of pgm. however, pgm was cloned and sequenced from four independent lines derived from the ives strain and the clones had the same dna sequence. the data therefore suggest that most, if not all, of the pgm alleles in these strains have the same sequence, making sequence heterogeneity an unlikely explanation of the present results. the results suggest several possibilities for the relationship of pgm allele frequency to the increased lifespan of the selected strains. the first is that pgm might in fact contribute to the increased lifespan of the selected strains as a result of an increased or decreased activity, but that the effect on lifespan is too small to be detected in these assays. the lifespan assay appears to have a variability with control strains of approximately -5% to + 5%, as seen in the data presented here and elsewhere (g.l., d. bhole and j.t. the second possibility is that pgm allele frequencies are altered because of selection for an unknown linked gene, and this possibility can not be ruled out at this time. the experiments presented here suggest that tet - on regulated overexpression and rnai of genes near pgm may be a promising approach. third, an interesting model is that pgm contributes to increased lifespan of the selected strains because of an alteration in its regulation and/or subcellular localization. the sequence analysis of the pgm alleles reveals a novel potential site in pgm for phosphorylation by pkc ; however, the potential significance of this change is unknown. the pgm allele was used for the overexpression experiments presented here, and presumably the overexpressed enzyme should be subject to any regulation or localization characteristic of pgm. however, the pgm enzyme was overexpressed in a ' wild - type ' pgm background, and it may be that any phenotypic consequences of pgm allele function are not apparent when pgm is also present. finally, another possible explanation for the absence of an effect on longevity is the method of assay. the selected o stocks were handled and assayed with males and females together, repeatedly mating during adult life. this was done because the segregated - sexes assay is sensitive to even small changes in lifespan, and has been used successfully in the past to detect the effects of other genes on lifespan. it is possible that an effect of pgm on lifespan might be identified with different lifespan assay conditions. however, even if this explanation were to be true, the data still indicate that pgm does not have a simple strong effect on longevity. further experiments will be required to distinguish between these interesting possibilities for the link between pgm allele frequency and lifespan. all transgenic drosophila stocks were generated by p - element germline transformation, using a modified microinjection technique. transgenic lines are named as the construct, followed by the chromosome of insertion in parentheses, followed by a unique line designation. for example, pgminvrpt(3)76 is pgminvrpt construct inserted on chromosome 3, independent transgenic line 76. multiple strains homozygous for each of the pgm, pgm and pgm alleles were generated by purifying third chromosomes from the ives stock, which is the precursor to the o and b selection stocks. homozygosity for a particular pgm allele was confirmed by starch - gel electrophoresis assay, as previously described (and data not shown). all stocks were grown on standard cornmeal - agar medium and were cultured at 25c. strains homozygous for the transgenic constructs were crossed to rtta(3)e2 strain, homozygous for the rtta transactivator construct, to generate progeny heterozygous for both constructs. control flies were rtta(3)e2/+, that is, lacking a target construct. to obtain adult flies of defined age, the crosses were cultured at 25c in urine specimen bottles. before eclosion of the majority of pupae, bottles were cleared of adults and newly eclosed flies were allowed to emerge over the next 48 h. most of the males will have mated during this time. the males only were then removed and were designated one day old, and were maintained at 25c at 40 per vial in culture vials with food, and passaged to new vials every 48 h. for certain experiments the adult males were maintained at 29c, as indicated. for certain experiments, those flies being fed dox were kept on food vials supplemented with 250 g / ml dox. all dna sequencing reactions were done using the dideoxy chain - termination protocol and t7 sequenase v2.0 (amersham). the clones were obtained by polymerase chain reaction (pcr) amplification of each allele using genomic dna template isolated from strains homozygous for each allele. the primer locations were assigned on the basis of their relative distance from the a in the atg start codon, which was defined as position + 1. pgm 5'-end-2 (-27 to -4) : 5'-agccagcagccggaaaactccagt-3 ' ; pgm 3'-end (+ 1706 to + 1727) : 5'-ggatgggttggtaatctcagtg-3 '. each pcr product was gel - purified and cloned into the ecorv site of pbluescript ks+ (stratagene). briefly, ten male flies were anesthetized with co2 and placed in 200 l ice - cold 0.1 m tris buffer solution (ph 7.6). an additional 200 l tris buffer was added for a total volume of 400 l. approximately 200 - 300 l supernatant was removed, taking care to avoid a top layer of oil and debris. for the assay, 30 - 40 l of this supernatant was used in a 3.1 ml assay mixture that contained : 1.5 mm mgcl2, 26 mm histidine solution (ph 7.6), 8 m glucose-1,6-diphosphate, 0.45 u / ml glucose-6-phosphate dehydrogenase, and 0.2 mm nadp. this solution was mixed at room temperature and then 5 mm glucose 1-phosphate was added. the solution was mixed again and placed in a spectrophotometer. to insure that readings were being taken in the linear range of the enzyme activity, readings were taken at t = 6 min at od340 and were expressed as od/g / min. all extracts were assayed in triplicate, and the averages and standard deviations are presented. means were compared using unpaired, two - side t - tests and p - values are presented. the enzyme assay measures the conversion of nadp to nadph rather than a direct measure of phosphoglucomutase itself. in other words, it is measuring a secondary, coupled reaction. all inserts for microinjection were cloned into a derivative of the 7t40 construct called usc1.0. usc1.0 was generated as follows : first, 7t40 was digested with ecori, thus liberating the hsp70 polyadenylation signal sequence. next, the psti site in polylinker 1 was destroyed by partial psti digestion followed by t4 dna polymerase fill - in and ligation. clones were screened for destruction of the correct psti site to generate usc1.0 (figure 2a). the reverse transcription was carried out on total rna isolated from a strain of flies homozygous for the pgm allele, using the following primer : dtbibo : 5'-taacccgggtctacaaagtgatactgcgtaactgactatattttttttttttttttt-3 '. a nested pcr strategy was then used to amplify the pgm cdna using the following primers : first primer set : bi : 5'-taacccgggtctacaaagtg-3 '. the resulting pcr product was cloned into the ecorv site of pbluescript ks+ and clones were screened for the correct orientation. the pgm cdna was then liberated by ecori complete digest followed by psti partial digest and cloned into the ecori to psti sites of usc1.0 to generate construct pgmsense (figure 2b). the pgminvrpt construct was made as follows : because the final insert would be a large inverted repeat, pgm was pcr - amplified in two pieces, in opposite orientations. the two sets of primers used for the amplifications were : first set : pgm - sense (3 ') (+ 1,773 to + 1,794) : 5'-agctgaattccacaaactttaataaatccgaaac-3 '. second set : pgm - anti not (+ 711 to + 731) : 5'-agctgcggccgccctgaaccggttgggtgccac-3 '. each of these pcr products was cloned individually into the ecorv site of pbluescript ks+, and then restriction digested with ecori and noti (first - set fragment) or noti and psti (second - set fragment). each of the liberated fragments was gel purified and triple - ligated into the psti to ecori sites of usc1.0 to generate construct pgminvrpt (figure 2c). rna was extracted using trizol reagent (gibco brl) and the amount of rna was quantitated by uv spectrophotometry. approximately 5 - 10 g rna per lane after blotting, the rna was fixed to genescreen nylon membrane (dupont) using a uv crosslinker (stratagene). probes were p - labeled using the prime - it ii kit (stratagene). to determine fold increases / decreases, northern blots were exposed onto a phosphor screen and results were analyzed using a phosphorimager (imagequant, molecular dynamics). ribosomal protein 49 gene (rp49) was used as a loading control, and all pgm northern data is normalized to rp49 rna levels. all transgenic drosophila stocks were generated by p - element germline transformation, using a modified microinjection technique. transgenic lines are named as the construct, followed by the chromosome of insertion in parentheses, followed by a unique line designation. for example, pgminvrpt(3)76 is pgminvrpt construct inserted on chromosome 3, independent transgenic line 76. multiple strains homozygous for each of the pgm, pgm and pgm alleles were generated by purifying third chromosomes from the ives stock, which is the precursor to the o and b selection stocks. homozygosity for a particular pgm allele was confirmed by starch - gel electrophoresis assay, as previously described (and data not shown). all stocks were grown on standard cornmeal - agar medium and were cultured at 25c. strains homozygous for the transgenic constructs were crossed to rtta(3)e2 strain, homozygous for the rtta transactivator construct, to generate progeny heterozygous for both constructs. control flies were rtta(3)e2/+, that is, lacking a target construct. to obtain adult flies of defined age, the crosses were cultured at 25c in urine specimen bottles. before eclosion of the majority of pupae, bottles were cleared of adults and newly eclosed flies were allowed to emerge over the next 48 h. most of the males will have mated during this time. the males only were then removed and were designated one day old, and were maintained at 25c at 40 per vial in culture vials with food, and passaged to new vials every 48 h. for certain experiments the adult males were maintained at 29c, as indicated. for certain experiments, all dna sequencing reactions were done using the dideoxy chain - termination protocol and t7 sequenase v2.0 (amersham). the clones were obtained by polymerase chain reaction (pcr) amplification of each allele using genomic dna template isolated from strains homozygous for each allele. the primer locations were assigned on the basis of their relative distance from the a in the atg start codon, which was defined as position + 1. pgm 5'-end-2 (-27 to -4) : 5'-agccagcagccggaaaactccagt-3 ' ; pgm 3'-end (+ 1706 to + 1727) : 5'-ggatgggttggtaatctcagtg-3 '. each pcr product was gel - purified and cloned into the ecorv site of pbluescript ks+ (stratagene). briefly, ten male flies were anesthetized with co2 and placed in 200 l ice - cold 0.1 m tris buffer solution (ph 7.6). an additional 200 l tris buffer was added for a total volume of 400 l. approximately 200 - 300 l supernatant was removed, taking care to avoid a top layer of oil and debris. for the assay, 30 - 40 l of this supernatant was used in a 3.1 ml assay mixture that contained : 1.5 mm mgcl2, 26 mm histidine solution (ph 7.6), 8 m glucose-1,6-diphosphate, 0.45 u / ml glucose-6-phosphate dehydrogenase, and 0.2 mm nadp. this solution was mixed at room temperature and then 5 mm glucose 1-phosphate was added. the solution was mixed again and placed in a spectrophotometer. to insure that readings were being taken in the linear range of the enzyme activity, readings were taken at t = 6 min at od340 and were expressed as od/g / min. all extracts were assayed in triplicate, and the averages and standard deviations are presented. means were compared using unpaired, two - side t - tests and p - values are presented. the enzyme assay measures the conversion of nadp to nadph rather than a direct measure of phosphoglucomutase itself. in other words, it is measuring a secondary, coupled reaction. all inserts for microinjection were cloned into a derivative of the 7t40 construct called usc1.0. usc1.0 was generated as follows : first, 7t40 was digested with ecori, thus liberating the hsp70 polyadenylation signal sequence. next, the psti site in polylinker 1 was destroyed by partial psti digestion followed by t4 dna polymerase fill - in and ligation. clones were screened for destruction of the correct psti site to generate usc1.0 (figure 2a). the reverse transcription was carried out on total rna isolated from a strain of flies homozygous for the pgm allele, using the following primer : dtbibo : 5'-taacccgggtctacaaagtgatactgcgtaactgactatattttttttttttttttt-3 '. a nested pcr strategy was then used to amplify the pgm cdna using the following primers : first primer set : bi : 5'-taacccgggtctacaaagtg-3 '. the resulting pcr product was cloned into the ecorv site of pbluescript ks+ and clones were screened for the correct orientation. the pgm cdna was then liberated by ecori complete digest followed by psti partial digest and cloned into the ecori to psti sites of usc1.0 to generate construct pgmsense (figure 2b). the pgminvrpt construct was made as follows : because the final insert would be a large inverted repeat, pgm was pcr - amplified in two pieces, in opposite orientations. the two sets of primers used for the amplifications were : first set : pgm - sense (3 ') (+ 1,773 to + 1,794) : 5'-agctgaattccacaaactttaataaatccgaaac-3 '. second set : pgm - anti not (+ 711 to + 731) : 5'-agctgcggccgccctgaaccggttgggtgccac-3 '. each of these pcr products was cloned individually into the ecorv site of pbluescript ks+, and then restriction digested with ecori and noti (first - set fragment) or noti and psti (second - set fragment). each of the liberated fragments was gel purified and triple - ligated into the psti to ecori sites of usc1.0 to generate construct pgminvrpt (figure 2c). rna was extracted using trizol reagent (gibco brl) and the amount of rna was quantitated by uv spectrophotometry. approximately 5 - 10 g rna per lane after blotting, the rna was fixed to genescreen nylon membrane (dupont) using a uv crosslinker (stratagene). probes were p - labeled using the prime - it ii kit (stratagene). to determine fold increases / decreases, northern blots were exposed onto a phosphor screen and results were analyzed using a phosphorimager (imagequant, molecular dynamics). ribosomal protein 49 gene (rp49) was used as a loading control, and all pgm northern data is normalized to rp49 rna levels. m.j.a. was supported by a pre - doctoral training grant from the national institute on aging (ag00093). this research was supported by a grant from the department of health and human services to j.t. the amino acids that differ between the pgm, pgm, pgm and wild - type canton - s alleles are indicated in single - letter code, with the predicted pi values at the right. the genbank accession numbers for the sequences are af416982, af416981, af416983 and af416984, respectively. unique psti and ecori sites are located downstream of the tet - on promoter, enabling cloning of cdnas. (c) the pgminvrpt construct, containing an approximately 1 kb inverted repeat of pgm coding region, as indicated by inverted arrows. total rna was isolated from adult males of the indicated pgmsense lines, pgminvrpt lines, and control line, with or without dox treatment. (a) northern blot hybridized with a probe that will recognize both endogenous and transgenic pgm transcripts and with a probe specific for rp49 as a loading control. (b) northern blot hybridized with a probe that will recognize only the endogenous pgm transcript and with a probe specific for rp49 as a loading control. (c) northern blot of control strain hybridized with a probe that will recognize the endogenous pgm transcript and with a probe specific for rp49 as a loading control. | backgrounda tetracycline - regulated (conditional) system for rna interference (rnai) would have many practical applications. such a strategy was developed using rnai of the gene for phosphogluconate mutase (pgm). pgm is a candidate lifespan regulator : pgms allele frequency is increased by selection for increased lifespan, whereas pgmm and pgmf allele frequencies are decreased.resultsthe pgm alleles were cloned and sequenced and were found to differ by amino - acid substitutions consistent with the relative electrophoretic mobilities of the proteins. the ' tet - on ' doxycycline - regulated promoter system was used to overexpress pgms in a wild - type (pgmm) background. enzyme activity increases of two- to five - fold were observed in five independent transgenic lines. tet - on was also used to drive expression of an inverted - repeat fragment of pgm coding region. the inverted - repeat transcript was expected to form a dsrna hairpin, induce rnai, and thereby reduce endogenous pgm gene expression at the rna level. endogenous pgm rna levels in adult flies were found to be reduced or eliminated by doxycycline treatment in five independent inverted - repeat transgenic lines. our results show that doxycycline - regulated expression of inverted - repeat constructs can cause a conditional reduction in specific gene expression. the effect of sense and inverted - repeat construct expression on lifespan was assayed in multiple transgenic lines. under the conditions tested, altered pgm gene expression had no detectable effect on adult drosophila lifespan.conclusionsa system for conditional rnai in drosophila adults shows promise for assay of gene functions during aging. our results indicate that pgm does not have a simple strong effect on longevity. |
the west nile virus (wnv) is an arthropod - borne flavivirus that is associated primarily with epidemics of flu - like febrile illness. neurologic manifestations are uncommon with overlapping clinical syndromes including encephalitis, meningitis and acute flaccid paralysis. wnv has been endemic in israel since the 1950s usually causing a mild, self - limiting disease but an isolated epidemic occurred in the year 2000 with 428 hospitalized cases of neurological disease and 42 deaths. the observation of a patient with chronic lymphocytic leukemia and wnv encephalopathy who made a prompt and complete recovery after receiving high dose intravenous immunoglobulin (ivig) lead to the finding that immunoglobulin preparations in israel contain high titers of antibodies to wnv (1:1600). since then, case studies have continued to suggest that ivig therapy can be effective in some patients including immunosuppressed post - transplant patients. recent reviews have called for randomized controlled trials and a multicenter randomized, placebo - controlled trial sponsored by the national institute of health is in progress. the results of such studies are urgently needed because of the high morbidity and mortality rates of neuroinvasive wnv. nevertheless until results of randomized controlled trials are available, an accumulated series of cases can provide a higher degree of evidence than isolated case reports. in this report, we summarize the clinical course of all patients known to have received intravenous immunoglobulin for wnv neuroinvasive disease in israel until the end of 2007. we also characterized the prevalent genotypes found in mosquitoes (highly correlated with human infection) according to time and place in order to determine if there is a relationship to the outcome according to the various genotypes. we contacted the infectious disease consultants of israeli hospitals to request details of the clinical course of patients with serious wnv neuroinvasive disease who received treatment with at least one day of high dose intravenous immunoglobulin. serological testing was performed in the ministry of health s national center for zoonotic viruses, at the central virology laboratory by using an igm - capture enzyme - linked immunosorbent assay (elisa). diagnosis of primary wnv infection was made on the basis of clinical symptoms and signs, together with laboratory confirmation of the presence of immunoglobulin m (igm) antibodies, with or without igg antibodies, and low igg avidity. diagnosis was also made on the basis of a significant rise in antibody level between paired samples (4 fold), and a specific reaction calculated from the reaction to viral antigen over the reaction to mock antigen (2 fold). all tests were developed in house and performed with a local wnv isolate (as antigen) either genotypically similar genotype to the new york 1999 strain or a genotype similar to both the new york 1999 and to the romania 1997 strain. for identification of the circulating wnv genotypes in the country, rna was extracted from mosquito pools, as part of the yearly routine mosquito surveillance program. rna was then amplified by real - time rt - pcr with specific primers of the env gene. virus isolates were identified by standard rt - pcr methods, amplified, sequenced, and the gene compared with published sequences in the gene bank. serological testing was performed in the ministry of health s national center for zoonotic viruses, at the central virology laboratory by using an igm - capture enzyme - linked immunosorbent assay (elisa). diagnosis of primary wnv infection was made on the basis of clinical symptoms and signs, together with laboratory confirmation of the presence of immunoglobulin m (igm) antibodies, with or without igg antibodies, and low igg avidity. diagnosis was also made on the basis of a significant rise in antibody level between paired samples (4 fold), and a specific reaction calculated from the reaction to viral antigen over the reaction to mock antigen (2 fold). all tests were developed in house and performed with a local wnv isolate (as antigen) either genotypically similar genotype to the new york 1999 strain or a genotype similar to both the new york 1999 and to the romania 1997 strain. for identification of the circulating wnv genotypes in the country, rna was extracted from mosquito pools, as part of the yearly routine mosquito surveillance program. rna was then amplified by real - time rt - pcr with specific primers of the env gene. virus isolates were identified by standard rt - pcr methods, amplified, sequenced, and the gene compared with published sequences in the gene bank. there were 12 patients who received ivig ; three patients died, 6 had partial recovery, and 3 recovered completely (table 1). eleven of the 12 patients were infected with israeli genotypes that are highly homologous to europe / africa viruses. a standard dose of ivig of 0.4g / kg / day was used, for a variable number of days (table 1). the effects of therapy were often dramatic and occurred within 48 hours in 4 patients. table 1clinical characteristics, treatment and outcome.casegender/agesignificantpast historypresentationdaysof rxdaysto responseresponse / residualdiseaserecovery1f/72dementia - meningiomastupor, seizures, weakness2-nodied2f/86dementiaseizures, paralysis, loss of consciousness22completecomplete3f/37noneloc, paralysis, ventilated13awake/ weaknesspartial4m/74diabetes mellitusstupor, paralysis, ventilated5<5awake/ weaknesspartial5m/76diabetes mellitusloss of consciousness, paralysis, ventilated5<20awake / tracheotomypartial6f/65high grade nhlloss of consciousness, paralysis, ventilated4-nodied7f/83toxic goiterstupor, weakness11completecomplete8m/40alcoholicstupor, muscle weakness12completecomplete9f/41noneparalysis51weakness / ataxiapartial10m/67thymomastupor, muscle weakness, tremor1<3awake/ weaknesspartial11f/45lung transplant- ipfstupor, muscle weakness, ventilated3-nonedied12f/87dementiastupor, weakness3<20completecompleterx, treatment ; cll, chronic lymphocytic leukemia ; nhl, non - hodgkin s lymphoma ; loc, loss of consciousness ; weakness means objective muscle weakness. rx, treatment ; cll, chronic lymphocytic leukemia ; nhl, non - hodgkin s lymphoma ; loc, loss of consciousness ; weakness means objective muscle weakness. the major finding of our study is the prompt response to treatment with ivig observed in some patients with wnv neuroinvasive disease. our findings are consistent with the response observed in 10 patients reported previously with israeli genotypes that are highly homologous to american viruses and with cases reported outside of israeli. eleven of the 12 patients reported here had genotypes homologous to the europe / africa viruses. furthermore although none of the 5 new cases ventilated had complete recovery, there were 3 of 5 such patients reported previously with complete recovery. this is in contrast to the prolonged recuperation and recovery, and lack of complete recovery in 21 patients who did not receive ivig reported in a review of the literature. animal data indicate an important role for humoral immunity in controlling west nile virus infection, and treatment with antibodies is still used in certain viral illnesses such as disseminated vaccinia after small pox vaccination. recent studies in mice have shown that antibodies present in israeli plasma are effective in reducing morbidity in mice. we conclude that it is warranted to treat wnv neuroinvasive disease with ivig because of the rapid response to treatment observed in some patients, and the favorable outcome in patients requiring respiratory support. | the aim of the study was to determine whether intravenous gamma globulin (ivig) treatment is effective in patients with west nile virus (wnv) neuroinvasive disease.we contacted hospital based infectious disease experts in israeli hospitals to identify patients with wnv neuroinvasive disease who were treated with ivig. the main outcome measure was neurological response after treatment. there were 12 patients who received ivig and four improved within 48 h. three patients died, 6 had partial recovery, and 3 recovered completely. eleven of the 12 patients were infected with israeli genotypes that are highly homologous to europe / africa viruses. the rapid response in some patients suggests that ivig is effective, and might be used to treat patients with wnv neuroinvasive disease with ivig. |
the immune system has evolved for several million years to ameliorate the defenses of our organism from any kind of pathogens, including those causing sepsis, a life - threatening condition in which almost all components of the innate and adaptive immune system have to work together in a coordinated manner. however, in septic hosts, a deregulated response to infection may lead to a sustained systemic inflammation that causes the failure to clear primary pathogens and alters immune responses. the study by monserrat and colleagues in the previous issue of critical care adds one more piece in this complex puzzle by evaluating the early subset distribution and activation of circulating b - lymphocytes in patients with septic shock. but what is the relevance of the b - cell compartment in this puzzle ? to better understand this issue, a brief and schematic description of the immune response is desirable. the first is the activation of several toll - like receptors present on cells of the innate immunity (for example, monocytes, granulocytes, dendritic cells, and natural killer cells) that recognize pathogen - associated molecular patterns (pamps) present in or released by the invading microorganisms. the activation of these cells results in the production of pro - inflammatory cytokines - that is, interleukin (il)-1, il-6, il-10, and tumor necrosis factor - alpha (tnf-) - and vasoactive peptides, complement, and reactive oxygen species that together start a generalized inflammation. a second response mechanism involves the activation of cd4 t lymphocytes that bear particular amino - acid sequences in the variable region of the -chain (v) of their t - cell receptor (tcr). each t cell bearing a given v tcr can bind bacterial (or even viral) products in a non - major histocompatibility complex - restricted manner (that is, outside from the region where the clonal - specific recognition of a unique antigen occurs) ; for this reason, such molecules, which activate a large percentage of t cells that are in fact specific for other molecules, are defined as superantigens (sags). an sag like the staphylococcal enterotoxin b is massively released during sepsis and can activate up to 30% of t cells, which produce several cytokines, including il-17a, il-2, interferon - gamma, and tnf-. however, activated cells have a high tendency to develop mitochondrial damage and undergo apoptosis, and this clearly results in a further impaired immune response. a family of b - lymphocytes (known as b-1 cells, which are unable to further differentiate into mature b cells) located mainly in the peritoneal and pleural cavities can produce immuno globulin m (igm) and il-10 and modulate the systemic inflammatory response. studies in mice have identified the presence of an effector b - cell population that protects against microbial sepsis. these cells, defined as innate response activators, depend on pamp receptors and produce granulocyte - macrophage colony - stimulating factor. their deletion impairs bacterial clearance, elicits a cytokine storm, and can precipitate septic shock. the above brief description highlighted the role and the importance of the b - cell compartment in the initial immune response to infection ; hence, the data provided by monserrat and colleagues are really helpful in providing more insight in this issue. in 52 septic shock patients who were admitted to the intensive care unit (icu), the authors observed an early (that is, within 24 hours) and sustained circulating b - cell reduction that was associated with a significant redistribution of b - cell subsets compared with healthy subjects. the cd19cd23b cells (activated regulatory b cells) were lower in non - survivors than in survivors for the 7 days of the follow - up, whereas cd19cd69(early activated b cells) and the expression of critical antigens on b cells (cd80, a t - cell co - stimulation molecule, and cd95, an apoptosis susceptibility marker) were higher in non - survivors. moreover, lymphopenia and an increased expression of cd80 persisted in survivors for all 28 days of the study period. the authors concluded that, in patients with septic shock, the circulating b - cell compartment is early and deeply altered in both quantitative and qualitative terms and that there is a close relationship between these alterations and patient outcome. the final message from the article is sound and in accordance with previous studies on the other components of the immune response (that is, dendritic cells, natural killer cells, and t - lymphocytes) that in patients with septic shock revealed a significant decrease in the number and function of the immune cells with early signs of apoptosis and cell exhaustion in non - survivors [6 - 10 ]. however, the reasons for this different pattern between survivors and non - survivors remained to be clarified. a recent article by gogos and colleagues indicated that the alterations in natural killer cells and t - lymphocytes depend on the severity of sepsis and the site and type of infection. the time length from infection to the onset of septic shock (and to its treatment) may also have an important role : the longer the time, the larger the expected activation and exhaustion of the inflammatory and immune response. lastly, genetic factors could also be connected to the expression of different immune patterns among patients with similar types of infection and sepsis severity. unfortunately, owing to the low number of patients studied and the difficulties to define the time length from infection to icu admission, the article by monserrat and colleagues can not offer a proper analysis of these issues and leaves the question open. whatever the reasons for the differences observed in the immune system, the study by monserrat and colleagues emphasizes once again that immunosuppression is common even in the early stages of sepsis and that patients with sepsis constitute a complex and heterogeneous population requiring specific monitoring and individualized therapeutic approaches. immune - modulatory therapies tailored to the immune state of the patients will be the object of future clinical trials. in the meantime, as in other pathologies with variable disorders of the immune system (for example, cancer and rheumatologic diseases), monitoring the immune - inflammatory state in septic shock patients admitted to the icu could be useful for identifying the high - risk patients and the fluctuating inflammatory - immune response. icu : intensive care unit ; il : interleukin ; pamp : pathogen - associated molecular pattern ; sag : superantigen ; tcr : t - cell receptor ; tnf- : tumor necrosis factor - alpha ; v : variable region of the beta - chain. | impairment of the inflammatory - immune response is currently accepted as a hallmark of severe sepsis even in the early stages of the disease. in this context, the alterations of the circulating b - lymphocytes have never been described in detail. the study by monserrat and colleagues in the previous issue of critical care indicated that, in patients with septic shock, the b - cell compartment is early and deeply altered with different patterns in subset distribution and activation between survivors and non - survivors. |
all clinical microbiology laboratories culturing campylobacters in finland were asked to collect domestic campylobacter isolates detected from human clinical fecal samples. isolates collected on july 1 through september 30, 1999, from patients who had not been abroad for 2 weeks before becoming ill, were considered of domestic origin and were included in the study. information on foreign travel, received by the physicians, was collected by the local clinical microbiology laboratories when culture results were reported to the clinical unit. the isolates were sent to helsinki university central hospital (huch) laboratory diagnostics along with information about the patient (date of birth, sex, recent travel history) and the isolate (date of stool sampling, hippurate hydrolysis result). hippurate - positive (c. jejuni) isolates were subsequently serotyped based on heat - stable penner s (pen) antigens by passive hemagglutination using a serotyping set including 25 antisera (campylobacter antisera seiken set, denka seiken co., tokyo) as earlier described (9). a total of 3,303 campylobacter cases in finland in 1999 were reported to nidr ; of these, 1,412 (43%) cases were diagnosed during our study period. in the present study, a total of 551 campylobacter isolates were collected from patients who had presumably acquired their infections in finland. the absence of case linkage between the two data sources prevents exact correlation between them ; however, the number of domestic cases from the isolate collection is approximately 40% of the number of cases in nidr for the same period. of the strains collected, 533 (97%) the 533 case - samples of domestic c. jejuni infection collected within a population of 5.17 million yields a rate of 41.2 domestic c. jejuni cases per 100,000 inhabitants for the 3-month period the rate of domestic c. jejuni infections by age group varied from 19.6 to 72.8 per 100,000 inhabitants for the 3-month period, with the highest rates observed among those > 60 years old, among young adults (2034 years of age), and among children < 5 years of age (figure 1). domestically acquired campylobacter jejuni infections in finland by age and sex, july based on the municipality of the clinical unit from which the stool culture had been sent, several eastern hospital districts had high rates of domestic campylobacter cases during the study period ; the 95 % confidence intervals for these cases did not overlap those in several southern and western hospital districts with low rates (figure 2). in some eastern districts, domestically acquired campylobacter infections comprised even more than 80% of all cases reported to nidr in july. in the other hospital districts, a peak was also demonstrated in july ; then the number of domestic c. jejuni cases declined in august, and the number of isolates collected in september comprised only 10% of all strains isolated during the 3-month study period. september 1999. the seasonal distribution of the different serotypes is presented in the table. the predominant serotypes (pen 1,44, pen 2, pen 4-cluster, pen 6,7, pen 12, and pen 27) comprised 61% of the isolates (table). no clear geographic differences in the distribution of serotypes were found. none of the strains reacted with the antisera against serotypes pen 10, 32, 38, or 45. reactions with serotype pen 3 and pen 8 antisera were only present in complex serotypes. serotypes pen 5, 11, 18, 19, 31, 52, and 55 (not included in the table) were so uncommon that they together represented approximately 5% of all the isolates. uncommon serotypes pen 5, 11, 18, 19, 31, 52, and 55 not included. 4-cluster, serotypes 4, 13, 16, 43, 50. the mean age of all case - patients was 41.4 years. patients infected with c. jejuni serotype pen 15 (mean age 23.5 years, 95% ci 6.0, 41.0) or serotype pen 2 (mean age 32.7 years, 95% ci 26.3, 39.0) were significantly younger than those infected with serotype pen 27 (mean age 49.6 years, 95% ci 42.7, 56.4) or serotype pen 6,7 (mean age 46.8 years, 95% ci 42.2, 51.4). the age distribution of serotypes pen 2 and pen 27 is shown in figure 3. differences between the age distributions of case - patients infected with other serotypes were not significant. pen 2 and pen 27 serotypes among domestically acquired campylobacter jejuni infections in finland by age, july all hospital districts in finland participated in this study, and the rate of domestically acquired c. jejuni infections, as measured by the number of isolates sent, was highest in several eastern hospital districts. the higher rates can not be explained by urban versus rural lifestyle alone since the western districts in the study included rural and urbanized areas in an approximately similar proportion as in the eastern districts. we have studied human domestic campylobacter infections in finland since 1995 (911) and found the same serotypes to be common among c. jejuni isolates collected during july through september, although annual variation in the relative proportion of predominant serotypes is evident. serotypes pen 1,44, pen 2, and pen 4-cluster have been found relatively often in human fecal samples in denmark (4,12), england (13), and new zealand (14), but serotypes pen 6,7, pen 12, and pen 27 have been less common (< 3% each) in those studies. from may to september 1999, a total of 1,132 chicken flocks, representing most of the chicken meat produced and consumed in finland, were monitored for campylobacters. thirty - one c. jejuni positive flocks were detected ; the most common penner serotypes identified were pen 6,7 and pen 12, found mainly from july through august (15). these particular serotypes were also commonly found in human infections in the present study in july and in august. serotypes pen 27 and pen 4-cluster were also found in the chickens (15). humans and chicken may share a common source for c. jejuni, or humans may acquire the infection from contaminated chicken meat. the highest rate of domestic c. jejuni infection was found among patients in the age group of 7074 years, accompanied by an above average rate among patients 6569 years old. this is the first report of such an age peak for c. jejuni (1,3,4). furthermore, the age distribution of patients infected with isolates of certain serotypes suggests that older people in finland may have somewhat different sources of infection than younger people. serotypes pen 6,7 and pen 27, in particular, appeared to be relatively common among the elderly patients. in our study, serotype pen 2 was more common than pen 27 in all age groups < 50 years of age (excluding the age group 2529 years) but less common in all older groups. since serotypes pen 6,7 and pen 27 are also common in chicken (15), there may be a link between raw poultry handling or chicken consumption practices and c. jejuni in the elderly. during the 1999 seasonal peak, domestically acquired cases of c. jejuni in finland were caused by the same serotypes that are most commonly found in other developed countries, yet some common serotypes in our study seem to be less common elsewhere. we recommend further study to determine if the results were specific to the time period or representative of a persistent phenomenon. | campylobacter jejuni isolates (n = 533) from domestic cases diagnosed in finland during a 3-month peak period were studied. the highest rate was observed among those 7074 years of age. domestic c. jejuni isolates were especially frequent in the eastern districts. six serotypes covered 61% of all c. jejuni isolates. |
we complied with the institutional review boards of the centers for disease control and prevention (cdc) (protocol 4797) and the broad institute of mit and harvard. denv was obtained from human serum received through the passive surveillance system administered by cdc. each sample was accompanied by a form that captured geographic and clinical information maintained for this study without patient identifiers. primary or secondary status of infection was inferred by absence or presence of serum immunoglobulin g (15). selection of 3 isolates per year in the 5 municipalities with the highest reporting of denv-2 cases resulted in 253 isolates, of which 140 were successfully sequenced and are representative of our virus repository with respect to patient age (27.7 vs. 22.6 years), sex (54.4% vs. 47.4% male), and history of infection (84.6% vs. 77% secondary infections). we extracted rna from tissue culture supernatant using the m48 or mdx biorobot (qiagen, valencia, ca, usa). cdna was generated by using sensiscript rt (qiagen) with random hexamers (applied biosciences, foster city, ca, usa). presence of cdna was confirmed by pcr by using pfuultraii (stratagene, la jolla, ca, usa) or itaq (bio - rad, hercules, ca, usa) dna polymerase and specific oligonucleotides (cdc, atlanta, ga, usa). pcr at cdc (san juan, pr) and sequenced at the broad institute (cambridge, ma, usa) by bidirectional sanger by using an abi 3730 after pcr with 96 m13-tailed serotype - specific primers. resulting reads were trimmed of the primer sequences, filtered for high quality, and assembled by using algorithms developed by the broad institute. all coding sequences for the poliproteins (10,173 nt) and parts of the 5 and 3 noncoding regions were deposited in genbank. coding sequences for the unprocessed polyprotein (5 and 3 noncoding regions excluded) were aligned by clustalw software (www.ebi.ac.uk/tools/clustalw/index.html) in mega 4 (www.megasoftware.net). maximum likelihood analysis and bootstrapping tests were performed in paup (16) under the best - fit substitution model estimated by modeltest v3.07 (14) (parameters available on request). mean rates of nucleotide substitution and relative genetic diversity (net, where t is the generation time) were estimated by using bayesian markov chain monte carlo (mcmc) from beast v1.4.7 (http://mbe.oxfordjournals.org/content/25/7/1459). general time reversible substitution model with strict and relaxed molecular clocks and constant population size or bayesian skyline coalescent analysis was used. all mcmc chains were run for sufficient length ensuring stationary parameters, with statistical error reflected in values of the 95% highest probability density. amino acid differences were mapped by using parsimony methods in macclade v4.08 (17). we determined dn / ds ratios with the single likelihood ancestor counting method using hyphy and accessed through the datamonkey server (13). associations between phylogeny and geographic data were investigated by using bayesian tip - association significance testing (http://evolve.zoo.ox.ac.uk/evolve/bats.html) with the posterior sample of trees calculated by beast. for the parsimony score, association index, and monophyletic clade size we complied with the institutional review boards of the centers for disease control and prevention (cdc) (protocol 4797) and the broad institute of mit and harvard. denv was obtained from human serum received through the passive surveillance system administered by cdc. each sample was accompanied by a form that captured geographic and clinical information maintained for this study without patient identifiers. primary or secondary status of infection was inferred by absence or presence of serum immunoglobulin g (15). selection of 3 isolates per year in the 5 municipalities with the highest reporting of denv-2 cases resulted in 253 isolates, of which 140 were successfully sequenced and are representative of our virus repository with respect to patient age (27.7 vs. 22.6 years), sex (54.4% vs. 47.4% male), and history of infection (84.6% vs. 77% secondary infections). we extracted rna from tissue culture supernatant using the m48 or mdx biorobot (qiagen, valencia, ca, usa). cdna was generated by using sensiscript rt (qiagen) with random hexamers (applied biosciences, foster city, ca, usa). presence of cdna was confirmed by pcr by using pfuultraii (stratagene, la jolla, ca, usa) or itaq (bio - rad, hercules, ca, usa) dna polymerase and specific oligonucleotides (cdc, atlanta, ga, usa). pcr at cdc (san juan, pr) and sequenced at the broad institute (cambridge, ma, usa) by bidirectional sanger by using an abi 3730 after pcr with 96 m13-tailed serotype - specific primers. resulting reads were trimmed of the primer sequences, filtered for high quality, and assembled by using algorithms developed by the broad institute. all coding sequences for the poliproteins (10,173 nt) and parts of the 5 and 3 noncoding regions were deposited in genbank. coding sequences for the unprocessed polyprotein (5 and 3 noncoding regions excluded) were aligned by clustalw software (www.ebi.ac.uk/tools/clustalw/index.html) in mega 4 (www.megasoftware.net). maximum likelihood analysis and bootstrapping tests were performed in paup (16) under the best - fit substitution model estimated by modeltest v3.07 (14) (parameters available on request). mean rates of nucleotide substitution and relative genetic diversity (net, where t is the generation time) were estimated by using bayesian markov chain monte carlo (mcmc) from beast v1.4.7 (http://mbe.oxfordjournals.org/content/25/7/1459). general time reversible substitution model with strict and relaxed molecular clocks and constant population size or bayesian skyline coalescent analysis was used. all mcmc chains were run for sufficient length ensuring stationary parameters, with statistical error reflected in values of the 95% highest probability density. amino acid differences were mapped by using parsimony methods in macclade v4.08 (17). we determined dn / ds ratios with the single likelihood ancestor counting method using hyphy and accessed through the datamonkey server (13). associations between phylogeny and geographic data were investigated by using bayesian tip - association significance testing (http://evolve.zoo.ox.ac.uk/evolve/bats.html) with the posterior sample of trees calculated by beast. for the parsimony score, association index, and monophyletic clade size during 19862007, dengue cases in puerto rico ranged from 2,000 to 16,000 per year (figure 1, panel a), with major epidemics (> 8,000 cases) reported in 1986, 1992, 1994, 1998, and 2007 (24,18,19). despite major fluctuations in serotype circulation, denv-2 circulated predominantly for 10 years (figure 1, panel b), alternating with denv-1 through 2 periods of resurgence during the 1990s and cocirculation of denv-4 (figure 1, panel b). denv-2 declined markedly after the 1998 epidemic and the dissemination of denv-3 concomitant to the disappearance of denv-1 and -4. however, denv-2 continued to cause a low number of cases during 19992003 and reemerged in 20042007 (figure 1, panel b). samples from every year of the 22-year study period (figure 1, panel c) comprised our analysis. a) number of suspected, clinically defined cases of dengue fever / dengue hemorrhagic fever by year reported to the centers for disease control and prevention s dengue branch. b) percentage of identifications of each serotype relative to the total of positive serotype identifications by using tissue culture isolation or reverse transcription pcr per year. numbers in parenthesis indicate numbers of dengue virus (denv) serotype 2 identifications each year. black, denv-1 ; blue, denv-2 ; white, denv-3 ; red, denv-4. c) number of partially sequenced (e gene) autochthonous puerto rican isolates reported by previous studies (14,20) and whole genome sequences obtained in the present study by year of their corresponding case presentation. d) bayesian coalescent inference of population dynamics and genetic diversity by using the bayesian skyline plot. sampling procedures were used to estimate posterior distribution of denv-2 genetic diversity in an effective population through the study period on the basis of full genome sequence data. x axis, time in years through the study period ; y axis, product of the effective population size (relative genetic diversity) and generation length in years ; black line, median estimate ; blue shadow, 95% highest probability density. the bayesian skyline analysis (figure 1, panel d) of the autochthonous viral sequences (figure 2 ; clades ib and ii figure a1) showed a gradual increase in the genetic diversity of denv-2 during 19871991 that corresponds to a period of high transmission and dominance (figure 1, panel b). this increase was followed by 9 years of high genetic diversity that coincided with a period of denv-1 and denv-4 cocirculation. the genetic diversity of denv-2 declined sharply during 19992003, coinciding with a period of minimal denv-2 transmission. maximum likelihood phylogeny of the 140 puerto rico and 20 international isolates of denv-2 (see number of isolates by year below). names of clades (i, ii, and iii) and subclades (ia, ib) are shown at the base of their respective branches on the phylogeny tree. clade ii (dark blue) circulated during 19861996 and clade i (light blue) during 19942007. subclade ia and clade iii represent foreign, transient reintroductions throughout the 22-year study period. black dots indicate 18 isolates from puerto rico with phylogenetic associations closer to foreign isolates than to other puerto rico viruses. two - letter geographic codes indicate origin (pr, puerto rico ; jm, jamaica ; sj, saint john ; sh, saint thomas ; ve, venezuela ; cl, colombia ; nc, nicaragua ; mx, mexico ; cu, cuba ; si, saint kits ; dr, dominican republic ; sx, saint croix ; br, brazil). pr code is accompanied by a 2-digit number that indicates geographic location (municipality) on the island. the 2-digit numbers between the geographic codes and the genbank accession numbers indicate the year of the corresponding case of each isolate. bootstrap values are shown for all clades (i, ii, and ii), subclades (ia, ib), and most immediate lineages. twelve amino acid changes associated with ib / ii differences are shown on the table ; 6 other selected changes across subclade ib are shown on the left at the base of the branches exhibiting the respective changes. relevant epidemiologic events are highlighted to the right with pie charts showing the relative levels of each serotype isolated for that period black, denv-1 ; blue, denv-2 ; white, denv-3 ; red, denv-4. the densely populated island of puerto rico (3,808,610 population ; 3,508 square miles) is divided into 78 municipalities grouped in 8 regions. the 140 denv-2 genomes from 37 municipalities represented all 8 regions and ranged from 220 isolates per year (figure 1, panel c ; figure 2). the number of puerto rico sequences is proportional to the epidemic level or the relative proportion of denv-2 identifications in the municipalities with highest denv-2 reporting per year. the phylogeny of the 160 denv-2 genomes showed 2 major clades (i and ii) and a smaller clade (iii) (figure 2). subclade ia (19982007) contains 13 puerto rico and 12 caribbean sequences, including 1 from st. subclade ib (19942007) contains 90 sequences mostly of local origin, but the presence of 6 foreign and 1 local basal sequences confirms its foreign origin. these genetically distinct isolates do not fit in clade i or ii, but a separate analysis with publically available envelope gene sequences pointed to possible caribbean origin (k.l. first, a mixture of foreign and local strains at the base of subclades ia and ib provides evidence of multiple introductions. these years also are associated with a distinct subgroup basal to subclade ib concomitant with the extinction of clade ii in 1997. third, a period of limited circulation of denv-2 reflected in low levels of genetic diversity (19992003) coincided with the expansion of denv-3 and decline of denv-1 and -4. forty - nine amino acid differences mapped to the phylogeny were detected across the major internal branches of the tree. twenty of these comprise major differences between clades i and ii and between subclades ia and ib, as well as substitutions that arose during the continuous evolution of subclade ib (figure 2). only 1 aa substitution distinguished isolates in clades i / ii from iii : a hydrophilic glutamine to a hydrophobic leucine at position 131 in the e protein. excluding pr79_1995_eu569708 as a possible foreign introduction, 18 aa differences distinguish isolates across clade i, 12 of which separate subclade ib from clade ii and potentially could have been involved in the 19941997 lineage turnover (figure 2). the remaining differences between isolates in subclades ib and ii were present in nonstructural (ns) genes and are preponderantly conservative mutations, with the exception of position 31 in ns3, which was nonconservative. among the additional changes, the only nonconservative mutation was a hydrophobic alanine to hydrophilic threonine at position 137 in ns4b that originated with pr40_1999 eu482730, and most changes were found in the ns genes. using bayesian mcmc and dn / ds analyses, we estimated the mean substitution rates for the full genomes at 9 10 to 1.1 10 for all clades, consistent with previously published rates (20,21). the low dn / ds ratios (0.070.08) provide evidence of a low percentage of substitutions that have been fixed along independent lineages, possibly indicating purifying, negative selection. bats analysis shows that lineages often correlated with the corresponding region of origin of the isolates. seven of the 8 regions had > 4 isolates in subclade ib or clade ii. the most significant geographic correlation of lineages were found in the san juan (19861990 and 19941996), ponce (19871989), and mayaguez (1989 and 1993) (figure 3, panel a). in addition, isolates clustered geographically for san juan (19971999 and 20012006), caguas (19982001, 2004, and 2005), ponce (19951997 and 2005), mayaguez (19961998 and 2006), aguadilla (19961998), and arecibo (19941995, 2004, and 2006) (figure 3, panel b). considering the denv-2 historical data, we recognize that high - reporting municipalities usually are located in regions where we identified significant phylogenetic clustering (figure a1). for example, in 1987, most denv-2 cases originated from the ponce and san juan regions, where we identified lineages of clade i. for19941996, denv-2 cases in san juan, ponce, mayaguez, and arecibo regions may reflect the coexistance of subclades ib and clade ii. correlation estimated by using bats. bats, bayesian tip - association significance testing ; hpd, highest probability density ; ci, confidence interval. b) maximum - likelihood phylogeny of subclade ib shows isolates by year and genbank accession numbers. six regions had > 5 isolates (san juan, caguas, ponce, mayaguez, aguadilla, and arecibo). c) eight regions of puerto rico with colors corresponding to isolates in panel a and year for the 3 regions with more isolates of that clade : san juan, mayaguez, and ponce. d) eight regions of puerto rico, showing colors and years corresponding to isolates in panel b. correlation between phylogeny and geographic location of isolation for the isolates in this study was estimated by using bayesian tip - association significance testing. association index 6.51 (95% confidence interval 6.037.22) ; parsimony score statistic 52.27 (95% confidence interval 5154). monophyletic clade size bayesian tip - association significance estimates are shown for 6 regions of puerto rico with > 5 isolates represented in at least 1 subclade and statistically representative geographic associations (p<0.05). we investigated other possible associations with the denv-2 phylogeny, including age and df / dhf status, but found none. most denv-2 infections were secondary (84.6% and 77% of denv-2 infections in the cdc collection and this study, respectively). however, we found no relationship between phylogeny and incidence of primary or secondary infection in patients. the year 1999 began a period of low circulation and low genetic diversity of the caguas lineage of subclade ib (figure 1, panel d ; figure 2 ; figure 3, panel b) that lasted until 2003. during these 4 years, most denv-2 cases originated from only 4 municipalities in eastern puerto rico (figure 4, panel a) ; < 20 additional denv-2 cases were reported during that period in 12 other neighboring municipalities (figure 4, panel a). because phylogenetic lineages are geographically and temporally clustered, (figure 2), we illustrated these associations on the map of puerto rico (figure 4). this map shows that denv-2 descendants from western puerto rico emerged in san juan in 19971998 (figure 4, panel b, top), then appeared and persisted within the refuge in 19992002 (figure 4, panel c, middle) to then disseminate across the island in 20032005 (figure 4, panel d). in the 4 municipalities with uninterrupted denv-2 transmission, denv-2 incidence increased 2 years after the islandwide increase (figure 5). denv-3 incidence within this denv-2 refuge was minimal during the period of high denv-2 incidence but peaked 2 years later concomitant with an increase across the rest of the island epidemiology of dengue virus (denv) serotype 2 in puerto rico, 19972006. a) municipalities with persistent denv-2 transmission (caguas, juncos, las piedras, carolina) versus those with discontinuous transmission (morovis, toa alta, toa baja, catao, guaynabo, cidra, san lorenzo, canvanas, humacao, naguabo, ceiba, fajardo), 19982002. inset shows satellite view ; red dot indicates national capital (san juan), and yellow box indicates region where denv-2 took refuge during 20002002. white pins point to specific geographic locations where denv-2 isolates were collected during the specified time period. b) denv-2 traveled to the san juan region from the west during 19971999 ; c) denv-2 transmission retracted to the eastern, refuge region with restricted dispersion patterns during 20002002 ; d) denv-2 reemerged focused on the san juan region and later dispersed throughout the island during 20032006. incidence of dengue virus (denv) serotypes 2 and 3 in puerto rico, 19962005. solid blue line, incidence of denv-2 within the refuge region ; dashed blue line, incidence of denv-2 in the rest of the island outside the refuge reason ; solid black line, incidence of denv-3 within the denv-2 refuge region ; dashed black line) incidence of denv-3 in the rest of the island outside the refuge region. incidence was calculated as number of confirmed, positive cases of each serotype per thousand residents. puerto rico is a model for fine - scale studies on denv evolution in the americas. the long - term persistence of denv-2 and its ability to reemerge after transient periods of low circulation is a remarkable aspect of the epidemiology of dengue in the region. the fact that 13% of denv-2 isolates represent importations or close descendants from importations brings new insights to our understanding of denv long - term circulation. foreign viruses were identified in 8 years (1987, 1989, 1991, 1995, 1998, 1999, 2005, and 2007), of which only 1991 and 1998 had been previously sampled (14). ten of the 18 introductions occurred during periods of high denv-2 predominance : 19871991, 1995, and 20052007 (figure 1, panel b ; figure 2). the other 8 introductions originated from the 1998 epidemic or shortly thereafter (1999). therefore, denv-2 seems to be introduced mainly during periods of favorable preponderance, not necessarily epidemic transmission of this serotype. these assessments showed a previously unknown feature of denv-2 persistence : the endemic strain is recalcitrant to influences from frequent foreign introductions. denv-2 to persist in the presence of the dominant subclade ib viruses is not well understood. the puerto rico strain might be highly adapted and thus have a fitness advantage, the frequently introduced strains might be simply underrepresented, or introduced strains may have disappeared through genetic drift. isolate pr76_1995_eu569708, which lies basal to this subclade in the phylogeny (figure 2), is more closely related to south american denv-2 viruses than to other puerto rico viruses, and this lineage does not appear to have progressed, supporting the foreign origin of subclade ib. our findings then show that subclade ib resulted from an introduced strain, as previously suggested by bennett. (14), and successfully penetrated during a period of proportionally high incidence of foreign introductions. interestingly, this clade replacement was completed in 1997, less than a year before the finding of denv-3 and the concomitant decline of denv-1, -2, and -4. the early portion of subclade ib is seen as a period of short - lived lineages ending in 1997, therefore, the rise and expansion of this subclade mainly occurs in coexistence with denv-3, a different epidemiologic scenario from that of the now extinct clade ii a decade earlier. the dominance of conservative amino acid changes that segregated the viruses by clade hinders the assessment of phenotypic changes. compensatory mutations might have conferred replicative advantages that could have influenced the displacement of clade ii or the persistence of subclade ib in puerto rico ; however this hypothesis has not been tested. others have not detected positive selection and attribute lineage extinctions or clade replacements to stochastic events rather than natural selection (25). more analysis to detect site - specific selection is needed to corroborate whether positive selection is not at play in these populations of viruses. the period 19992003 represents historically low rates of denv-2 circulation (figures 1, 2, 4), and the epidemiologic and phylogenetic aspects of this transient retrieval had not been studied previously. we show that the genetic variability of denv-2 decreased during these 4 years when the virus was transmitted in only a subset of municipalities. denv-2 represented 29% of the cases in this area but only 5% island - wide. the reason this region became a refuge of denv-2 for 4 years remains unclear, but the low incidence of denv-2 in prior years compared with the rest of the island suggests susceptibility for infection in this population (figure 5). studies in thailand showed serotype displacement affecting population diversity and lineage turnover (26). short - term serotype cross - protection has been suggested to contribute to serotype displacements (2729), implying that as denv-3 infected a large susceptible population, cross - protective antibodies momentarily impeded transmission of other serotypes and dissemination of denv-2 outside the eastern refuge. our study confirms the utility of systematic sampling and genome sequencing in large - scale surveillance systems as ways to understand the dynamics of dengue transmission and endemicity. | to study the evolution of dengue virus (denv) serotype 2 in puerto rico, we examined the genetic composition and diversity of 160 denv-2 genomes obtained through 22 consecutive years of sampling. a clade replacement took place in 19941997 during a period of high incidence of autochthonous denv-2 and frequent, short - lived reintroductions of foreign denv-2. this unique clade replacement was complete just before denv-3 emerged. by temporally and geographically defining denv-2 lineages, we describe a refuge of this virus through 4 years of low genome diversity. our analyses may explain the long - term endurance of denv-2 despite great epidemiologic changes in disease incidence and serotype distribution. |
multiport laparoscopic donor nephrectomy has become an established technique of organ harvesting.[14 ] in an attempt to further improvise donor outcomes, laparoendoscopic single - site donor nephrectomy (less - dn) has been recently introduced.[57 ] though prospective randomized trials with this technique are lacking, less - dn has been shown to be associated with decreased donor morbidity and better cosmesis. this surgical approach is still in evolution, as only three case series with small number of patients have been reported so far.[68 ] the techniques for less - dn reported by canes. and ganpule. described the use of disposable transumbilical single port access devices, digital laparoscopes with an integrated camera head along with curved and articulating working instruments. preformed the procedure through a pfannenstiel incision, using extra long instruments and a flexible tip laparoscope. utilization of such instruments and disposable access ports can impose a significant cost burden on patients in developing countries. with a prior experience of more than 300 conventional laparoscopic donor nephrectomies by one of us (d.d.), we have successfully performed five transumbilical less donor nephrectomies using conventional laparoscopy instruments, without the need for single port laparoscopy access devices. we herein report our modifications and feasibility of this cost - effective technique of less - dn. between january 2010 and april 2010, five kidney donors were selected to undergo less - dn. all patients underwent left - sided less - dn, and all had a single renal artery and vein. warm ischemia was defined as the time between the application of the first hemolok clip on the renal artery and the initiation of cold perfusion of the kidney on the bench. demographics of donors the patient is placed in a conventional 45 lateral position as for multiport laparoscopic donor nephrectomy. the upper and lower edges of the skin incision are marked 1 cm above and below the umbilicus. pneumoperitoneum is established using a verees needle inserted through the rectus fascia in the midline. three separate vertically aligned ports (one 10 mm and two 5 mm ports) are inserted through this single incision [figures 1 and 2 ]. a 46 cm long, 45 (bariatric) laparoscope (karl storz) is used through the 10 mm camera port. a fascial (port) closure needle (karl storz gmbh and co, tuttlingen, germany) the upper part of an infant feeding tube is cut and fed into the open end of a plastic needle cap. the cap is inserted into the abdomen through one of the 10 mm ports.the port closure needle is passed into the open end of the needle cap through the rubber of the feeding tube. this arrangement allows for a snug and secure placement of the port closure needle into the needle cap. this assembly is used to provide traction to the kidney and adjacent organs [figure 3 ]. the descending colon is mobilized along the white line of toldt, exposing the ureter and the gonadal vein packet. this is dissected off the psoas and lifted up to (and) expos (ing) the renal vein. the renal upper pole is freedfrom the adrenal gland and other posterior abdominal wall structures. (a) skin incision marked from 1 cm above to 1 cm below umbilicus, (b) vertical transumbilical incision, (c) rectus sheath dissected under skin flaps. veress needle (arrow) inserted for pneumoperitoneum, (d) final arrangement of working ports. central camera port (10 mm), working ports (5 mm) 2 schematic diagram showing arrangement of working ports at the site of umbilical skin incision (a) fascial closure (port closure) needle, inserted through the left subcostal area, being fitted into a plastic needle cap, (b) needle - cap assembly used for traction of the renal hilum at this point, a window is made in the lower pole fat and a no. 1 vicryl suture is tied around the fat including the gonadal vein. this suture is exteriorized through one of the ports and used for kidney extraction [figure 4 ]. (a) schematic diagram demonstrating extraction suture taken through the lower pole fat and gonadal vein and exteriorized through a 5 mm port, (b)laparoscopic view of extraction suture (s) taken through lower pole fat and gonadal vein (g) note ureter (u) is medial to the suture prior to renal hilar clamping, the lower 5 mm port is exchanged for a standard metallic 10 mm port. the vicryl thread tied to the lower pole fat is pulled and kidney delivered along its transverse axis into the wound. after kidney retrieval, the port closure needle is brought into the umbilical wound and needle cap is removed. the skin incision is closed with interrupted no.1 vicryl sutures and working ports are reinserted in between sutures. the patient is placed in a conventional 45 lateral position as for multiport laparoscopic donor nephrectomy. the upper and lower edges of the skin incision are marked 1 cm above and below the umbilicus. pneumoperitoneum is established using a verees needle inserted through the rectus fascia in the midline. three separate vertically aligned ports (one 10 mm and two 5 mm ports) are inserted through this single incision [figures 1 and 2 ]. a 46 cm long, 45 (bariatric) laparoscope (karl storz) is used through the 10 mm camera port. a fascial (port) closure needle (karl storz gmbh and co, tuttlingen, germany) the upper part of an infant feeding tube is cut and fed into the open end of a plastic needle cap..the port closure needle is passed into the open end of the needle cap through the rubber of the feeding tube. this arrangement allows for a snug and secure placement of the port closure needle into the needle cap. this assembly is used to provide traction to the kidney and adjacent organs [figure 3 ]. the descending colon is mobilized along the white line of toldt, exposing the ureter and the gonadal vein packet. this is dissected off the psoas and lifted up to (and) expos (ing) the renal vein. the renal upper pole is freedfrom the adrenal gland and other posterior abdominal wall structures. (a) skin incision marked from 1 cm above to 1 cm below umbilicus, (b) vertical transumbilical incision, (c) rectus sheath dissected under skin flaps. veress needle (arrow) inserted for pneumoperitoneum, (d) final arrangement of working ports. central camera port (10 mm), working ports (5 mm) 2 schematic diagram showing arrangement of working ports at the site of umbilical skin incision (a) fascial closure (port closure) needle, inserted through the left subcostal area, being fitted into a plastic needle cap, (b) needle - cap assembly used for traction of the renal hilum at this point, a window is made in the lower pole fat and a no. this suture is exteriorized through one of the ports and used for kidney extraction [figure 4 ]. (a) schematic diagram demonstrating extraction suture taken through the lower pole fat and gonadal vein and exteriorized through a 5 mm port, (b)laparoscopic view of extraction suture (s) taken through lower pole fat and gonadal vein (g) note ureter (u) is medial to the suture prior to renal hilar clamping, the lower 5 mm port is exchanged for a standard metallic 10 mm port. the renal artery is clipped with two hemolok clips and divided. a rectus fascial incision is made, connecting the three laparoscopic ports. the vicryl thread tied to the lower pole fat is pulled and kidney delivered along its transverse axis into the wound. after kidney retrieval, the port closure needle is brought into the umbilical wound and needle cap is removed. the skin incision is closed with interrupted no.1 vicryl sutures and working ports are reinserted in between sutures. the patient is placed in a conventional 45 lateral position as for multiport laparoscopic donor nephrectomy. the upper and lower edges of the skin incision are marked 1 cm above and below the umbilicus. pneumoperitoneum is established using a verees needle inserted through the rectus fascia in the midline. three separate vertically aligned ports (one 10 mm and two 5 mm ports) are inserted through this single incision [figures 1 and 2 ]. a 46 cm long, 45 (bariatric) laparoscope (karl storz) is used through the 10 mm camera port. a fascial (port) closure needle (karl storz gmbh and co, tuttlingen, germany) the upper part of an infant feeding tube is cut and fed into the open end of a plastic needle cap..the port closure needle is passed into the open end of the needle cap through the rubber of the feeding tube. this arrangement allows for a snug and secure placement of the port closure needle into the needle cap. this assembly is used to provide traction to the kidney and adjacent organs [figure 3 ]. the descending colon is mobilized along the white line of toldt, exposing the ureter and the gonadal vein packet. this is dissected off the psoas and lifted up to (and) expos (ing) the renal vein. the renal upper pole is freedfrom the adrenal gland and other posterior abdominal wall structures. (a) skin incision marked from 1 cm above to 1 cm below umbilicus, (b) vertical transumbilical incision, (c) rectus sheath dissected under skin flaps. veress needle (arrow) inserted for pneumoperitoneum, (d) final arrangement of working ports. central camera port (10 mm), working ports (5 mm) 2 schematic diagram showing arrangement of working ports at the site of umbilical skin incision (a) fascial closure (port closure) needle, inserted through the left subcostal area, being fitted into a plastic needle cap, (b) needle - cap assembly used for traction of the renal hilum at this point, a window is made in the lower pole fat and a no. this suture is exteriorized through one of the ports and used for kidney extraction [figure 4 ]. (a) schematic diagram demonstrating extraction suture taken through the lower pole fat and gonadal vein and exteriorized through a 5 mm port, (b)laparoscopic view of extraction suture (s) taken through lower pole fat and gonadal vein (g) note ureter (u) is medial to the suture prior to renal hilar clamping, the lower 5 mm port is exchanged for a standard metallic 10 mm port. the renal artery is clipped with two hemolok clips and divided. a rectus fascial incision is made, connecting the three laparoscopic ports. the vicryl thread tied to the lower pole fat is pulled and kidney delivered along its transverse axis into the wound. after kidney retrieval, the port closure needle is brought into the umbilical wound and needle cap is removed. the skin incision is closed with interrupted no.1 vicryl sutures and working ports are reinserted in between sutures. the mean intraoperative blood loss was 150 cc (range, 135180 cc) and the mean warm ischemia time was 3.2 minutes (range, 34 minutes) [table 1 ]. all recipients had brisk urine output on completion of anastomosis, and mean serum creatinine at discharge was 1.14 mg% [table 1 ]. all donors commenced fluid intake on the night of the operation and solid diet on the first postoperative day. all donors received one to two doses of inj tramadol 50 mg intravenously on the day of the operation. on the second postoperative day, oral analgesia none of the patients required postoperative administration of narcotics or any form of analgesia from postoperative day 2 onwards. all donors were able to return to work / and or resume normal physical activity by two weeks following the operation. the cosmetic outcome was excellent [figure 5 ] frontal view of patient 's abdomen 2 weeks following trans - umbilical less - dn in developing countries like india where cadaveric organ donation is still in its infancy, living organ donation remains the prime driver for renal transplantation. laparoscopic donor nephrectomy has become the gold standard for harvesting kidneys, as numerous trials have shown that it is associated with improved outcomes in comparison with open donor nephrectomy.[13 ] laparoendoscopic single - site surgery for nephrectomy was first described in 2007. subsequently, gill. demonstrated its feasibility for donor nephrectomy in their initial report of four cases. there have been three further case series of less - dn from centers performing high volume of urological laparoscopy cases.[68 ] these studies have demonstrated that less - dn can be safely performed by urologists who are proficient in laparoscopy. our less - dn programme was started with a background of one of us (d.d.) having performed more than 300 conventional laparoscopic donor nephrectomies and the second (s.g.) having an experience of more than 50 less gastrointestinal surgical procedures. before embarking on less - dn, we developed our less technique for a variety of urological operations like simple and radical nephrectomy, nephroureterectomy, adrenalectomy, heminephrectomy, and pyeloplasty. our technique of less - dn differs from that reported by others and we herein discuss its advantages. canes. and ganpule. used the r - port (tri - port and quadport - advanced surgical concepts, dublin, ireland) to provide access for less - dn. our technique of port insertion is similar to the one described by raman., where 5 or 10 mm working ports are directly inserted through the rectus fascia, under the umbilical skin flaps. less access ports are meant for single use and can be expensive for patients in developing countries. the current cost of the triport and quadport ranges between rs 25 000 and rs 40 000. moreover, we feel that these ports are associated with limited maneuverability of working instruments, as the fulcrum for movement is at the level of the access port. there is greater freedom of movement of working instruments when laparoscopy ports are directly inserted through the fascia. one of us (s.g.) has performed gastrointestinal less procedures using both the sils port (covidien plc, dublin, ireland) and direct port insertion, and found the latter technique to be more surgeon friendly. it is well - accepted that retraction of structures like the spleen, pancreas, kidney, and large bowel provides tissue traction, which facilitates laparoscopic dissection. in 11 of their cases, ganpule. used either a 3 or 5 mm port for retraction, whereas canes. used a 2 mm instrument for the same purpose. in our opinion, also, 2 or 3 mm instruments have sharper ends and have the potential to cause injury when used for traction. we have used the port closure needle (diameter 1.5 mm) fitted with a plastic needle cap intracorporeally to provide retraction. this obviates the need of any extra skin incision and the plastic cap (which has a blunt wide rounded end) that can provide atraumatic, robust retraction of organs like the spleen, upper pole of the kidney, large bowel, and pancreas during renal and hilar mobilization. we have found this to be an extremely useful adjunct to renal dissection. we used a 10 mm, 46 cm long bariatric 45 bariatric laparoscope lens fitted with a hd camera. due to its extra length, this allows the camera driver to sit comfortably and prevents clashing of hands between the camera driver and the operating surgeon. andonian. used a 5 mm flexible tip laparoscope and found the vision to be inferior in comparison with standard 10 mm rigid laparoscopes. gill. and ganpule. used a 5 mm digital laparoscope with an integrated camera head. all three reported studies of less - dn described the used of articulating, extra long and curved working instruments. these instruments are high cost, high maintenance devices and require familiarization to work with. in an experimental study, stolzenburg. found prebent and flexible instruments to be more time - consuming in comparison with standard laparoscopic instruments for the performance of laparoscopic tasks we have used standard rigid laparoscopy working instruments along with a standard length harmonic scalpel, thereby decreasing the amount of investment required for setting up infrastructure for starting a less program. the warm ischemia time in our first case was 4 minutes and in the subsequent four cases, it has consistently been 3 minutes. we feel our technique of kidney extraction is safe, as the suture around the lower pole fat, when pulled, aligns the transverse diameter of the kidney in line with the incision on the rectus sheath and allows for quick extraction of the kidney. the potential for losing the graft in the abdomen during retrieval is also annulled. have used an endocatch bag for kidney retrieval and reported a mean warm ischemia time of 6 minutes. in one of their patients, a large kidney could not be prebagged before hilar clamping. this required the kidney to be bagged under warm ischemic conditions, extending the warm ischemia time to 10.2 minutes. the median warm ischemia time in the series of andonian. was 5 minutes and that reported by ganpule. was 6.79 minutes. in a matched pair retrospective comparison of conventional laparoscopic donor nephrectomy and less - dn, canes. demonstrated significantly reduced pain requirement in the period after discharge and earlier to return to normal physical activity in the less group. we too have observed these benefits in our less donors, as all of them were able to return to work by 2 weeks after the operation. the cosmetic results were also gratifying. in a comparison of patients undergoing less nephrectomy and conventional laparoscopic nephrectomy, raman. similarly, in a small retrospective series, andonian. demonstrated only a cosmetic advantage for less donors compared with donors undergoing conventional multiport laparoscopic donor nephrectomy. it is evident that randomized prospective trials are required to clearly delineate the role of less - dn for kidney donation. our technique does not impose an added cost burden on patients, is ergonomically balanced, and can be performed with minimal added inconvenience to surgeons. encouraged by our early experience, we have embarked on a prospective randomized trial to compare less - dn with conventional multiport laparoscopic donor nephrectomy. used the r - port (tri - port and quadport - advanced surgical concepts, dublin, ireland) to provide access for less - dn. our technique of port insertion is similar to the one described by raman., where 5 or 10 mm working ports are directly inserted through the rectus fascia, under the umbilical skin flaps. less access ports are meant for single use and can be expensive for patients in developing countries. the current cost of the triport and quadport ranges between rs 25 000 and rs 40 000. moreover, we feel that these ports are associated with limited maneuverability of working instruments, as the fulcrum for movement is at the level of the access port. there is greater freedom of movement of working instruments when laparoscopy ports are directly inserted through the fascia. one of us (s.g.) has performed gastrointestinal less procedures using both the sils port (covidien plc, dublin, ireland) and direct port insertion, and found the latter technique to be more surgeon friendly. it is well - accepted that retraction of structures like the spleen, pancreas, kidney, and large bowel provides tissue traction, which facilitates laparoscopic dissection. in 11 of their cases, used either a 3 or 5 mm port for retraction, whereas canes. used a 2 mm instrument for the same purpose. in our opinion, also, 2 or 3 mm instruments have sharper ends and have the potential to cause injury when used for traction. we have used the port closure needle (diameter 1.5 mm) fitted with a plastic needle cap intracorporeally to provide retraction. this obviates the need of any extra skin incision and the plastic cap (which has a blunt wide rounded end) that can provide atraumatic, robust retraction of organs like the spleen, upper pole of the kidney, large bowel, and pancreas during renal and hilar mobilization. we have found this to be an extremely useful adjunct to renal dissection. we used a 10 mm, 46 cm long bariatric 45 bariatric laparoscope lens fitted with a hd camera. due to its extra length, this allows the camera driver to sit comfortably and prevents clashing of hands between the camera driver and the operating surgeon. andonian. used a 5 mm flexible tip laparoscope and found the vision to be inferior in comparison with standard 10 mm rigid laparoscopes. gill. and ganpule. used a 5 mm digital laparoscope with an integrated camera head. all three reported studies of less - dn described the used of articulating, extra long and curved working instruments. these instruments are high cost, high maintenance devices and require familiarization to work with. in an experimental study, stolzenburg. found prebent and flexible instruments to be more time - consuming in comparison with standard laparoscopic instruments for the performance of laparoscopic tasks. we have used standard rigid laparoscopy working instruments along with a standard length harmonic scalpel, thereby decreasing the amount of investment required for setting up infrastructure for starting a less program. the warm ischemia time in our first case was 4 minutes and in the subsequent four cases, it has consistently been 3 minutes. we feel our technique of kidney extraction is safe, as the suture around the lower pole fat, when pulled, aligns the transverse diameter of the kidney in line with the incision on the rectus sheath and allows for quick extraction of the kidney. the potential for losing the graft in the abdomen during retrieval is also annulled. canes. have used an endocatch bag for kidney retrieval and reported a mean warm ischemia time of 6 minutes. in one of their patients, this required the kidney to be bagged under warm ischemic conditions, extending the warm ischemia time to 10.2 minutes. the median warm ischemia time in the series of andonian. was 5 minutes and that reported by ganpule. was 6.79 minutes. in a matched pair retrospective comparison of conventional laparoscopic donor nephrectomy and less - dn, canes. demonstrated significantly reduced pain requirement in the period after discharge and earlier to return to normal physical activity in the less group. we too have observed these benefits in our less donors, as all of them were able to return to work by 2 weeks after the operation. the cosmetic results were also gratifying. in a comparison of patients undergoing less nephrectomy and conventional laparoscopic nephrectomy, raman. similarly, in a small retrospective series, andonian. demonstrated only a cosmetic advantage for less donors compared with donors undergoing conventional multiport laparoscopic donor nephrectomy. it is evident that randomized prospective trials are required to clearly delineate the role of less - dn for kidney donation. our technique does not impose an added cost burden on patients, is ergonomically balanced, and can be performed with minimal added inconvenience to surgeons. encouraged by our early experience, we have embarked on a prospective randomized trial to compare less - dn with conventional multiport laparoscopic donor nephrectomy. used the r - port (tri - port and quadport - advanced surgical concepts, dublin, ireland) to provide access for less - dn. our technique of port insertion is similar to the one described by raman., where 5 or 10 mm working ports are directly inserted through the rectus fascia, under the umbilical skin flaps. less access ports are meant for single use and can be expensive for patients in developing countries. the current cost of the triport and quadport ranges between rs 25 000 and rs 40 000. moreover, we feel that these ports are associated with limited maneuverability of working instruments, as the fulcrum for movement is at the level of the access port. there is greater freedom of movement of working instruments when laparoscopy ports are directly inserted through the fascia. one of us (s.g.) has performed gastrointestinal less procedures using both the sils port (covidien plc, dublin, ireland) and direct port insertion, and found the latter technique to be more surgeon friendly. it is well - accepted that retraction of structures like the spleen, pancreas, kidney, and large bowel provides tissue traction, which facilitates laparoscopic dissection. in 11 of their cases, ganpule. used either a 3 or 5 mm port for retraction, whereas canes. used a 2 mm instrument for the same purpose. in our opinion, also, 2 or 3 mm instruments have sharper ends and have the potential to cause injury when used for traction. we have used the port closure needle (diameter 1.5 mm) fitted with a plastic needle cap intracorporeally to provide retraction. this obviates the need of any extra skin incision and the plastic cap (which has a blunt wide rounded end) that can provide atraumatic, robust retraction of organs like the spleen, upper pole of the kidney, large bowel, and pancreas during renal and hilar mobilization. we have found this to be an extremely useful adjunct to renal dissection. we used a 10 mm, 46 cm long bariatric 45 bariatric laparoscope lens fitted with a hd camera. due to its extra length, this allows the camera driver to sit comfortably and prevents clashing of hands between the camera driver and the operating surgeon. andonian. used a 5 mm flexible tip laparoscope and found the vision to be inferior in comparison with standard 10 mm rigid laparoscopes. gill. and ganpule. used a 5 mm digital laparoscope with an integrated camera head. all three reported studies of less - dn described the used of articulating, extra long and curved working instruments. these instruments are high cost, high maintenance devices and require familiarization to work with. in an experimental study, stolzenburg. found prebent and flexible instruments to be more time - consuming in comparison with standard laparoscopic instruments for the performance of laparoscopic tasks. we have used standard rigid laparoscopy working instruments along with a standard length harmonic scalpel, thereby decreasing the amount of investment required for setting up infrastructure for starting a less program. the warm ischemia time in our first case was 4 minutes and in the subsequent four cases, it has consistently been 3 minutes. we feel our technique of kidney extraction is safe, as the suture around the lower pole fat, when pulled, aligns the transverse diameter of the kidney in line with the incision on the rectus sheath and allows for quick extraction of the kidney. the potential for losing the graft in the abdomen during retrieval is also annulled. canes. have used an endocatch bag for kidney retrieval and reported a mean warm ischemia time of 6 minutes. in one of their patients, this required the kidney to be bagged under warm ischemic conditions, extending the warm ischemia time to 10.2 minutes. the median warm ischemia time in the series of andonian. was 5 minutes and that reported by ganpule. was 6.79 minutes. in a matched pair retrospective comparison of conventional laparoscopic donor nephrectomy and less - dn, canes. demonstrated significantly reduced pain requirement in the period after discharge and earlier to return to normal physical activity in the less group. we too have observed these benefits in our less donors, as all of them were able to return to work by 2 weeks after the operation. the cosmetic results were also gratifying. in a comparison of patients undergoing less nephrectomy and conventional laparoscopic nephrectomy, raman. similarly, in a small retrospective series, andonian. demonstrated only a cosmetic advantage for less donors compared with donors undergoing conventional multiport laparoscopic donor nephrectomy. it is evident that randomized prospective trials are required to clearly delineate the role of less - dn for kidney donation. our technique does not impose an added cost burden on patients, is ergonomically balanced, and can be performed with minimal added inconvenience to surgeons. encouraged by our early experience, we have embarked on a prospective randomized trial to compare less - dn with conventional multiport laparoscopic donor nephrectomy. we have demonstrated the feasibility of less - dn with conventional laparoscopic instrumentation, without compromising on the surgical principles of donor nephrectomy. our early experience suggests a definitive cosmetic advantage and earlier further prospective trials will be required to confirm the true place of less - dn. | introduction : laparoendoscopic single - site donor nephrectomy (less - dn) is a procedure in evolution. currently described techniques utilize single port access devices and articulating, flexible, and bent working instruments. we describe a modified technique of transumbilical less - dn with conventional laparoscopic instruments in five kidney donors.materials and methods : three standard laparoscopic ports (10 mm 1, 5 mm 2) were placed through a 4.5 cm vertical transumbilical incision. a 10 mm 45, long bariatric lens (karl storz) was used. renal mobilization was performed using conventional rigid laparoscopy instruments. a port closure needle loaded with a blunt plastic needle cap was used for traction. after hilar clamping, an incision was made connecting the three ports, and the kidney was extracted using a preplaced suture over the lower pole fat. all data were prospectively recorded.results:less-dn was performed successfully in all five patients. the mean operative time was 157.2 minutes (range, 134184) and the mean warm ischemia time was 3.2 minutes (range, 34). all donors were discharged on postoperative day 3 and were able to resume normal physical activity by 2 weeks after the procedure. all donors had an excellent cosmetic outcome. the mean serum creatinine (recipient) at discharge was 1.14 mg% (range, 0.91.4).conclusions : transumbilical less - dn can be cost - effectively performed using conventional laparoscopy instruments and without the need for a single port access device. warm ischemia times with this technique are comparable with that during conventional multiport laparoscopic donor nephrectomy. |
insulin, a key hormone in blood glucose homeostasis, has been detected in human colostrum at supraphysiological levels (114306 mu / l) before decreasing to similar concentrations to those found in blood in the fasting state by day 5 postpartum in healthy mothers [1, 2 ]. has demonstrated that insulin is also present in the milk of mothers with type 1 diabetes mellitus, who lack any endogenous insulin secretion from the pancreas. they also demonstrated that levels of insulin are significantly higher (p < 0.001) in milk from type 1 diabetic mothers than that of nondiabetic control mothers, at both days 3 to 7 postpartum and 3 months postpartum. interestingly, insulin levels in the milk of control mothers vary greatly between studies, while those reported by shehadeh. showed higher milk insulin concentrations than those reported by tiittanen., suggesting that significant variation exists between mothers regardless of the presence of diabetes mellitus. however, the work by tiittanen. and shehadeh. does not further the time of collection or fasting state of mothers, which may account for some of the variability observed. in addition, kulski and hartmann have demonstrated that the levels of insulin in breastmilk may vary with the stage of lactation, with high levels present in colostrum and levels decreasing to a plateau in established lactation. despite this, current medical guides state that insulin is a peptide that is too large to be secreted into milk [57 ]. given the established role of dietary insulin in the maturation of intestinal epithelium in rats, it can be postulated that insulin present in milk may play a similar role in the development of human intestinal epithelium [8, 9 ]. additionally, work in rats has shown that a trypsin inhibitor present in milk acts to preserve insulin function [10, 11 ]. furthermore, epithelial cell insulin receptors exist in the intestine of both piglets and calves, and insulin has been suggested to play a role in influencing growth and development of the small intestine [12, 13 ]. this would suggest that insulin could retain biological activity when ingested by the infant and that intact insulin may pass over from the gastrointestinal tract of infants into blood. this suggestion is further supported and extensively discussed in a review of the in vivo and in vitro effects of insulin on the gastrointestinal tract by shehadeh.. endogenous insulin typically exists in blood in equilibrium between a free monomeric state and a hexamer with zinc ; it is only able to bind to receptors in the free state and has a half - life of ~20 minutes. a recombinant human protein is typically used for therapy. due to the relatively short half - life, a number of short- or long - acting variants have been developed, either by modifying the amino acid sequence to prevent hexamer formation, or through complexing to zinc or protophane to shift the equilibrium towards the hexameric state [6, 16 ]. due to these modifications to the amino acid sequence, specific assays can be used to identify whether the insulin present in a fluid sample is endogenous or a modified exogenous form of insulin [17, 18 ].. demonstrated that human dietary insulin in milk may have a tolerogenic effect against bovine insulin in formula and downregulate the production of igg antibodies to bovine insulin, protecting against the development of autoimmunity, as shown with decreased levels of igg to insulin in breast - fed children compared to those fed with cow 's milk formula containing bovine insulin. despite this beneficial effect at lower milk insulin levels, they also noted that high endogenous insulin levels in human milk were associated with a significantly increased incidence of -cell autoimmunity in children, in both control mothers (p = 0.030) and mothers with type 1 diabetes (p = 0.045), which may suggest that exposure to high levels of endogenous insulin can also promote autoimmunity. however, it must be noted that this does not imply that development of type 1 diabetes mellitus is similarly promoted. in contrast to this, other recent work has demonstrated that administration of oral insulin may help protect residual -cell function in children and adults with recent - onset type 1 diabetes [4, 19, 20 ]. other work has supported the possible role of breast milk in the prevention of type 1 diabetes mellitus, since there is a lower incidence of type 1 diabetes in breast - fed children, but this is still a controversial area and contended by other sources [2224 ]. the effects that type 2 and gestational diabetes mellitus may have on insulin concentration in milk have not yet been identified. given that the only known source of insulin in mothers with type 1 diabetes is injected exogenous insulin, this allows the hypothesis that either insulin is being transported into human milk, in contrast to current literature, or that it is being synthesised in the mammary gland and then secreted into the milk. there are currently no studies comparing the levels of exogenous insulin in milk compared to that produced endogenously in mothers with or without diabetes mellitus. the purpose of this study was to identify the origin of the insulin present in the milk of mothers with type 1 diabetes mellitus as either endogenous or exogenous by determining the levels of total insulin, endogenous insulin, and c - peptide in the milk compared to mothers without diabetes mellitus. this study also aimed to compare the levels of insulin in breast milk from mothers with type 2 diabetes and then investigate any relationship between blood glucose, milk insulin, glucose, c - peptide, and sodium content in breast milk in each of the groups. mothers with type 2 diabetes mellitus have been included in order to compare insulin concentrations and as they should have no exogenous insulin present in their milk or blood. samples from five control mothers (without diabetes mellitus) all in established lactation between 1 to 6 months were randomly selected from 24-hour production milk stock collected by the lactation research group. exact state of lactation is not further specified, as it was not relevant to the aims of this study in identifying the origin of insulin in milk. mothers with diabetes mellitus were recruited from the diabetes clinic at king edward memorial hospital, western australia. four mothers with type 1 diabetes mellitus were recruited, and all mothers in this group were on insulin replacement therapy. five mothers with type 2 diabetes mellitus were recruited, and all mothers in this group was on dietary, exercise or metformin treatment, or a combination of these. one mother in the type 1 group (t1 - 04) was lost to followup. all mothers in the three groups were breast feeding at the time and within 4 months postpartum. all mothers supplied written informed consent to participate, and the study was approved by the human research ethics committee, king edward memorial hospital. this involved collecting 1 to 2 ml of fore and hind milk from each breast used at each feed for a period of 24 hours. there were a varying number of feeds per day per mother, depending on infant hunger and the breast used (single versus both). samples were collected before and after a feed from each breast used for each feed. other time - points involved a breastfeed from each breast so samples were collected before and after a feed from both the left and right breast. sample analysis in this and previous studies has shown no difference in milk composition between left and right breasts so this data was analysed collectively. all samples were frozen by the mothers, and then transported to the laboratory and stored at 30c prior for analysis. samples were then thawed to 37c, mixed, and centrifuged at 3,000 rpm (05pr-22 refrigerated centrifuge, hitachi) for 10 minutes at 4c in 300 l polypropylene tubes to separate the fat from the skim layer. defatted milk was either immediately used for analysis or stored in appropriately labeled eppendorf tubes (0.5 to 1.5 ml) and frozen at 30c for later analysis. milk samples were analysed for glucose content using the spectrophotometric method described by arthur.. defatted milk samples were analysed for total insulin content (endogenous + exogenous) using a chemiluminescent microparticle immunoassay (cmia), which was performed at pathwest (pathwest laboratory medicine wa, qeii medical complex, sir charles gairdner hospital, western australia). c - peptide content of milk samples was similarly determined using the mercodia ab c - peptide elisa kit. the isoinsulin assay detects endogenous insulin, along with the exogenous insulin formulations (insulin aspart, detemir, glargine, glulisine, and lispro), compared to the standard insulin assay, which is specific for endogenous insulin only. exogenous insulin content was calculated by subtracting endogenous insulin content from total (endogenous and all modified forms) insulin content. two techniques were used to analyse total insulin content (cmia + elisa) in order to assess suitability of the cmia method for milk analysis ; if appropriate, it could then be used in a larger - scale study as a more cost + time - effective approach to analysis of milk insulin content. the recovery, detection, and interassay coefficient of variation are presented in table 2. insulin content was measured for all samples, including fore + hind milk for each feed (n = 199). total fat content of the milk samples was assessed using the creamatocrit method described by lucas.. finally, milk sodium analysis was performed using an atomic absorption spectroscopy approach, using a varian techtron absorption spectrophotometer model a100. haemoglobin a1c (hba1c) was also documented from routine analysis in pregnancy for those mothers with diabetes. although only a small number of mothers were present in each group, the large number of milk samples collected by each mother over the 24 hours periods allows for statistical analysis of differences between groups. differences between types of diabetes and between mothers were calculated using a repeated measures linear mixed - effects model, in order to take into account different numbers of feeds per day and differing numbers of mothers in each group. this was performed using the statistical functions of r (version 2.5.1, the r foundation for statistical computing). total insulin content of fore and hind milk samples was measured in all samples (n = 199) using the cmia method and showed good correlation between the content of fore and hind milk samples when all mothers were considered, as shown in figure 1. analysis of individual groups showed no significant difference in insulin content of fore and hind milk between groups (p = 0.20, p = 0.98, and p = 0.14 for control, type 1, and type 2, resp.). based on this, fore and hind milk insulin levels were averaged for the purpose of investigating any circadian variation ; and this analysis showed no significant variation in insulin levels during different time points during the day. while there was circadian variation evident for all mothers, there was no consistent trend evident when analyzing as a group (data not shown). in order to determine whether the insulin present in the milk of mothers with type 1 diabetes is synthesised in the mammary gland or is transported from blood, total and endogenous insulin levels were measured in all milk samples in this group (n = 53). mean sem exogenous insulin levels in the milk of mothers with type 1 diabetes were 12.22 1.34 mu / l for mother t1 - 01, 31.54 4.95 mu / l for mother t1 - 02, and 17.86 4.34 mu / l for mother t1 - 03. there was no detectable endogenous insulin content in the milk of mothers with type 1 diabetes ; therefore, all insulin detected in the milk of mothers with type 1 diabetes was of exogenous origin. in order to investigate variations in insulin concentration between control mothers and mothers with diabetes, total insulin content of each milk sample (fore + hind milk individually) from each mother there was no significant difference between fore and hind milk insulin content, so further data algorithmically treats each data point as the mean of fore + hind milk for each feed (time point). total insulin content from all samples is presented in figure 2, along with the mean insulin content of each group. the control mother t0 - 03 demonstrated significantly higher milk insulin content than the other control mothers (78.23 3.96 versus 15.46 1.03 there was no significant difference in mean insulin content between control mothers and mothers with type 1 diabetes (p = 0.74), control mothers and mothers with type 2 diabetes (p = 0.93), or between mothers with type 1 and type 2 diabetes (p = 0.62). insulin is produced by cleaving insulin and c - peptide from the precursor molecule proinsulin. hence, c - peptide content of the milk from two mothers with type 1 diabetes and 3 control mothers was measured using the elisa method described (n = 61). c - peptide was detected in all samples ; however, levels in the milk of control mothers were significantly higher than those in mothers with type 1 diabetes (21.09 1.00 versus 14.49 1.47 pmol / l, p < 0.01). there was no significant correlation between c - peptide and total insulin content in the milk of control mothers (p = 0.79) or mothers with type 1 diabetes (p = 0.85). in order to investigate the transport of insulin into human milk, the sodium content of the milk from all mothers was measured using atomic absorption spectroscopy (n = 199) as a marker of permeability of the paracellular pathway. there was significantly lower sodium content in the milk of mothers with type 1 diabetes compared to control mothers (p < 0.05), but no significant difference in mean sodium content between control mothers and mothers with type 2 diabetes (p = 0.09) or between types of diabetes (p = 0.54). there was no significant relationship between sodium and insulin content of milk for control mothers (p = 0.17) or mothers with type 1 diabetes (p = 0.37), but a mild negative correlation existed for mothers with type 2 diabetes (r = 0.34, p < 0.05). additionally, for the outlier control mother t0 - 03 who demonstrated significantly elevated insulin levels in milk, there was no significant relationship between sodium and insulin content in milk (p = 0.61). in order to investigate any correlation between blood glucose levels and insulin content in the milk of mothers with type 1 and 2 diabetes, mothers were asked to record their blood glucose levels at least 4 times over the course of the same 24-hour period as their milk sample collection. there was a great deal of variation within mothers for each group over a 24-hour period for both milk insulin and blood glucose ; however, no significant consistent trends were identified. however, there was no significant difference (p = 0.15) in mean blood glucose levels between mothers with type 1 and type 2 diabetes. mean blood glucose levels in mothers with type 1 diabetes were 8.52 0.57 mmol / l, and levels for mothers with type 2 diabetes were 7.45 0.26 mmol / l (mean sem). there was no significant relationship between mean blood glucose levels and mean milk insulin levels in mothers with type 1 diabetes (p = 0.72) or mothers with type 2 diabetes (p = 0.16). by comparing blood glucose levels and milk insulin concentration over time, there appears to be an inverse relationship between the two ; a reduction in blood glucose levels is mirrored by an increase in milk insulin content. mother t2 - 05 (type 2) demonstrated only small variability in milk insulin content, and mother t2 - 03 (type 2) demonstrated large variability in milk insulin content with only small variation in blood glucose levels. limited blood glucose data was provided by mothers due to sample collection difficulties, and further comparison is not possible within the scope of this paper. as the glucose present in human milk derives from blood glucose, there may be fluctuations in milk glucose content that correlate with either blood glucose levels or insulin content of milk. glucose content of each sample (fore and hind milk) was measured using the spectrophotometric method described. there was significantly higher glucose content in the milk of mothers with type 1 diabetes compared to control mothers (p < 0.05) and in the milk of mothers with type 2 diabetes compared to control mothers (p < 0.05), but no significant difference in milk glucose content between types of diabetes (p = 0.68). there was no significant correlation between milk and blood glucose levels for mothers with type 1 diabetes (p = 0.60) or mothers with type 2 diabetes (p = 0.33). there was no significant relationship between glucose and insulin content of milk in control mothers (p = 0.17) or mothers with type 2 diabetes (p = 0.11), but there was a slight negative correlation between glucose and insulin content in milk from mothers with type 1 diabetes (r = 0.41, p < 0.01). most current medical references state that insulin is too large to cross over from blood into milk [57 ]. however, there have been continuous references in the scientific literature to the presence of insulin in milk and the presence of elevated levels in the milk of mothers with diabetes compared to mothers without diabetes [13, 30 ]. based on this conflicting evidence, it is proposed that insulin could either be transported into milk or be synthesised in the mammary gland and directly secreted into milk. firstly, the type of insulin present in the milk of mothers with type 1 diabetes mellitus was assessed. these mothers should have no endogenous insulin production from the pancreas, and all insulin present in their blood should result from injections of exogenous insulin. analysis of the milk of these mothers revealed that there was no endogenous insulin present, suggesting that it was all derived from injected insulin present in blood. secondly, the presence of c - peptide in the milk of two mothers with type 1 diabetes and three control mothers was measured. as the endogenous elisa used is highly specific for human insulin, it was possible that any insulin synthesised in the mammary gland may be sufficiently structurally different to insulin synthesised in the pancreas as to not be detected by the elisa, as is seen the two human isoforms of apolipoprotein b (apob48, synthesized in the small intestine, and apob100, synthesized in the liver). as insulin and c - peptide are cleaved from proinsulin in equimolar quantities, if insulin was synthesised in the mammary gland, then there should be equimolar quantities of both present in milk. c - peptide was identified in milk mothers with type 1 diabetes, but at 15 to 20x lower than reference blood concentrations, despite the common view that patients with type 1 diabetes do not produce insulin. however, many patients with type 1 diabetes still retain some residual pancreatic -cell function, and insulin is secreted at subphysiological levels alongside c - peptide. these factors indicate that the insulin present in milk is derived from insulin in blood. insulin content in milk for all groups was found to be similar to known reference levels for blood. this is the first time insulin levels have been analysed in milk from mothers with type 2 diabetes and is in concordance with previous work investigating insulin concentration in the milk of control mothers and mothers with type 1 diabetes [13 ]. in contrast to other serum proteins of similar size, which are normally present in milk at up to 100x lower concentration than found in blood, these results suggest that insulin does not diffuse into milk via the paracellular pathway. this is supported by the lack of any correlation between insulin and sodium content of milk for control mothers and mothers with type 1 diabetes ; as sodium content of milk is an indicator of the permeability of the paracellular pathway, a positive correlation between insulin and sodium content of milk would be expected if insulin was entering via the paracellular pathway. additionally, sodium content was significantly lower in mothers with type 1 diabetes than in controls, suggesting that the paracellular pathway is closed in these mothers. there was no significant difference in sodium content of milk between mothers with type 2 diabetes and control mothers. these findings suggest that insulin must be actively transported into milk in order to achieve these higher concentrations. two insulin transporters have currently been identified ; an active transporter present on the blood brain barrier (bbb) and receptor - mediated endocytosis of insulin occurring in skeletal muscle endothelium [36, 37 ]. as the bbb transporter is only capable of establishing a cerebral concentration of less than 10% of the systemic blood concentration, it is unlikely to be involved in transport of insulin in the mammary gland. however, the transport system in the skeletal muscle endothelium is capable of transporting insulin at up to 50% of blood levels. while this is still lower than the ~100% transfer seen in the mammary gland, as milk is synthesised over a period of time, this could potentially account for the high concentrations present in milk. further consideration must be given to the supraphysiological insulin concentrations demonstrated in human colostrum, and how these are achieved. while this study did not assess colostrum insulin content, it can be postulated that the potential milk trypsin inhibitor postulated in rat models may act to preserve and stabilize insulin within milk, leading to these higher concentrations [10, 11 ]. alternately, active transport of insulin into milk may increase in the colostrum phase, particularly if insulin does indeed play a large role in maturation of the infant digestive system [13, 14 ]. further research into insulin transport mechanisms in both colostrum and normal milk is required to better investigate this possibility. there was no significant difference between the levels of insulin in the milk of control mothers, mothers with type 1, and mothers with type 2 diabetes, despite the insulin present in the type 1 mothers being artificial in nature. this suggests that the exogenous insulin used for treatment of type 1 diabetes is transported into milk with similar affinity to endogenous insulin in mothers without diabetes. as the exact transport mechanism has yet to be scientifically quantified, the significantly elevated milk insulin levels in control mother t0 - 03 can not be readily explained. if milk insulin concentration does reflect or mirror blood insulin concentrations, then the elevated insulin levels seen would typically represent a woman with undiagnosed, untreated type 2 diabetes. however, as the transporter has yet to be identified, the possibility remains that the elevated milk insulin levels in this mother are due to altered insulin transport, rather than elevated blood insulin levels ; as two isoforms of the insulin receptor exist, it is possible that the high specificity isoform may be preferentially expressed in the mammary gland of this mother, thus resulting in increased insulin transport into her milk. varying data exists in the literature as to the glucose content of milk in mothers with diabetes. it has been established by neville. that glucose transport into milk is insulin independent and dependent upon blood glucose concentrations. given this, it would be expected that patients with diabetes would have elevated milk glucose levels, reflecting their typically elevated blood glucose levels. this study identified significantly higher milk glucose concentrations in mothers with type 1 and type 2 diabetes compared to control mothers, as was predicted. there was no correlation with maternal blood glucose levels, in contrast to data reported by jackson. however, this may be a result of only being able to compare the mean blood and milk glucose levels for each mother ; the study by jackson. compared the direct relationship between blood glucose and milk glucose at time of expression. this finding and the inverse relationship have been included as a potential important course of future investigation. this study demonstrated that both exogenous and endogenous insulin, are actively transported into human milk in similar concentrations to those found in blood, in comparison to other serum proteins of similar or smaller size, such as c - peptide. presumably milk insulin must therefore play a functional or developmental role in the infant. studies in a rat model have demonstrated that oral insulin in milk is involved in maturation of intestinal epithelium and induces pancreatic amylase development at weaning [8, 10, 11 ]. it has also been demonstrated to influence lactase and saccharase activity in the small intestine in a piglet model. a series of papers reported by koldovsk show that human infants demonstrate decreased blood glucose levels in response to milk insulin in early development, suggesting that intact insulin is crossing into the bloodstream of the infant.. also showed that suckling rats exhibit decreased blood glucose levels in response to oral insulin, whereas weaned rats do not. this is of particular importance in neonatal care, as infants of mothers with diabetes are frequently retained in the neonatal intensive care unit and fed expressed breast milk from their mothers. these infants could potentially then remain hypoglycaemic for longer periods, in contrast to the intended aim of the protocol. tiittanen. showed that dietary nonhuman insulin can potentially act as an immunogen and predispose the infant to formation of anti - insulin antibodies, while low levels of human insulin in milk can act as a tolerogen to downregulate any immune response to foreign dietary insulin. their study investigated the presence of bovine insulin in formula as the potential immunogen, which is only three amino acids different in sequence to human insulin. given this potential immunogenic role of nonhuman insulin, it is important to consider the potential implications of the presence of artificial insulin in human milk. artificial recombinant human insulins that are currently used as treatment for patients with type 1 diabetes are typically also only one to three amino acids different in sequence to human insulin. it can therefore be hypothesised that the presence of artificial insulin in the milk of mothers with type 1 diabetes may be acting as an immunogen, which could potentially increase the risk of the infant developing auto - immune diseases in later life. as this immunogenic effect was demonstrated in both early and late lactation, it is possible that insulin is being degraded and only peptide fragments are entering the blood from the intestine. however, given the difference noted in immunogenicity, it is likely that those breakdown products that enter blood still contain the sequence regions that differ between recombinant and endogenous insulin. however, the data in the literature of the role of breastfeeding on the development of type 1 diabetes mellitus in control mothers and those with type 1 diabetes mellitus is still controversial and that the rate of diabetes mellitus in children of type 1 diabetic mothers is lower than in the general population, further suggesting a potential tolerogenic effect of milk insulin [2224 ]. a number of limitations must be taken into consideration when interpreting the results of the above study. firstly, as a pilot study, there are only a small number of participants and hence a low sample size. a repeated measures model and a large number of samples per participant are used to counter this, but the low sample size may limit the interpretation of trends of other milk components. however, this should not affect interpretation of human milk insulin origin as exogenous or endogenous. further, the specificity of the elisa assays used for identification of human pancreatic insulin compared to modified exogenous insulin forms means that if there were any structural differences between human pancreatic and a hypothetical mammary source, then the mammary insulin may not be detected with this assay. finally, there was a significant lack of blood glucose concentration data to compare to milk glucose and milk insulin, due to collection difficulties with mothers involved in this study. this study has identified that both endogenous and exogenous insulin is transported from blood into human milk. studies in a rat model with labelled artificial insulin could potentially be used to identify the source of transfer into milk, and use of a hyperinsulinaemic clamp model may be used to determine (a) the rate of transfer and (b) the transport maximum for the mammary gland transporter. given that insulin is actively transported into human milk and is protected from degradation, it presumably plays a functional or developmental role in the infant. given this, the presence of insulin in formula products needs to be reassessed ; as not all mothers will breast - feed their infants, if insulin plays a functional or developmental role then the addition of insulin to formula could potentially act to improve the similarities of formula to human milk and thus be beneficial to the health and development of formula - fed infants. | despite the important role that insulin plays in the human body, very little is known about its presence in human milk. levels rapidly decrease during the first few days of lactation and then, unlike other serum proteins of similar size, achieve comparable levels to those in serum. despite this, current guides for medical treatment suggest that insulin does not pass into milk, raising the question of where the insulin in milk originates. five mothers without diabetes, 4 mothers with type 1, and 5 mothers with type 2 diabetes collected milk samples over a 24-hour period. samples were analysed for total and endogenous insulin content and for c - peptide content. all of the insulin present in the milk of type 1 mothers was artificial, and c - peptide levels were 100x lower than in serum. this demonstrates that insulin is transported into human milk at comparable concentration to serum, suggesting an active transport mechanism. the role of insulin in milk is yet to be determined ; however, there are a number of potential implications for the infant of the presence of artificial insulins in milk. |
programmed dna - reorganization and dna - elimination processes are frequently observed in differentiating eukaryotic cells. examples include the mating type switch in yeast, the antigen variation in trypanosomes and other parasitic flagellates, the specific dna elimination observed during embryogensis of nematodes, cyclops and sciara, and the processing of mammalian immunoglobulin and t - cell receptor genes. however, these processes are most extreme in hypotrichous ciliates, which therefore serve as model systems to study the molecular basis of programmed dna reorganization, dna elimination and specific dna fragmentation during cellular differentiation. one characteristic of ciliated protozoa is the occurrence of two morphologically and functionally different nuclei in one cell : the generative micronucleus and the somatic macronucleus, which is responsible for transcription during vegetative growth. after sexual reproduction, a new macronucleus is formed as a derivative of the micronucleus while the old macronucleus degenerates. the stages of macronuclear development in hypotrichous ciliates, such as oxytricha, euplotes or stylonychia, are summarized in figure 1. first, a dna - synthesis phase leads to the formation of polytene chromosomes. during this developmental stage, transposon - like elements, as well as short internal eliminated sequences (ies), are excised in the form of dna circles. the polytene chromosomes then disintegrate and the dna to be eliminated is enclosed into vesicles where dna degradation takes place ; depending on the organism up to 95% of the dna is eliminated. the remaining dna is specifically fragmented into small dna molecules and telomeric sequences are added de novo. in a second dna - synthesis phase, each molecule is amplified to a specific copy number, resulting in the somatic macronucleus containing millions of individual dna molecules, each carrying one gene and all control sequences required for replication and expression (for reviews see). dna from injected cells was prepared either about 6 hours after injection or from vegetative cells ; the arrowheads show these two time points. the molecular mechanisms of these processes are still not well understood, but in the holotrichous ciliate tetrahymena, and lately in the hypotrichous ciliate euplotes, conserved cis - acting sequences have been identified that are involved in directing the process of specific fragmentation. in tetrahymena thermophila, a 15 base pair (bp) sequence, the chromosome breakage sequence (cbs), sequence comparisons in euplotes crassus have identified a 5 bp sequence, the proposed e - cbs, that either resides inside the macronuclear precursor at position 18, or is located in the flanking micronuclear - specific dna 12 bp from the macronuclear precursor. a model for chromosome fragmentation in e. crassus was proposed on the basis of the positions of the e - cbs and the finding of overlapping sequences, involving a 6 bp staggered cut on both sides of the macronuclear precursor. to date, no consensus sequence has been found at defined positions near the fragmentation sites in stylonychia lemnae. we therefore decided to identify cis - acting sequences involved in dna fragmentation and telomere addition by injecting modified macronuclear precursor sequences into the developing macronuclei. recently, we demonstrated that injection of such a construct (pce5) into the developing macronucleus resulted in correct fragmentation and de novo telomere addition. this construct contained two macronuclear precursor sequences homologous to a 1.1 kb and a 1.3 kb macronuclear dna molecule (; genbank accession numbers x72955 and x72956). they are separated by an 11 bp spacer and are flanked by micronuclear - specific sequences. to distinguish between the injected precursor sequence and the endogenous macronuclear dna molecule, moreover, we showed that neither sequences of the neighboring 1.1 kb macronuclear precursor sequence nor flanking micronuclear - specific sequences are required for specific fragmentation and telomere addition. deletion of 70 bp of the 3 ' end of the 1.3 kb precursor sequence resulted in no detectable processing, however, indicating that a sequence located in this subtelomeric region is indeed required for fragmentation and/or telomere addition. in contrast, deletion of 69 bp of the 5 ' end still led to a processed product. surprisingly, this processed product contained the subtelomeric sequences that were deleted in the construct, suggesting the presence of a so far uncharacterized proofreading mechanism during macronuclear development. only after deletion of 520 bp of the 5 ' end was a processed product no longer observed. here, we show that both subtelomeric regions are required for correct dna fragmentation but, at least in the case of the 1.3 kb precursor sequence, the distance of cis - acting sequences from the fragmentation sites are different in the 3 ' and 5 ' region. in addition, we show that the subtelomeric regions of the 1-tubulin gene are sufficient for correct dna processing. sequence analysis of all these regions revealed the presence of an inverted repeat with a sequence almost identical to the core e - cbs described in euplotes crassus. all deletion constructs used in this study are based on the construct pce5 (figure 2a). as shown previously by pcr and southern analysis, the concentration of the modified 1.3 kb macronuclear dna molecule derived from the injected construct was always very low compared with the copy number of the endogenous 1.3 kb dna molecule. this may be due to a very tight copy number control mechanism occurring during macrouclear development, which allows only a very limited amplification of the injected material, independent of the amount injected. all results presented in this study are therefore based on pcr analysis as described previously. (a) vector pce5 contains two macronuclear precursor sequences homologous to a 1.1 and a 1.3 kb macronuclear dna molecule. the 1.1 kb homologous precursor sequence contains 2 ies, and the 1.3 kb homologous sequence contains 3 ies. the two precursor sequences are separated by an 11 bp spacer and are flanked by micronuclear - specific sequences on both sides. a 500 bp polylinker was inserted to modify the 1.3 kb homologous macronuclear precursor sequences. primers used for construction of the deletion vectors and for pcr analysis are shown above and below the maps. the failure to detect a processed product from injected deletion constructs can be due to either the deletion of cis - acting sequences required for dna fragmentation and/or telomere addition, or the deletion of sequences required for subsequent amplification and replication of the dna molecule. we have shown that a sequence located within the first 70 bp of the 3'-subtelomeric region of the 1.3 kb macronuclear precursor sequence is required for dna fragmentation. after deletion of this region, fragmentation does not occur at either the 3 ' or the 5 ' end. this implied that either the 3'-subtelomeric sequence is the only cbs present or it has to interact specifically with other sequences located in the 5'-subtelomeric region. we therefore tested construct pce18, in which 520 bp of the 5'-subtelomeric region were deleted for these two possibilities : failure to become fragmented versus deletion of sequences required for amplification and replication. pce18 and control constructs pce5 and pce12, in which 70 bp of the 3'-subtelomeric sequences were deleted, were each injected into the macronuclear anlagen of 20 - 25 exconjugants in the polytene chromosome stage. the injected cells were allowed to continue macronuclear development for another 6 - 10 h until fragmentation was completed. dna was prepared and a pcr analysis was performed using primer combinations in which either both primers were derived from the modified macronuclear molecule (p9/p548 ; p20/p548), or only one primer came from the modified macronuclear molecule while the other primer hybridized to puc19 sequences in the case of pce5 (pucp1/p20 ; pucp2/p548) or to pgem sequences in the case of pce12 and pce18 (pgemp1/p20 ; pgemp2/p548) and to micronuclear - specific sequences (pucp2/p8 ; pgemp2/p8 ; p20/p8) flanking the insert. as a control, all primer combinations were tested using the different constructs as templates (figure 3c, d). in pce5-injected cells, a pcr product was obtained only when both primers were derived from the modified 1.3 kb macronuclear molecule (figure 3b, lane 5), whereas no products were obtained if one primer was derived from the flanking vector dna (lanes 1 - 3) or from the 3'-flanking micronuclear specific sequences (lane 4). these results indicate that, at this stage of macronuclear development, pce5 was fragmented completely. after injection of pce12 and pce18, however, all primer combinations led to amplified products (figure 3a, lanes 1 - 3 ; figure 3b, lanes 6 - 10), showing that some unfragmented plasmid remains in the cells, suggesting that chromosome breakage is reduced or does not occur at all. sequences required for this process must therefore have been deleted. in uninjected cells no primer combination, with the exception of p20/p8, led to pcr products. the signal obtained was always very weak and 500 bp smaller than that derived from the polylinker - containing processed construct. from these results we concluded that sequences required for fragmentation of the 1.3 kb macronuclear molecule are located not only in the first 70 bp of the 3'-subtelomeric region, but also in a region between 69 bp and 520 bp downstream of the 5 ' end of the 1.3 kb precursor molecule. similar to the observation made with the 3'-deletion construct pce12, no fragmentation was observed in pce18, either at the 5 ' or at the 3 ' end, strongly suggesting that an interaction between sequences located in both subtelomeric regions is required for dna fragmentation. the effect of 3 ' and 5 ' deletions in the 1.3 kb macronuclear precursor sequence on dna fragmentation. pce18 and, as controls, pce5 and pce12 were injected into the macronuclear anlagen in the polytene chromosome stage of 20 - 30 exconjugant cells. dna was isolated from these cells 6 - 10 h later and a pcr analysis was performed. (a) pcr products from cells injected with pce12 using the primer combinations p9/p548 (lane1), pgemp1/p20 (lane 2) and pgemp2/p23 (lane 3). (b) pcr products from cells injected with pce5 (lanes 1 - 5) using the primer combinations pucp1/p20 (lane 1) ; pucp2/p8 (lane 2) ; pucp2p548 (lane 3) ; p20/p8 (lane 4) and p20/p548 (lane 5). lanes 6 - 10 : pcr products from cells injected with pce18 using the primer combinations pgemp1/p20 (lane 6) ; pgemp2/p8 (lane 7) ; pgemp2/p548 (lane 8) ; p20/p8 (lane 9) and p20/p548 (lane 10). (c, d) control pcr using pce12 (c), pce5 and pce18 (d) construct dna as templates. pcr reactions in (c) are identical to those shown in (a), and pcr reactions in (d) are identical to those shown in (b). to characterize this region further, we constructed two more deletion vectors (pce19 and pce20 ; figure 2b). in pce19, 230 bp are deleted from the 5'-subtelomeric region of the 1.3 kb precursor molecule, whereas 350 bp are missing in pce20. again, dna of these constructs was injected into cells of the polytene chromosome stage and the injected cells were allowed to finish macronuclear development. macronuclear dna of 30 - 40 vegetative cells derived from single injected cells was analyzed using pcr for the presence of modified macronuclear sequences from the injected construct. by using one primer hybridizing to the 1.3 kb dna molecule and a second primer homologous to the inserted polylinker sequence, only pcr products are obtained from macronuclear dna molecules processed from the injected construct. as shown in figure 4, this was only the case with macronuclear dna of pce19 injected cells (figure 4a, b ; lane 5). no pcr product was detectable using macronuclear dna either of uninjected cells as a control (figure 4a, b ; lane 3) or of cells after injection of pce20 (figure 4a, b ; lanes 7). these results suggest that, in addition to sequences located in a 70 bp region at the 3 ' end, sequences located between 230 bp and 350 bp downstream of the 5 ' end are necessary for processing of this precursor sequence. different deletion constructs were injected into the developing macronucleus and the cells were allowed to finish macronuclear development. the presence of the injected constructs in the macronuclear dna and their processing was analyzed by pcr. all constructs were injected into the polytene chromosome stage of the macronuclear anlagen ; injection conditions were as described earlier. a pcr analysis was performed using dna of 30 - 40 vegetatively growing cells as template. primers used were p9 and p7, both derived from endogenous 1.3 kb macronuclear sequences, and p548, derived from the inserted polylinker. (a) pcr products from uninjected and injected cells were separated on a 1% agarose gel. as a control a pcr using vector pce5 as template was performed. m, molecular weight marker (1 kb ladder, gibco brl). lane 1, pcr product from pce5 vector dna using the primer combination p9/p548. lanes 2 - 3, pcr products from uninjected cells using the primer combinations p9/p7 (lane 2) and p9/p548 (lane 3). lanes 4 - 5, pcr products from cells injected with pce19 using the primer combinations p9/p7 (lane 4) and p9/p548 (lane 5). lanes 6 - 7, pcr products from cells injected with pce20 using the primer combinations p9/p7 (lane 6) and p9/p548 (lane 7). (b) the gel was hybridized with a dig - labeled probe of the polylinker inserted into the 1.3 kb macronuclear precursor sequence. while in the case of pce19 ((a) lane 5) no pcr product using the primer combination p9/p548 is visible in the ethidium bromide stained gel, a clear signal is observed after hybridization ((b) lane 5). it is still noteworthy that whenever processing was obtained from a 5'-deletion construct the processed product again contained the previously deleted sequences, suggesting the presence of a correction mechanism during macronuclear development as postulated before. to rule out that these results are due to pcr artefacts by template switching, we performed control experiments in which various amounts of construct dna were mixed with total cellular dna from uninjected cells. under our experimental conditions, the ratio of injected construct dna to total cellular dna is below 10. in our control experiments, however, we obtained non - specific pcr amplification only by increasing the ratio of construct dna to cellular dna to at least 1:10 and at dna concentrations above 50 g / ml, as described previously. these results therefore make a pcr artefact by template switching very unlikely, although homologous recombination events can not be completely ruled out with these control elements. we examined the 70 bp of the 3'-subtelomeric region and the 5'-subtelomeric regions from position 230 - 350 for the presence of palindromic sequences, direct and inverted repeats, and found a six - base inverted repeat of the sequence 5'-ttgaaa at position 267 - 272 in the 5'-subtelomeric region and at position 20 - 25 in the 3'-subtelomeric region. this sequence is almost identical to the core of the e - cbs, 5'-ttgaa, described in euplotes and can be found only once in each subtelomeric region. in order to demonstrate that this sequence is indeed required for specific dna processing, we mutagenized the 5'-ttgaaa present in the 3'-subtelomeric region into 5'-ttcaga. thirty exconjugant cells were injected with this construct, and the same number of cells was injected with pce5 as a control. in 23 cells injected with pce5, a processed product could be obtained using the primer combination p9/p548. however, a processed product could not be detected in any of the 30 cells injected with the mutagenized construct pce5mut (figure 5). the failure to detect such a product can not be explained by a variation in the success rate of injection but rather provides significant evidence that this inverted repeat acts as a cbs. cells were injected with either pce5 (lane 1) or pce5mut (lane 2). cells were allowed to finish macronuclear development and a pcr reaction was performed using the primer combination p9/p548 for specific detection of the modified precursor sequence. our experiments using the 1.3 kb macronuclear precursor sequence showed that subtelomeric regions are required and are probably sufficient to direct dna fragmentation and telomere addition. to test whether this is also true for other dna molecules, a vector was constructed that contains only 195 bp of the leader- and 240 bp of the trailer sequences but no telomeric sequences of the stylonychia 1-tubulin macronuclear molecule. the 1-tubulin open reading frame (orf) was replaced by the 717 bp orf of the enhanced green fluorescent protein (gfp) (figure 6a). again, the injected cells were allowed to finish macronuclear development and a pcr analysis with dna of 30 - 40 vegetative cells was performed. to detect the processed macronuclear molecules derived from the injected construct, a primer combination was used in which one primer (p27) hybridized to the 5'-1-tubulin leader sequences while the second primer (gfpp2) was derived from gfp sequences (figure 6a). as a pcr product could be obtained from cells injected with the 1-gfptel construct (figure 6b, c ; lanes 3), sequences required for fragmentation of the 1-tubulin macronuclear molecule must be located in the subtelomeric regions of the precursor sequences. no amplification was observed with primer gfpp2 and one primer from the flanking puc vector sequences as primer combination using either total cellular dna isolated from exconjugant cells 6 - 10 h after injection of 1-gfptel or from dna isolated from injected vegetative cells as template, demonstrating that fragmentation had occurred (data not shown). these subtelomeric sequences therefore seem to be sufficient for correct processing of the macronuclear molecule. as in the case with other injected constructs, the copy number of the processed construct was very low compared with endogenous macronuclear tubulin genes, suggesting that not only sequences required for processing but also for regulating amplification are contained in the subtelomeric regions. the copy number of this product was obviously too low to allow detection of gfp expression using fluorescence microscopy. the presence of the injected construct in the macronuclear dna of injected cells and its processing was analyzed by pcr. after injection, the cells were allowed to finish macronuclear development, then a pcr analysis was performed using dna of 30 - 40 vegetatively growing cells as templates. primers used were p27, derived from the endogenous 1-tubulin macronuclear molecule, and pgfpp2, derived from gfp sequences. (b) pcr products from the construct, from uninjected and injected cells were separated on an 1% agarose gel. lane 1, pcr product from construct dna ; lane 2, pcr product from uninjected cells ; lane 3, pcr product from cells injected with 1-gfptel. the gel was hybridized with a dig - labeled probe of the 1-gfptel vector dna. we analyzed whether the inverted repeats found in the subtelomeric region of the 1.3 kb dna molecule are also present in the subtelomeric regions of the 1-tubulin macronuclear dna molecule. in both subtelomeric regions, the core inverted sequence 5'-tgaa could be identified and was found to be 5'-ttgaa at position 62 - 66 in the 5'-subtelomeric region and 5'-tgaaa at position 45 - 49 in the 3'-subtelomeric region. again, these repeats were only found once in the subtelomeric regions of this dna molecule. here we describe an experimental approach to characterize cis - acting dna sequences involved in dna processing during macronuclear development in stylonychia lemnae. by sequence comparison, a putative fragmentation sequence (e - cbs) has previously been located at defined positions either in the subtelomeric regions of macronuclear dna molecules or in flanking micronuclear - specific sequences of euplotes crassus. in contrast, by sequence analysis of a large number of stylonychia macronuclear dna molecules no such sequences could be detected at defined positons in the subtelomeric regions of these molecules. we therefore decided to use a biological approach to identify such sequences by injecting various constructs carrying a modified macronuclear precursor sequence into the macronuclear anlagen. for the 1.3 kb macronuclear precursor sequence, we had already demonstrated that no micronuclear specific sequences are required for correct fragmentation and we localized a cbs within the first 70 bp of the 3'-subtelomeric region. deletion of the same number of base pairs from the 5 ' end still led to a processed product, however. only after deletion of more than 500 bp from the 5 ' end these results suggested that either the 3 ' sequence is the only cbs required for fragmentation at both ends or that a second cbs resides further downstream in the 5'-subtelomeric region. in fact, we now show that the 500 bp 5'-deletion construct was not fragmented, and the failure to detect a processed product from this construct was not just due to deletion of sequences required for amplification and replication later during macronuclear development. to characterize this putative cbs in the 5'-subtelomeric region, we analyzed further deletion constructs for their ability to become processed. from these results control experiments from an earlier study, and those made within this study, make a pcr artefact by template switching very unlikely. homologous recombination does not seem sufficient to produce the products with the restored sequences because they were not obtained from all deletion constructs, but only from those containing the putative cbs. perhaps some as yet uncharacterized proofreading mechanism occurs during macronuclear development, possibly using transcripts from the old macronucleus as templates. all experiments described so far were performed with only one macronuclear precursor sequence. to determine whether subtelomeric regions are not only required but are also sufficient for dna processing in stylonychia, a vector was constructed carrying only the subtelomeric regions of the dna molecule encoding the 1-tubulin gene ; the tubulin orf was replaced by enhanced gfp. correct processing of this construct could be demonstrated by pcr analysis, showing that in this case subtelomeric regions are required and indeed are sufficient for correct fragmentation and telomere addition. this again may be explained by the low copy number of the macronuclear molecule derived from this vector as indicated by the pcr analysis (figure 6a, b ; lane3) and would imply that sequences required for copy number control also reside within the subtelomeric regions. a sequence comparison of the subtelomeric regions of the 1.3 kb precursor sequence containing the putative cbs revealed the presence of a six - base inverted repeat resembling the core of the e - cbs described in euplotes crassus. a subsequent search showed that a similar sequence is also present in the subtelomeric regions of the 1-tubulin macronuclear dna - molecule and in the subtelomeric region of many other (over 80%) macronuclear dna molecules from stylonychia lemnae (data not shown). as in the case of the 1.3 kb macronuclear molecule, this sequence could only be found once in each subtelomeric region. however, in contrast to euplotes it never occurred at the same position in both subtelomeric regions. although it was always found within the first 60 bp of the 3 ' end, the position at the 5 ' end was more variable and in all cases examined it is localized further downstream than at the 3 ' end. to analyze whether this sequence is indeed involved in dna processing we mutagenized this sequence in the 3'-subtelomeric region of the 1.3 kb precursor sequence. the fact that a processed product was never observed after injection of this construct is a strong indication that this sequence is required for specific dna processing during macronuclear development ; we therefore now call it st - cbs. the occurrence of cbs at different locations in the two subtelomeric regions could be explained by the presence of several nucleases, each with different binding and cutting characteristics, a possibility that seems very unlikely. moreover, we showed that already only one deletion, either in the 3'- or 5'-subtelomeric region, resulted in the lack of fragmentation on both ends. this strongly suggests that an interaction between two cbs located in the two subtelomeric region is a necessary prerequisite for the fragmentation process to occur. on the basis of these and our earlier results, we would like to suggest a working hypothesis for the mechanism of dna processing in stylonychia (figure 7), which is a modification and extension to our previously suggested model. the six - base inverted repeat occurring in the subtelomeric regions required for dna fragmentation could determine a loop structure stabilized by dna - binding proteins and probably by the appropriate chromatin structure. an endonuclease could bind to this complex or to the six - base inverted repeat cutting the stucture at the base of the loop. as the 3'-inverted repeat is always found within the first 60 bp of the subtelomeric region, whereas the position of the 5'-inverted repeat seems quite variable, this would imply that initially a precise cut is made only at the 3'-end and parts of the 5'-subtelomeric regions would become lost. we continously observed a proofreading mechanism at the 5 ' end, which we can not explain at present but excluded that these observations are due to an experimental artefact. similar proofreading mechanisms have been postulated recently by herbert and rich and comparable phenomena were described in paramecium. it still has to be determined, however, whether these processes also occur with the endogenous precursor sequences. finally, de novo addition of telomeric sequences to the resulting gene - sized dna molecules occurs. although this model is still very hypothetical, using our experimental approach it should be possible to verify it experimentally and also eventually to characterize the postulated proofreading machinery. inverted repeats acting as cis - acting dna sequences required for dna processing are localized in both subtelomeric regions but not necessarily at similar positions. by interaction of these sequences, a loop - like structure is formed, which is then resolved on its base by a specific nuclease. this implies that a precise cut is made only at the 3 ' end, the 5 ' end has to be filled in by a so far unknown proofreading mechanism. our data indicate that no micronuclear specific dna sequences are required for specific dna fragmentation during macronuclear development of the hypotrichous ciliate stylonychia. instead they are found in both subtelomeric regions of macronuclear precursor sequences, although they do not have to be localized at identical positions with respect to the breakage site. we show that these sequences are not only required but also sufficient for dna fragmentation. moreover, a functional analysis of an inverted repeat found in this region revealed that it functions as a st - cbs. stylonychia lemnae were grown in neutral pringsheim solution and fed daily with the alga chlorogonium elongatum. to achieve conjugation, cells of two different mating types were mixed and the stages of macronuclear development were determined by phase microscopy. dna at concentrations between 5 - 20 g / ml of the various constructs was injected into the developing macronucleus of the polytene chromosome stage using the procedure described earlier. cells were allowed to finish macronuclear development and, after 15 - 20 cell divisions, dna was isolated from vegetative macronuclei. sometimes dna was isolated from exconjugant cells only 6 - 10 h after injection. to characterize the dna of injected cells, total cellular dna isolated from 30 - 40 cells was dissolved in 40 l and aliquots of 10 l were used for each pcr reaction carried out as described by saiki.. the gels were blotted onto nylon membranes (amersham pharmacia biotech) and hybridized with random primed probes labeled with dig - oxigenin - dutp (boehringer mannheim). primers for construction of the vectors and for analysis of the dna from injected cells are summarized in table 1. all deletion constructs based on pce5 were pcr fragments cloned into pgem-7zf+ (promega) (pce12 ; pce18), in pcr2.1-topo (invitrogen) (pce19) and in puc57(t) (fermentas) (pce20). stgfp was constructed by ligation of the leader and trailer sequences (including telomeres) of the 1-tubulin gene of stylonychia with the open reading frame of the gfp (s65 t) as described previously. from this construct, a pcr fragment was amplified using p27 and p28 (table 1 and figure 6a) as primers. this pcr fragment was cloned into puc57(t) (fermentas), resulting in the construct 1-gfptel. primers used for construction of the deletion vectors and for pcr analysis sequences of the primers used for construction of the different deletion vectors and for pcr analysis after injection of these constructs into the developing macronucleus. the 5 ' and 3 ' primers used for construction of each deletion vector are indicated. for mutagenesis of the sequence 5'-ttgaaa present in the 3'-subtelomeric region of the 1.3 kb macronuclear precursor sequence, a megaprimer was generated in a pcr reaction using the primer combination p20/pmut2 (table 1 and figure 2b). this pcr product was then used as megaprimer for amplifying the whole mutagenized pce5mut using pce5 as a template. by simultaneously inserting a second mutation in pce5mut to ensure correct amplification the pcr reactions were performed using pfu - polymerase (stratagene). this work was supported by the deutsche forschungsgemeinschaft and the alfried krupp von bohlen und halbach foundation. | background : programmed dna - reorganization and dna - elimination events take place frequently during cellular differentiation. an extreme form of such processes, involving dna reorganization, dna elimination and dna fragmentation, is found during macronuclear differentiation in hypotrichous ciliates. ciliated protozoa can therefore serve as a model system to analyze the molecular basis of these processes during cellular differentiation in eukaryotic cells.results:using a biological approach to identify cis - acting sequences involved in dna fragmentation, we show that in the hypotrichous ciliate stylonychia lemnae sequences required for specific dna processing are localized in the 3'- and the 5'-subtelomeric regions of the macronuclear precursor sequence. they can be present at various positions in the two subtelomeric regions, and an interaction between the two regions seems to occur. sequence comparison revealed a consensus inverted repeat in both subtelomeric regions that is almost identical to the putative euplotes chromosome breakage sequence (e - cbs), also identified by sequence comparison. when this sequence was mutagenized, a processed product could no longer be detected, demonstrating that the sequence plays a crucial role in dna processing. by injecting a construct into the developing macronucleus, which exclusively contains the subtelomeric regions of the stylonychia l - tubulin gene, we show that subtelomeric regions are not only required but are also sufficient for dna processing in stylonychia.conclusions:our results indicate that an inverted repeat with the core sequence 5'-tgaa present in both subtelomeric regions acts as a cbs in stylonychia. the results allow us to propose a mechanistic model for dna processing in this ciliate. |
the purpose of this study is to report the use of intravitreal triamcinolone for treatment of cancer - associated retinopathy (car) refractory to systemic therapy. a 67-year - old man presented with cancer - associated retinopathy with antibodies against a 46-kda retinal protein, alpha enolase. serial intravitreal injections of triamcinolone resulted in restoration of photoreceptor anatomy on optical coherence tomography and visual improvement. the patient s vision was preserved at 20/40 od and 20/32 os until his death from lung cancer 31 months after car diagnosis. cancer - associated retinopathy (car) is a paraneoplastic process caused by autoantibodies against retinal proteins, including recoverin and alpha enolase [1, 2 ]. approximately 50 % of patients with car present with visual symptoms before the diagnosis of a malignancy is made. car is rare, and there are currently no standard treatment regimens for the condition. systemic corticosteroids, steroid - sparing immunosuppression, and non - traditional immunomodulatory therapies including intravenous immunoglobulin (ivig) have been shown to variably preserve or improve vision [57 ]. cystoid macular edema (cme) has been reported in patients with non - paraneoplastic autoimmune retinopathy (npair) but is less common with car. we report a patient with car in whom serial intravitreal steroid injections resulted in maintenance of visual function for more than 2 years until his death. examination revealed best corrected visual acuities of 20/32 in the right eye and 20/25 in the left eye. fluorescein angiography (fa) revealed petalloid leakage at the fovea, perivascular leakage, and disk staining bilaterally (fig. multifocal and full - field electroretinography (erg) revealed severely decreased scotopic and photopic responses but no negative erg pattern. serologic testing revealed the presence of antibodies to a 46-kda retinal protein that was confirmed to be alpha enolase. the patient was initially treated with intravenous methylprednisolone, 1 g / day for 3 days, followed by oral prednisone (initial dose of 80 mg / day and then a taper over 2 months) with decreased retinal vascular leakage and cme.fig. b fluorescein angiography showing petalloid leakage in the macula, perivascular leakage, and disk staining a fundus photos showing macular edema and arteriolar narrowing. b fluorescein angiography showing petalloid leakage in the macula, perivascular leakage, and disk staining the patient s visual acuities worsened to 20/125 od and 20/60 os with tapering of the prednisone, and fa showed worsening retinal vasculitis. steroid - sparing treatment was attempted first with mycophenolate mofetil (2 g / day) and then with four doses of ivig, with no response to either therapy. intravitreal triamcinolone (ivta) was administered bilaterally, with vision improving to 20/40 od and 20/32 os and resolution of cystic changes. over the ensuing 2.5 years when the ivta effect waned, his vision diminished to as low as 20/70 in the right eye and hand motions in the left eye. after the initial injection, there were no retinal cystic changes or significant retinal thickening on optical coherence tomography (oct) at the subsequent visits, yet the patient periodically reported visual deterioration and had objectively diminished visual acuity with disruption of photoreceptor anatomy on oct (fig. his visual acuities were 20/40 od and 20/32 os after the most recent ivta injections. the patient developed steroid response intraocular pressure rises in both eyes that decreased with topical antihypertensive therapy. he passed away from complications of his lung cancer 31 months after his car diagnosis.fig. 2a oct of left eye showing loss of the inner segment / outer segment (is / os) junction subfoveally (arrow) and hyperreflective areas along the photoreceptor layer (arrowheads), possibly indicative of foci of active inflammation. the is / os junction has been restored subfoveally (arrow) and the hyperreflective areas have largely resolved, with vision improving to 20/32 a oct of left eye showing loss of the inner segment / outer segment (is / os) junction subfoveally (arrow) and hyperreflective areas along the photoreceptor layer (arrowheads), possibly indicative of foci of active inflammation. the is / os junction has been restored subfoveally (arrow) and the hyperreflective areas have largely resolved, with vision improving to 20/32 this report describes the successful treatment of car with intravitreal steroids. the treatment of car is often challenging. treatment of the underlying malignancy alone does not alter the course of the ocular disease. various treatment modalities have been tried in patients with car, including oral and intravenous steroids, plasmapheresis, ivig, rituximab, azathioprine, cyclosporine, and mycophenolate mofetil [2, 68 ]. despite treatment with these systemic medications, our patient responded initially to systemic steroids but did not have an alteration of his disease course with mycophenolate mofetil or ivig. vision improvement in car is unusual ; vision stabilization is often the best that can be hoped for. in this patient, there was strong evidence for a causal relationship between the injections and visual improvement. when the intravitreal triamcinolone wore off, his vision declined. with his initial injections, the improvement in vision could be attributed, at least in part, to treatment of the cme. but subsequent injections were given when there were no retinal cystic changes or thickening present on oct and yet reinjection led to marked subjective and objective improvement. the improvement was likely due to suppression of inflammation around the photoreceptors as evidenced by restoration of inner segment / outer segment junction and resolution of hyperreflective lesions along the photoreceptor layer on oct after the injections. cme and periphlebitis can be seen in patients with npair but is less common with car. there is one case report of retinal periphlebitis associated with car in a patient with ovarian cancer. in the presence of cme, the unique presentation of car with cme and vasculitis in our patient motivated the use of intraocular triamcinolone, but there was evidence for an effect on the course of car directly given the visual improvements even when ivta injected in the absence of cme. the presence of anti - retinal antibodies in the sera of patients with car suggests the need for systemic intervention in general, but this patient s course suggests that a subset of car patients might be successfully treated with local therapy. the oct photoreceptor findings in this patient pre- and post - injection indicate that intravitreal steroids may be able to successfully suppress inflammation and apoptosis induced by the autoantibodies at the level of the photoreceptors despite ongoing systemic production and/or circulation of antibodies. some authors caution that readministration of intravitreal steroid for recurrent cme in npair and car is best done early when cystic changes are just starting to recur to avoid cycles of retinal cyst expansion and collapse which may cause retinal damage [2, 10 ]. similarly, car patients without cme being treated with intravitreal steroids should be followed closely with oct imaging and retreated promptly when evidence of photoreceptor layer disruption becomes apparent to avoid permanent photoreceptor damage. in summary, intravitreal steroids should be considered in patients with car who do not respond to systemic therapy. in our patient 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 source are credited. | purposethe purpose of this study is to report the use of intravitreal triamcinolone for treatment of cancer - associated retinopathy (car) refractory to systemic therapy.methodsthis was a retrospective chart review study.resultsa 67-year - old man presented with cancer - associated retinopathy with antibodies against a 46-kda retinal protein, alpha enolase. there was disease progression despite therapy with mycophenolate and intravenous immunoglobulin. serial intravitreal injections of triamcinolone resulted in restoration of photoreceptor anatomy on optical coherence tomography and visual improvement. the patient s vision was preserved at 20/40 od and 20/32 os until his death from lung cancer 31 months after car diagnosis.conclusionsintravitreal triamcinolone may be beneficial for maintenance of vision in patients with car. |
host resistance in protozoan infections is dependent on both innate and acquired cell - mediated immune responses. in addition, several studies have implicated b cells and antibodies (abs) in host survival and protozoan parasite clearance [13 ]. b cells can function as ab - producing cells but they can also modulate immune responses through critical ab - independent mechanisms that include secretion of cytokines and chemokines as well as antigen presentation [46 ]. furthermore, b cells can directly modulate dendritic cells and t - cell subsets, and, consequently, they can influence adaptive immunity and the progression of the infection. accordingly, in protozoan infections b cells may play a protective and a pathological role. in malaria and trypanosome infections, abs appear to play a famajor role in immunity. in trypanosoma cruzi and t. brucei gambiense infections, ab - dependent cytotoxic reactions against the parasite several studies demonstrated that abs are responsible for the survival of susceptible animals in the initial phase of t. cruzi infection and for the maintenance of low levels of parasitemia in the chronic phase [9, 10 ]. although abs were shown to be responsible for clearing the african trypanosomes from the blood of infected animals, recent evidence suggests that the survival time of infected mice does not necessarily correlate with the ability of the animal to produce trypanosome - specific antibody. in general, the parasite - specific immune response mounted during protozoan infections is insufficient to completely eradicate the pathogen, allowing chronic infection. in fact, they are required for the development of th2 cell response and, consequently, for the susceptibility to infection with leishmania major. balb / c umt mice infected with l. major lv39 mount a th1 response and present restricted lesion development and contained parasite replication. adoptive transfer of b cells from balb / c mice in b - cell - deficient balb / c umt mice before infection restores susceptibility to l. major lv39 and th2 cell development in resistant mice. given the role for b cells in conditioning the progression of protozoan infections, it is important to understand the kinetics and regulation of the whole b - cell cycle from the development to the differentiation into mature and memory b cells and plasma cells. the humoral immune response has been shown to be two branched providing an innate - like response (involving b1 and marginal zone (mz) b cells) and an adaptive immune response (involving conventional b2 cells). in the adult, b cells are generated in the bone marrow (bm) and migrate to the periphery at the transitional b - cell stage, when they are still short lived and functionally immature [12, 13 ]. conventional b2-cell development occurs via a series of bm stromal cell - facilitated processes that begin within the hematopoietic stem cell pool and proceed in hierarchical steps of lineage commitment. b lymphopoiesis yields several developmental stages of pre - pro - b, pro - b, pre - b, and, eventually, immature b cells, which show a high expression of the igm form of the antigen receptor and low or no expression of the igd maturation marker. to complete their development, immature b cells migrate through the periphery ; however, only 10% reach the spleen as transitional b cells of the t1 type. in the spleen, b1 cells are efficiently generated in fetal life and during the first few weeks after birth. the fetal liver is an efficient source of b1 cells ; however, it is not the only one as a recent study identified a b1-cell precursor in adult bm. interestingly, protozoan parasite infections can affect the different compartments of b - cell development (summarized in figure 1), influencing the generation of new mature b cells or their survival and, consequently, the cellular immune response. bm is the main hematopoietic organ of an adult organism and is able to provide cells of immune systems rapidly in cases of infection. immature b - cell reduction in bm during an infection would limit the ab source cells and favour parasite replication and chronicity, so the identification of the mechanisms ruling b - cell depletion represents an important challenge in biomedical research. we have reported that t. cruzi infection induces a marked loss of immature b cells in the bm and also compromises recently emigrated b cells in the periphery. the depletion of immature bm b cells was associated with an increased rate of apoptosis, and we established that t. cruzi trypomastigotes failed to directly induce immature b - cell apoptosis. we proved that this cell death process occurs in a fas / fasl - independent fashion but depends on the presence of cd11b myeloid cells that secrete a product of the cyclooxygenase pathway that depletes immature b cells. in fact, infections with neospora caninum and t. brucei also cause a general decrease in bm cells. recently, the t. brucei infection upshot on b lymphopoiesis has been examined using a c57bl/6 mouse t. brucei antat 1.1e infection model. using this model, bockstal. observed that the number of hematopoietic stem cells was minimally affected, but bm b lymphopoiesis was severely affected in t. brucei - infected mice, starting with the common lymphoid progenitor fraction. the pre - pro - b - cell population showed a 50% reduction by day 20 after infection, while the subsequent b - cell maturation stage, that is, the pro - b, pre - b, and immature b - cell populations reached more than 95% depletion by day 10 after infection and failed to recover throughout the further course of infection. in t. brucei infection, mice do not present increased apoptosis of bm b - cell precursors nor alteration in the expression of b - cell - development - specific transcription factors like icaros, pu.1, ebf and e2a and the il-7. however, t. brucei - infected mice show a reduction in bm cxcl12 levels, indicating that during early t. brucei infections, b - cell precursors prematurely migrate out of the bm as a result of the initiation of inflammation. similarly, cxcl12 decreased production by bm cells was determined in plasmodium chabaudi infection. furthermore, the significant reduction in cxcl12 expression in the bm of 10 days p. chaubaudi - infected mice correlates with a reduction in b - cell precursor cells. at days 20 and 30 of infection, a significant recovery in cxcl12 expression in bm is detected, coinciding with a slow recovery of b lymphopoiesis. among the mature b cells, mz and b1 b cells appear to be evolutionarily selected and maintained to facilitate prompt ab responses. due to this, they provide a bridge between the innate and the adaptive arms of the antipathogen immune response. b1 cells, distinguished from b2 cells by their phenotype (b220 cd5 cd11b) and anatomic location and functional properties, are the dominant population of b cells in the pleural and peritoneal cavities, but represent only a small fraction of splenic b cells. b1 cells produce most of the natural serum igm and much of the gut iga and express a bcr repertoire that is enriched for highly polyspecific receptors with low affinities to a broad range of antigens. despite the fact that b1 cells are very efficient in the control of bacterial and viral infections, they apparently do not play a role in the control of protozoan parasite replication. indeed, balb / c xid mice carrying an x - linked mutation (that prevents b1 cell development) infected with t. cruzi display poor b - cell responses to the infection, accompanied by low levels of specific and nonspecific immunoglobulins in the serum. surprisingly, xid mice infected with t. cruzi were able to control parasitemia and did not show the wasting syndrome observed in wild - type mice. the resistance of these mice to experimental chagas disease was associated with the absence of il-10-secreting b1 cells and high levels of ifn - gamma. these results suggested that b1 cells play a pathological rather than protective role in chagas ' disease. additionally, in t. cruzi infection, we observed a disappearance of peritoneal b1 cells, due to an enhanced differentiation into a particular type of plasma cells, the mott - like cells. nevertheless, the specific role of these cells in the experimental chagas disease has not been elucidated yet ; their association with autoimmune manifestations in cd22-deficient mice and lupus [31, 32 ] suggests that these cells may be involved in the autoimmune responses observed in t. cruzi infection. we and others have reported that the peritoneal b - cell response observed in t. cruzi infection is almost not specific for the invading pathogen [29, 33 ]. however, b1 cells are not only implicated on ab secretion ; in fact, they may modulate t - cell response. in this sense, o'garra. have reported that b1 cells secrete large amounts of il-10 and, consequently, can contribute to the susceptibility of balb / c mice to l. major infection by skewing the t - helper cell network towards a th2 phenotype. in this way, it has been observed that l. major infection of b - cell - defective balb / c xid mice induces a less severe disease compared to wild - type control mice. another report indicates that the behavior of l. major - infected xid mice can be explained more in relation to the high endogenous ifn - gamma production than to the lack of b1 cells. indeed, b1-cell - depleted irradiated mice showed similar or even worse disease progression compared to control balb / c mice. b1 cells would also be implicated in the pathogenesis of toxoplasmosis through the production of abs against the heat shock protein 70 of t. gondii that also recognize mice hsp70. these abs seem to have a pathogenic role in toxoplasmosis as their injection in t. gondii - infected mice clearly increases the number of parasites in mice brain. moreover, il-10 produced by b1 cells could, in turn, favor t. gondii survival. then, fine tune regulation of the exacerbated th1 response by il-10 is important during t. gondii infection. mz b cells are also considered innate - like cells that can be induced to differentiate into short - lived plasma cells in the absence of bcr ligation. splenic mz b cells can be distinguished from the other splenic b cells by cd24, igm, igd, cd23 expression, as well as by their higher expression of cd21. it is known that these b cells mediate humoral immune responses against blood - borne type 2 t - independent antigens but their role in parasite infection has been scarcely studied. induction of a t - independent anti - trypanosome igm response has been shown to be a crucial factor in t. brucei parasite elimination. even when increased splenic cellularity occurs after t. brucei infection, a significant reduction of splenic igm+ mz b - cell numbers takes place right after the first week of infection. the infection - associated disappearance of the mz b cells from the spleen could be explained by two independent mechanisms, namely, cell differentiation and/or cell death. supporting the first possibility is the observation that the rapid disappearance of mz b cells coincided with the temporary accumulation of igm+ plasma cells. the analysis of mz b cells that remain in the spleen in the days following the clearance of the first peak of parasitemia revealed that these cells upregulated annexin v expression. in addition, these cells exhibit caspase 3 gene expression as well as the conversion of procaspase 3 into the cleaved 12 kd and 17 kd caspase 3 activation products suggesting the induction of trypanosomiasis - associated apoptosis in the splenic mz b - cell population. as in african trypanosomes, p. chabaudi chabaudi infection also caused a severe depletion of mz b cells in the spleen and this loss is mainly the result of the highly increased rate of apoptosis. as mz b cells serve as an important source for t - cell independently generated igm+ plasma cells during early stages of infection, apoptosis induction of mz b cells can be used by parasites as strategy to avoid early igm protective response and, consequently, prolong their survival. whereas the bm of some protozoan - infected mice suffers from a strong b - lineage - cell depletion, the spleens show a marked cellular hyperplasia as a consequence of an intense b - cell response. a detailed analysis of splenic b - cell response was performed in experimental chagas disease and malaria [10, 41 ]. an extrafollicular ab response, in mice infected with t. cruzi, is evident a few days after infection and reached a peak after 18 days of infection. this extended kinetics of the extrafollicular response could be characteristic of infections caused by blood circulating protozoan parasites since in p. chabaudi chabaudi infection extrafollicular plasmablasts are visible from day 4, and by day 10 they are unconventionally sited in the periarteriolar region of the white pulp. in this region, in both t. cruzi and plasmodium infection, the kinetics of the appearance of gcs during t. cruzi and plasmodium infection are similar to those observed after immunization with classical haptenated proteins, where gcs are visible within the 8 days of immunization. the gcs in the spleens of t. cruzi - infected mice persisted for at least 32 days resembling the kinetics of the response seen in p. chabaudi and l. amazonensis infections. we observed that even though t. cruzi infection induces early, persistent, and massive extrafollicular and follicular plasmablast responses together with classical and ectopic gcs, infected mice have a delayed parasite - specific ab response. a key finding of our study is that, while an important amount of abs is rapidly secreted during infection, antigen specific antibodies were not detected until the third week of infection. the consequence of the massive extrafollicular and follicular b - cell response is the polyclonal b - cell activation that leads to hypergammaglobulinemia with serum abs specific for the parasite and self- and/or nonrelated ags [4446 ]. in leishmaniasis, t. congolense infection also results in a strong production of non - parasite - specific abs characterized by the predominance of igg2a- and igg2b isotypes. all the mouse strains infected with t. congolense present a marked increase in splenic b cells resulting in a nonspecific polyclonal activation of lymphocytes that affects primarily b cells. in strains of t. congolense mice which survived longest, that is, c57b1/6j and akr / a, the increase in splenic b cells is less marked. different roles are proposed for polyclonal b - cell activation, which can be crucial for early host defense by contributing with abs specific for a spectrum of conserved structures present in the pathogens. additionally, polyclonal activation can be a mechanism triggered by microorganisms to escape the host - specific immune response by diluting pathogen - specific abs while increasing irrelevant antibodies. accordingly, recently it has been reported that c57bl/6 mice, resistant to t. cruzi infection, had improved parasite - specific humoral responses that were associated with decreased polyclonal b - cell activation. in the context of parasite infection, study shows that th2 cytokine responses were associated with amplified polyclonal b - cell activation and diminished specific humoral immunity. this report demonstrate, that polyclonal b - cell activation during acute experimental chagas disease is not a generalized response and suggests that the nature of humoral immunity during t. cruzi infection contributes to host susceptibility. in leishmaniasis visceral, at early times after infection, there is a marked b - cell expansion in the draining lymph nodes of the site of the infection, which persists throughout infection. as early as day 7 after infection, polyclonal antibodies (tnp, ova, chromatin) were observed in infected mice and the levels appeared comparable to the specific antileishmania response. although b - cell - deficient jhd balb / c mice are relatively resistant to infection, neither b - cell - derived il-10 nor b - cell antigen presentation appears to be primarily responsible for the elevated parasitemia. interestingly, passive transfer and reconstitution of jhd balb / c with secretory immunoglobulins (igm or igg ; specific or nonspecific immune complexes) results in increased susceptibility to l. infantum infection. another potential deleterious role for polyclonal activation is that it could potentially turn on anti - self - responses and lead to autoimmune manifestations during chronic infections. igg autoantibodies to brain antigens are increased in p. falciparum - infected patients and correlate with disease severity in african children. autoreactive abs against endocardium and nerves can be detected in mice and humans infected with t. cruzi [51, 52 ] and are thought to be responsible for much of the chagas ' disease pathological damage. recently, we reported that baff - baff - r signaling in t. cruzi infection partially controls polyclonal b - cell response but not parasite - specific class - switched primary effectors b cells. baff (tnf superfamily b lymphocyte stimulator), a crucial factor for the survival of peripheral b cells [5355 ] associated to the development of autoimmune disorders, is produced early and persists throughout the infection with t. cruzi. by baff blockade we observed that this cytokine mediates the mature b - cell response and the production of non - parasite - specific igm and igg and influences the development of antinuclear igg. in addition, polyclonal b - cell activation can be responsible for maintenance of memory b - cell responses because of the continuous, unrestricted stimulation of memory b cells whose ab production may be sustained in the absence of the antigens binding - specific bcr. besides being the precursors of the ab - secreting cells, b cells are committed to do other immune functions such as ag presentation to t cells or cytokine / chemokine production. it has been widely studied that cd8 ctl are important for protective t. cruzi immunity [59, 60 ] but generally they are not induced by soluble protein vaccines. however, a mechanism known as cross - priming has been described whereby certain professional apc can induce cd8 t - cell responses after the uptake of exogenous ag. have demonstrated that the apc functions of b cells may be important for the induction of optimal vaccine - induced responses in mice immunized with a mix of cpg and t. cruzi transsialidase, an enzyme involved in parasite infectivity. they also demonstrate that mice deficient in b cells (umt mice) fails to induce protective immunity when they were immunized with cpg and t. cruzi transsialidase. this failure of umt mice to be protected was associated with the absence of t. cruzi transsialidase - specific cd8 t cell response, suggesting that b cells may be important for the cross - priming of cd8 ctl. in addition, it has been reported that t. cruzi - infected b - cell - deficient mice have reduced numbers of cd8 splenic t cells and impaired generation of central or effector splenic memory t cells. t. gondii - infected c57bl/6 mice develop a robust and uncontrolled th1 response, and it has been reported that t. gondii - primed b cells, but not naive b cells, were able to increase ifn gamma production by splenic t cells in vivo. the mechanisms involved may be linked to the presence of membrane - bound tnf on b - cell surface. the fine tune regulation of migratory cells during infection is an important event in which chemokines and their receptors play a leading role. different reports have demonstrated that the chemokine receptor ccr5 plays a role in systemic protection and cardiac inflammation during t. cruzi infection [65, 66 ]. ccr5 cells migrate to both mucosal and systemic sites in response to the chemokines ccl3 (mip-1a), ccl4 (mip-1b), and ccl5 (rantes). in line with these reports, sullivan. showed that neutralization of ccl5 in ccr5 t. cruzi - immune mice results in decreased levels of t. cruzi - specific b cell responses and decreased mucosal protection in these mice. they also showed that ccl5 produced by b cells acts in an autocrine manner to increase b cell proliferation and total igm secretion. a hallmark of adaptive immunity is the ability to generate humoral immunological memory by which memory b cells could respond more rapidly and robustly to re - exposure to a new infection. interestingly, it has been reported that t. brucei infection is capable of abrogating the efficacy of the vaccine - induced protective responses against non - related pathogens such as b. pertussis. in the same line, strickland and sayles showed that t. gondii infected mice which were immunized with srbc had a depression not only in the primary, but also in the secondary humoral immune response, since they showed less igm and igg splenic ab - secreting cells than non - infected control mice. all the data discussed in the present review indicate that protozoan parasites not only affect the development of the cells involved in ab production but also affect an already established humoral response against other pathogens. then, the identification of mechanisms able to improve b cell response and, consequently, parasite control will also be beneficial to avoid the deterioration of a memory response to other pathogens. | in this review, we discuss how protozoan parasites alter immature and mature b cell compartment. b1 and marginal zone (mz) b cells, considered innate like b cells, are activated during protozoan parasite infections, and they generate short lived plasma cells providing a prompt antibody source. in addition, protozoan infections induce massive b cell response with polyclonal activation that leads to hypergammaglobulnemia with serum antibodies specific for the parasites and self and/or non related antigens. to protect themselves, the parasites have evolved unique ways to evade b cell immune responses inducing apoptosis of mz and conventional mature b cells. as a consequence of the parasite induced - apoptosis, the early igm response and an already establish humoral immunity are affected during the protozoan parasite infection. moreover, some trypanosomatides trigger bone marrow immature b cell apoptosis, influencing the generation of new mature b cells. simultaneously with their ability to release antibodies, b cells produce cytokines / quemokines that influence the characteristic of cellular immune response and consequently the progression of parasite infections. |
the scans were sourced from a particular cbct machine (planmeca promax 3d mid, of idea dental, chennai.) using the following set acquisition parameters as follows : tube volume, 90 kv ; tube current, 10 ma, acquisition period, 13.88 seconds, and image size of 400 mm 400 mm. a single operator worked with all the files from a single desktop personal computer (pentium dual - core cpu e 5700 @ 3.00 ghz with a ram of 2 gb). the images were analyzed and the measurements were done using the tools given in the proprietary software (planmeca romexis viewer). first the image was viewed on the screen, and the presence or absence of the canal on both sides was noted. the nerve tracking tool was used to measure the length from the origin to the end of the visible nerve canal [figure 1 ]. a sagittal section of the cbct was used to measure the distance of the nerve (both at the origin and the apex) from the inferior border of the mandible [figure 2 ]. the coronal plane was used to measure the distance (both at the origin and the apex) of the nerve from the buccal and lingual cortices, respectively [figures 3 and 4 ]. nerve tracking tool used to measure the length from the origin to the end of the visible nerve canal nerve to inferior border of the mandible. a sagittal section of the cone beam computer tomography used to measure the distance of the nerve from the inferior border of the mandible nerve to buccal and labial cortex at the origin. the coronal plane used to measure the distance of the nerve from the buccal and lingual cortices at the origin nerve to buccal and labial cortex at the apex. the coronal plane used to measure the distance of the nerve from the buccal and lingual cortices at the apex not of indian originany anatomical or pathological abnormalitiessubjects below 30 and above 50 years. the scans were sourced from a particular cbct machine (planmeca promax 3d mid, of idea dental, chennai.) using the following set acquisition parameters as follows : tube volume, 90 kv ; tube current, 10 ma, acquisition period, 13.88 seconds, and image size of 400 mm 400 mm. a single operator worked with all the files from a single desktop personal computer (pentium dual - core cpu e 5700 @ 3.00 ghz with a ram of 2 gb). the images were analyzed and the measurements were done using the tools given in the proprietary software (planmeca romexis viewer). first the image was viewed on the screen, and the presence or absence of the canal on both sides was noted. if present, it was noted if present unilaterally or bilaterally. the nerve tracking tool was used to measure the length from the origin to the end of the visible nerve canal [figure 1 ]. a sagittal section of the cbct was used to measure the distance of the nerve (both at the origin and the apex) from the inferior border of the mandible [figure 2 ]. the coronal plane was used to measure the distance (both at the origin and the apex) of the nerve from the buccal and lingual cortices, respectively [figures 3 and 4 ]. nerve tracking tool used to measure the length from the origin to the end of the visible nerve canal nerve to inferior border of the mandible. a sagittal section of the cone beam computer tomography used to measure the distance of the nerve from the inferior border of the mandible nerve to buccal and labial cortex at the origin. the coronal plane used to measure the distance of the nerve from the buccal and lingual cortices at the origin nerve to buccal and labial cortex at the apex. the coronal plane used to measure the distance of the nerve from the buccal and lingual cortices at the apex a total of 144 canals were identified and studied in 120 subjects [table 1 ]. total number of cases surveyed and those with and without incisive nerve the various dimensions of the incisive nerve when examining the cbct images in the viewer, the incisive nerve canal is easily identifiable, similar to inferior alveolar nerve canal as roughly circular radio - opaque rim in a translucent medullary mandibular bone. presence of incisive nerve was not readily identified using panoramic radiograph (11.2%) as against cbct (88%), in the study of pires ca. hence, it is logical to assume that, the study of the inter - foraminal area of the mandible using cbct, becomes mandatory. in this study, the nerve canal was detected till about 10.173 4.682 mm anterior to the mental foramen. this is very much into the perceived safety zone in the inter - foraminal region. the canal runs an approximately horizontal course parallel to the base of the mandible, from the origin (9.425 0.7798 mm) to the apex (9.095 0.6534 mm). medio - laterally, the canal runs a more medial course, increasing its distance from the buccal cortex. the knowledge of the course of the incisive canal is as important as that of the inferior alveolar nerve canal in the mandible, as it might lead to injury of the nerve and lead to postoperative myalgia and other nerve related problems. it should be acknowledged that more number of patients is opting for prosthodontic treatment that includes the use of implants. in recent years, the concept of all on four and similar treatment modalities have gained in popularity. one of the most easy and relevant location for placing an implant is the anterior mandible. in the past, fewer complications were reported while placing implants in the anterior mandible as they were less in number. in recent times hence, with an increase in use, knowledge of the anatomy of this particular region has to be refined. with the mapping of the incisive nerve canal, it can be used as a roadmap to plan safely and negotiate this region. | aim : the aim of this study was to find the prevalence of the mandibular incisive canal, evaluate its location and dimensions using cone beam computer tomography (cbct) in indian population.materials and methods : cbct scan images of 120 subjects were analyzed for the presence of the mandibular incisive canal, its location, size, and its length. the distance between the incisive canal and the buccal and lingual plate of the alveolar bone, and the distance from the canal to the inferior border of the mandible were also measured to position the canal in the mandible.results:about 71.66% of the cbct scans of indian subjects examined showed the presence of the incisive canal, of which 48.33% exhibited canals bilaterally and 23.33% showed unilateral canals. 28.33% of the subjects cbct scans did not exhibit the presence of incisive nerve canal. the average length of the incisive canal was 10.173 mm. the average diameter of the incisive canal in the cbct scans was 2.578 mm. the distance from the inferior border of the mandible to (a) the origin of the incisive canal was 9.425 mm and (b) to the apex of the incisive canal was 9.095 mm. the distance from the buccal cortex of the mandible to (a) the origin of the incisive canal was 1.48 mm and (b) to the apex of the incisive canal was 4.476 mm. the distance from the lingual cortex of the mandible to (a) the origin of the incisive canal was 4.464 mm and (b) to the apex of the incisive canal was 5.561 mm.conclusion:the presence, location, and dimensions of the mandibular incisive canal are an additional required data that needs to be elicited before planning an inter - foraminal placement of implants. |
matrix metalloproteinases (mmps) are zn - dependent endopeptidases that are implicated in numerous physiological processes such as reproduction, wound repair, development, and immune function as well as pathological conditions such as cancer, inflammation, neurodegenerative disorders, and autoimmune diseases [14 ]. the expression of many mmp family members is enhanced in endotoxemia [57 ] and cecal ligation and puncture (clp) [810 ], two mouse models of sepsis, a highly mortal infectious disease, which still lacks an efficient therapeutic treatment. additionally, studies using mmp inhibitors and mmp knockout mice indicate that mmps play a detrimental role in sepsis [9, 1113 ]. moreover, several human data have directly correlated higher levels of active mmp with lower rates of survival in sepsis patients and some mmps are considered as valuable biomarkers in sepsis [8, 9, 14, 15 ]. their detrimental role in several inflammatory diseases justifies the development of specific mmp inhibitors, which have been intensely sought after for over two decades but with poor success [4, 16 ]. most mmp inhibitors (mmpi) consist of two distinct features : a peptidomimetic backbone that interacts noncovalently with specific subsites of the catalytic domain that govern substrate interaction and a zn - chelating moiety or zinc - binding group (zbg) that binds to the hydrolytic zn - ion via coordinate - covalent bonds, rendering the enzyme inactive [16, 17 ]. the majority of mmpi were initially synthesized containing hydroxamic acid as zbg which, although very potent, resulted in poor specificity, oral availability, or biocompatibility. to circumvent these problems, pyrone - based inhibitors emerged as superior mmpi with known biocompatibility, good aqueous solubility, and synthetic versatility [19, 20 ]. based on this strategy, a specific mmp-3 pyrone - based inhibitor (am-6) was developed. however, the specificity of am-6 was only characterized for mmp-1, mmp-2, and mmp-3 and therefore, in this study, we synthesized the same compound and screened it against a broader range of mmps to firmly ascertain mmp selectivity. surprisingly, the compound displayed specificity and potency towards mmp-12. to accommodate these findings, we show that the inhibitor protects against inflammation - induced disruption of the blood - cerebrospinal fluid barrier both in vitro and in vivo. the target compound was synthesized according to the literature with some modifications (scheme 1). the omission of an aqueous recrystallization step leads to near - quantitative yields of the hydrolytically unstable chlorokojic acid (1) without sacrificing purity. furthermore, 4-iodobenzylamine could be replaced with 4-bromobenzylamine (g), which is less costly and not sensitive towards light and air. the hcl / acetic acid protocol as written in the original publication did not provide us with the final inhibitor in good yield (some 39% crude yield). reductive debenzylation (step i) did prove to be a very reliable method to obtain the deprotected molecule. proton nmr spectra were recorded using a bruker avance 300 (300 mhz) device using tetramethylsilane as internal standard for cdcl3 and dmso - d6. c - nmr spectra were recorded using a bruker avance 300 (75 mhz) device, using the deuterated solvent as an internal standard. reagents were purchased from various companies, most notably tci, sigma - aldrich, and acros. kojic acid (tci, 15 g, 0.105 mol) is placed in a round bottom flask equipped with a magnetic stirring bar. the flask is cooled in an ice bath and thionyl chloride (60 ml) is slowly added after which the ice bath is removed and the mixture is placed under an argon atmosphere. after stirring for 1.5 h a yellow mass is filtered off and washed extensively with petroleum ether. the product was an off - white solid (16.6 g, 98%) : h - nmr (dmso - d6) 4.6 (s, 2h, cl - ch2 - 6), 6.6 (s, 1h, 5-h), and 8.1 (s, 1h, 2-h-) in agreement with the literature. chlorokojic acid 1 (2.5 g, 0.0156 mol, 1 eq) is dissolved in water (8 ml) and heated to 50c under an argon atmosphere in a flask equipped with a magnetic stirring bar. zinc dust (2.1 g, 0.031 mol, 2 eq) is added. an aqueous solution of hcl (4.7 ml, 0.055 mol, 3.5 eq) is slowly added to the reaction mixture under vigorous stirring, during which the temperature is increased to 70c. after 4 h the excess zinc dust is filtered off while being hot, and the resultant liquid is extracted with dichloromethane (320 ml). the combined organic phase is dried over magnesium sulfate and the solvent is removed by evaporation under reduced pressure. the crude material is purified via recrystallization from isopropanol to obtain an off - white solid (1.2 g ; 52%) : h - nmr (cdcl3) 2.3 (s, 3h), 6.2 (s, 1h), and 7.8 (s, 1h) in agreement with the literature. sodium hydroxide (0.696 g, 0.017 mol, 1.1 eq) is dissolved in water (16 ml). to this solution 2 (2 g, 0.016 mol, 1 eq) a magnetic stirring bar is added. a 35% aqueous formaldehyde solution (0.36 ml, 0.017 mol, 1.1 eq) is added and the mixture is allowed to stir overnight. after acidification to ph 1 with 35% aqueous hydrochloric acid the mixture is cooled to 2c and allowed to stand overnight. the crystalline mass is filtered off, washed with 5 ml of water, and dried via lyophilisation. an off - white solid is obtained (1.71 g, 71%) : h - nmr (dmso - d6) 2.26 (s, 3h), 4.38 (s, 2h), 5.36 (b, s, 1h), 6.21 (s, 1h), and 8.86 (b, s, 1h) in agreement with the literature. sodium hydroxide (0.56 g, 8.93 mmol, 1.1 eq) is dissolved in water (2.4 ml) and added to a solution of 3 (2 g, 8.12 mmol, 1 eq) in methanol (1.2 ml) in a two - necked flask under argon equipped with a reflux condenser, a septum, and a magnetic stirring bar. benzyl bromide (1.7 ml, 8.93 mmol, 1.1 eq) is slowly added and the mixture is allowed to stir overnight. the mixture is concentrated in vacuo and taken up in dichloromethane (approximately 20 ml). is then extracted with 5% aqueous sodium hydroxide (220 ml) and brine (120 ml). the organic phase is dried with anhydrous sodium sulfate after which the solvent is removed under reduced pressure. the solid is dissolved in as little dichloromethane as possible after which heptane (or petroleum ether) is added until no more precipitation occurs. the precipitate is filtered off and dried to yield a white to off - white powdery solid (2.3 g, 73%) : h - nmr (cdcl3) 2.25 (s, 3), 2.284.30 (d, 2h, 6.8 hz), 5.18 (s, 2h), 6.19 (s, 1h), and 7.37 (s, 5h) in agreement with the literature. compound 4 (2 g, 1 eq, 8.18 mmol) is dissolved in 37.8 ml chloroform and 10.2 ml dmso. to this mixture triethylamine (7 ml, 0.050 mol, 6 eq) and pyridine (0.1 ml) are added. the mixture is stirred in an ice bath under argon using a magnetic stirring bar until a temperature of 35c is reached. sulfur trioxide - pyridine complex (6.48 g, 0.040 mol, 5 eq) is subsequently added and the mixture is allowed to stir overnight. it is then extracted (220 ml water, 120 ml brine) and dried over anhydrous sodium sulfate. the solvent is removed under reduced pressure using a rotavapor equipped with a cold trap (dry ice / isopropanol) to obtain the crude product as orange oil. this is diluted with 10 ml diethyl ether and transferred onto a flash column packed with silica / diethyl ether. after eluting with diethyl ether the solvent is removed under reduced pressure using a rotavapor equipped with a cold trap. a white to yellow crystalline solid is obtained (1.38 g, 69%) : h - nmr (cdcl3) 2.32 (s, 3h), 5.49 (s, 2h), 6.31 (s, 1h), 7.35 (s, 5h), and 9.85 (s, 1h) in agreement with the literature ; ft - ir 1690.33 cm (c = o aldehyde), 1641.24 cm (c = o pyrone), 1613.12 cm, and 1583.88 cm (c = c pyrone). (6) preparation of compound 6 (3-(benzyloxy)-6-methyl-4-oxo-4h - pyran-2-carboxylic acid). compound 5 (1.57 g, 6.4 mmol, 1 eq) is dissolved in acetone / water (20 ml/20 ml) with a magnetic stirring bar. sulfamic acid (0.86 g, 8.9 mmol, 1.4 eq) and sodium chlorite (80%, 0.76 g, 6.7 mmol, 1.05 eq) are added and the mixture is allowed to stir for 1 h in an open vessel. the mixture is subsequently evaporated under reduced pressure to remove the acetone after which the crude product is filtered off and washed with a small amount of ethanol. the product is then dried in vacuo to obtain a stark white powder (1.3 g, 78%) : h - nmr (dmso - d6) 2.29 (s, 3h), 5.10 (s, 2h), 6.40 (s, 1h), and 7.327.45 (m, 5h) in agreement with the literature ; ft - ir 1720.37 cm (c = o acid), 1625.50 cm (c = o pyrone), 1554.48 cm and 1496.36 cm (c = c pyrone), and mp (precipitated from water / acetone) 180c. compound 6 (0.1 g, 0.38 mmol, 1 eq) is dissolved in dry tetrahydrofuran (4.16 ml) in a flame - dried round bottom flask equipped with a magnetic stirring bar under an argon atmosphere. n - hydroxysuccinimide (0.045 g, 0.39 mmol, 1.02 eq) is added and the mixture stirred for 30 min. dicyclohexylcarbodiimide (0.079 g, 0.38 mmol, 1 eq) is added as a solid and the mixture is stirred for an additional 3 h at rt under an argon atmosphere. dicyclohexylurea is filtered off and the precipitate is washed with tetrahydrofuran (2 ml). 4-bromobenzylamine (0.05 ml, 0.385 mmol, 1.01 eq) is added to the filtrate in a dried round bottom flask equipped with a magnetic stirring bar and a reflux condenser under an argon atmosphere. this solution is transferred to a chromatography column (silica, eluent chloroform / methanol 1%). after chromatography, the product is obtained as an off - white solid (0.15 g, 91%) : h - nmr (cdcl3) 2.37 (s, 3h), 4.34.38 (d, 2h, j = 5.6), 5.33 (s, 2h), 6.28 (s, 1h), 6.97.5 (m, 9h), and 8.06 (b, s, 1h) ; ft - ir 3363.82 cm ((nh)), 1677.31 cm ((c = o) pyrone), 1643.58 cm (amide i band), 1621.41 cm ((c = c) pyrone), 1584.93 cm ((c = c) pyrone), 1536.21 cm (amide ii band), and mp (recrystallized from methanol) 118c. (8) preparation of compound 8 (n-([1,1:4,1-terphenyl]-4-ylmethyl)-3-(benzyloxy)-6-methyl-4-oxo-4h - pyran-2-carboxamide). compound 7 (90 mg, 0.21 mmol, 1 eq) is dissolved in toluene in a round bottom flask equipped with a reflux condenser, magnetic stirring bar, and an argon atmosphere. to this mixture are added aqueous potassium carbonate (2 m solution, 10 ml), triphenylphosphine (5.5 mg, 0.021 mmol, 0.1 eq), [1,1-biphenyl]-4-ylboronic acid (41.5 mg, 0.21 mmol, 1 eq), and palladium acetate (4.6 mg, 0.02 mmol, 0.1 eq) after which the mixture is heated to 135c with vigorous stirring. the mixture is allowed to stir for 24 h before being extracted with toluene (320 ml) and dried over sodium sulfate and the solvent is removed under reduced pressure. the crude product is purified via column chromatography (silica, chloroform/1 - 2% methanol) : h - nmr (cdcl3) 2.37 (s, 3h), 4.444.5 (d, 2h, j = 5.7 hz), 5.34 (s, 2h), 6.29 (s, 1h), 7.107.75 (m, 18h), and 8.12 (b, 1h). compound 8 (186.5 mg) is dissolved in dichloromethane / methanol (70/30, 35 ml) in a thick - walled glass vessel. after flushing with argon, the vessel is placed in a shaker - type hydrogenation apparatus and placed under 40 psi hydrogen gas for 20 h after flushing with hydrogen gas to remove traces of oxygen. the catalyst is filtered off and the filtrate washed with 10 ml dichloromethane / methanol (50/50). the combined organic phase is evaporated in vacuo to yield an orange solid (138.5 mg, 90%) : h - nmr (cdcl3) 2.24 (s, 3h), 4.6 (s, 2h), 6.16 (s, 1h), and 77.8 (m, 13h) ; ft - ir 2339.5 cm (oh), 1633.1 cm (amide i), 1591.8 cm (c = o pyrone), 1537.5 cm (amide ii), 1483.9 cm (c = c pyrone), and mp (recrystallized from toluene) 253270c. the specificity of the synthesized inhibitor was determined in vitro against a panel of ten human recombinant mmp catalytic domains using a fluorescent assay kit (bml - ak016, enzo life sciences). briefly, the compound was dissolved in dimethyl sulfoxide (dmso) and further diluted in assay buffer (50 mm hepes, 10 mm cacl2, 0.05% brij-35, and ph 7.5). ten mmps (mmp-1, mmp-2, mmp-3, mmp-7, mmp-8, mmp-9, mmp-10, mmp-12, mmp-13, and mmp-14) were individually incubated with varying concentrations of inhibitor for one hour at 37c, followed by addition of a quenched fluorogenic substrate (omnimmp fluorogenic substrate mca - pro - leu - gly - leu - dpa - ala - arg - nh2 [mca is (7-methoxycoumarin-4-yl)-acetyl ; dpa is n-3-(2,4-dinitrophenyl)-l---diaminopropionyl ], 4 m in assay). the assays were performed in black, clear bottom, nbs greiner 96-well plates (655906, greiner bio - one) and fluorescence (ex = 325 nm, em = 405 nm) was measured at 60 sec intervals for 30 min with a flexstation ii microplate reader running softmax pro 5.3 (molecular devices). after each fluorescent measurement the well plates were shaken for 2 sec to agitate the reagents. in each experiment we included a blank, a substrate control (substrate only in assay buffer), a positive control (nngh, 1.3 m final concentration), and negative control (only substrate and inhibitor in assay buffer). the latter was implemented to exclude possible interference of the inhibitor with the substrate. to confirm the potency against mmp-3 and mmp-12, separate assays were performed using human recombinant mmp-3 (72006, anaspec) and mmp-12 catalytic domains (55525 - 1, anaspec) and quenched fluorescent substrate qxl 520-pro - leu - ala - tyr - trp - ala - arg - lys(5- fam)-nh2 (ex = 485 nm, em = 538 nm, 60577 - 01, anaspec). the ic50 values were determined in a single experimental run in duplicate. data analysis and curve fitting for determination of ic50 values were performed in graphpad prism 6. female c57bl/6j (810 weeks old) mice were purchased from janvier labs (france) and mmp-12 knockout mice (mmp-12) (in c57bl/6j background together with littermate mmp-12 controls) were obtained from dr. c57bl/6 and mmp-12 mice were housed with 46 mice / cage with ad libitum access to food and water and with a 14-hour light/10-hour dark cycle in a specific pathogen - free animal facility and conventional facility, respectively. all experiments were approved by the ethics committee of the faculty of sciences of ghent university. injection of lipopolysaccharide (lps) from salmonella enterica serotype abortus equi (sigma) dissolved in pbs. the dose was 200 g/20 g body weight (the ld100 dose for c57bl/6 mice). 100 g mmp-12i (1 g/l) or vehicle control was administered intraperitoneally at different time points : 15 min before and 3 and 6 hours after lps administration. for isolation of total rna from in vivo choroid plexus samples, mice were anesthetized with ketamine / xylazine and perfused with pbs supplemented with bromophenol blue. brains were dissected from the skull and choroid plexus from the third and fourth ventricles were dissected under a dissection microscope. total rna was isolated at different time points after lps injection with the rneasy kit (qiagen). rna concentration and purity were determined spectrophotometrically using the nanodrop nd-1000 (nanodrop technologies). cdna was made by using iscript cdna synthesis kit (bio - rad) with 500 ng starting material and qpcr was done using the sensifast sybr no - rox kit (bioline) on the light cycler 480 system (roche). expression levels of mmp-12 were normalized to the expression of the two most stable housekeeping genes, ubc and gapdh, which were determined using the genorm housekeeping gene (hkg) selection software. one hour before csf isolation, mice were injected i.v. with 75 mg / kg body weight of fitc - labeled dextran (4 kda, sigma). csf was obtained using the cisterna magna puncture method as described previously. in brief, borosilicate glass capillary tubes (b100 - 75 - 15, sutter instruments) were used to pull needles on the sutter p-87 flaming micropipette puller (pressure 330 pa, heat index 300). before sampling csf, mice were sedated with ketamine / xylazine. the incision site was sterilized with 70% ethanol, and cisterna magna was exposed by cutting skin and muscle tissue on the posterior side of the skull. the head of the mouse was placed at an angle of 135 degrees and csf was collected by inserting the trimmed needle into the fourth ventricle by piercing the cisterna magna. a csf sample of 2 l was diluted 25-fold in sterile pbs and leakage into this brain compartment was determined by measurement of fluorescence with ex/em = 488/520 nm. culturing primary mouse choroid plexus epithelial cells was done as described. in brief, 29-day - old pups were decapitated and brains were isolated. the cells were dissociated enzymatically by incubating them for 57 min with pronase (isolated from streptomyces griseus, sigma). digestion was stopped by adding an excess of hbss buffer and the cells were washed twice with hbss. the cell pellet was resuspended in dmem - f12 culture medium and plated on laminin - coated plates. two days later, they were shifted to dmem - f12 containing cytosine arabinoside (ara - c) to eliminate growth of fibroblasts. purity of the cultures was confirmed by checking the levels of transthyretin (ttr) both by qpcr and by immunostaining. primary cpe cells retained the epithelial and the barrier properties which were confirmed by staining for e - cadherin, zo-1, and occludin. the electric cell - substrate impedance (ecis) technique (applied biophysics) was used to monitor barrier properties as described before. 250.000 cells per well were seeded onto laminin - coated 8w10e array plates (applied biophysics) and the array holder was placed in a standard cell culture incubator (37c, 100% humidity, and 5% co2). after ~16 hours, a stable impedance at low and high frequency was obtained and cells were incubated with vehicle or 1 m mmp-12i, followed 1 hour later by vehicle or lps (1000 ng / ml). compound 9 (figure 1(a)) was assessed for its inhibitory properties towards an array of ten different mmps using a fluorescent in vitro assay. for all tested mmps, the compound is poorly potent against mmp-1, mmp-3, mmp-7, mmp-9, mmp-10, and mmp-13, reflecting ic50 values higher than 50 m (figure 1(b)). the compound displays slightly better inhibitory activity towards mmp-2, mmp-8, and mmp-14 as the apparent ic50 values are situated between 2 m and 50 m. the initial screen revealed an apparent ic50 value for mmp-12 below 2 m and when performing a detailed dose - response experiment using a more specific substrate for mmp-12 (substrate xiii, anaspec) a nonlinear regression analysis indicated that at higher inhibitor concentrations an inhibitory plateau (72 1.75%) is reached with a relative ic50 of 177 nm (figure 1(c)). although the use of a different substrate can potentially influence the kinetic readout, a comparison between the fluorescent in vitro assay kit and the use of substrate xiii resulted in similar inhibitor potencies (data not shown), which justifies the use of the more specific substrate for mmp-12. taken together, these data indicate that the synthesized compound is a potent and fairly specific inhibitor of mmp-12 and therefore from section 3.2 onwards it will be referred to as mmp-12i. the discrepancy between puerta. and this study regarding the inhibitory selectivity towards mmp-12 over mmp-3 could in part be explained by the strong ph - dependence of inhibitor potency for human mmp-3. both mmp-3 and mmp-12 have a deep pocket s1 subsite in which the terphenyl can reside. however, mmp-3 is uniquely characterized by a sharp optimum at ph 6 for efficient catalysis and inhibition and this is drastically reduced at neutral ph by structural changes in the s1 pocket and ionization of inhibitor residues. in contrast to the study of puerta and colleagues, the compound was tested in physiologically relevant ph 7.5 to accommodate a large array of mmps. importantly, the ph - dependence of inhibitor - mmp-3 interactions typically gives rise to a change in ic50 values not larger than one order of magnitude as opposed to the three orders of magnitude we established here, so other factors might be at play. because of this and the observed potency towards mmp-12 we further validated the biological relevance of the compound in the context of mmp-12. endotoxemia, that is, systemic administration of lipopolysaccharide (lps), induces systemic inflammation and eventually lethality in mice. we have previously shown that mice can be protected from lps - induced lethality by administration of a broad spectrum mmp inhibitor bb-94 and several mmps appeared to play a role in the lps - induced lethality [8, 1113 ]. here, we studied the effect of mmp-12 deficiency on the lps sensitivity by using mmp-12 mice. as shown in figure 2(a), mmp-12 mice display a partial protection to an ld100 dose of lps, indicating that mmp-12 plays a detrimental role in the lps - induced lethality. to verify this, we treated wild type mice with the mmp-12i and challenged them with lps (figure 2(b)). mmp-12i treatment resulted in a significant reduction in lps - induced lethality compared with vehicle treated mice, which again demonstrates that the compound most likely targets mmp-12. systemic inflammation is known to be associated with loss of blood - brain barrier integrity and this has been correlated with lethality. next to the most studied endothelial blood - brain barrier (bbb), we have previously shown that the blood - cerebrospinal fluid barrier (bcsfb) which is formed by the choroid plexus epithelium (cpe) is also affected in response to systemic lps administration. mrna expression analysis of choroid plexus tissue revealed 500-fold upregulation of mmp-12 upon systemic lps administration (figure 2(c)), associated with an increase in bcsfb permeability (figure 2(d)). interestingly, the loss of bcsfb permeability was significantly reduced when endotoxic mice were treated with our mmp-12i (figure 2(d)). therefore, primary cpe cells were isolated and plated onto electrical cell impedance sensing (ecis) arrays containing gold electrodes. ecis is a real - time, label - free, impedance - based method used to study the barrier properties of cells grown in culture. after reaching confluency, cells were incubated with lps or lps supplemented with mmp-12i and compared to untreated cells. lps resulted in a drop in normalized resistance compared to control and this was significantly reduced in the presence of mmp-12i (figure 2(e)). these data clearly show that mmp-12 plays a role in the observed lps - induced loss of bcsfb integrity. in summary, we report the synthesis and biological evaluation of a hydroxypyrone - based mmpi with excellent potency and specificity towards mmp-12. both in vivo and in vitro studies on the effects of the target compound on endotoxemia and bcsfb integrity clearly indicate a role for mmp-12 in these inflammatory processes and establish the use of this compound in a biological context. | a hydroxypyrone - based matrix metalloproteinase (mmp) inhibitor was synthesized and assayed for its inhibitory capacity towards a panel of ten different mmps. the compound exhibited selective inhibition towards mmp-12. the effects of inhibition of mmp-12 on endotoxemia and inflammation - induced blood - cerebrospinal fluid barrier (bcsfb) disruption were assessed both in vitro and in vivo. similar to mmp-12 deficient mice, inhibitor - treated mice displayed significantly lower lipopolysaccharide- (lps-) induced lethality compared to vehicle treated controls. following lps injection mmp-12 mrna expression was massively upregulated in choroid plexus tissue and a concomitant increase in bcsfb permeability was observed, which was restricted in inhibitor - treated mice. moreover, an lps - induced decrease in tight junction permeability of primary choroid plexus epithelial cells was attenuated by inhibitor application in vitro. taken together, this hydroxypyrone - based inhibitor is selective towards mmp-12 and displays anti - inflammatory activity in vitro and in vivo. |
in patients undergoing percutaneous coronary intervention (pci) for the treatment of acute coronary syndrome, administration of glycoprotein (gp) iib / iiia receptor antagonists has been shown to improve their clinical outcomes. however, the associated hemorrhagic risk may evoke serious complications, such as acute profound thrombocytopenia.1) we present a successfully managed case of a rare complication of acute profound thrombocytopenia after the use abciximab, an intra - aortic balloon pump (iabp) for the treatment of no - reflow phenomenon, and consecutive cardiogenic shock during primary pci in a patient with st - segment elevated myocardial infarction (stemi). a 65 year - old man with hypertension visited the local hospital with resting chest pain for 3 hours. he was transferred to our hospital due to the abnormal electrocardiogram (ecg) findings showing an elevated st - segment in the inferior wall territory with a reciprocal change, which was suspicious of stemi. in the emergency room, blood pressure was 140/80 mm hg and chest radiography showed no definite abnormal findings, which was graded as killip class 1. it was decided to have the patient undergo primary pci for the treatment of stemi. conventional medical treatments that include a bolus injection of 5000 iu of heparin, a loading dose of aspirin (300 mg), and clopidogrel (300 mg) was applied before primary pci. coronary angiography (cag) showed a totally occluded lesion of the proximal right coronary artery (rca) with collateral flow grade 1 from the left anterior descending artery (fig. after predilatation with a 2.020 mm conventional balloon, a bare metal stent 5.024 mm liberte (boston scientific, natick, ma, usa) was implanted in the proximal rca lesion (fig. 1e) and blood pressure decreased with findings of a complete atrioventricular - block and re - elevation of the st - segment on ecg monitoring. the patient was treated with intracoronary injection of nitrate and adenosine, and intravenous bolus consecutive injection of abciximab with maintenance (intravenous bolus of 0.25 mg / kg, 10 to 60 minutes during the procedure, followed by 0.125 g / kg / min infusion for 12 hours). although an improvement was observed in the thrombolysis in myocardial infarction flow grade from grade 0 to grade 3 (fig. the patient was moved to the coronary care unit for intensive monitoring with maintenance of abciximab and iabp. although the baseline platelet count was 167000/l before pci, a routine check determined a complete blood count showing a decreased platelet count of 6000/l at 22 hours after bolus administration of abciximab, followed by a platelet count of 3000/l 4 hours later. it was considered that the patient had acute, profound thrombocytopenia as a complication of abciximab. heparin induced thrombocytopenia (hit) was excluded because hit typically develops after 6 to 10 days of heparin use with no previous exposure to heparin. in this case, the patient had no history of exposure to heparin. therefore, the patient was started on clopidogrel 75 mg / day and aspirin 100 mg / day were started to prevent stent thrombosis. the patient was withdrawn from heparin treatment in consideration of additional hemorrhagic risk, but not hit. the next followed count of platelet increased to 24000/l without clinical evidence of stent thrombosis, such as chest pain and a change of st - segment on ecg. vital signs were stabilized the next day and iabp was removed successfully using the closing device, perclose (abbott, redwood city, ca, usa) due to the hemorrhagic risk of the access site and a decreased platelet count of 26000/l. after additional transfusions of 4 units of platelet concentrates, the platelet count increased to 49000/l without complications. a 1-year follow up of cag showed good flow of rca without chest pain or thrombocytopenia. the benefit of platelet inhibition by gp iib / iiia inhibitors, as adjunctive treatment in patients undergoing pci, is well established. abciximab, a potent antiplatelet agent that blocks the final pathways to platelet aggregation,2) improves the outcomes of high risk procedures and decreases the incidence of major adverse cardiac events. however, the associated hemorrhagic risk may be a major safety concern. according to previous studies, the incidence of thrombocytopenia (platelet count of < 100000/mm) is 0.5% to 5.6%.3 - 5) in particular, acute profound thrombocytopenia, defined as a platelet count of < 20000/mm, is a rare and serious complication, since it increases the risk for significant bleeding. however, an increased risk of stent thrombosis, due to the need for antithrombotic drugs discontinuation and platelet transfusion should be considered to determine the proper therapeutic approach. although little is known about the pathophysiological mechanisms that lead to thrombocytopenia induced by gp iib / iiia antagonists, preexisting platelet surface antibodies and gp iib / iiia antagonists may induce the immune response of platelets by conformational changes in the gp iib / iiia receptors on the platelet surface and the expression of new epitopes recognized by the antibodies.6) the platelet count is necessary to detect thrombocytopenia, and should be done before starting treatment with abciximab, 1 - 4 hours later and, if it decreases, once a day until resolution (for platelet count < 20000/mm it should be checked twice a day until the count rises to 50000/mm). several drugs, such as heparin and clopidogrel, could induce thrombocytopenia, thus should also be considered as possible causative factors. it is important to exclude hit, because both heparin and abciximab are frequently infused.7) there is no test that can distinguish this syndrome from hit. however, abciximab induced thrombocytopenia occurs rapidly after the administration of abciximab, while hit develops over a period of days in patients without previous exposure to heparin. hit is not acute and it may be associated with bleeding and thrombosis.8) in this case, thrombocytopenia was identified the next day after administration of abciximab and heparin without a previous exposure history of heparin. acute profound thrombocytopenia as a complication of abciximab is a more problematic situation in this case. it can create hemorrhagic complications. however, the following should be considered : microvascular thromboembolic condition of no - reflow phenomenon with poor clinical outcomes, such as a lack of recovery of the left ventricular function of affected myocardial segments, increased in - hospital mortality, a poor long - term prognosis,9 - 11) and the need for additional usage of heparin for the maintenance of iabp. in addition, the management of abciximab - induced profound thrombocytopenia is controversial, since there is no previous evidence of this in the medical literature. therefore, clopidogrel 75 mg / day and aspirin 100 mg / day were started and only heparin was withdrawn. in addition, it was difficult to expect a value of transfusion of platelet concentrates in the condition of no - reflow phenomenon. transfusion of platelet concentrates was decided based on the operator 's experience in the present case. in summary, we report a case of a 65-year - old man with a rare complication of acute profound thrombocytopenia after administration of abciximab for the treatment of no - reflow phenomenon during primary pci. although hit should be differentiated and the removal of iabp was an issue due to the risk of increased bleeding, our patient was managed successfully with transfusion of platelet | glycoprotein iib / iiia antagonists are well established for their effectiveness in improving clinical outcomes in acute coronary syndrome patients undergoing percutaneous coronary intervention. acute profound thrombocytopenia is a rare complication of abciximab. we present a case which was managed successfully for the rare complication of acute profound thrombocytopenia after using abciximab and an intra - aortic balloon pump for the treatment of a no - reflow phenomenon and consecutive cardiogenic shock during primary percutaneous coronary intervention. |
the search for alternative growth models in these years of financial and economic difficulties has aroused increasing interest in the green growth paradigm as a path towards economic growth and sustainable development, limiting environmental degradation while conserving biodiversity and responsible use of natural resources. according to the europe 2020 strategy1 for smart, sustainable and inclusive growth, innovation and better use of natural resources constitute a driver for the creation of new jobs and services. recent estimates2, 3 show an increase in so - called green jobs defined as work in agricultural, manufacturing, research and development (r&d), administrative, and service activities that contributes substantially to preserving or restoring environmental quality. in 2008, the united nations environment program (unep)3 estimated there were 11 million green jobs worldwide and statistics suggest they can be expected to rise to 20 million by 2030. renewable energy alone has generated 2.3 million new jobs, mainly in the biomass and solar thermal industries. the european union (eu) is in the lead as regards energy from renewable sources4 : 950,000 new jobs were created in 2010 and 1.4 million are forecast for 2020. in italy the number of people in green jobs is estimated at 850,000 to 950,000, and should reach 1.3 to 1.5 million by 2020, 250,000 of them in renewable energy5. however, the transition towards a greener economy poses new challenges for occupational health and safety (ohs). on the one hand, green jobs aim to safeguard environmental quality and/or produce green goods or services but, on the other, it is not yet clear whether they are safe for workers. besides traditional work - related risks (chemical, physical, biological, etc.), green workers could potentially be exposed to emerging risks related to the introduction of new technologies, substances, processes, workforce changes6, 7, etc. in addition, the rapid expansion of the green economy could pose further training requirements, leaving some unskilled workers involved in procedures for which they have not been adequately trained, and putting their health and safety at risk8, 9. forecasts and estimates as far as possible of potential new and emerging risks related to ohs are therefore needed before they arise, particularly for green jobs. predict new and emerging risks ; the second community strategy, 20072012, defined the risks related to the new technologies as a field where risk prediction needs to be improved10, 11. research is needed to assess traditional and new ohs risks in different situations and combinations within green jobs. this would facilitate the transfer of existing ohs knowledge to green technologies, and the development of job - specific risk assessment for green jobs, as well as identifying ohs training needs. however, an analysis of the literature on the potential health and safety risks for green workers, referring to the period 19982009, issued by the european agency for safety and health at work (eu - osha) in the second phase of the project12foresight of new and emerging risks to occupational safety and health associated with new technologies in green jobs by 2020, found very few studies or detailed research into the impact of this type of work on health and safety, particularly in the renewable energy sector. our analysis of the literature on this topic for the period 2009 to date confirmed the interest in investigating certain basic aspects of green jobs ; these included definition and measurement3, economic impact4, consequences for occupation13, environmental protection and management14, and others. there was also a growing focus on emerging issues directly linked to technological innovation ; these included the promotion of decent work in the green economy15,16,17, the importance of industrial relations and social dialogue18, identification of new green skills19,20,21, the importance of new education and training strategies22, 23, possible scenarios related to new and emerging risks in new green technologies24. although the number of scientific publications focused on identifying the potential risks for the health and safety of workers in single renewable sectors and/or specific aspects is growing, there is still no systematic, thorough systematic approach to ohs in the green economy. the main investigation to date has been done by eu - osha12, 24, 25, which provided a detailed forecast and analyzed the possible development of green jobs up until 2020, and the challenges these might involve in ohs. the study envisaged a series of future scenarios related to the development of green technologies, in different economic and social circumstances. two literature reviews consolidated by 50 phone interviews and two online surveys resulted in the selection of key drivers of change that could shape green jobs and key new technologies likely to have an impact on ohs in green jobs. this was followed by eight technology workshops that explored potential new and emerging ohs risks for each of the key technologies and discussed ways in which to address them. these workshops showed the value of the scenarios in engaging with different groups of stakeholders and generating strategic discussions between them. the aim was to call attention to potential ohs risks in this sector and provide those responsible for eu policies with tools to help create jobs where european workers health and safety could be guaranteed. in italy therefore, with the aim of drawing attention to ohs in the green economy, the department of occupational and environmental medicine, epidemiology and hygiene at the italian workers compensation authority (inail) has conducted a survey among some italian stakeholders, social partners, institutions and green businesses to gather their perceptions of the potential effects of green jobs on ohs, with particular regard to the renewable energy sector, taking as its starting point the eu - osha study12, 24, 25 and two previous inail investigations on emerging risks such as work - related stress26 and nanotechnologies27, 28. the survey was the first stage of an inail study mainly aimed at obtaining a thorough, detailed picture of the work - related risks of green jobs, and fostering the development and adoption of tools to limit or prevent them. the survey, started in may 2013 and completed in march 2014, analyzed the potential impact of the transition to a green economy from the viewpoint of the health and safety of italian workers in the sector of renewable energy, where in recent years the numbers of workers have been growing constantly29. first of all it was simple for us to identify and involve italian stakeholders who had taken part in previous surveys26,27,28 organized by the department of occupational and environmental medicine, epidemiology and hygiene and related to this survey ; there was also the fact that right now there are no studies of ohs in the green economy in italy. the survey was conducted in several steps, described below. before drafting the questionnaire we made a critical comparative analysis of international and european questionnaires with a view to identifying their weak and strong points in dealing with new and emerging risks for workers health and safety in the green sector. most of the international and italian questionnaires looked at general aspects of green work (for instance, definition and quantification of green jobs, costs, benefits and barriers to the development of renewable energy, adoption of regulatory measures, and energy policies), and neglected ohs matters in this sector. we therefore used as a reference point the eu - osha questionnaire as part of their project12foresight of new and emerging risks to occupational safety and health associated with new technologies in green jobs by 2020. this generated a series of scenarios relating to the use of new technologies in green work and their potential effects on workers health and safety13. once the questionnaire had been drafted, it was administered in the pilot stage to a restricted sample of people to test its applicability and efficacy. the final version was then posted on the research activity section of the inail website (http://www.ispesl.it/greenjobs/). the questionnaire is brief and easy to complete, comprising 15 questions, under four headings : 1. the web page provides a short description of the survey and a glossary with brief definitions of the main entries in order to make sure responders fully understood the terminology used in the questionnaire. on the basis of the nature of research design and aims and objectives, we select units to be sampled based on their skills, knowledge and opinion (judgmental sampling). it is the only viable sampling technique in obtaining information from a very specific group of people. firstly, for each category we selected the people holding the main roles in ohs management and sustainable development policies. then, we looked for the same numbers of people for each category, to form a homogeneous sample. we identified five categories : institutions, trade unions, employers organizations, businesses, research. regarding the first category, representatives belonging to institutions that play a central role in the development and management of ohs and renewable energy policies were chosen (e.g. ministry of environment, ministry of economic development, ministry of health, ministry of labour, lombardia region, lazio region, tuscany region, inail). in the case of employers and trade unions, osh experts who have a decision - making role in the development of ohs policies were identified. with respect to companies, groups of companies and professionals with expertise in the design, the sector of research is represented by members with different expertise in the field of ohs and renewable energy (e.g. engineers, occupational physicians, researchers), belonging to universities, associations and research centers for the promotion of renewable energy sources, associations for industrial research or of industrial hygienist. the 61 stakeholders from the different categories are listed in table 1table 1.survey participants grouped by categorycategorynumber of participantsinstitutions11trade unions11employers organizations13businesses11research 15total 61. the individual stakeholders were sent access codes by e - mail to enable them to complete the questionnaire on line. replies to the questionnaire were uploaded into a database in microsoft excel format and analyzed using spss version 16.0 for windows. in a preliminary analysis we calculated the response rates for the multiple - choice questions, analysis was done on the following : valid cases (those who selected at least one response) ; valid responses (the number of times responders selected a specific answer, which could therefore be more than 100% of cases) ; and missing cases (no responses). contingency tables were used to analyze the percentages in each category. to check for associations between the groups and the responses, we used the test to verify the significance of differences between the observed and expected or theoretical frequency distributions, i.e. those expected if there is no relation between the variables investigated. we ensured that all responses would be considered confidential in the data analysis, as required by the italian deontological code and code for good conduct in the treatment of personal data for statistical and scientific purposes (provvedimento del garante no. 2 del 16 giugno 2004, gazzetta ufficiale 14 agosto 2004, no. 190). before drafting the questionnaire we made a critical comparative analysis of international and european questionnaires with a view to identifying their weak and strong points in dealing with new and emerging risks for workers health and safety in the green sector. most of the international and italian questionnaires looked at general aspects of green work (for instance, definition and quantification of green jobs, costs, benefits and barriers to the development of renewable energy, adoption of regulatory measures, and energy policies), and neglected ohs matters in this sector. we therefore used as a reference point the eu - osha questionnaire as part of their project12foresight of new and emerging risks to occupational safety and health associated with new technologies in green jobs by 2020. this generated a series of scenarios relating to the use of new technologies in green work and their potential effects on workers health and safety13. once the questionnaire had been drafted, it was administered in the pilot stage to a restricted sample of people to test its applicability and efficacy. the final version was then posted on the research activity section of the inail website (http://www.ispesl.it/greenjobs/). the questionnaire is brief and easy to complete, comprising 15 questions, under four headings : 1. the web page provides a short description of the survey and a glossary with brief definitions of the main entries in order to make sure responders fully understood the terminology used in the questionnaire. on the basis of the nature of research design and aims and objectives, we select units to be sampled based on their skills, knowledge and opinion (judgmental sampling). it is the only viable sampling technique in obtaining information from a very specific group of people. firstly, for each category we selected the people holding the main roles in ohs management and sustainable development policies. then, we looked for the same numbers of people for each category, to form a homogeneous sample. we identified five categories : institutions, trade unions, employers organizations, businesses, research. regarding the first category, representatives belonging to institutions that play a central role in the development and management of ohs and renewable energy policies were chosen (e.g. ministry of environment, ministry of economic development, ministry of health, ministry of labour, lombardia region, lazio region, tuscany region, inail). in the case of employers and trade unions, osh experts who have a decision - making role in the development of ohs policies were identified. with respect to companies, groups of companies and professionals with expertise in the design, construction, installation and maintenance of energy production from renewable sources were selected. the sector of research is represented by members with different expertise in the field of ohs and renewable energy (e.g. engineers, occupational physicians, researchers), belonging to universities, associations and research centers for the promotion of renewable energy sources, associations for industrial research or of industrial hygienist. the 61 stakeholders from the different categories are listed in table 1table 1.survey participants grouped by categorycategorynumber of participantsinstitutions11trade unions11employers organizations13businesses11research 15total 61. the individual stakeholders were sent access codes by e - mail to enable them to complete the questionnaire on line. replies to the questionnaire were uploaded into a database in microsoft excel format and analyzed using spss version 16.0 for windows. in a preliminary analysis we calculated the response rates. for the multiple - choice questions, analysis was done on the following : valid cases (those who selected at least one response) ; valid responses (the number of times responders selected a specific answer, which could therefore be more than 100% of cases) ; and missing cases (no responses). descriptive analysis was based on the percentages in frequency tables and graphs. contingency tables were used to analyze the percentages in each category. to check for associations between the groups and the responses, we used the test to verify the significance of differences between the observed and expected or theoretical frequency distributions, i.e. those expected if there is no relation between the variables investigated. we ensured that all responses would be considered confidential in the data analysis, as required by the italian deontological code and code for good conduct in the treatment of personal data for statistical and scientific purposes (provvedimento del garante no. 2 del 16 giugno 2004, gazzetta ufficiale 14 agosto 2004, no. 190). in all, 34 questionnaires were completed, giving a response rate of 55.7% (table 2table 2.response rates grouped by categorycategoryresponses (no.)responses (%) institutions763.6trade unions763.6employers organizations646.2businesses763.6research 746.7total3455.7). the majority of responders were male, with 20.6% female ; the median age was 52 yr, with 33.3% aged between 55 and 64 yr ; just under two thirds (61.8%) reported a college degree. as regards the weight participants attached to the green economy, 73.5% considered it an important issue, 20.6% felt it was still marginal but could be expected to become more important in the short and long terms, and only 5.9% considered it altogether marginal. analysis of the results based on the stakeholder categories brought to light significant differences (p<0.05) (table 3table 3.how do you rate the importance of the green economy in italy ? categorymarginalimportance (%) still marginal butgrowing importance (%) currentimportance (%) total (%) institutions14.342.842.9100.0trade unions14.3085.7100.0employers orgs. p=0.037). among employers organizations (100%), businesses (100%) and trade unions (85.7%), the proportions of people indicating that the green economy is an important issue were larger than responders from institutions and research (42.9%). among the aspects considered fundamental for a fair and equitable transition towards a green economy, sustainable development ranked first (30.1% of responses, 82.4% of cases), followed by investment in research (29.0% of responses, 79.4% of cases). trade unions and businesses were equally interested in enhancing workers protection in the green economy (ohs issues), both with 42.9% of cases (17.6% of responses). the employers association did not consider ohs issues important for ensuring a fair and equitable transition towards the green economy (no responses) (fig. 1.aspects rated most important for ensuring a fair and equitable transition to the green economy : total sample and categories (percentage responses).). aspects rated most important for ensuring a fair and equitable transition to the green economy : total sample and categories (percentage responses). on the question of health and safety at work, one fifth of the cases (20.6%, 12.7% of responses) believed that, compared to traditional energy sources, renewable energies entailed new risks that could not be managed with current risk management approaches. however, most responders (61.8% of cases, 38.2% of responses) maintained that these new risks could in fact be managed with current procedures ; or else they thought that renewables entailed the same types of risks as other forms of energy but called for new risk management procedures (44.1% of cases, 27.3% of responses). in the first case, the percentage frequency were highest from trade unions (35.7%) and businesses (30.0%) ; in the second case employers organizations (57.1%) and research (41.7%) gave the highest percentage frequency (fig. 2.risks more likely to be present in the renewable energy sector than in traditional energy areas : total sample and categories (percentage responses).). risks more likely to be present in the renewable energy sector than in traditional energy areas : total sample and categories (percentage responses). looking at ohs risks associated with various sustainable energy sources, wind, solar photovoltaic and solar thermal energy (41.2%) entailed low health and safety risks. medium risks were identified for geothermal (47.1%), hydroelectric (44.1%) and biomass (38.2%) (fig. 3fig. 3.perception of the level of risk for workers health and safety in several renewable energy sectors.). in this case, there were no significant differences between organization types and levels of risk associated with renewable sources ; this explains the similar percentage frequency from the various organizations surveyed. perception of the level of risk for workers health and safety in several renewable energy sectors. most of the total sample70.6%believed that micro - enterprises would have more problems / difficulties in managing ohs in the green economy. as regards the adequacy of the current ohs legal framework for specific risks associated with new technologies in the green sector, 38.2% of responders claimed there were still deficiencies ; the highest proportions were from trade unions (71.4%) and institutions (57.1%). in contrast, more than 50% of employers organizations and businesses found no shortcomings. frequency analysis indicated that the best means to enhance the protection of workers health and safety in the green sector were to include ohs issues in the early planning and design of plant, materials and equipment (33.8%) and to establish ad hoc risk management systems (32.4%). in the first case, the percentage frequency was highest among businesses (42.9%) ; in the second, trade unions (35.7%), institutions (31.2%) and research (46.7%) gave the highest percentage frequency (fig. 4.main measures to be taken to safeguard the health and safety of green sector workers (percentages of responses for each category).). main measures to be taken to safeguard the health and safety of green sector workers (percentages of responses for each category). promotion of ohs culture (79.4%) and investment in education and training (70.6%) were considered the best development strategies for the enhancement of ohs in the green sector, followed by the development of good practice (55.9%), investment in research (47.1%) and incentives to enterprises (26.5%). looking closely, the institutions (85.7%), businesses (85.7%) and employers organizations (83.3%) were those who assigned high priority to promoting an ohs culture as a means of safeguarding workers conditions in green jobs. research (85.7%), the employers organizations (83.3%) and the institutions (71.4%) gave high priority to education and training for effectively safeguarding ohs in green work. the other categories trade unions and businesses also expressed interest in education and training, as shown by the fact that more than half of them responded. for good practice from the ohs viewpoint, the trade unions (71.4%) and employers organizations (66.7%) assigned the highest priority. incentives to business were given high priority from the ohs angle by employers organizations (50.0%) and trade unions (42.9%). businesses tended to consider this item much less important (14.3%) ; more than half 57.0%gave incentives medium priority, 28.6% low. the institutions and trade unions, accounting for 57.1% of responses, were the main categories that assigned high priority to research for improving the health and safety of green workers. a majority of those interviewed (82.4%, 41.2% of responses) indicated that the green skills necessary for the workforce to perform their duties adequately were technical, followed by ohs - related competence (32.4%). finally, half the responders believed that specific training programs had to be planned by public prevention institutions (such as the regions, or inail) to create adequate green skills ; the rest opted more or less in equal proportions for social dialogue and university curricula including ohs issues related to the green economy. as the possible implications of green work for workers health and safety are an emerging question still involving a certain degree of ambiguity, the present survey employed a method based on a subjective approach, questioning various stakeholders about their perceptions of ohs risk in the green sector. this can be considered the first level of analysis, to obtain information on the level of knowledge about this issue in italy. the approach based on risk perception, and the sample of stakeholders with a general understanding of ohs, but no detailed knowledge of the emerging risks in the green economy might be the main reasons why the findings differ from the results reported by other groups. many studies, for instance, found particularly critical ohs points in the photovoltaic sector30, 31 on account of the harmful chemicals employed in manufacturing the cells ; these include crystalline silica (x - si), amorphous silica (-si), cadmium telluride (cdt), copper indium sellenide (cis), gallium arsenide (gaas). then in the wind sector32, 33 there is the risk of falling from heights and exposure to resins, styrene, solvents, hazardous gases, vapors and dusts. despite these limitations, the lack of statistically significant responses (apart from the first question) by the single stakeholders indicates a certain uniformity of opinion. this brought to light quite clearly the main critical points as regards the ohs implications of green work, and pointed to the principal policies to be adopted to safeguard workers health and safety. a point in favor of the method is that the categories sampled represented various interests in ohs, enabling us to analyze the topic from several viewpoints. although the green economy is a matter of current interest, the question of health and safety in the workplace has still not been adequately examined. this is borne out by the low perception of its importance as part of the fair and equitable transition towards the green economy. italian productive, scientific and political circles are also being slow to analyze possible critical points for workers health and safety arising with the introduction of new green technologies. this might be a consequence of the stakeholders medium / low perception of the risks, especially in the renewables industry, which therefore leads them to believe they will be easily managed. probably they are not fully aware or not informed about the risks involved in each of the renewables sectors, as indicated by the number of no answers in the questionnaire. the total lack of interest in the ohs aspect of green work by employers organizations is worrying, whereas businesses and trade unions were the categories most sensitive to the topic. this confirms other investigations that have found the small italian firms most willing to invest in the green economy34. these findings highlight the need for systematic assessment of the ohs factors involved in any new technology, new product or development process, not only in the early stages but throughout its lifespan ; this comprises planning, manufacture, transport, installation, operation, maintenance, demolition and disposal, etc. it will also be essential to foster regular contact between the various disciplines and social partners, so as to ensure ohs is properly integrated into innovative and technological developments, and to generate further knowledge and new skills with a view to identifying future challenges better and the related ohs requirements. here again, the replies to this survey mirror the results of the main studies to date on this subject2, 35. a top - down approach based on consultation with the stakeholders is probably the most appropriate way to define nation - wide policies to strengthen safeguards for the health and safety of workers dealing with green technologies. the promotion of ohs culture, and education and training will play a decisive role in boosting awareness of the conditions for health and safety of green workers. to sum up, this survey indicated that one way to ensure what the ilo defines as decent work17 must involve at least three categories : research, education and training, and policy. research, for example, can identify the risks arising with the introduction of new green technologies and investigate alternative planning methods. policy - makers can develop standards on the knowledge of certain ohs features, or on partnerships between health and safety professionals, businessmen, environmentalists, workers, and any other stakeholders. | despite all the emphasis laid today on the green economy, occupational health and safety (ohs) issues have still been talked only limitedly, as already noted in previous studies and literature reviews. the department of occupational and environmental medicine, epidemiology and hygiene of the italian workers compensation authority (inail) has conducted a survey among some italian stakeholders, social partners, institutions and green businesses to gather their perceptions of the potential effects of green jobs on ohs, particularly in the renewable energy sector. the survey involved a sample of 61 stakeholders in the following categories : institutions (11), trade unions (11), employers organizations (13), businesses (11), research (15). participation in this survey of national stakeholders who have a central role in the development and management of policies on renewable energy and ohs, allowed to analyze in depth the fundamental aspects for a fair transition towards green economy. also, the good agreement among respondents brought to light quite clearly the main critical points as regards the ohs implications of green work in italy, and pointed to the principal policies to be adopted to safeguard workers health and safety. |
the maxilla and mandible are the most commonly affected bones of the facial skeleton, followed by the zygoma, the orbit, and the condyle. fifty two cases of venous malformation (vm) of the zygoma have been reported in the literature. intraosseous venous malformation (iovm) presents a rare yet unique clinical challenge to the surgeon. the keys for treatment are : (1) hemorrhage control, (2) en - bloc resection of the bony lesion including the normal bone, and (3) reconstruction after resection. because of significant hemorrhage risk, intraosseous vascular anomalies can be life - threatening entities. twenty five deaths by spontaneous hemorrhage have been reported in the literature. in these literature, preoperative angiography was performed to confirm the diagnosis followed by embolization to reduce the risk of bleeding during the operation. a good cosmetic result was achieved using autologous calvarial bone for reconstruction of the zygoma defect. a 59-year - old female presented with a 3-month history of progressive painless swelling of the left cheek computed tomography (ct) showed a round, well - defined expansile bony lesion measuring 5 cm in the left zygoma. the mass had an overall intermediate t1 signal intensity and a high t2 signal intensity on magnetic resonance imaging (mri). within the mass were areas of no signal that corresponded to the trabeculae seen on the ct study. selective bilateral external carotid artery angiograms displayed markedly hypertrophied branches of the left facial artery and left internal maxillary artery (fig. after microcatheter embolization, en - bloc resection of the zygoma including the vm was performed with a subcilial and intraoral approach to avoid postoperative facial nerve palsy caused by preauricular approach. anatomical reconstruction of the zygoma and orbital floor was performed by using a 5 5 cm split parietal calvarial bone for the zygoma and a split iliac bone for the orbital floor. histology of the affected area (hematoxylin and eosin staining) revealed thin - walled, enlarged vascular channels with a single layer of flat, quiescent endothelium between bony trabeculae. the endothelium showed no signs of proliferation, mitotic figures, atypia, or tufting, and the bony margins did not show any sign of vascular anomaly. follow - up at 6 months demonstrated no significant deformity of the left zygoma, with good contour of the midface on clinical examination (fig. ct scan at 6 month postoperatively, revealed volume maintenance and anatomical continuity of the calvarial bone grafts (fig. hemangiomas (hs) and cavernous hemangiomas (chs) are included types of iovm. the incidence of intraosseous venous malformations of the zygoma occurs in a female to male ratio of 4.5:1. in 7% of cases, preoperative arterial embolization followed by total resection of the lesion was performed. with regard to reconstruction, 40% were with autologous calvarial bone graft and 40% were with non - autologous substances, such as silicone, polyethylene, titanium plate, orhydroxyapatite paste. there was one recurrence after 6 months, and infection was reported in two cases that were reconstructed with hydroxyapatite. historically, the reporting of vascular anomalies has been misrepresented in the literature through the indiscriminate use of the term hemangioma to describe a variety of distinct vascular lesions. this ambiguity has led to misconceptions and inaccuracies regarding the appropriate diagnostic approach and therapeutic management of vascular lesions. the international society for the study of vascular anomaly (issva) classification is a reasonable classification that leads to appropriate clinical diagnosis and treatment strategies with the differentiation of vascular tumors and vm using simple, easily comprehensible nomenclature. although vms have been described differently in the who classification as well as in the prominent textbook of enzinger ans weiss, the issva classification is the most logical and clear classification system. prior nomenclatures including cavernous hemangioma, venous hemangioma, intramuscular hemangioma, and synovial hemangioma are all categorized as vm in the issva classification. the frontal bone is the most common site (31%) followed by the temporal (13%) and zygomatic bones (12%). the ct appearance most commonly shows a characteristic sharply marginated expansile lesion with intact inner and outer tables and a sunburst pattern of radiating trabeculae. soap bubble and honeycomb configurations may also occur. in the mri, vm shows low to iso - intense areas within the muscle on t1 imaging with high intensity areas on t2 imaging. in our case, the diagnosis of iovm of the zygoma was suggested on the basis of the morphologic similarity of the lesion to a calvarial hemangioma. intraoperatively, the lesions looked to be hard, blue violet domed masses lying beneath the periosteum, and the bone seemed spongy. angiography is essential not only to confirm the diagnosis, but also to aid in the preoperative management of the tumors. vms often show dilations of the vascular supply and venous drainage, pooling of contrast in ectatic vascular spaces, and shunting. because multiple arteries supply the lesion, embolization needs to be performed before the operation to prevent hemorrhage. the indications for treatment include mass effect, high risk of hemorrhage, and cosmesis. different types of treatments have been described including radiotherapy, embolization of the main afferent vessels, curettage, and resection. radiotherapy may stop the progress of the lesion but does not address the cosmetic deformity and has the risks of tissue necrosis, retardation of growth of bones and teeth, telangiectasias, and malignant degeneration. although embolization or ligation of the external carotid artery has been described, this strategy not only can lead to incomplete remission of the lesions, but also carries with it a risk of recurrence. en - bloc resection including a margin of normal bone around the lesion can decrease the recurrence and simultaneously avoid disastrous bleeding. the honeycomb appearance of these lesions on ct is the result of reactive osteoblastic bone remodeling in response to the stress created by the enlarging vm, and the lesions are continuous with the normal surrounding bone. it is important to distinguish the margins of lesion in order to resect normal bone around it. temporary facial palsy has not found in among the literature regarding with iovm of zygoma. reconstruction after resection is of great importance to preserve aesthetics and for ocular functionality of the zygoma orbital region. various reconstructive methods have been used including repair with a surgical pack, silicone implants, titanium mesh implants hydroxyapatite cement, and autologous graft with rib and iliac bone. we chose autologous parietal calvarial bone over other autologous bone grafts for primary reconstruction because the parietal curve has a similar contour to the zygomatic bone and also calvarial free bone grafts are well preserved in the recipient site, particularly in the face. experimental studies have demonstrated enhanced volume maintenance for calvarial bone as compared with other traditional bone graft sources. because the ratio of cortical bone is higher in calvarial bone than in other bone grafts, such as iliac and rib bones and its dense cortical nature limits revascularization jinz described that the use of autologous bone graft is better than using non - autologous materials like methylmethacrylate - based decalcified allograft bone or bone substitutes like hydroxyapatite cement because these can become infected or resorbed, particularly in the case of hydroxyapatite cement. historically, the reporting of vascular anomalies has been misrepresented in the literature through the indiscriminate use of the term hemangioma to describe a variety of distinct vascular lesions. this ambiguity has led to misconceptions and inaccuracies regarding the appropriate diagnostic approach and therapeutic management of vascular lesions. the international society for the study of vascular anomaly (issva) classification is a reasonable classification that leads to appropriate clinical diagnosis and treatment strategies with the differentiation of vascular tumors and vm using simple, easily comprehensible nomenclature. although vms have been described differently in the who classification as well as in the prominent textbook of enzinger ans weiss, the issva classification is the most logical and clear classification system. prior nomenclatures including cavernous hemangioma, venous hemangioma, intramuscular hemangioma, and synovial hemangioma are all categorized as vm in the issva classification. the frontal bone is the most common site (31%) followed by the temporal (13%) and zygomatic bones (12%). the ct appearance most commonly shows a characteristic sharply marginated expansile lesion with intact inner and outer tables and a sunburst pattern of radiating trabeculae., vm shows low to iso - intense areas within the muscle on t1 imaging with high intensity areas on t2 imaging. in our case, the diagnosis of iovm of the zygoma was suggested on the basis of the morphologic similarity of the lesion to a calvarial hemangioma. intraoperatively, the lesions looked to be hard, blue violet domed masses lying beneath the periosteum, and the bone seemed spongy. angiography is essential not only to confirm the diagnosis, but also to aid in the preoperative management of the tumors. vms often show dilations of the vascular supply and venous drainage, pooling of contrast in ectatic vascular spaces, and shunting. because multiple arteries supply the lesion, embolization needs to be performed before the operation to prevent hemorrhage. the indications for treatment include mass effect, high risk of hemorrhage, and cosmesis. different types of treatments have been described including radiotherapy, embolization of the main afferent vessels, curettage, and resection. radiotherapy may stop the progress of the lesion but does not address the cosmetic deformity and has the risks of tissue necrosis, retardation of growth of bones and teeth, telangiectasias, and malignant degeneration. although embolization or ligation of the external carotid artery has been described, this strategy not only can lead to incomplete remission of the lesions, but also carries with it a risk of recurrence. en - bloc resection including a margin of normal bone around the lesion can decrease the recurrence and simultaneously avoid disastrous bleeding. the honeycomb appearance of these lesions on ct is the result of reactive osteoblastic bone remodeling in response to the stress created by the enlarging vm, and the lesions are continuous with the normal surrounding bone. it is important to distinguish the margins of lesion in order to resect normal bone around it. temporary facial palsy has not found in among the literature regarding with iovm of zygoma. reconstruction after resection is of great importance to preserve aesthetics and for ocular functionality of the zygoma orbital region. various reconstructive methods have been used including repair with a surgical pack, silicone implants, titanium mesh implants hydroxyapatite cement, and autologous graft with rib and iliac bone. we chose autologous parietal calvarial bone over other autologous bone grafts for primary reconstruction because the parietal curve has a similar contour to the zygomatic bone and also calvarial free bone grafts are well preserved in the recipient site, particularly in the face. experimental studies have demonstrated enhanced volume maintenance for calvarial bone as compared with other traditional bone graft sources. because the ratio of cortical bone is higher in calvarial bone than in other bone grafts, such as iliac and rib bones and its dense cortical nature limits revascularization, the calvarial bone resorption rate is low. jinz described that the use of autologous bone graft is better than using non - autologous materials like methylmethacrylate - based decalcified allograft bone or bone substitutes like hydroxyapatite cement because these can become infected or resorbed, particularly in the case of hydroxyapatite cement. the field of vascular malformations has long been hampered by non - uniformity of language, which has frustrated attempts at research, diagnosis, and treatment. recent advances in research reveal the underlying genesis of these lesions and have led to a coherent, reliable issva classification system that facilitates uniformity in communication and efficacy of treatment. no single medical specialty in isolation can deliver high - quality care to these patients. collaboration and mutual co - operation between surgeons, radiologists, and pathologists are necessary for accurate diagnosis and management. superselective arteriography and embolization are extremely important diagnostic and therapeutic tool in arteriovenous and venous forms. the combination of surgery and embolization makes it possible to achieve definitive results. for primary reconstruction of the zygoma, the use of autologous calvarial bone can maintain the volume and reconstruct the natural malar contour via a relatively simple technique. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request. | highlightsintraosseous venous malformations of the zygoma is rare.venous malformations are often misrepresented as hemangiomas.preoperative arteriography and embolization are necessary for diagnosis and hemorrhage control during operation.en-bloc resection and primary reconstruction of the lesion is important to prevent recurrence and postoperative malar irregularity.for reconstruction, calvarial bone graft is optimal for maintaining malar contour. |
the classification of lupus nephritis (ln) was revised by the international society of nephrology and the renal pathology society (isn / rps) in 2003. the increasingly recognized phenomenon of apparent minimal change disease (mcd) and focal segmental glomerulosclerosis (fsgs) without significant immune deposits in a patient with systemic lupus erythematosus (sle) is termed as lupus podocytopathy (lp). the diagnosis of lp in a patient with sle and nephrotic syndrome is based on the finding of severe foot process effacement in the absence of peripheral capillary wall immune deposits. a 22-year - old female presented with fever, symmetrical arthritis involving small and medium joints of 3 months duration. she had oral ulcers, malar rash, and pedal edema urinanalysis revealed 3 + protein, 24 red blood cells / hpf, 12 white blood cells (wbcs)/hpf, no casts or crystals. hemogram showed hemoglobin 9.2 g / dl, total wbc count 4800/l, and platelets 2.2 lakhs/l. serum protein and albumin were 6 g / dl and 3.0 g / dl, respectively. serum c3 was 60 mg / dl (normal 90180 mg / dl), c4 - 10 mg / dl (normal 1040 mg / dl). she was admitted after 3 months for persistent edema and underwent renal biopsy that showed morphologically normal glomeruli with normal appearing capillary loops, mesangium, tubulointerstitium, and blood vessels. immunofluorescence study was negative for igg, igm, iga, c3, c1q, kappa, and lambda light chains. electron microscopy (em) showed diffuse foot process effacement with no electron dense deposits in mesangial, capillary walls, or tubulointerstitial compartment [figure 1 ]. she was started on oral prednisolone 50 mg / day, and attained complete remission in 4 weeks. electron micrograph of a portion of a glomerulus shows diffuse effacement of foot processes of podocytes. note that there are no electron dense deposits in the subepithelial, subendothelial or mesangial areas the second case is a 36-year - old male who presented with anasarca and arthritis involving small and large joints. there was no history of fever, rash, oral ulcer, photosensitivity, or hair loss. clinical examination revealed mild anemia, anasarca, hypertension, and evidence of active small joint arthritis. his creatinine was 2.63 mg / dl with serum albumin of 2.2 g / dl and cholesterol 330 mg / dl, and triglycerides of 380 mg / dl. serology was positive for ana and anti - double - stranded dna with low c3 (46 mg / dl) and borderline low c4 (8.43 mg / dl). serology was negative for rheumatoid factor, antibodies to cyclic citrullinated peptide, hepatitis b virus, hepatitis c virus and hiv as well as anti - proteinase 3 and anti - myeloperoxidase. nephrotic range proteinuria, anemia, arthritis, ana positive serositis and serology profile satisfied slicc diagnostic criteria and patient was subjected to renal biopsy. segmental sclerosis with adhesions to the bowmans capsule was seen in two glomeruli [figure 2 ]. immunofluorescence study was negative for igg, igm, iga, c3, c1q, kappa, and lambda light chains. there was diffuse foot process effacement with no electron dense deposits in the mesangial region, capillary walls or tubular compartment on em. the above features were consistent with fsgs, not otherwise specified, in a patient with sle. he was treated with pulse methylprednisolone for 3 days and iv diuretic with albumin, followed by oral steroids after 3 days 1 mg / kg body weight, with rapid resolution of edema and normalization of serum creatinine. at 2 weeks, proteinuria came down to 4 g / day and by 12 weeks he was in partial remission with < 2 g proteinuria. after 12 months follow - up, he remains in complete remission, but on 5 mg of prednisolone per day. immune complex aggregation that is thought to be a key feature of sle was conspicuously absent in our sle patients. the light microscopic findings with absence of immune deposits on immunofluorescence study and diffuse foot process effacement on em with rapid remission of nephrotic syndrome with prednisolone support the diagnosis of mcd and fsgs associated with active extra renal lupus. it is important to follow - up these cases whether this proteinuria though steroid responsive, has a potential to flare or change class to typical ln in later days. the first question remains whether this entity is a chance occurrence in sle and if it is not, to explain this by pathogenic mechanism common to sle. the presence of minimal or no capillary wall immune deposits with or without mesangial proliferation associated with nephrotic proteinuria is collectively termed as lp. in literature, so far 22 cases have been reported. the association of mcd or fsgs without immune deposits in sle is of a rarity. the chance occurrence of mcd and sle in the same individual is expected to be < 1 in 10,000. dube. in a series of seven patients with lp, reported two patients with features consistent with mcd akin to our case. reported the case of a 29-year - old sle patient with remitting and relapsing nephrotic syndrome. her renal biopsy showed normal glomeruli with effacement of foot processes compatible with mcd, which completely remitted without further relapse. kraft. evaluated a series of 18 renal biopsies from a series of 470 patients, eight with nephrotic range proteinuria and 10 from nonnephrotic proteinuria. seven of eight biopsies with nephrotic range proteinuria showed more than 80% foot process effacement while no biopsy for nonnephrotic range proteinuria had exhibited more than 20% foot process effacement. in this study, only eight biopsies fit into lp associated with nephrotic syndrome. to explain the pathogenesis, the majority of authors favor a circulating toxin hypothesis, though the exact agent has never been proved. interleukin (il)-13 has been suggested on the basis of overexpression in rats causing apparently inducing minimal change picture on em. the il-13 pathway has also been implicated to have a role in hodgkin 's lymphoma where mcd has been found in high prevalence. abnormal release of il-13 from aberrant t - cells and a cross talk between the renal dendritic cells and the th cells may directly damage the podocytes. animal studies with rats showed il-13 overexpression leading to podocyte injury with downregulation of nephrin, podocin and dystroglycan and concurrent upregulation of b7 - 1 in the glomeruli inducing mcd. t cell function plays an important role in the pathogenesis of sle by altering the expression of adhesion molecules as well as inducing b cells to produce autoantibodies. ineffective immune regulation in sle is due to a functional imbalance of t lymphocyte subsets. activated th2 subset overproduces il-6, il-10, il-13, and tumor necrosis factor- whereas th1 subset under produces il-2, interferon- and transforming growth factor-. il-10 and il-13 play an important role in th2 cell differentiation and production of autoantibodies in sle. the t - cell abnormalities in both the disorders could be the unifying pathogenic mechanism in the occurrence of mcd or fsgs in sle. in our patients, proteinuria responded to steroid therapy but one of them is still on minimal steroid dose. these patients need longer follow - up, with the need for repeat biopsy with a nephrotic or nephritic flare if any. diffuse podocyte effacement without immune complex deposition on em is the sine qua non for this entity. clinicians and renal pathologists need to be aware of this entity as a cause of nephrotic syndrome in patients with sle. importantly, this lesion should be recognized as highly steroid responsive but a vigilant follow - up is needed keeping in mind the natural history of flare and quiescence of this disease. | some patients with systemic lupus erythematosus (sle) present with sudden onset of nephrotic syndrome and biopsy findings may be of minimal change disease or focal segmental glomerulosclerosis with diffuse foot process effacement on electron microscopy but without significant immune deposits. this entity is termed lupus podocytopathy. clinicians and renal pathologists need to be aware of this condition. though steroid sensitive, it needs follow - up to recognize flare and class change, thereby optimizing therapy. |
glomerulonephritis occurring as a consequence of mrsa infection was first reported by koyama in 1995. subsequently more cases were reported that elaborated the histopathology and pathogenesis including role of staphylococcal cell envelope antigen.[24 ] however, there are only a few case reports that focus on the treatment of this entity. the roles of steroids have been controversial with regard to their indications, timing and long - term complications including relapse of infection. we report a case of post - mrsa iga dominant pign that required treatment with steroids in addition to antibiotic therapy. a 39 year - old caucasian man was transferred from an outlying facility for management of a psoas abscess diagnosed on computerized tomography (ct) scan of abdomen and pelvis. he denied any fever, trauma, tingling, numbness, bladder, or bowel disturbances. past medical history was unremarkable except for an abscess on his right forearm and right scrotal area following a tick bite which was draining a couple of days before he started having back pain. laboratory data revealed white blood cell count 33.8 thousand / mm (neutrophils count 31,900/mm and bands 24%), hemoglobin 11.9 gm / dl, sodium 124 mmol / l, potassium 4.5 mmol / l, blood urea nitrogen 24 mg / dl, creatinine 1.1 mg / dl, total protein 7.3 gm / dl, albumin 1.5 gm / dl. urinalysis was significant for small amount of blood, red blood cells (8/hpf), white blood cells (10/hpf) and occasional white blood cell casts. urine color, specific gravity and ph were within normal limits. a ct scan of the abdomen without contrast done at an outlying facility revealed possible psoas abscess and left renal abscess [figure 1 ]. magnetic resonance imaging (mri) of the lumbar spine confirmed the findings including multiple abscesses in the psoas muscles bilaterally and spondylitis involving l1l4. ct guided biopsy of the left paravertebral mass as well as blood culture grew mrsa. patient was started on vancomycin and levofloxacin with a plan to continue these antibiotics for at least 46 weeks. ct scan of abdomen with contrast showing bilateral psoas abscesses during the hospital course, his condition started improving clinically ; however his renal function started deteriorating and he developed acute renal failure. his blood urea nitrogen and creatinine increased from a baseline of 12 mg / dl and 1.1 mg / dl, respectively, to 24 and 2.4 mg / dl within 20 days of hospitalization and these values continued to increase. within the next 2 weeks his blood urea nitrogen and creatinine were increased to 43 and 7.4 mg / dl, respectively. vancomycin and levofloxacin were stopped and the patient was started on linezolid, which also was stopped after six days as the patient developed an apparent allergic skin reaction. serum creatinine time course and antibiotics administered during the hospital stay during this time period patient was evaluated for the cause of acute renal failure. his urine analysis at this point revealed red blood cells 56/hpf, a few dysmorphic red blood cells, wbcs < 1/hpf, no eosinophils, proteinuria with urine protein to creatinine ratio 2.94, fractional excretion of sodium 7.67, and fractional excretion of urea 124.5 ; urine microscopic exam revealed few muddy brown casts. considering the possibility of acute glomerulonephritis, the patient underwent ct guided biopsy of the right kidney, which revealed iga dominant pign with extensive acute tubular injury and acute interstitial nephritis. light microscopy showed glomeruli with mild to moderate increase in the mesangial matrix and cellularity, with cellular crescents in 2 of the 3 glomeruli sampled, and fibrinoid necrosis in one of these. in addition, rare hump shaped deposits were visualized overlying the glomerular basement membrane in the capillary notch area [figure 3a ]. correspondingly, immunofluorescence (if) microscopy showed 2 + mesangial and segmental capillary loop staining for iga and c3 while igg was negative [figure 3b ]. electron microscopy showed scattered mesangial deposits and rare subepithelial hump type deposits [figure 3c ]. the biopsy also showed acute tubular injury and a hypersensitivity drug induced acute interstitial nephritis, with widespread tubultitis and interstitial nephritis in areas away from the crescents. (a) glomerulus with small cellular crescent, fibrinoid necrosis (top) and small hump - shaped deposit (arrow) (jones silver stain, 400). (b) immunofluorescence showed 2 + iga predominantly mesangial and segmental capillary wall staining (anti - iga if, 400). (c) by electron microscopy, rare hump - type subepithelial deposits were seen, protruding out from the basement membrane, in addition to scattered mesangial deposits (transmission electron microscopy, 10,000) the patient received pulse doses of steroids followed by oral steroids. his creatinine at the onset of the steroid therapy was 5.5 mg / dl, which improved after 2 weeks of treatment, at which point he was removed off hemodialysis. on follow up at three months and at one year, his creatinine was 1.7 and 1.3 mg / dl, respectively. post - mrsa infection glomerulonephritis has been well documented in japan. however, as the incidence of mrsa infection increases globally and the association between mrsa infection and iga dominant glomerulonephritis is now clear, more cases are being reported from all countries including the united states. clinical manifestations of pign following mrsa infection include acute renal failure developing within 410 weeks after the onset of infection. kidney biopsy should be considered in all patients with arf and documented infection when pign is suspected to confirm the diagnosis and determine the need for steroid therapy especially when renal function does not improve with antibiotic treatment as patients with presence of crescents on renal biopsy usually require treatment with steroids. it may be difficult to differentiate pign from iga nephropathy. in classic acute pign cases, there is an acute exudative proliferative response with frequent polymorphonucler leucocytes (pmns) infiltrating the glomeruli. in more chronic cases, electron microscopy can then be helpful in demonstrating the typical subepithelial humps characteristic of postinfectious glomerulonephritis, contrasting the lack of such deposits in usual iga nephropathy. the coexisting ain may also have contributed to renal dysfunction, as in our patient. in such instances it is always difficult to quantify the response obtained in terms of improvement in renal function with respect to different disease processes affecting the kidneys. there have been case reports of successful treatment of pign following mrsa infection with antimicrobial agents only. a study done in japan included eight patients ; six of which were treated with antibiotics only and the rest of the patients received steroids. the patients who received steroids only had poor outcome secondary to relapse of the infection. two of the six patients who received steroid therapy were diagnosed after five months and three months of their presentations, respectively, and were not on antibiotic treatment when they received steroid therapy. as these patients developed glomerulonephritis a few months after the initial infection therefore, these cases do not reflect the refractory cases that fail to respond to antimicrobial agents only. on the other hand, there are several case reports of successful treatment of resistant cases of pign following mrsa infection with steroids without any relapse of the infection. the role of steroids in these patients could be explained by the pathogenesis of pign, which involves the interaction of the host immune system with bacterial superantigen. based on our experience, we would like to suggest a possible role of steroid therapy in pign who fail to respond to antibiotic therapy alone and in patients with crescentic form of pign. it is also possible that the presence of ain contributed to steroid responsiveness in our patient. the benefits of steroid therapy in terms of impact on quality of life may be considered significant as it may alleviate the need for long - term hemodialysis. however, more studies are needed to define the role of steroids and compare the benefits with the risks associated with such therapy. | the role of steroids in treatment of postinfectious glomerulonephritis (pign) has been controversial. the reason for such controversy is the risk of infection relapse associated with steroid therapy. steroids may have a place in the treatment of resistant cases where renal function does not improve despite aggressive antibiotic therapy as well as in patients with crescentic form of pign. we report a case of a 39 year - old caucasian man who was diagnosed with methicillin - resistant staphylococcus aureus (mrsa) bacteremia resulting in acute iga dominant pign that failed to respond to antibiotic treatment alone, but responded significantly to steroids in addition to antibiotics. this anecdotal experience suggests that steroids could be considered in conjunction with antibiotic therapy for the treatment of refractory cases of pign or crescentic form of pign. more studies with long - term follow - up of patients treated with steroids in addition to antimicrobial agents are required to quantify the risk of infection relapse with steroid therapy. |
congenital hypothyroidism causes mental retardation and has irreversible consequences if not treated soon after birth. routine testing of newborns allows a rapid diagnostic and permits to initiate a therapeutic intervention, th supplementation, which usually restores most cognitive functions. however, iodine deficiency is still considered to be one of the most frequent causes of preventable mental retardation in children worldwide [2, 3 ], prompting to reinforce salt iodization in all countries. upon thyrotropin (tsh) stimulation, thyroid gland it is usual to assume that measuring tsh, t4, and t3 levels in blood is sufficient to estimate t3 level in brain. furthermore, even after the onset of the fetal thyroid gland function, which occurs only at midgestation in humans, fetal brain relies on maternal th. the predominant function of transport across the brain - blood barrier has been shown by using pregnant female rats, depleted of both maternal and fetal t4 and t3 [6, 7 ]. in these conditions, administration of excess of t4 during the second half of gestation increases t4 and t3 levels in the maternal serum, and restores the t3 level in both fetal liver and fetal cortex. by contrast, maternal administration of t3 leads to a similar increase in t3 level for the fetal liver, implying its transport through the placenta, but to partial restoration of t3 level in fetal cortex. this confirms the importance of differential t4 and t3 transport, and outlines the requirement of local conversion of t4 by deiodination, performed by type 2 deiodinase (d2), which is already present in glial cells at fetal stage. it has been calculated that, in physiological situation, 80% of t3 in rodent brain might be produced by local deiodination of t4. the differential transport of t4 and t3 has an important consequence : maternal hypothyroxinemia, that is, low t4 level in maternal serum with t3 and tsh levels within normal range, is a cause of neurodevelopmental disorders. even mild maternal hypothyroxinemia was recently found to be associated with a higher risk of delay in the onset of expressive language of children. this situation is much more frequent than overt hypothyroidism in geographical areas with low iodine uptake. reporter mice have been made, in which lacz expression is controlled by a gal4-tr1 artificial fusion protein, to visualize t3 signaling in brain. second t3 signaling is highly heterogeneous, and large variations of reporter gene expression are observed depending on brain areas. this signaling pattern in adults is well correlated with t3 distribution measured in various brain areas [10, 11 ], suggesting that local t3 concentration is the main determinant of signaling and that unknown mechanisms maintain an heterogeneous distribution of t3 in brain. a number of transporters have been identified, which might participate to the transfer of th to the brain. however, knockout of individual transporters does not seem to affect neurodevelopment, suggesting a cooperation between several transporters. one transporter that received considerable attention is the monocarboxylate transporter 8, encoded by an x - linked mct8 (slc16a2) gene. human mutations of mct8 increase rather than suppress th circulating levels but have dramatic consequences on neurodevelopment, therefore suggesting a predominant function of mct8 for th transport across the brain - blood barrier. however, the knockout of mct8 does not have any obvious influence on mouse brain development and the reasons for this discrepancy are unclear. two alternative possibilities can be proposed : the first would be that human mct8 is able to transport not only th but also some unknown signaling molecule required for proper brain development. this hypothesis is consistent with the clinical observations of patients with mct8 mutations, which display a syndrome that is apparently distinct and more severe that congenital hypothyroidism. the other possible explanation for the mild consequences of mct8 mutation in mice could be that other transporters, for example, the oatp14, lat1, and lat2 transporters, are present at earlier stages in the rodent brain, and can compensate for mct8 deficiency in this species [1416 ]. knocking - out the dio2 gene, which encodes d2, allowed to precisely address the function of t4 deiodination during fetal brain development, but led to surprisingly mild phenotypic alterations. no major change in t3 level the reduction of t3 level at postnatal day 15 (p15) was only 50% in cerebellum and hypothalamus, and not significant in cortex [17, 18 ]. gene expression analysis and neurobehavior testing confirmed a very mild neurodevelopmental phenotype. crossing with findt3 reporter mice indicated that the spatial pattern of th signaling was not obviously affected throughout development (l. quignodon unpublished data). type 1 deiodinase (d1 encoded by dio1) is the other enzyme able to convert t4 into t3, and is mainly expressed in liver. surprisingly, elimination of both dio1 and dio2 did not aggravate the neurobehavioral phenotype. although dio1/dio2 combined knockout increased t4 level, t3 level was maintained in serum and adult brain. dio2 knockout was also combined to mct8 knockout [19, 20 ]. for a set of genes that are positively regulated by t3 in cortex this suggests that t3 is produced by local deiodination of t4, or directly transported by mct8, and that blocking both pathways leads to a status close to hypothyroidism in cortex. looking at genes that are found to be upregulated in hypothyroid cortex revealed, however, that the situation is more complex, as, for these negatively regulated genes, only dio2 knockout had a influence on expression level. since the molecular mechanism underlying negative regulation of gene expression by t3 is unknown, this observation is difficult to explain, but probably suggests that the entry route of t3 in brain is somehow an important information to define individual gene regulation status. the third known deiodinase, type 3 deiodinase (d3, encoded by dio3), is responsible for t3 catabolism and is expressed in several brain areas. as dio3 gene expression in brain is upregulated in case of hyperthyroidism, local t3 catabolism can act as a safety pathway to protect brain against excess of t3. this revealed a slow accumulation of t3 in the anterior cortex, indicating that catabolism is required to regulate t3 level in adult cortex. dio3 knockout did not eliminate the spatial heterogeneity of t3 signaling, at early and late stages, ruling out a major function of d3 in defining the spatial distribution of t3 in brain. in adult hypothalamus, local control of t3 level by deiodinases appears to be used to sense and react rapidly to external signals. local th metabolism participate to hypothalamic response to feeding status, inflammation, seasonal change [25, 26 ], and trh feedback regulation. this example might prompt to reexamine the possibility that rapid metabolism could also influence local environment during development. in conclusion, t3 signaling level greatly varies in brain, depending on brain areas, cell type, and developmental stage and measuring th levels in blood is poorly informative. although the system generating this heterogeneity is not understood, it appears as a very robust and insensitive to genetic mutation affecting th transport and metabolism. the thra and thrb genes encode a number of isoforms [28, 29 ]. among these only tr1, tr1, and tr2 are considered as t3-dependent activators of nuclear transcription. tr1 mrna is ubiquitous in brain, both in neuronal and glial cell types whereas tr1 expression is more restricted with prominent expression in zones of neuroblasts proliferation such as the germinal trigone and the cortical ventricular zone. tr2 expression in brain is limited to the developing hippocampus and striatum [3032 ]. to more precisely address thra expression pattern, a mouse strain that expresses tr1 and green fluorescent protein (gfp) as a chimeric protein from the thra locus has been recently created by homologous recombination. immunocytochemistry against gfp confirmed a broad expression, but also indicates important variations in expression level. although very reliable, this method is probably of limited sensitivity, and absence of detection should not be taken as a proof for absence of expression. for example, gfp is detected first at embryonic day 13.5 (e13.5) whereas in situ hybridization indicates earlier expression. one interesting case is the purkinje neurons in cerebellum, in which tr1 expression decreases overtime while tr1 gradually increases, suggesting a series of postnatal maturation events enrolling the two receptors sequentially. due to the difficulty to detect endogenous proteins by western blotting or immunocytochemistry, thra and thrb expression studies in brain this is regrettable because tr1 mrna can encode several proteins, beside the canonical receptor. among these, the p43 protein is translated from a downstream aug codon, providing a 43 kda protein lacking the n - terminus of the tr1 canonical receptor. this p43 isoform has been proposed to be targeted to mitochondria and to regulate mitochondrial genome transcription, providing a possible complement to the well - known nuclear activation of genes encoding mitochondrial enzymes which is relevant to neurodevelopment [39, 40 ]. the involvement in neurodevelopment of p43, as well as other so - called nongenomic pathways for t3 action would merit specific investigation. t3 is also a precursor of thyronamines, which are present in brain, and activate a different class of membranes receptors also expressed in brain, but which neurodevelopmental function has not yet been evaluated. thra knockout has no visible consequence on cerebellum, a posterior brain structure which postnatal development is highly sensitive to th deficiency, and is the most classical model to study congenital hypothyroidism in rodents. it induces increased anxiety in adults, which probably involves hippocampus function, but does not necessarily reflect a developmental defect. thrb knockout affects hearing and vision but not the central nervous system development. no developmental defect has been reported after combining thra and thrb knockout [46, 47 ]. the inability of thra / thrb knockout to phenocopy congenital hypothyroidism was explained by depleting thra knockout newborns of t3 by potassium perchlorate and 1-methyl-2-mercaptoimidazole treatment. in the cerebellum of these mice, the persistence of the external granular layer, a typical sign of congenital hypothyroidism normally induced by t3 depletion, was absent. this strongly suggests that the manifestations of congenital hypothyroidism in brain are mainly due to the presence of unliganded tr1, which is bound to dna and represses transcription. knockin mutations in thra have been produced to introduce point mutations in the tr1 reading frame and compromise its ability to transactivate upon t3 binding, without impairing its ability to repress transcription. as expected, such mutations, unlike the thra knockout, lead to a cerebellum phenotype resembling congenital hypothyroidism [49, 50 ], and these defects are not limited to cerebellum [51, 52 ]. whether tr1 is the only active t3 receptor during cerebellum development remains unclear. one thrb knockin mutation also produces a cerebellum phenotype resembling hypothyroidism, with some important differences, perhaps due to the associated increase in circulating th levels [53, 54 ]. when given to hypothyroid pups, tr selective ligands seem to have an effect limited to purkinje neurons [48, 55 ]. however, the action of t3 on in vitro purkinje cells dendritogenesis appears to be depending on tr1 rather that tr1. finally, many human germline mutations have been reported in the human thrb, but not thra, gene, responsible for a syndrome of resistance to th. although these mutations can be a cause of iq deficit, they do not have major neurological consequences. most detailed studies are focused on cerebellum postnatal development in rodents, where each cell type is sensitive in some respect to th deficiency : the inward migration of the granular cell precursors (gcps) present in the granular layer cells (egl) is inhibited, the development of the dendritic arborization of purkinje neurons is impaired, and the maturation of the gabaergic interneurons is delayed. the proliferation and differentiation of glial cells, including astrocytes [60, 61 ], oligodendrocyte precursors, and microglia are affected. the morphology of bergmann glia, a specific type of radial glia found only in cerebellum, is abnormal. the diversity of these effects raises several possibilities for the mode of action of t3. the first would be that the repertoire of t3 target genes is completely different in different cell types, and that genetic programs governing cell migration, cell proliferation, and cytological maturation are regulated in a cell - specific manner. in this case, the chromatin status in a given cell type would exert a predominant influence to define the repertoire of tr target genes. the alternative would be that t3 exerts similar effects in different cell types, controlling a shared repertoire of tr target genes, but that this cell - autonomous response would represent only a small fraction of the observed effects of t3. th deficiency is known to affect the level of several neurotrophic and growth factors in brain, which are required for proper development [65, 66 ]. cell culture can be used to distinguish between these two possibilities, and to address the cell autonomous response of purified or enriched cell populations to t3, isolated from their natural environment. purkinje cells, oligodendrocyte precursors, and astrocytes were all shown to respond to in vitro stimulation by t3. by contrast, impaired migration of gcps, which is a main sign of congenital hypothyroidism, is possibly an indirect effect for several reasons. second, microarray analysis, performed on primary cultures of gcps, identified very limited changes in gene expression after t3 exposure, and failed to establish a direct link between t3 and known mechanism of neuronal migration. third, according to in situ hybridization, gcps do not express thrb, but their migration is affected by the tr mutation. finally, gcps do not produce the t3-regulated neurotrophic factors (neurotrophin 3, insulin - like growth factor-1, sonic hedgehog, and brain - derived neurotrophic factor) but need these neurotrophic factors for their proliferation, inward migration, and differentiation [7072 ]. as purkinje cells are the main source of sonic hedgehog and insulin - like growth factor-1, they probably play a central role in a local network of cellular interactions. however, if this local effect is plausible, a more indirect, systemic, influence of t3 can not be ruled out. in that respect, it is remarkable that a metabolic disease, which is clearly restricted to the liver, has been found to impact gcps inward migration. all these observations suggest that gcps migration, which is required to give rise to more than 90% of the cerebellum neurons, does not result from a cell autonomous action of t3. a direct genetic demonstration of this hypothesis would be possible using the cre / loxp technology, for which thra and thrb alleles are available. it is striking that genome - wide analysis performed by a number of groups over the years, using a variety of protocols, identified only few direct t3 targets in neural cell types, defined as genes for which transcription is under the direct control of tr1 or tr1 bound to neighboring regulatory sequences. microarray analysis showed that in vivo t3 treatment has a very limited effect on transcriptome of whole cerebellum [7476 ], or fetal cortex, compared to adult striatum or other tissues. comparison between these studies also suggests that t3 response is very different in different brain areas and at different developmental stages. the effect of graded degrees of th deficiency, induced by propyl - thio - uracyl, on gene expression has been studied in postnatal hippocampus and cortex. interestingly, transcriptome is affected in a dose - dependent manner, specific gene clusters being sensitive to only mild hypothyroidism. whereas many putative t3 target genes have been listed in these broad surveys, most changes are not rapidly reversed when t3 is given to hypothyroid animals, and might reflect changes in the cell composition of the considered area, or in the differentiation status or the cell types. this is probably the case, for example, for genes expressed in mature oligodendrocytes, as differentiation of this cell type is known to be regulated by t3. the actual demonstration of direct regulation by t3/tr requires deeper investigations, which have been done in only few cases. first, transcriptional regulation in a primary culture system, where environmental parameters can be controlled, can be used to reinforce the hypothesis for a direct regulation. second, transient expression assays can be used to test the ability of gene promoter regions to confer t3 responsiveness to reporter plasmid constructs. however, this approach is often of little physiological relevance and is hardly feasible in appropriate cell types. finally, chromatin immunoprecipitation, now considered as a gold standard, can demonstrate actual binding by tr1 or tr1 on putative response elements in their natural genomic environment. this method appears to be much more reliable than in vitro protein / dna interaction assays, which do not account for the complexity of the cellular chromatin context but remains technically challenging for several reasons. first, data obtained in other systems suggest that the distance between tr binding sites and transcription start site can be very large, making it difficult to make a selection among many putative response elements, and to ensure that tr occupancy is responsible for the observed regulation. second, it is hardly feasible to perform such studies on purified cerebellar cell populations. preparing chromatin from whole cerebellum mainly addresses chromatin occupancy in granular neurons, which represent a large majority of the cerebellum cells. one attempt has been made to identify new t3 target genes by the so - called chip - on - chip high - throughput analysis. ironically, hairless (hr), encoding a transcriptional corepressor of several nuclear receptors, continues to be the best - characterized t3 target gene in neurons, fifteen years after its identification as a t3 target gene in rat postnatal cerebellum. its expression is rapidly induced in a number of neuronal cell types, and tr occupancy in the promoter region has been confirmed by chromatin immunoprecipitation. among other likely tr1/tr1 direct target genes in neurodevelopment, are genes encoding a krppel - like transcription factor (klf9), a poorly studied synaptotagmin - related gene (srg1), a kinase (prka) anchor protein 1 (akap1), neurogranin (rc3/nrgn), and, in the fetal cortex, ca2+/calmodulin - dependent protein kinase iv (camk4). the paucity in well - characterized t3 target genes in brain mainly stems from the high cellular heterogeneity of neural tissues, and the lack of suitable in vitro systems. it remains a bottleneck for the analysis of the mechanisms underlying the neurodevelopmental function of t3. the above considerations have significant consequences for a matter of growing public concern : a number of chemicals found at low concentration in the water and food are suspected to exert a chronic toxicity by interfering with t3 signaling, and might thus be considered as putatively neurotoxic. the main suspected compounds are bisphenol a flame retardants (tetra - bromo - bisphenol a, polybrominated diphenyl ethers) and the persistent polychlorobiphenyls (pcbs). acute exposure of rats to these compounds can lower or raise the circulating level of th. xenopus tadpole metamorphosis, which is fully dependent on th, provides a basis for an in vivo assay recognized by the organisation for cooperation and development (oecd) to define th disruptors. the recently developed assay, based on transgenic xenopus tadpoles is a promising alternative, as it is rapid, and might better address tr1 function. in some cases, transactivation assays performed in transfected cell lines, or primary cultures of neural cells, confirm a possible direct influence of chemicals on t3 signaling. although some compounds, like coplanar pcbs, display some structural similarities with t3, they do not seem to fit into the t3 binding pocket and are thus unlikely to act as high - affinity tr1/tr1 ligands. recent data suggests, however, noncompetitive binding to tr, preventing interactions with dna. in most cases, a systemic influence on t3 metabolism and transport can account for most of the observed in vivo toxicity after acute exposure. few experiments address the possibility that th disruptors can interfere with the neurodevelopmental function of t3, although significant effects on gene expression in brain have been reported after exposure to a high dose of pcbs [95, 96 ] or bisphenol a. one could argue that the powerful compensatory mechanisms, revealed by the knockout of genes encoding transporters and deiodinases, would be sufficient to maintain a stable level of t3 in brain in most cases. chronic exposure to a low dose of chemicals, acting on t3 transport and metabolism, would thus be unlikely to have neurotoxic consequences, unless other signaling pathways are involved. in fact, t3 level in brain is maintained in case of mild th deficiency, a condition which already requires exposure to high doses of th disruptors. by contrast, the results of thra knockin mutations suggest that exposure to any chemical that would act as a tr1 antagonist would be expected to significantly impair neurodevelopment. if such a hypothetical chemical was tr1 selective, it would not change the circulating level of th in serum, as this regulation is mainly exerted by tr1 and tr2. defining the neural cell types that respond in a cell autonomous manner to t3 during neurodevelopment, and then identifying direct tr1 target genes in these cells, appears therefore as a prerequisite to the definition of relevant endpoints for future toxicity assay development. recent advances in our understanding of the neurodevelopmental function of t3 heavily rely on mouse genetics. a complex landscape in which t3 probably exerts a number of different functions in different cell types has been uncovered. coordinating the network of interactions, mediated by direct contacts and exchanges of diffusible factors, t3 synchronizes neuronal and glial cells differentiation to ensure the onset of functional neuronal structures. whereas the transport and metabolism of t3 is a robust system, which enables to maintain t3 level within physiological range in many situations, signal transduction in the developing brain mainly relies on the tr1 isoform. single amino - acid substitutions can have dramatic consequences on neurodevelopment and phenocopy congenital hypothyroidism. this is probably the reason why no thra germline mutation has been reported, and why single nucleotide polymorphism in the human locus are only found in noncoding sequences. | thyroid hormones (th, including the prohormone thyroxine (t4) and its active deiodinated derivative 3,3,5-triiodo - l - thyronine (t3)) are important regulators of vertebrates neurodevelopment. specific transporters and deiodinases are required to ensure t3 access to the developing brain. t3 activates a number of differentiation processes in neuronal and glial cell types by binding to nuclear receptors, acting directly on transcription. only few t3 target genes are currently known. deeper investigations are urgently needed, considering that some chemicals present in food are believed to interfere with t3 signaling with putative neurotoxic consequences. |
radiotherapy is an important therapeutic modality in the treatment of advanced (uicc stage iii and iva / ivb) head neck squamous cell carcinoma (hnscc) in curative intent and is used either as adjuvant or primary treatment modality. it has been proven that concomitant chemotherapy improves overall survival as well as loco - regional control both in the primary and in the adjuvant situation (bauchaud. ; browman. 2001 ; cooper. 2004 ; el - sayed and nelson 1996 ; pignon. common regimens besides several others are cisplatin 100 mg / m on days 1, 22, and 43 of radiotherapy (rades. 2008) ; 40 mg / m weekly during radiotherapy (geeta. 2006), or 20 mg / m for 4 days in week 1 and 5 of radiotherapy (lau.. a relatively infrequent used regimen in radiochemotherapy for head and neck cancer is the use of daily low dose cisplatin, which includes the administration of cisplatin 6 mg / m on each radiotherapy day. however, this application form is well established in the treatment of locally advanced non small cell lung cancers with primary radiochemotherapy (pradier. 2005 ; schaake - koning. 1992 ; semrau. 2007 ; takiguchi. 2005). the experiences obtained from these studies regarding the treatment of nsclc show that good treatment outcome with acceptable toxicity can be achieved with low dose cisplatin compared to radiotherapy alone. so far, there are only few reports on efficacy and toxicity of low dose cisplatin in radiochemotherapy for advanced hnscc (hoebers. 2007 ; jeremic. 2004 ; jeremic and milicic 2008). therefore, the purpose of this study was to evaluate the toxicity in patients treated with this regimen for locally advanced hnscc either in the primary or adjuvant situation in our department. from october 2003 to october 2006, all stage iii / iva / b hnscc patients treated in our department (primary or adjuvant) were designated to receive concomitant cisplatin 6 mg / m on each radiotherapy day. no other cisplatin regimens were used in our department during the respective time period as patients, who were not able to receive cisplatin due to a reduced creatinine clearance (60 ml / min), which was determined in every case before starting treatment, were either selected to receive radiotherapy alone or radiotherapy plus cetuximab in the primary situation. in summary, 50 patients with locally advanced hnscc received concomitant radiochemotherapy with cisplatin 6 mg / m on each radiotherapy day in the respective time period and therewith qualified for inclusion in the presented study. all tumors were histologically determined as squamous cell carcinoma (41 histologic grade 2 and 9 grade 3). forty - eight patients were males and two females, patients age ranged from 43 to 80 years (median 61 years). tumors were localized as follows : oral cavity (6), oropharynx (29), hypopharynx (7), and larynx (8). disease was staged according to the union internationale contre le cancer / american joint committee on cancer (uicc / ajcc) criteria. eight patients were in stage iii, 35 patients were in stage iva, and 7 patients were in stage ivb. thereby, in 2 patients the primary tumor was staged as t1, in 7 patients as t2, in 15 patients as t3, and in 26 patients as t4. in summary, 43 patients presented with histologically proven positive cervical lymph nodes (6 patients n1, 30 patients n2, and 7 patients n3). one day before the start of radiochemotherapy, the hemoglobin level was determined in each patient, and its median was 8.9 mmol / l (13.9 g / dl) [range 611.5 mmol / l (9.317.9 g / dl) ]. four male patients presented with an anemia grade 1 according to the ctc score before radiochemotherapy. pretreatment characteristics of patients entered in study are concluded in table 1.table 1pretreatment characteristics of patients entered in studycharacteristicno. patients (%) gender male48 (96) female2 (4)tumor localization oral cavity6 (12) oropharynx29 (58) hypopharynx7 (14) larynx8 (16)stage iii8 (16) iva35 (70) ivb7 (14)histologic grade 10 (0) 241 (82) 39 (18)hemoglobin level before treatment > 8.32 mmol / l (> 13.9 g / dl)23 (54) < 8.32 mmol / l (< 13.9 g / dl)27 (46)surgery yes38 (76) no12 (24) pretreatment characteristics of patients entered in study initial examinations before treatment included medical history, clinical ent (ear - nose - throat) examination (magnifying laryngoscopy, upper bronchoscopy, esophagoscopy, ear - nose - throat endoscopy) with biopsies in potential mucosal primary sites, complete blood counts, biochemical analysis including creatinine clearance, electrocardiogram, chest x - rays, abdominal ultrasound, and ct scans of the thorax and the head and neck with contrast medium. a total of 32 patients underwent curative surgery as a primary treatment followed by adjuvant radiochemotherapy up to a total dose of 64 gy. twenty - five of these patients had histologically proven involved lymph nodes ; extracapsular extension of lymph nodes was detected in six of these patients. eighteen inoperable patients were treated with primary radiochemotherapy to a total dose of 70 gy. thereby, all patients received conventional fractionated 3-d conformal external beam radiotherapy (2 gy per fraction, five times a week, no alternative fractionation schemes like hyperfractionation were applied in our patient population). the first phase delivered a dose of 50 gy to the primary tumor and associated nodal drainage sites. subsequently, a boost was applied : in the primary setting, the boost included the primary tumor and macroscopic involved lymph node areas to a total dose of 70 gy. in the adjuvant situation, the boost was applied to the primary tumor region and tributary lymph node regions including such lymph nodes with extranodal spread to a total dose of 64 gy. treatment was delivered with a varian clinac 600 c / d accelerator (varian, palo alto, ca, usa). the prescribed dose was defined in accordance with the international commission on radiation units and measurement report (international commission on radiation units and measurements prescribing, recording and reporting photon beam therapy. simultaneous chemotherapy was given as follows to all patients in the study : intravenous infusion of cisplatin 6 mg / m was combined with 1l nacl with facultative antiemetic medication on every radiotherapy day. toxicity was monitored weekly during radiochemotherapy and every second week following therapy until disappearance of acute toxicity. 2000) and according to the lent scoring system for chronic toxicity (rubin. after radiochemotherapy, remission was evaluated by clinical ent - examination (ear - nose - throat endoscopy, magnifying laryngoscopy, whenever necessary upper bronchoscopy, and esophagoscopy) and a computed tomography with contrast medium. afterwards, patients underwent quarterly clinical ent - examination, complete blood counts, biochemical analysis, chest x - rays, abdominal ultrasound or a computed tomography of the head and neck, if necessary. meier product - limit method was used to determine overall survival and loco - regional control. loco - regional control was defined as the absence of local or regional recurrence or progression (kaplan and meier 1958). the impact of possible prognostic factors was estimated by univariate analysis (log - rank test) for gender, patient age, primary site, tumor stage, histological grading, surgery, and preradiotherapeutic hemoglobin levels. acute grade 3 sole mucositis / dysphagia was seen in 11 patients (22%), sole hematologic toxicity grade 3 occurred in 7 patients (14%) [5 leukopenia (3 of these patients with grade 4 toxicity), 1 patient thrombopenia grade 3 and one patient leukopenia and additionally anemia grade 3 ]. in another 4 patients (8%) mucositis / dysphagia and hematologic toxicity grade 3 (2 leukopenia and 2 leukopenia and thrombopenia) was observed. overall, 94% of our patients received the intended dose of radiotherapy (in 2 patients receiving adjuvant treatment radiotherapy dose was reduced from 64 to 60 gy and from 64 to 62 gy, respectively and in one patient receiving primary radiochemotherapy from 70 to 62 gy). more than 80% of the intended cumulative chemotherapy dose could be applied in 90% of our patients (chemotherapy break for more than one week was necessary in 8 patients, only). during follow - up, high grade chronic toxicity (grade 3) was also infrequent and occurred in nine patients (18%), only (3 patients xerostomia, 2 patients subcutaneous fibrosis, 2 patients lymphedema, and 2 patients subcutaneous fibrosis and lymphedema, respectively). all patients, who underwent surgery in curative intent, were at least macroscopically completely resected (28 patients r0-resection, 4 patients r1-resection). complete remission after primary radiochemotherapy was seen in 12/18 patients (66%). in the other six patients, the median duration of follow - up was 24.2 months (range, 7.848.7 months). death has occurred in 12 patients (24%) ; 9 of these died from tumor, and 3 died from intercurrent disease (secondary primary tumor, pulmonary embolism and one not known). collectively, the 2- and 3-year overall survival rates were 72.7 and 67.1%, respectively. loco - regional relapse (recurrence after complete or progress after partial remission) occurred in ten patients (in 7 patients after adjuvant and 3 patients after primary radiochemotherapy) ; the median time to relapse was 9 months (range 0.813.9). in nine patients, the initial site of relapse was local and in one patient regional. distant metastases occurred in two patients (one hepatic and one pulmonal), in both cases not associated with a loco - regional recurrence. collectively, the 2- and 3-year loco - regional control rates were 78%. to evaluate the prognostic value of individual factors, univariate subgroup analyses concerning gender, patient age, primary site, tumor stage, histological grading, surgery, and preradiotherapeutic hemoglobin levels have been done. subgroup analysis of tumor stage (stage iii versus stage iva / b) showed that disease stage iii at the time of diagnosis is associated with a statistical significant (p = 0.02) higher 2- and 3-year year overall survival compared to the patients staged iva / b (100 vs. 65.3%, and 100 vs. 57%, respectively). in contrast, subgroup analyses concerning gender, patient age, primary site, histological grading, surgery, and preradiotherapeutic hemoglobin levels showed no statistically significant influence on overall survival or loco - regional control. still, this may be due to the small sample size. acute grade 3 sole mucositis / dysphagia was seen in 11 patients (22%), sole hematologic toxicity grade 3 occurred in 7 patients (14%) [5 leukopenia (3 of these patients with grade 4 toxicity), 1 patient thrombopenia grade 3 and one patient leukopenia and additionally anemia grade 3 ]. in another 4 patients (8%) mucositis / dysphagia and hematologic toxicity grade 3 (2 leukopenia and 2 leukopenia and thrombopenia) was observed. overall, 94% of our patients received the intended dose of radiotherapy (in 2 patients receiving adjuvant treatment radiotherapy dose was reduced from 64 to 60 gy and from 64 to 62 gy, respectively and in one patient receiving primary radiochemotherapy from 70 to 62 gy). more than 80% of the intended cumulative chemotherapy dose could be applied in 90% of our patients (chemotherapy break for more than one week was necessary in 8 patients, only). during follow - up, high grade chronic toxicity (grade 3) was also infrequent and occurred in nine patients (18%), only (3 patients xerostomia, 2 patients subcutaneous fibrosis, 2 patients lymphedema, and 2 patients subcutaneous fibrosis and lymphedema, respectively). all patients, who underwent surgery in curative intent, were at least macroscopically completely resected (28 patients r0-resection, 4 patients r1-resection). complete remission after primary radiochemotherapy was seen in 12/18 patients (66%). in the other six patients, the median duration of follow - up was 24.2 months (range, 7.848.7 months). death has occurred in 12 patients (24%) ; 9 of these died from tumor, and 3 died from intercurrent disease (secondary primary tumor, pulmonary embolism and one not known). collectively, the 2- and 3-year overall survival rates were 72.7 and 67.1%, respectively. loco - regional relapse (recurrence after complete or progress after partial remission) occurred in ten patients (in 7 patients after adjuvant and 3 patients after primary radiochemotherapy) ; the median time to relapse was 9 months (range 0.813.9). in nine patients, distant metastases occurred in two patients (one hepatic and one pulmonal), in both cases not associated with a loco - regional recurrence. to evaluate the prognostic value of individual factors, univariate subgroup analyses concerning gender, patient age, primary site, tumor stage, histological grading, surgery, and preradiotherapeutic hemoglobin levels have been done. subgroup analysis of tumor stage (stage iii versus stage iva / b) showed that disease stage iii at the time of diagnosis is associated with a statistical significant (p = 0.02) higher 2- and 3-year year overall survival compared to the patients staged iva / b (100 vs. 65.3%, and 100 vs. 57%, respectively). in contrast, subgroup analyses concerning gender, patient age, primary site, histological grading, surgery, and preradiotherapeutic hemoglobin levels showed no statistically significant influence on overall survival or loco - regional control. the data show that our radiochemotherapy regimen using low dose cisplatin is associated with relatively low high grade (grade 3) toxicity. thus, 94% of the patients received the intended dose of radiotherapy and in 90% of the patients 80% of the intended cumulative chemotherapy dose could be applied. other common chemotherapy regimens (cisplatin 100 mg / m on days 1, 22, and 43 of radiotherapy, 50 mg / m weekly during radiotherapy, or 20 mg / m for 4 days in week 1 and 5 of radiotherapy) are associated with equal or considerably more high grade acute toxicity : as shown in table 2, the incidence of grade 3 mucositis / dysphagia / hematologic toxicity in these studies varies from 41 to 89%. high acute toxicity may reduce quality of life in patients, and toxicity related changes of the therapy concept may be necessary which, however, may adversely influence prognosis. (2004) described 2004 that the third cycle of their chemotherapy (cisplatin 100 mg / m on days 1, 22, and 43 of radiotherapy) could be administered on time without delay in only 49% of their patients.table 2toxicity of different primary and adjuvant radiochemotherapy regimens for locally advanced head and neck tumorsstudypatients (n)primary / adjuvant rctrt - dose (gy)cisplatin regimefull ct appliedfull rt dose appliedacute toxicity iii / iv (%) chronic toxicity iii / iv (%) present study50rctands + rct70646 mg / m on every day of rt90% of patients received more than 80% of planned dose94%mucositis / dysphagia 22%;hematologic 14%;mucositis + dysphagia + hematologic 8%xerostomia 6%;cutaneous fibrosis 4%;lymphedema (2008)61rctands + rct6072100 mg / m on day 1, 22, 43 or52%87%hematologic 39%nausea / vomiting 28%mucositis 40%skin 24%xerostomia 7%fibrosis 7%67rctands + rct607220 mg / m and 600 mg / m 5-fu on days 15 and 293390%not givenhematologic (2006)57rct7020 mg / m during days 14 of weeks 1 and 562%100%63%not givencastro. mg / m on day 1, 22, 4349%96% with more than 60 gy41%fibrosis 10%, xerostomia 14%, lymphedema 7%, bone 1%, skin 1%cooper. (2004)206s + rct66100 mg / m on day 1, 22, 4361%80%77%esophagus 15%xerostomia 7%, bone 6%, skin 5%, renal 2%bauchaud. (1996)39s + rct657050 mg / m once per week82% of patients received more than 66% of planned dose100%41%fibrosis 10%osteonecrosis 3%hoebers. (2007)47rct706 mg / m daily for 20 shotsmean no. of cisplatin shots : 17.396%mucositis 65%hematologic 44%fibosis 9% osteradionecrosis 4%jeremic. m on every day of rt92%92%stomatitis 13%esophagitis 3%xerostomia 6%, fibrosis 3%, bone 2%, skin 2%rct primary radiochemotherapy, s + rct curative surgery and adjuvant radiochemotherapythird cycle of chemotherapy could be administered on time without delay in 49% of patients toxicity of different primary and adjuvant radiochemotherapy regimens for locally advanced head and neck tumors rct primary radiochemotherapy, s + rct curative surgery and adjuvant radiochemotherapy third cycle of chemotherapy could be administered on time without delay in 49% of patients our data are in accordance with other studies using daily low dose cisplatin in radiochemotherapy for locally advanced hnscc : jeremic. (2007) also demonstrated that concurrent normofractionated chemoradiation with daily low dose cisplatin for advanced head and neck cancer patients is feasible and effective. 2004) reported similar data concerning toxicity compared to our study, hoebers. 2008) considered this treatment scheme as a relatively safe protocol with respect to ototoxicity. our data show a 2-year/3-year overall survival rate of 72.7%/67.1%, and a 2-year/3-year loco - regional - control rate of 78%, respectively. however, as both, patients with surgery and postoperative radiochemotherapy and patients with radiochemotherapy alone as definitive treatment are regarded, conclusions concerning oncological efficacy in comparison to the literature can not be drawn from the data. whereas in the adjuvant situation conventional fractionated radiotherapy is the standard approach, overall survival and loco - regional control may be improved by alternative fractionation like acceleration or hyperfractionation when using radiotherapy as primary therapy for inoperable advanced hnscc : it has been shown that hyperfractionation with moderate dose escalation leads to a significant improvement of loco - regional control and overall survival if radiation therapy is used as single modality. in contrast, accelerated radiation therapy alone, especially when given as split course radiation schedule, does not increase overall survival (budach. however, no evidence proofs that hyperfractionation is better compared to conventional fractionation if chemotherapy is given concomitantly (welz. furthermore, it has to be considered that alternative fractionation is associated with higher acute toxicity and may lead to toxicity related changes of the therapy concept resulting in negative impact on prognosis. potentially, daily low dose cisplatin is appropriate to reduce acute toxicity in hyperfractionated radiotherapy without compromising tumor control compared to regimens using higher single doses of cisplatin for radiosensitizing. in summary, despite hundreds of clinical trials in patients with advanced disease, there is no absolute consensus about patient selection for altered fraction regimens, type of chemo - radiotherapy association, radiation or chemotherapy dose schedule (corvo 2007). the data show that our radiochemotherapy regimen using low dose cisplatin is associated with relatively low high grade (grade 3) toxicity. thus, 94% of the patients received the intended dose of radiotherapy and in 90% of the patients 80% of the intended cumulative chemotherapy dose could be applied. other common chemotherapy regimens (cisplatin 100 mg / m on days 1, 22, and 43 of radiotherapy, 50 mg / m weekly during radiotherapy, or 20 mg / m for 4 days in week 1 and 5 of radiotherapy) are associated with equal or considerably more high grade acute toxicity : as shown in table 2, the incidence of grade 3 mucositis / dysphagia / hematologic toxicity in these studies varies from 41 to 89%. high acute toxicity may reduce quality of life in patients, and toxicity related changes of the therapy concept may be necessary which, however, may adversely influence prognosis. (2004) described 2004 that the third cycle of their chemotherapy (cisplatin 100 mg / m on days 1, 22, and 43 of radiotherapy) could be administered on time without delay in only 49% of their patients.table 2toxicity of different primary and adjuvant radiochemotherapy regimens for locally advanced head and neck tumorsstudypatients (n)primary / adjuvant rctrt - dose (gy)cisplatin regimefull ct appliedfull rt dose appliedacute toxicity iii / iv (%) chronic toxicity iii / iv (%) present study50rctands + rct70646 mg / m on every day of rt90% of patients received more than 80% of planned dose94%mucositis / dysphagia 22%;hematologic 14%;mucositis + dysphagia + hematologic 8%xerostomia 6%;cutaneous fibrosis 4%;lymphedema (2008)61rctands + rct6072100 mg / m on day 1, 22, 43 or52%87%hematologic 39%nausea / vomiting 28%mucositis 40%skin 24%xerostomia 7%fibrosis 7%67rctands + rct607220 mg / m and 600 mg / m 5-fu on days 15 and 293390%not givenhematologic (2006)57rct7020 mg / m during days 14 of weeks 1 and 562%100%63%not givencastro. mg / m on day 1, 22, 4349%96% with more than 60 gy41%fibrosis 10%, xerostomia 14%, lymphedema 7%, bone 1%, skin 1%cooper. (2004)206s + rct66100 mg / m on day 1, 22, 4361%80%77%esophagus 15%xerostomia 7%, bone 6%, skin 5%, renal 2%bauchaud. (1996)39s + rct657050 mg / m once per week82% of patients received more than 66% of planned dose100%41%fibrosis 10%osteonecrosis 3%hoebers. (2007)47rct706 mg / m daily for 20 shotsmean no. of cisplatin shots : 17.396%mucositis 65%hematologic 44%fibosis 9% osteradionecrosis 4%jeremic. m on every day of rt92%92%stomatitis 13%esophagitis 3%xerostomia 6%, fibrosis 3%, bone 2%, skin 2%rct primary radiochemotherapy, s + rct curative surgery and adjuvant radiochemotherapythird cycle of chemotherapy could be administered on time without delay in 49% of patients toxicity of different primary and adjuvant radiochemotherapy regimens for locally advanced head and neck tumors rct primary radiochemotherapy, s + rct curative surgery and adjuvant radiochemotherapy third cycle of chemotherapy could be administered on time without delay in 49% of patients our data are in accordance with other studies using daily low dose cisplatin in radiochemotherapy for locally advanced hnscc : jeremic. (2007) also demonstrated that concurrent normofractionated chemoradiation with daily low dose cisplatin for advanced head and neck cancer patients is feasible and effective. 2004) reported similar data concerning toxicity compared to our study, hoebers. 2008) considered this treatment scheme as a relatively safe protocol with respect to ototoxicity. our data show a 2-year/3-year overall survival rate of 72.7%/67.1%, and a 2-year/3-year loco - regional - control rate of 78%, respectively. however, as both, patients with surgery and postoperative radiochemotherapy and patients with radiochemotherapy alone as definitive treatment are regarded, conclusions concerning oncological efficacy in comparison to the literature can not be drawn from the data. whereas in the adjuvant situation conventional fractionated radiotherapy is the standard approach, overall survival and loco - regional control may be improved by alternative fractionation like acceleration or hyperfractionation when using radiotherapy as primary therapy for inoperable advanced hnscc : it has been shown that hyperfractionation with moderate dose escalation leads to a significant improvement of loco - regional control and overall survival if radiation therapy is used as single modality. in contrast, accelerated radiation therapy alone, especially when given as split course radiation schedule, does not increase overall survival (budach. however, no evidence proofs that hyperfractionation is better compared to conventional fractionation if chemotherapy is given concomitantly (welz. furthermore, it has to be considered that alternative fractionation is associated with higher acute toxicity and may lead to toxicity related changes of the therapy concept resulting in negative impact on prognosis. potentially, daily low dose cisplatin is appropriate to reduce acute toxicity in hyperfractionated radiotherapy without compromising tumor control compared to regimens using higher single doses of cisplatin for radiosensitizing. in summary, despite hundreds of clinical trials in patients with advanced disease, there is no absolute consensus about patient selection for altered fraction regimens, type of chemo - radiotherapy association, radiation or chemotherapy dose schedule (corvo 2007). we found concurrent chemoradiation with daily low dose cisplatin to be feasible with advantage of low acute and chronic toxicity. therefore, use of low dose cisplatin should be evaluated in future clinical trials testing conventional or alternative fractionated radiotherapy for locally advanced hnscc. | purposeto evaluate toxicity of radiochemotherapy schedule using daily - low - dose - cisplatin in radiochemotherapy of locally - advanced head - and - neck - cancer (hnscc).methods and patientsfrom october 2003 to october 2006, 50 patients with hnscc (stage iii / iva / ivb) were treated. in 32 patients, surgery and adjuvant radiotherapy(64 gy), in 18 patients definitive radiotherapy(70 gy) was performed. low - dose - cisplatin was applied concomitantly (6 mg / m2/every radiotherapy - day).resultsacute toxicity grade 3 was observed in 22 patients (11 patients mucositis / dysphagia, 7 hematologic toxicity, 4 mucositis / dysphagia / hematologic toxicity). 90% of our patients received > 80% of the planned cumulative chemotherapy dose, 94% the intended dose of radiotherapy. after median follow - up of 24.2 months, 3-year overall survival and loco - regional control rates were 67.1 and 78%. during follow - up, chronic toxicity grade 3 (xerostomia, subcutaneous fibrosis, or lymphedema) was observed in nine patients.conclusionwe found chemoradiation with daily - low - dose - cisplatin to be feasible with advantage of low acute and chronic toxicity. therefore, use of low - dose - cisplatin should be evaluated in future clinical trials. |
we present a case of disseminated cysticercosis with extensive central nervous system (cns) involvement and extracranial dissemination in a young woman, which presented as a case of affective mood disorder with the chronic tension type headache. the extra - cranial dissemination involved atypical head and neck regions such as posterior pharyngeal wall, masseter, pterygoids, paravertebral neck muscles and tongue. though cysticercosis is the most common helminthic infection of the, cns disseminated cysticercosis is rare. a few cases of neurocysticercosis presenting with psychiatric complaints have been reported, but there have been no reported cases of disseminated neurocysticercosis presenting with depression. a 21-year - old woman residing in new delhi presented to the department of medicine with a history of insidious onset, progressive headache on and off for the past 6 months. the headache was bilateral, dull aching in nature occurring at any time of the day and had no special character or precipitating factors. it was associated with fatigue, low mood, irritability and loss of appetite, multiple somatic pains and crying spells. initially, the headache was relieved by over the counter analgesics, but it used to recur after a few days. after 2 months of persistent symptoms, she had consulted a physician where she had been diagnosed to have tension type headache. a formal psychiatric evaluation done at that time revealed minor depressive episode. when her symptoms did not abate she was started on amitriptyline 25 mg once daily for prophylaxis of chronic tension type headache. there was no history of blurring of vision, diplopia, squint, pain, redness or watering of eyes. her elder sister had a history of seizure disorder for which she had been taking anti - epileptic drugs for the past 2 years. a contrast enhanced magnetic resonance imaging was done, which showed multiple cystic lesions with eccentric nodules in bilateral basal ganglia, cerebellum, brainstem, cerebral hemispheres ; a few of them indenting the lateral ventricle. similar lesions were seen in the scalp, posterior pharyngeal wall, masseter, pterygoids, paravertebral neck muscles and tongue [figure 1 ]. there was a single lesion in left orbit abutting the superior rectus and a single lesion in the right orbit abutting the optic nerve [figures 2 and 3 ]. x - ray of the thigh showed multiple cigar shaped calcifications [figure 4 ]. her complete blood count, liver function test, kidney function test were within the normal limits. based on the imaging findings a definite diagnosis of neurocysticercosis with extra - cranial dissemination was made. she was started on albendazole with oral steroids and neuroimaging follow - up was planned. after 2 months follow - up, her headache had decreased and she did not complain of any myalgia. magnetic resonance imaging showing cystic lesions in masseter, pterygoids, tongue and cerebral hemispheres magnetic resonance imaging brain showing multiple hypodense lesions in bilateral brain parenchyma with scolices and surrounding edema. single cystic lesion in left orbit abutting optic nerve magnetic resonance imaging brain with cystic lesion in right orbit compressing optic nerve and superior oblique muscle plain x - ray of thigh showing multiple cigar - shaped calcifications consumption of measly pork containing cysticerci leads to intestinal teniasis, but does not directly cause cysticercosis. when cysticerci involve the cns it leads to neurocysticercosis. cases of disseminated cysticercosis have been reported from india with involvement of subcutaneous tissues, skeletal muscles, heart, lungs, spleen, pancreas and liver. cysticerci are uncommon in the head and neck region apart from the orbit but cases of cysticerci of tongue, masseter, mylohyoid, lower lip, soft palate and sternocleidomastoid have been reported previously. neurocysticercosis can present with seizures, headache, focal neurological deficit, encephalitis, dementia. neurocysticercosis presenting as extrapyramidal disease (parkinsonism and focal dystonia), kluver - bucy syndrome, weber 's syndrome and cortical blindness have also been reported. hamed and el - metaal reported a case of neurocysticercosis presenting with depressive manifestations together with disturbance of writing, mild deep sensory loss, limb - kinetic apraxia, lack of coordination and dystonia of the right upper limb. disseminated cysticercosis presenting as a mood disorder and mimicking a primary headache disorder has not been reported before. in our patient our patient was treated with albendazole and oral steroids according to the consensus guidelines published by garca. traditionally, tension type headache has been believed to have a psychological basis. even though studies have proved the neurobiological basis of these headaches, the psychological connotations of this diagnosis remain in the minds of physicians and it is easy to leap to this diagnosis in a clinical background of low mood, crying spells and other psychiatric complaints. routine use of imaging modalities may be justified to rule out neurocysticercosis in endemic areas before making a diagnosis of a primary headache disorder. an early diagnosis of cysticercosis and cysticidal treatment can prevent considerable morbidity in this setting. | neurocysticercosis is a common parasitic infection in india presenting usually with seizues, headache, focal neurological deficits. neurocysticercosis presenting as a psychiatric illness is rare. disseminated cysticercosis with involvement of central nervous system and head and neck muscles is rare even in endemic areas. we present a case of disseminated cysticercosis, which presented with chronic tension type headache and affective mood disorder. treatment with cysticidal drugs led to complete remission of psychiatric complaints. in endemic areas history suggestive of mood disorder should not be used as supportive evidence of a primary headache syndome like tension type headche without ruling out secondary causes. making an early diagnosis can prevent morbidity. |
the prognosis is affected by various factors such as the size, location, and time of perforation as well as the ability of the material used to seal the defect. these perforations can be repaired nonsurgically with suitable biocompatible, nontoxic, radiopaque, nonabsorbent material, thus preventing bacterial contamination. in permanent teeth, several materials have been suggested for perforation repair such as amalgam, calcium hydroxide, reinforced zinc oxide - eugenol cements, mineral trioxide aggregate (mta), calcium - enriched mixture (cem) cement, and biodentine. mta was introduced by lee. in 1993 for repair of lateral root perforations. it consists of dicalcium silicate, tricalcium silicate, tricalcium aluminate, and tetracalcium aluminoferrite. although mta has certain drawbacks such as long setting time, poor handling, and relatively high price, it has a superior sealing ability compared to other restorative materials when used for repairing perforations. furcal repair in primary teeth has become more essential than extraction, to prolong the longevity of the tooth. oliveira. showed that the tooth with furcal perforation treated with mta was asymptomatic after 20 months and also concluded that bone formation was seen in the furcation area. concluded that cem and mta have similar sealing ability in furcal perforation repair of primary molar teeth. biodentine is relatively new calcium silicate - based material introduced as a dentine substitute by septodont in 2009. it is mainly composed of highly pure tricalcium silicate, which regulates the setting reaction, calcium carbonate (filler), zirconium dioxide (radiopacifier), calcium chloride (setting accelerator), water reducing agent (superplasticizer), and water. it has been claimed that this material can be used for pulp capping, pulpotomy, apexification, root perforation, internal and external resorption and also as a root - end filling material in periapical surgery. literature search showed no studies in assessing the effect of biodentine in primary tooth perforations. hence, the present in vitro study was planned to compare the sealing ability of mta and biodentine when used to repair the furcal perforations in primary molars using scanning electron microscope (sem). a randomized controlled in vitro trial was planned, and the study protocol was approved by the institutional review board. the exclusion criteria included the samples with root resorption where the furcal area could not be involved. the samples were stored in 5.25% sodium hypochlorite for 24 h for the removal of tissue remnants. after 24 h, the samples were washed and stored in saline (0.9% w / v, nirlife, nirma limited, gujarat, india) until the preparation was done. 2, the hindustan dental products, hyderabad, india). a 0.5 mm round diamond bur was initially used to prepare the access cavity. a standard access cavity was prepared in each tooth using a diamond bur and non - end cutting bur in high - speed handpiece with water spray. a 0.5 mm round bur was used to standardize the size of furcal perforation, and the furcal involvement was made on the center of the pulpal floor. after the furcal perforation, the blocks were randomly divided into two groups : group a (n = 20) and group b (n = 20). group a mta group (angelus, angelus industries, brazil). the powder and liquid were dispensed in a glass slab and mixed in circular motion. the powder and liquid in a capsule were manipulated using triturator for 30 s. the material was scooped and applied on the perforation site. all the sealed perforations were compacted using a moist cotton pellet, and the samples were stored in a closed container for 24 h to allow the repair materials completely set. after 24 h, the samples were sectioned using a hard tissue microtome and the perforated portion of the teeth was taken for examination. the samples were gold sputtered and viewed under sem in different magnifications (50, 500, 750, 1000, 3000) for evaluating the sealing ability and the intra - molecular space between the materials as shown in [figures 14 ]. the microleakage was evaluated by measuring the gap (in m) between the pulpal floor and the material used for the furcal repair. sealing ability of biodentine with pulpal floor intramolecular space in biodentine material sealing ability of mineral trioxide aggregate with pulpal floor intramolecular space in mineral trioxide aggregate material wilcoxon - signed ranks test was used for statistical analysis using spss statistical package (spss statistics for windows, version 17.0, spss inc., the overall results showed that the microleakage was lesser in biodentine (0.149 0.097) when compared to that of mta (0.583 0.24). table 1 shows that biodentine had more sealing ability than mta in 18 samples whereas mta had only 1 sample which showed better sealing ability than biodentine. one sample in both the groups revealed equal ability. there was a statistically significant difference in the sealing ability between the two groups (p < 0.01). statistical ranks for microleakage in two groups comparison of the mean microleakage values in m of biodentine and mineral trioxide aggregate the success of the furcation repair is always dependent on the effective seal between the root canal and the periodontal ligament. this can be achieved by a suitable material which should stop the microleakage and communication between the tooth and periodontal ligament. to obtain success, the perforation repair material should ideally result in formation of new bone, periodontal ligament and cementum. previous studies have shown that cementogenesis is a vital process in dentoalveolar formation and the newly formed cementum acts a biological barrier against the spread of microbial irritants within the root canal system. mta and biodentine are capable of causing complete regeneration of the adjacent dentoalveolar tissue in permanent teeth and are hence used in furcal perforation repairs. various techniques such as bacterial leakage, fluid filtration method radioisotopes, and dye penetration were used to measure the sealing ability of repairing materials. orosco. stated that for evaluation of marginal adaptation, the samples can be directly viewed under sem after gold sputtering and there is no need for creation of resin replicas as direct sem evaluation of the samples did not result in artificial gap formation ; hence, we sectioned the samples and examined its interface directly under sem. the search for alternative materials has been aimed to overcome the drawbacks of previously used materials to reduce the cost and to increase the feasibility to both professionals and patients. this present study is the first of its kind to compare the sealing ability of biodentine and mta in repairing the furcal perforation in primary molars using sem. the manufacturers claim that the addition of setting accelerators and softeners, a new predosed capsule formulation for use in a mixing device predominantly improves the physical properties of the material, making it more user - friendly. biodentine does not require two - step obturation as the setting is faster and thus is lower risk of bacterial contamination making it superior to mta. evaluated the marginal adaptation of biodentine in comparison with mta and intermediate restorative material (irm) using sem. they conducted the study using thirty permanent central incisors and stated that in overall comparison, mta and irm were significantly superior when compared to biodentine in terms of marginal adaptation when used as retrograde filling material. compared the solubility, microhardness, radiopacity, and setting time of biodentine and proroot mta. they stated that the proroot mta showed lower solubility with higher setting time and the radiopacity of biodentine which did not fulfill the requirements as per the iso 6876:200. kokate and pawar evaluated the microleakage of three root - end filling materials : mta, glass ionomer cement, and biodentine using dye penetration method in thirty permanent central incisors and concluded that microleakage was found to be significantly less in biodentine. the results of the present study also showed less microleakage in the biodentine group compared to mta group. from this in vitro study, it can be concluded that biodentine showed lesser microleakage compared to mta and may be a good alternative to mta in sealing the furcal perforations in primary molars, thereby increasing the life of the tooth. | objective : the aim of this study was to compare the sealing ability of mineral trioxide aggregate (mta) and biodentine when used to repair the furcal perforations in primary molars using scanning electron microscope (sem).study design : the study sample comprised forty recently extracted primary molars. these teeth were placed in a 5.25% sodium hypochlorite solution for 24 h and washed with tap water. access cavities were made using a round bur in high - speed handpiece. perforations were made in the center of the floor of the pulpal chamber using a 0.5 mm round bur. the teeth were randomly assigned into two experimental groups based on the material used to seal the perforation : group a mta and group b biodentine. the packed materials were allowed to set for 24 h. the samples were sectioned longitudinally and the extent of marginal adaptation was measured by sem. wilcoxon - signed rank test was used for statistical analysis using spss software.results:all teeth exhibited microleakage, but biodentine showed significantly less leakage (0.149) compared to mta (0.583).conclusion : based on the results of this study, biodentine showed lesser microleakage compared to mta and thus may be a good alternative to mta. |
scientific information is neutral and unbiased since it reflects and describes the objective real world in a specific form using specialized concentrated language. acquiring the reliable and valuable information requires nowadays more and more sophisticated equipment, resources and highly qualified researchers. however, the new knowledge is becoming emotionally attenuated upon interference with human society or specific human beings at definite periods of time. considering unidirectional time axis and changing human factor we get temporarily positive and negative results depending on the posed questions and expectations. the clear examples of well - established knowledge are laws of mechanics discovered by sir isaac newton three centuries ago, laws of thermodynamics, maxwell equations and quantum mechanics ; they make the theoretical basement for modern technology being practically confirmed within centuries under certain physico - chemical limitations (range of speed, temperature, temporal and spatial resolution etc.). more recent new knowledge may not be neutral ; for example, data about global warming due to increased economic activity of human civilization are rather negative since it requires essential recourses to overcome the fast consequences and potentially revert the processes. results of short - term (usually one to three years) projects in biomedicine are often considered negative or positive especially for evaluation of specific chemicals to treat diseases (e.g., clinical trials). scientific information is expressed in publications, databases and books ; necessity of publishing results is important for sharing the new knowledge within the research community, reporting the results for funding bodies, getting funding to continue the research, for intellectual and professional development of researchers. time scale with scheduled periods is important to integrate the science into the social and economic flux of the society. time scale and high demand for robust and reliable initial results are vital for medical research. applicable to scientific research is a problem of optimal allocation of resources with maximum information outputs. it appears and has to be solved by a researcher or a group of researchers ; the problem is changing with time and depends on many factors. an analog for methods to solve the problem is variational principle, which is widely used in science (especially in physics). variational principle is applied to functional f depending on several variable functions and uses variational methods to find extremum of the functional. assuming new knowledge being f (resources ; funding ; time ; errors) would require to find the best functions achieving highest result with zero level of errors while aiming to minimal funding and time. the description sounds unnecessarily sophisticated ; however, it is useful to set the upper level of abstraction and exclude emotional component. it s conceivable to introduce research efficiency depending on the real and ideal trajectories for obtaining the new knowledge. assessing the value of the knowledge is the next step involving experts and taking longer time, so the parameter of research efficiency is not absolute ; it is convenient for initial consideration. the opinion paper compares biological and social systems describing the changes occurred in biomedical research within the past 60 years, and proposes short- and long - term measures to reduce errors and increase efficiency. the main part of the paper is aimed at applied biomedical research and prepared to stimulate further discussion. within the past 60 years since the description of the three dimensional structure of dna by watson and crick the landscape of biomedical sciences has drastically changed, however, not being completely supported by the corresponding changes in scientific traditions, lifestyle and, especially, in medical philosophy. traditional reliable biochemistry and physiology with relatively simple methods are becoming complemented and often substituted by cell culture, gene sequencing, microarrays with robotic stations for data acquisition, computer modeling of single molecules and ensembles of molecules, online databases and publications. adding the ideal requirement of absolute reproducibility and long - term checks for medical research worldwide, variable opportunities and situation in different countries, we get the existing developing situation. similar situations and specific transformations for physics and chemistry centuries ago had been considered in more detail by philosopher of science thomas kuhn who proposed concept of scientific revolutions for shifting from one scientific paradigm and state of science - world - society to another one. the sort of digital (computer) and molecular (switch to the level of single molecules) revolution is occurring in biomedicine and has significant influence on research and verification of biological theories and paradigms. a complex way of introducing, changing and educating the human factor under the conditions depends on many parameters ; it is reasonable to mention that, for example, computers were widely introduced to research only about 20 years ago, they accelerated data processing by many orders of magnitude. in parallel, large scale methods of data acquisition with robots emerged, they enormously increased the volume of information from an experiment and influenced the ways of thinking. the huge informational explosion is seen in the number of biomedical publications, it rose about 10 times since 1951 to about one million publications per year, over 90% of articles are in english nowadays (vs 50% in 1951). some areas are developing faster : the number of publications related to cell cycle - regulating protein p53 (data from pubmed http://www.ncbi.nlm.nih.gov/pubmed/advanced) increased over 100 times since the beginning of the 1980s reaching saturation by the next millennium. the aforementioned fast changes of rapidly developing branches of biology, especially related to practically important and well - funded biomedical aspects, may bring a load of experimental errors due to high demand for fast results, high pressure and changing human factor (which expresses at small time scales and due to increasing speed of research). time and reproducibility will clarify the stream of results, while sometimes a good point better not to do at the moment could be used to save biological material, time and financial resources. since the changes are not always gradual and consistent in science / methods / technology from one side and research community / society / medicine from the other side, the mismatches (or failed gaps under extreme situations) have to be filled with fast available (sometimes improper) solutions or even medical errors. under a better outcome, the mismatches may result in extra resources and funding wasted. a disputable example includes pesticide ddt, which was awarded nobel prize in medicine in 1948 since it helped to fight typhus during the second world war, but later proved to have some toxicity for humans (increasing cancer and neurological diseases and reducing reproductive health) and was banned in 1970s (e.g., reviewed in). it is worth mentioning high inconsistency of results between first microarrays for analysis of altered gene expression. three similar microarrays from different companies showed overlap just in 4 genes from 24 to 93 detected. it was proposed later that the start from the beginning could be the best way to continue with the technology. recent rise in nanoscience promises big discoveries, however, needs a more thoughtful approach and a consideration of multiple explanations and experimental design. for example, nanoparticles simply interact with proteins under biological environment ; hence biological environment of organisms with proteins should be carefully considered in nanoscience research. some areas are more prone to errors. for example, recent indications for sampled 53 publications in cancer research show that up to 90% of them may be incorrect. the most serious problem is that over 60% of retracted papers in biomedical sciences are due to fraud or suspected fraud including plagiarism and duplicate publication, twice more than due to error. moreover, percentage of retraction gradually rose 10 times since 1975 with the average time before retraction being about 3 years. the rise appears to reflect the changes in behavior of authors and institutions over the time. it poses serious questions about scientific ethics, research community and funding in the direction. the indications coincide with the drop in efficiency of research and development in the pharmaceutical industry, where expenses rose about 80 times for a new medical drug since 1950. this might be a reason why money and production move out of developed european countries to new growing economies with a cheaper though less regulated and less qualified labor force. it seems reasonable to compare biological and social systems trying to find specific features and peculiarities. a comparison of 1) earth with human population consisting of seven billion human beings in 2013 (about 2.5 times more than in 1953) and speaking several thousand languages in about 200 countries with, for example, 2) a usual eukaryotic yeast cell containing only about 50 million of individual proteins of several thousand types in less than ten compartments demonstrates the extent of complexity of the systems (fig. 1). however, the presumed attractive coefficients (or attractive properties) of the systems could be different from the complexity. simplified comparison of a cloud of proteins in a eukaryotic yeast cell with human population demonstrates the comparative complexity of the two systems. attractive coefficient of a system for a definite researcher could be introduced from the point of emotionally attenuated information enclosed in the system. the sort of advanced theory would vary between discrete and continuous systems ; the theory does nt seem to have been developed yet. a few obvious simple linear equations could be written for a discrete system with n = n elements : where k = k is the number of connections k between n = n elements, then where r is the total attractive coefficient, while and are specific coefficients demonstrating informational value of the corresponding element or connection between elements, respectively. pondering ideal researcher (without physiological demands and financial interest), it s conceivable to introduce minimal requirements expressed in certain specific coefficients and (demand for unique equipment, high expectation time to get parameters, necessity of multiple efforts from groups of researchers, novelty and uniqueness of results, high social or biomedical demand for results etc.) for a relatively simple biological system to supersede the attractiveness of surrounding social system. increase in number of elements starting from a eukaryotic yeast cell to one milliliter of yeast cell suspension with tens million of cells or considering multicellular organisms with trillions (10) of cells of different types leads to a more common situation for a biomedical research. moreover, modern society is essentially based on known technology with nearly absolute and predictable (when compared with biology) established laws of physics and chemistry while the social life is tightly regulated by law and traditions ; so different ways of thinking and better explanations with fewer promises for society are needed for biology. applying simple digital logic and hard laws of economics to biology and further to medicine, when estimating the conditions of norm and pathology or assessing the results, is not correct. it would seem that essential part of biomedical research is not sufficiently scheduled and predictable for being planned, realized and ever funded. however, biological objects and medical problems, not the society, determine the mainstream and intrinsic logic of research (fig. 1.) the new complex of biomedical science - research community - society nowadays compared with situation 60 years ago needs a series of novel measures to exclude errors and balance the further development. it requires short - term measures at the timescale of years and efforts of the whole scientific society for the timescale of decades. research culture and values, ethics of researchers are slightly modifying according to the dynamic changes of scientific environment. several obvious simple measures starting from the level of publications could make the research more efficient and pondered. they could be as follows : 1) include fast publishing of negative results and 2) publish detailed and descriptive methods (a good example is journal of visualized experiments http://www.jove.com/, which can essentially help with the reproducibility of the results though may restrict the creative potential of researchers), 3) exclude strong correlation between career progression and publication activity (especially for younger scientists in biomedical research), 4) provide more opportunities to express opinion and share results for students. it costs more to the whole community when fast incorrect, but highly desired results are spreading further (fig. 2). the list is not comprehensive and points to reducing or neutralising unavoidable emotional component arising from human factor and novelty of research. figure 2. increase in number of publications correlates with the faster increase of retracted publications. data are normalized to the maximum of the corresponding curve (corresponding data point in 20062010) and taken with 5 year intervals. data for total number of publications and publications for p53 protein are from pubmed ; data for retracted publications are from the supplementary table. assuming average retraction time being 3 years for incorrect publications, it s clear that faster growing areas may have higher load of incorrect publication and need extra measures from authors and publishers. from the other point, it might be reasonable to reclassify a large part of biomedical research, (not related to medicine) to animal and cell science research and shortcut recourses and experience from practical medicine with the rest of the field. an additional factor to increase research efficiency an important feature of the modern biomedical research is the increasing role of theoretical and systems biology. recent development of systems biology attracted large numbers of mathematicians to biology, raising the level of quantitative understanding in experiments and mathematical culture of biologists. briefly, systems biology could be considered in a number of ways : 1) philosophy and way of integrative thinking for biological systems, 2) methodology for processing huge arrays of experimental data and dealing with exploding new information and 3) predictive models for experiments and biological objects. obviously, new unexpected knowledge exists and complex biological systems may have multiple unpredictable responses and networks of reactions to the same stimulus, so systems biology is a powerful tool to help for experimental research, not a substitution of experiments. the initial sparkling promises of systems biology had not been realized so far, however, the development continues and also provides educational and recourse - saving influence. a shift to theoretical aspects of biology is an additional way to stabilize the emerging rapid flow of scientific biomedical research. an approach of using several different experimental methods for biological experiments (building experimental dimensions) aimed at solving a biological problem is another good way to increase research efficiency (fig. the proof of concepts based on overlapping results obtained using several methods provides higher reliability. figure 3. simple scheme of using several experimental dimensions (also depicted by overlapping circles) for achieving reliable results with multiple experimental lines of evidence. an additional factor to increase research efficiency is to make it smarter, more pondered. an important feature of the modern biomedical research is the increasing role of theoretical and systems biology. recent development of systems biology attracted large numbers of mathematicians to biology, raising the level of quantitative understanding in experiments and mathematical culture of biologists. briefly, systems biology could be considered in a number of ways : 1) philosophy and way of integrative thinking for biological systems, 2) methodology for processing huge arrays of experimental data and dealing with exploding new information and 3) predictive models for experiments and biological objects. obviously, new unexpected knowledge exists and complex biological systems may have multiple unpredictable responses and networks of reactions to the same stimulus, so systems biology is a powerful tool to help for experimental research, not a substitution of experiments. the initial sparkling promises of systems biology had not been realized so far, however, the development continues and also provides educational and recourse - saving influence. a shift to theoretical aspects of biology is an additional way to stabilize the emerging rapid flow of scientific biomedical research. an approach of using several different experimental methods for biological experiments (building experimental dimensions) aimed at solving a biological problem is another good way to increase research efficiency (fig. the proof of concepts based on overlapping results obtained using several methods provides higher reliability. simple scheme of using several experimental dimensions (also depicted by overlapping circles) for achieving reliable results with multiple experimental lines of evidence. | this brief opinion proposes measures to increase efficiency and exclude errors in biomedical research under the existing dynamic situation. rapid changes in biology began with the description of the three dimensional structure of dna 60 years ago ; today biology has progressed by interacting with computer science and nanoscience together with the introduction of robotic stations for the acquisition of large - scale arrays of data. these changes have had an increasing influence on the entire research and scientific community. future advance demands short - term measures to ensure error - proof and efficient development. they can include the fast publishing of negative results, publishing detailed methodical papers and excluding a strict connection between career progression and publication activity, especially for younger researchers. further development of theoretical and systems biology together with the use of multiple experimental methods for biological experiments could also be helpful in the context of years and decades. with regards to the links between science and society, it is reasonable to compare both these systems, to find and describe specific features for biology and to integrate it into the existing stream of social life and financial fluxes. it will increase the level of scientific research and have mutual positive effects for both biology and society. several examples are given for further discussion. |
acute disseminated encephalomyelitis (adem) can represent a diagnostic challenge for clinicians, as many disorders (inflammatory and noninflammatory) have a similar clinical and radiologic presentation. the diagnosis of adem depends on the history, physical examination, and supplemental neuroimaging. acute disseminated encephalomyelitis is an immunologically mediated inflammatory disease of the central nervous system (cns) resulting in multifocal demyelinating lesions affecting the gray and white matter of the brain and spinal cord. acute disseminated encephalomyelitis should be adequately defined and distinguished from other diseases affecting the white matter. in particular, a diagnostic challenge lies in distinguishing multiphasic forms of adem from multiple sclerosis (ms). available diagnostic criteria do not reliably distinguish it from first presentations of relapsing diseases such as ms and neuromyelitis optica. criteria for adem presented at a mean age of 7.1 years versus a mean of 12.0 years for ms. however, complete differentiation between the two diseases is impossible on a single magnetic resonance imaging (mri) examination. this is especially important, not only for prognostic purposes, but for therapeutic purposes, since a diagnosis of ms carries the recommendation for early treatment with immunomodulators. some pediatric patients with inflammatory demyelinating cns disorders can not be classified under any of the established disease entities, making their treatment and prognosis difficult. guillain barr syndrome (gbs) is the most common cause of acute flaccid paralysis in healthy infants and children. most pediatric groups have used iv methylprednisolone (1030 mg / kg / day, maximum dose of 1 g / day) or dexamethasone (1 mg / kg) for 35 days followed by oral steroid for 46 weeks with full recovery reported in 5080% of patients. a 10-year - old boy presented with inability to walk since 2 days with difficulty in swallowing, dysphonia and drooling of saliva. there was no history of recent infection, fever, diarrhea, convulsions or trauma or recent immunization. neurological examination revealed the generalized hypotonia ; weakness started with lower limbs then progressed to involve both upper limbs. the anterior abdominal muscles were weak, and the patient could support himself with difficulty in sitting position. emergency computed tomography (ct) scan head was done which revealed vasogenic edema in bilateral fronto - parietal and left temporal regions and infarcts. further evaluation with mri study was suggested. magnetic resonance imaging brain study was done based on ct scan findings mri revealed white matter lesions in the brain, pons and thoracic levels of the spinal cord. it revealed peripherally enhancing lesions in right fronto - parietal and left fronto - temporo - parieto - occipital region involving grey and white matter, subcortical region and corpus callosum ; suggestive of adem. multiple altered signal intensity areas, largest size 5.3 cm 3.6 cm, were seen involving grey matter and bilateral periventricular deep white matter [figure 1 ]. magnetic resonance imaging showed peripherally enhancing lesions in right fronto - parietal and left fronto - temporo - parieto - occipital region involving grey and white matter, subcortical region and corpus callosum ; acute disseminated encephalomyelitis electro - diagnosis showed an important reduction of conduction velocity that is, mild generalized motor sensory axonal polyneuropathy (acute motor and sensory axonal neuropathy - gbs). fundus evaluation revealed bilateral optic neuritis after 3 weeks of illness [figure 2a and b ]. (a and b) visual evoked potential suggestive of bilateral optic neuritis based on clinical findings and electromyography - nerve conduction velocity, a diagnosis of gbs was made and based on mri findings and fundoscopy, concurrent diagnosis of adem and gbs was considered. hence, the treatment followed with intravenous immunoglobins (2 g / kg) for 2 days and intravenous methylprednisolone (30 mg / kg / day) for 3 days the patient showed significant clinical improvement after 4 weeks and could stand with support and walk with assistance. the patient was advised physiotherapy and discharged on oral steroids prednisolone at (1 mg / kg / day) in tapering doses over 4 weeks period. however, this could be considered as the first episode of ms with optic neuritis, therefore, our patient will be evaluated on follow - up for further episodes and treatment with immunomodulators. acute disseminated encephalomyelitis is an immunologically mediated inflammatory disease of the cns resulting in multifocal demyelinating lesions affecting the gray and white matter of the brain and spinal cord. to avoid further misdiagnosis and to develop a uniform classification, the international pediatric ms study group proposed a consensus definition for adem for application in both research and clinical settings [table 1 ]. international ms study group - monophasic adem criteria we have reported a case of the practical approach for management of patients suspected of having adem when the diagnosis is uncertain. building on clinicopathological and retrospective studies of adem, mikaeloff. have applied the most restricted definition of adem to date in a prospective study of children : the occurrence in a previously healthy child of acute symptoms associating the following at onset : more than one neurological deficit, change in mental state ; and any combination of alterations seen on mri, providing that these included white matter lesions. barr syndrome is an acute inflammatory polyradiculoneuropathy characterized by rapidly progressive, essentially symmetric weakness, areflexia, and the time to the full progression is 4 weeks. the main features of gbs are rapidly progressive bilateral and relatively symmetric weakness of the limbs with or without involvement of respiratory muscles or cranial nerve - innervated muscles. patients with gbs differ from each other regarding the extent and distribution of weakness, and the presence of autonomic dysfunction, sensory symptoms and cranial nerve deficits. multiple clinical factors should be considered in combination before arriving at a probable diagnosis of adem including : presenting signs and symptomsconventional and advanced mri abnormalitiessufficient follow - up (as long as 10 years) based on clinical and/or neuroimaging criterialack of alternative diagnosis, concurrent diagnosisbrain histopathology when available. presenting signs and symptoms conventional and advanced mri abnormalities sufficient follow - up (as long as 10 years) based on clinical and/or neuroimaging criteria lack of alternative diagnosis, concurrent diagnosis brain histopathology when available. concurrent adem and gbs are uncommon with few reported cases presenting with severe neurological morbidity. prompt recognition and treatment can hasten the recovery and, therefore, improve the neurological outcome. | acute disseminated encephalomyelitis (adem) and guillain barr syndrome (gbs) are distinct demyelinating disorders that share an autoimmune pathogenesis and prior history of viral infection or vaccination. our patient is a 10 years with acute flaccid paralysis, quadriparesis (lower limbs affected more than upper limbs), generalized areflexia and urinary retention. he had difficulty in speech and drooling of saliva. he also presented with raised intracranial pressure with papilledema ; then bilateral optic neuritis developed during the later course of illness. based on the temporal association and exclusion of alternative etiologies, diagnosis of the association between adem and gbs was made. electro - diagnosis (electromyography - nerve conduction velocity) and magnetic resonance imaging study supported our diagnosis. he improved remarkably after treatment with intravenous immunoglobulin and intravenous methylprednisolone. |
congenital atrichia refers to total absence of hair from birth. it may occur as an isolated defect or in association with a wide range of anomalies. congenital atrichia without associated ectodermal abnormalities is very rare and is inherited as an autosomal recessive trait. we report a rare case of congenital atrichia associated with situs inversus of the viscera and mesocardia which has not been earlier reported in the literature. a 2-year - old male child born of consanguineous marriage presented to pediatric dermatology opd for evaluation of the complete absence of hairs on scalp, eyebrows, and eyelashes since birth. there was history of sparse hair erupting after birth which eventually was shed in 2 months. there was no history suggestive of abnormalities in hearing, dentition, epilepsy, and mental retardation, intolerance to heat, or immunodeficiency. child 's maternal aunt had alopecia of scalp and was diagnosed as having keratosis follicularis spinulosa decalvans. past history was remarkable for bronchopneumonia and congenital lobar emphysema. cutaneous examination revealed total alopecia of the scalp, eyebrows, and eyelashes and all over the body [figure 1 ]. situs inversus of the viscera with mesocardia in the child had been confirmed by chest x ray and computed tomography of chest and abdomen [figure 2 ]. based on clinical presentation and histological findings, two - year - old male child with total alopecia of scalp, eyebrows, and eyelashes ct - scan of chest and abdomen showing mesocardia with liver on left side and spleen on the right side it can be inherited as an autosomal recessive or autosomal dominant or x linked pattern. the cases inherited as autosomal recessive are generally the most severe form and are present since birth. congenital atrichia presents as total alopecia at birth, but sometimes scalp hair can be present at birth which is later shed between the first few months, after which no further regrowth occurs. congenital atrichia may be associated with papular lesions which is a rare form of autosomal recessive syndrome characterized by numerous, small, horny papules on the face, neck, limbs, and trunk. other syndromes associated with congenital alopecia are moynahan 's syndrome (mental retardation, epilepsy), hidrotic ectodermal dysplasia (palmoplantar keratoderma, thickened nails) and aging syndromes. skin histology shows the total absence of hair follicles or a few scattered miniaturized follicles. alopecia universalis and vitamin d - dependent rickets type ii a induced alopecia should be considered in the differential diagnosis. the prevalence of situs inversus varies among different populations, but is less than 1 in 10,000 people. it is a congenital condition usually inherited as an autosomal recessive or x linked in which the major visceral organs are reversed or mirrored from their normal position. cardiac malpositions refer to heart that are located abnormally within the thoracic cavity or that are located outside the thoracic cavity. three major cardiac malpositions occur in the presence of right / left asymmetry : visceroatrial situs inversus with dextrocardia, visceroatrial situs solitus with dextrocardia and visceroatrial situs inversus with levocardia. the association of situs inversus and mesocardia with congenital atrichia has not been reported in literature. it is currently not known whether there is underlying genetic cause for the co - existence of these two condition in our patient or it constitutes a coincidence. | congenital alopecia includes a broad differential diagnosis and presents a diagnostic and therapeutic challenge for the physician. congenital atrichia is a rare form of irreversible alopecia that is usually inherited as an autosomal recessive pattern. we report a 2-year - old male child presenting with total alopecia of scalp, eyebrows, eyelashes, and body hair since birth. the child had cardiac malposition with situs inversus of the viscera. computed tomography of the chest and abdomen revealed median position of the heart with transposition of abdominal viscera. to our knowledge, this is the first case of congenital atrichia associated with situs inversus and mesocardia. |
sleep disturbances and cognitive dysfunction are prevalentphenomena in patients with breast cancer [1, 2 ]. these symptoms are often intertwined in a symptom cluster together with other common cancer - related symptoms including fatigue, depression, anxiety, and pain all having a negative influence on overall quality of life and performance status [35 ]. disturbed sleep and cognitive function may arise around the time of diagnosis, the time of surgery, or in the course or aftermath of adjuvant therapy, making the specific aetiology difficult to establish as many factors contribute to the development. postoperative cognitive dysfunction (pocd) is a deterioration of intellectual function postoperatively, typically presenting as impaired short - term memory or concentration [7, 8 ]. in the long - term, pocd has been shown to be associated with increased mortality and risk of leaving the labour market prematurely. among the many pathophysiological explanations for the development of pocd, postoperative sleep disturbances are one of them [7, 11 ]. it is well known that overall sleep loss and sleep fragmentation may have negative cognitive consequences [12, 13 ] and it has previously been shown that sleep disturbances may arise in the postoperative period after surgery for breast cancer and persist for months or even years. depression in itself has not been shown to be correlated with pocd in the short- or long - term postoperative period. exogenous melatonin can improve sleep quality by reducing sleep onset latency, increasing sleep efficiency, and increasing total sleep duration in healthy subjects and patients with primary sleep disorders. furthermore, some studies have shown that melatonin can influence cognition positively in healthy men exposed to a stress - test and in adults with mild cognitive impairment [1719 ]. therefore, we hypothesized that melatonin would have a beneficial effect on cognitive function and sleep after breast cancer surgery with less cognitive disturbances and better sleep in the patients treated with melatonin. our primary aim was to evaluate the effect of melatonin on cognitive function 2 weeks postoperatively. the melody trial was a randomized (1 : 1), double - blind, placebo - controlled trial that primarily sought to investigate depressive symptoms as reported in another paper. the study was approved by the local ethics committee (h-4 - 2011 - 007), the danish medicines agency (eudract no. 2010 - 022460 - 12) and the danish data protection agency (2007 - 58 - 0015/heh.750.89 - 12). the trial was registered on https://www.clinicaltrials.gov/(nct01355523) before inclusion of the first patient and the good clinical practice unit at copenhagen university hospital monitored the trial. the study was undertaken at the department of breast surgery, herlev hospital, copenhagen, denmark. eligible patients were women aged 3075 years, scheduled for lumpectomy or mastectomy for breast cancer, with american society of anesthesiologists (asa) classes i iii. we excluded pregnant patients and patients with signs of depression on the major depression inventory (mdi). when screening patients for enrollment, we used the mdi as a diagnostic instrument and we excluded those who had mild, moderate, or severe depression using the icd-10 criteria. the only change after trial commencement was that the upper limit of the age criteria was raised from 70 to 75 years on october 19, 2011, because of slow recruitment. this included administration of the mini - mental state examination, a neuropsychological test battery, and the mdi. on inclusion, patients were randomly assigned 1 : 1, in blocks of six, to melatonin or placebo. patients in the intervention group received 6 mg oral melatonin daily 1 hour before bedtime for 1 week preoperatively and 12 weeks postoperatively. the randomization list was computer generated using dedicated software (http://www.randomization.com/). to assure blinding, this procedure was completed by the hospital pharmacy, which received the medicine directly from pharma nord (vejle, denmark). the pharmacy packed the melatonin / placebo in identical, sequentially numbered, sealed boxes. the participants, the health care providers, the research staff, and the investigators assessing the outcomes were all blinded to allocation and the allocation sequence by taking the sequentially numbered sealed boxes. the melatonin / placebo was supplied by pharma nord and the tablets and packages were physically identical. after inclusion, patients kept a daily record of subjective sleep quality using a visual analogue scale (vas) and a sleep diary was completed daily. a visit was scheduled 2 weeks postoperatively where patients were tested with the neuropsychological test battery and the mdi (data not reported here). during the last 10 weeks of the study, patients were assessed with the mdi twice and every 2 weeks they completed vas and a sleep diary. at the final visit, 12 weeks postoperatively, patients were tested with the neuropsychological test battery and the mdi. the patients underwent neuropsychological testing using the ispocd test battery on 3 occasions : preoperatively and 2 and 12 weeks postoperatively. seven variables from 4 neuropsychological tests (visual verbal learning, concept shifting task, stroop color - word test, and letter - digit coding) were used in the analysis : cumulative numbers of words recalled in 3 trials and the number of words at delayed recall from the visual verbal learning test ; the time and number of errors in part c of the concept shifting test ; the time and error scores from the third part of the stroop color - word interference test ; and the number of correct answers from the letter - digit coding test. one was defined as the perioperative period, ranging from 3 days preoperatively till 8 days postoperatively and the other was defined as the long - term postoperative period, ranging from approximately 2 to 12 weeks postoperatively. sleep diary data consisting of sleep latency (min), number of awakenings, total sleep period (tsp) (min), and sleep efficiency (%) were recorded for both time periods in the 2 groups. sleep efficiency was defined as (tsp sleep latency minutes awake)/(tsp) and total sleep period as time in bed trying to sleep. equivalent to 100 mm were measured in the short- (daily) and long - term (every second week) postoperative period in the 2 groups. no interim analyses were done. for statistical analyses, ibm spss statistics for windows, version 20.0 (ibm corp., the sample size estimation was based on a conservative estimate of the incidence of depression of 30% being decreased to 15% with melatonin. the study was powered at 80% with a risk of type i error of 5% and a risk of type ii error of 20%. according to this, the analysis of the neuropsychological test data was based on normative data from 133 females aged 4060 years collected in another study. we evaluated changes from the preoperative baseline to the 2 postoperative test sessions. in controls we calculated mean and standard deviations (sd) of these differences. for the individual patients, we compared baseline scores with the 2- and 12-week postoperative test results, subtracted the average learning effect from the changes, and divided the result by the sd of the control group to obtain a z score for the 7 individual test outcomes. we defined a composite z score as the sum of the 7 z scores and normalized this using the sd for that sum in the controls. pocd was defined as a combined z score > 1.96 or a z score > 1.96 in at least 2 of the 7 subtests as previously described. if patients refused or were not able to complete a specific test, the z score for that test was considered a missing value. the outcomes were the incidence of pocd (yes / no) in the 2 groups at the test 2 weeks postoperatively and at follow - up 12 weeks postoperatively. fisher 's exact test was used to compare the incidence of pocd between the 2 groups. the 7 variables from the 4 neuropsychological tests are reported as median values with interquartile range (iqr). due to the nonnormal distribution of data, nonparametric statistics were used and data are presented as frequencies or median iqr, with the exception of the results from the bootstrapping used to analyze sleep diary data. sleep diary data were calculated as a median for each patient in the 2 time periods. finally, bootstrapping was used to calculate confidence intervals for the means and p values for the difference in means. this was performed using the smean.cl.boot function in the hmisc library in r version 3.0.1 (r foundation for statistical software, vienna, austria). bootstrapping was performed with 10000 bootstrap samples and p values were calculated by an unpaired t - test. patients were recruited fromjuly 2011 to december 2012 where the trial was terminated prematurely (n = 54) due to restructuring of surgery for breast cancer in the region resulting in an overall low inclusion rate and an inability to complete the trial. we tried to include other centres in other regions but this was not possible because of lack of funding. the consort trial profile (figure 1) shows the screening, randomization, and follow - up of the patients ; 703 patients were screened for eligibility, 649 were excluded, and the remaining 54 were randomized to either melatonin (n = 28) or placebo (n = 26). baseline, perioperative and demographic characteristics were similar between the two groups (table 1), although median duration of surgery was 92 and 125 min and duration of anaesthesia was 155 and 190 min in the melatonin and placebo groups, respectively. of the patients, 11 withdrew from the study : 10 in the placebo group2 lost to follow - up, 2 due to noncompliance, 2 due to adverse events, and 4 due to not being able to cope with participating in the trial and one in the melatonin group due to not being able to cope with participating in the trial (p = 0.002). the first neuropsychological test after baseline was completed at a median of 14 (iqr 1214) days after surgery and the second at a median of 85 (iqr 8390) days after surgery. pocd could be assessed in 46 patients at the first postoperative test after baseline (a combined z score could not be calculated in 1 patient due to dyslexia and 7 patients due to discontinued intervention) and in 42 patients at the test at 12 weeks postoperatively (4 more discontinued intervention). the incidence of pocd was 0% (0/20) [95% confidence intervals (ci) 0.0% ; 16.8% ] in the placebo group and 0% (0/26) [95% ci 0.0% ; 13.2% ] in the melatonin group 2 weeks postoperatively (p = 1.00) and 6.3% (1/16) [95% ci 0.0% ; 30.2% ] in the placebo group and 0% (0/26) [95% ci 0.0% ; 13.2% ] in the melatonin group 12 weeks postoperatively (p = 0.38) (table 2). data for the 7 variables from the 4 neuropsychological tests are presented in table 3. in the short - term perioperative period sleep efficiency was significantly greater in the melatonin group compared with placebo with a mean difference of 4.28% [95% ci 0.57 ; 7.82 ] (p = 0.02). other variables in the short - term perioperative period did not show any significant differences. in the long - term postoperative period the total sleep period was significantly greater in the melatonin group compared with placebo with a mean difference of 37.0 min [95% ci 3.6 ; 69.7 ] (p = 0.03). subjective sleep quality assessed by vas between the melatonin and placebo group was not significantly different in the short - term perioperative (mean difference 4.71 mm [95% ci 14.63 ; 5.45 ] (p = 0.37)) or long - term postoperative period (mean difference 0.78 mm [95% ci 10.20 ; 8.48 ] (p = 0.87)). common side effects in the melatonin group were dizziness (14%), headache (10%), and paresthesia in the mouth region, arms, or legs (10%). headache (27%), difficulty falling asleep (13%), and nausea (13%) were the most common side effects in the placebo group. of the patients in the melatonin group, 56% (15/27) experienced at least one side effect, whereas this applied to 50% (12/24) in the placebo group (p = 0.78). to our knowledge this is the first study to investigate the effect of oral bedtime melatonin on cognitive function and sleep in patients undergoing surgery for breast cancer. cognitive dysfunction was not detected as a significant problem but the sample size does not allow firm conclusions. melatonin increased sleep efficiency in the short - term postoperative period and increased total sleep period in the long - term postoperative period compared to placebo. however, there were no differences in subjectively assessed sleep quality at any time between the 2 groups. we anticipated that we would find a higher incidence of pocd, especially at the first measurement, as previous studies have shown that pocd was present approximately 1 week after surgery in 741% of patients depending on age, magnitude of surgery, type of anaesthesia, and type of hospitalization [9, 24, 3133 ]. an overall explanation could be the use of a more modern anaesthetic regimen, multimodal pain treatment, and shorter length of hospital stay than in the years these studies enrolled patients (19942002). our finding of a very low incidence of pocd may allude that this postoperative complication is not as prominent as it was in the past, but this needs to be confirmed in larger studies and in other categories of surgical patients. another explanation for the low incidence could be that the preoperative assessment underestimated cognitive function due to the negative influence of the psychological aspect on performance in neuropsychological tests, as the testing was done close to or on the actual day where the patients were diagnosed with cancer and also close to their day of surgery. furthermore no general consensus exists on the optimal duration of the intervals between surgery and test sessions. we evaluated pocd at a median of 14 and 85 days after surgery, where other studies [9, 24, 3133 ] used the same test battery earlier (59 days after surgery) or later (96117 days). possibly we tested too late to pick up the cognitive dysfunction in the immediate postoperative period and cognitive improvement had already occurred. another study in a population of patients with breast cancer did not find objective cognitive dysfunction either, at a mean of 34.5 (range 1975) days after surgery using the same neuropsychological test battery as in our study, although interestingly they found that the women perceived themselves as cognitively impaired, compared to healthy controls. in addition, it has been reviewed that there is a lack of a relationship between objective and subjective cognitive dysfunction in breast cancer patients. this emphasizes that even though we did not detect any objective difference in cognitive decline between the two groups, it could possibly be present subjectively and thereby have clinical relevance. sleep disturbances were less common than anticipated in the postoperative period and patients in both groups showed normal sleep on all 4 parameters when compared to a population of healthy subjects also using a sleep diary. this could be yet an explanation for the low incidence of pocd, as we had hypothesized that sleep disturbances were one of the contributing factors to the development of pocd [7, 11 ]. the well - known effects of melatonin on sleep with an increased sleep efficiency and total sleep duration measured by polysomnography or actigraphy were confirmed by our study. we showed these two changes in measurable sleep parameters, which we believe are large enough in size to be clinically relevant, although we did not find a corresponding change in subjective sleep quality (vas). another study investigating the effect of 5 mg melatonin for 3 nights postoperatively found a decrease in sleep latency in the melatonin group and this was not associated with improved subjective sleep quality (vas) either. a possible explanation for these findings could be that the measurement of subjective sleep quality by vas is too imprecise to detect a difference. it could be valuable to investigate what the minimal clinically relevant difference is on vas for sleep quality in future studies. based on an estimate of pocd of 30% in middle - aged patients 6 days after major surgery and an expectation of a reduction to 15% with melatonin treatment, we should have included around 260 patients, which corresponds to the original sample size calculation done on the incidence of depression. our study was not completed and together with a possible underestimation of cognitive function at baseline and the later timing of the first postoperative test, these major limitations could explain the negative findings. however, with the timing of our tests we found a very low incidence of cognitive dysfunction, which means that we would most likely not have been able to find a difference, even in 260 patients. we still believe that future studies are warranted and that melatonin could be a potentially useful treatment for pocd, despite the negative results of this study, most likely due to the very low incidence of pocd. the external validity of our study is limited in its present form as 61% (432/703) of the patients assessed for eligibility were excluded due to exclusion criteria and we could overall only include about 8% (54/703) of those assessed for eligibility. to improve the external validity regarding the internal validity of our study, it was highly influenced by the distribution of drop - outs, 10 : 1 (placebo : melatonin). due to the high rate of drop - outs, and therefore a missing data problem, which we assessed could not be sufficiently solved by any type of imputation, we chose not to complete intention - to - treat analyses. lost to follow - up or discontinuation of the intervention can be a possible cause of bias. this skewness in the distribution of drop - outs is interesting and can possibly be explained by a significantly lower risk of developing depressive symptoms in the melatonin group. patients in the placebo group might have dropped out due to more depressive symptoms, contributing to overall reduced mental well - being and possibly reduced cognitive function and/or poor sleep, although the latter was not quantifiable by the measurement instruments used in this study. this could contribute to an underestimation of cognitive function and sleep disturbances in the placebo group. future studies are needed to determine whether melatonin administration is associated with improved mental and physical functioning in cancer patients, giving them the ability to complete participation in a trial. in conclusion, melatonin increased sleep efficiency and total sleep time after breast cancer surgery, but we did not demonstrate an effect of melatonin on cognitive function after surgery. furthermore, the drop - out rate was significantly lower in the melatonin group than in the placebo group. | background. sleep disturbances and cognitive dysfunction are common in patients with breast cancer. disturbed sleep leads to poor cognitive performance and exogenous melatonin may improve sleep and attenuate cognitive dysfunction. we hypothesized that melatonin would improve sleep and cognitive function after surgery. methods. this study reports secondary endpoints from a randomized, double - blind, placebo - controlled trial. women, 3075 years, were randomized to 6 mg oral melatonin / placebo for 3 months. we assessed postoperative cognitive dysfunction (pocd) with a neuropsychological test battery, sleep with a diary, and sleep quality with vas. results. 54 patients were randomized to melatonin (n = 28) or placebo (n = 26) ; 11 withdrew (10 placebo, 1 melatonin, p = 0.002). the incidence of pocd was 0% (0/20) [95% ci 0.0% ; 16.8% ] in the placebo group and 0% (0/26) [95% ci 0.0% ; 13.2% ] in the melatonin group 2 weeks postoperatively (p = 1.00) and 6.3% (1/16) [95% ci 0.0% ; 30.2% ] in the placebo group and 0% (0/26) [95% ci 0.0% ; 13.2% ] in the melatonin group 12 weeks postoperatively (p = 0.38). sleep efficiency was significantly greater in the melatonin group ; mean difference was 4.28% [95% ci 0.57 ; 7.82 ] (p = 0.02). the total sleep period was significantly longer in the melatonin group ; mean difference was 37.0 min [95% ci 3.6 ; 69.7 ] (p = 0.03). conclusion. melatonin increased sleep efficiency and total sleep time but did not affect cognitive function. the dropout rate was significantly lower in the melatonin group. this trial is registered with clinicaltrials.gov nct01355523. |
their main distinction from other types of polymers lies in their densely crosslinked molecular structure. this crosslinking leads to a number of favorable thermal and mechanical properties including high strength and modulus, high creep resistance, high glass transition temperature, low shrinkage, and better chemical resistance. these properties in conjunction with ease of processing have made epoxy resins an attractive choice for use in many engineering components and structures. they have found huge applications in aerospace, automotive, packaging, coating, and microelectric industries. in recent years, researchers have developed and investigated polymer nanocomposites based on a wide variety of micro-/nanoscale fillers including clay particles [16 ], aluminum particles, tio2 particles, graphenes [9, 10 ], carbon nanotubes [1113 ], and halloysites [4, 14 ] to improve the mechanical properties of epoxy polymers. among the various reinforcements, large aspect ratio layered silicates or clays are especially attractive for enhancing the barrier properties and, hence, can be used to improve the resistance to moisture degradation. epoxy polymers are characteristically hydrophilic, which means that they have strong affinity towards water. this nature of epoxy resins makes them susceptible to high moisture absorption ; in general, depending on the nature of the epoxy resin, the equilibrium moisture uptake can be in the range of 17%. absorbed moisture usually degrades the functional, structural, and mechanical properties of the polymer matrix [4, 1619 ]. it has been reported that mechanical and thermal properties of epoxy - based systems are severely affected by moisture absorption in comparison to other matrix materials, such as bismaleimide (bmi), polyetheretherketone (peek), and cyanate ester. water absorption into a polymer matrix leads to change in both chemical and physical characteristics and affects the mechanical properties through different mechanisms such as plasticization, crazing, hydrolysis, and swelling. plasticization is the most important physical change that occurs through the interaction of the water molecules with polar groups in the matrix, which can severely depress the glass transition temperature [12, 18, 2124 ]. for example, high moisture absorption capability of tgddm / dds epoxy resin, which is about 7 wt%, reduces the system tg from 260c to 130c [2527 ]. in general, for most epoxy systems, tg is reduced by 20c/1 wt% of moisture intake. the decrease in modulus of epoxy has also been reported to be due to plasticization according to several studies [2932 ]. other studies showed that the decrease in modulus resulted from crazing [3335 ], where the absorbed water acted as a crazing agent continuously decreasing the mechanical strength of epoxies with exposure time in water. this was supported by sem micrographs of epoxies, which have shown cavities and fractured fibrils that could be explained by a moisture induced crazing mechanism. the aforementioned chemical changes mainly include chain scission and hydrolysis [22, 36 ]. plasticization is considered reversible upon drying, while other effects of moisture absorption are irreversible. in recent years, effect of moisture absorption on the mechanical properties of neat epoxy and clay - epoxy nanocomposite has been frequently studied. zhao and li reported that tensile strength and modulus decreased for both neat epoxy and nanocomposites upon moisture absorption, while the tensile strain increased significantly for moisture absorbed samples. although failure occurred in brittle manner, effect of plasticization was found in sem images, which showed shear yielding for both neat epoxy and nanocomposite samples after being exposed to moisture. alamri and low reported lower flexural strength and modulus for halloysite, nanoclay, and nanosilicon carbide nanocomposites as a result of moisture absorption. al - qadhi. studied the effect of moisture absorption on the tensile properties of clay - epoxy nanocomposites and found that tensile strength and modulus decrease as a result of water uptake. wang. investigated the effect of hydrothermal degradation on mechanical properties such as tensile strength, modulus, and fracture toughness with immersion duration. for dgeba epoxy systems, fracture toughness and modulus were not influenced much with immersion time, while tensile strength decreased for nanocomposites. tensile and flexural properties of nanoclay reinforced composite laminates and cnt reinforced nanocomposites have also been found to be affected adversely as a result of moisture absorption [12, 40 ]. according to the study conducted by buck., at elevated temperature, combination of moisture and sustained load significantly reduced ultimate tensile strength of e - glass / vinyl - ester composite materials. a study on elastic modulus of epoxy polymer after absorption - desorption cycle showed recovery of property from wet condition, although modulus remained at a lower value than the as - prepared samples for lower filler volume. for higher volume fraction of reinforcement, elastic modulus improved to a value which was more than the elastic modulus of the as - prepared samples. phua. also reported recovery of tensile properties after refrying omtt - pbs nanocomposites. a similar study conducted by ferguson and qu showed recovery of elastic properties from moisture saturated state after a desorption cycle. however, de'ne`ve and shanahan did not observe any recovery of elastic modulus after absorption - desorption cycle at elevated temperature. it is evident from the published works that moisture absorption can severely alter mechanical properties of epoxies by decreasing the elastic modulus [12, 29, 33 ], tensile strength [3, 12 ], shear modulus [30, 31 ], flexural strength and flexural modulus [40, 43 ], yield stress, and ultimate stress as water uptake increases. most of the research on polymer - clay nanocomposites has been mainly focused on investigating the effect of moisture absorption on mechanical properties such as elastic modulus and tensile strength. although fracture toughness is a very important property for these nanocomposites as these are used in various structural applications, the effects of moisture absorption on fracture toughness of polymer - clay nanocomposites have not been studied extensively. durability of polymer / clay nanocomposites is still needed to be studied in depth, particularly for hygrothermal aging in which the degradation of the mechanical properties and loss of integrity of these nanocomposites occur from the simultaneous action of moisture and temperature. this study on clay - epoxy nanocomposites was designed to investigate the effect of hygrothermal aging on mechanical properties of these nanocomposites. two different clay particles were used to investigate the effect of clay structure on the permanent property changes due to hygrothermal aging. a drying cycle was employed to quantify the recovery of the properties after hygrothermal aging. this was helpful to understand the extent of permanent degradation that occurred by the combined application of elevated temperature and moisture. mechanical properties in terms of fracture toughness, flexural strength, and flexural modulus are the properties that were studied. scanning electron microscopy and fourier transform spectroscopy were conducted to further elucidate the underlying fracture mechanisms of these preconditioned specimens. the epoxy resin used for this study is epon 862, which is a diglycidyl ether of bisphenol f. the curing agent used for this resin system was a moderately reactive, low viscosity aliphatic amine curing agent, epikure 3274. both of these chemicals were supplied by miller - stephenson chemical company, inc.,, arlington heights, il) modified with a quaternary amine (trimethyl stearyl). somasif mae (co - op chemical co., japan), which was the other clay particle used for this study, is a synthetic mica modified with dimethyl dihydrogenated tallow ammonium chloride. epoxy was preheated to 65c before desired amount of clay was introduced and mixed using mechanical mixer for 12 hours. to reduce the viscosity of the mixture and to facilitate mixing, temperature was maintained at 65c using a hot plate for the entire duration of mixing. the mixture was then degassed for around 30 minutes to remove any entrapped air bubbles. bubble - free mixture of clay and epoxy was then shear - mixed using a high - speed shear disperser (t-25 ultra turrax, ika works inc., north carolina, usa) for 30 minutes. during this process curing agent was added to the mixture at 100 : 40 weight ratio and carefully hand - mixed to avoid introduction of any air bubble. after it was properly mixed, the final slurry containing epoxy and clay was poured into an aluminum mold and cured at room temperature for 24 hours followed by postcuring at 121c for 6 hours. the final sample had a nominal dimension of 177.80 mm 152.50 mm 12.70/6.35 mm. to study the effect of loading percentage, the weight fraction of the clay in the nanocomposite was varied from 0.5 to 2.0%. after specimens were cut into the final required dimension according to the astm standards d5045 and d790, they were subjected to degradation. specimens from each nanocomposite were taken and submerged in purified deionized boiling water for 24 hours. water saturated specimens were dried in an oven at 110c for 6 hours to remove moisture from the samples leaving only permanent degradation in the form of bonded water. critical stress intensity factor, kic, was determined by single edge notch bend (senb) test as per the astm d5045 on universal testing machine (instron 5567, norwood, ma) in a displacement - controlled mode with fixed crosshead speed of 10 mm / min. nominal dimension for the senb test samples was 67.30 mm 15.20 mm 6.35 mm. a notch was created using precision diamond saw (mk-370, mk diamond products inc., torrance, ca, usa) and, afterwards, a sharp precrack with ratio of 0.45 < a / w < 0.55 was created by tapping a fresh razor blade into the notch. fracture toughness for the specimens was calculated in terms of critical stress intensity factor, kic. the crack length, a, was measured using an optical microscope (nikon l150) which has a traveling plate with graduations : (1)kic = pbwf(aw), where p = maximum applied force (n), b = thickness of the specimen (mm), w = width of the specimen (mm), and f(a / w) = geometry factor, and it is given by the following equation : (2)f(aw)=3(s / w)a / w2(1 + 2(a / w))(1a / w)3/2 [(2.153.93(aw)+2.7(aw)2)1.99(aw)(1aw)iiiiiii(2.153.93(aw)+2.7(aw)2) ], where s = support span (mm) and a = length of the precrack (mm). flexural strength and flexural modulus were determined using three - point bend (3pb) test according to astm d790 on universal testing machine (instron 5567, norwood, ma). the nominal dimension for the flexural test specimens was 55.90 mm 12.70 mm 6.35 mm. consider (3)r = zl26d, where r = rate of crosshead motion (mm / min), l = support span (mm), d = depth of beam (mm), and z = rate of straining of the outer fiber (mm / mm / min) = 0.01. the flexural strength and flexural modulus were calculated using the following equations, respectively : (4)f, max=3pmaxl2bd2,(5)eb = l3m4bd3, where f, max = flexural strength (mpa), eb = flexural modulus (mpa), pmax = maximum load on the load - deflection curve (n), l = support span (mm), b = width of beam tested (mm), d = depth of beam tested (mm), and m = slope of the tangent to the initial straight - line portion of the load - deflection curve (n / mm of deflection). surface morphology of the fractured specimens from senb tests was observed using scanning electron microscopy (hitachi s-4800 fesem, dallas, tx). as polymer materials are nonconductive to electrons, all fracture surfaces were sputtered with gold - palladium alloy before sem imaging. ftir spectroscopy measurements were performed using atr - ftir spectrometer (nicolet is10, waltham, ma) using 64 scans at a resolution of 2.0 cm. table 2 shows the relative weight changes that occurred in the specimens after 24 hours of boiling water absorption and 6 hours of high temperature desorption cycle. for the studied material systems, percentage weight gain after absorption cycle showed no change as a function of clay loading percentage. this observation was different from the findings reported by glaskova and aniskevich for clay - epoxy nanocomposites. according to their study, moisture absorption was found to have increased slightly with the increase of clay weight percentage. the reason why a different clay - epoxy nanocomposite system behaves differently in moisture absorption test is still not clear and further investigation is required to understand it. in this study, although both clays are structurally different, the observed percentage weight gain for both nanocomposite systems was found approximately to be the same. these two observations led to the conclusion that moisture diffusion process primarily depended on the polymer system under investigation. moisture desorption data showed that most of the absorbed water is free water, which can be driven out of the system by drying. for neat epoxy, this is possibly due to the fact that presence of clay hindered the moisture diffusion process in and out of the epoxy polymers. an increasing trend in the amount of retained moisture for higher clay loading nanocomposites also supported the aforementioned statement. amount of water retained after the desorption cycle has been found to be almost similar for both clay - epoxy nanocomposite systems. the critical stress intensity factors as a function of clay loading percentage for nanomer i.28e and somasif mae clay - epoxy nanocomposites are shown in figure 1. critical stress intensity factor, kic, increased 28% for the as - prepared 0.5 wt% nanomer i.28e clay - epoxy nanocomposite compared to neat epoxy. the reason behind this observation can be attributed to the layered structure of the clay. clay in a polymer material physically blocks, bifurcates, and deflects the crack path, compelling the crack to travel longer path, which in turn results in higher toughness in a clay - polymer nanocomposite. the toughening effect of clay on epoxy polymer started to decrease with any additional clay reinforcement. this is a common behavior for several types of epoxy - clay nanocomposites and has been reported in previous studies conducted on clay - epoxy nanocomposites [3, 6, 45, 46 ]. depending on the processing technique and epoxy - clay interaction, there is an optimum weight percentage for which the property enhancement can be maximized. any further addition of clay negates the positive effect by forming agglomerates due to improper exfoliation of the clay platelets and thus results in stress concentration forcing the material to fail at lower loads. for moisture saturated epoxy - clay nanocomposites, kic was found to be lower compared to the as - prepared nanocomposite samples for all the nanomer i.28e nanocomposites. as water molecules diffuse into the nanofiller - epoxy interface, debonding and weakening of the interface occur, resulting in poor stress transfer between the filler and the epoxy matrix [43, 47, 48 ]. redried neat epon 862, free of void - filling water, showed 29% reduction in fracture toughness compared to the as - prepared neat epon 862 samples. addition of 0.5 wt% of clay resulted in 16% reduction in fracture toughness when compared to the as - prepared samples, which was significantly less compared to 29% reduction of neat epoxy. for 2.0 wt% nanomer i.28e clay - epoxy nanocomposite, however, the standard deviation of the as - prepared sample was much higher, which could possibly mean that the dried and the as - prepared samples have no difference in toughness. for the as - prepared and moisture saturated samples, somasif mae nanocomposites showed comparable trend in fracture toughness data ; clay reinforcement successfully improved the baseline epoxy properties and moisture absorption degraded the mechanical properties for all clay percentages. however, the permanent degradation after absorption - desorption cycle was found to be more prominent in the case of somasif mae clay nanocomposites compared to nanomer i.28e clay nanocomposites. the recovery of property after 6 hours of drying was negligible for somasif mae clay nanocomposites, whereas nanomer i.28e nanocomposites showed significant recovery of property after drying. the difference in property recovery of these two clay - epoxy nanocomposites can be attributed to the structural differences of the two clay particles and has been further investigated through sem and ftir technique. flexural modulus for the neat epoxy and clay - epoxy nanocomposites was determined from 3pb test and has been plotted against clay loading percentage in figure 2 for nanomer i.28e and somasif mae clay. flexural modulus increased almost linearly for both clays as a function of clay loading percentage. according to previous studies on epoxy polymers, incorporation of hard substance such as clay in polymer matrix results in higher modulus. when a load is applied on epoxy, the polymer chains slide past each other and deform. once layered silicates such as clay particles are introduced in a polymer system, it restricts the motion of the polymer chain sliding and makes the matrix less pliable. as the clay content increases, it is more difficult for the polymer chains to untangle and move. this increase in restriction of polymer chains is responsible for the increase in modulus as the clay percentage increases. for the hygrothermally conditioned specimens, this behavior observed is mostly due to the presence of water inside the epoxy system, which increases the ductility of the epoxy system. water acts as an effective plasticizer and can diffuse into the nanofiller - polymer interface and weaken the bonding between them [40, 43 ]. presence of water in epoxy system also results in an increase in free volume through rupture of hydrogen bonding between polymer chains, which increases the chain mobility and eases the segmental motion when a load is applied to the composite. these physical changes can be attributed to the observed lower modulus for water absorbed specimens. other mechanisms affecting the polymer such as hydrolysis and chain scission may also be responsible for lowering the modulus. once hydrolysis and chain scission take place, less bonding between the polymers makes it more deformable resulting in lower modulus for aged samples [22, 36, 51 ]. the effect of hygrothermal aging was more severe in neat epoxy than in the nanocomposites. for neat epon 862, flexural modulus decreased 20% after hygrothermal aging, whereas it was only 13% and 11% for 0.5 wt% of nanomer i.28e and somasif mae clay - epoxy nanocomposites, respectively. the high aspect ratio of the clay platelets provides resistance against polymer chain mobility in a water absorbed ductile polymer leading to the observed lower degradation of flexural modulus values in comparison to neat polymer. for nanomer i.28e clay - epoxy samples conditioned at 110c for 6 h, recovery of flexural modulus once redried, free water residing in the microvoids was evaporated, and the effect of plasticization was not prominent anymore. as a result, the ductility of the polymer reduced and the recovery of mechanical properties from moisture absorbed state occurred. nevertheless, in case of somasif mae clay - epoxy samples, modulus recovery was negligible after the desorption cycle. due to the structural difference between the two clay particles, it is possible that the interface of somasif mae clay - epoxy is being more affected by the hygrothermal degradation than the nanomer i.28e clay - epoxy interface. flexural strengths for the epoxy and clay - epoxy nanocomposites were determined using three - point bend (3pb) test and are plotted against clay loading percentage in figure 3 for nanomer i.28e and somasif mae clay. figure 3 shows that the addition of nanomer i.28e clay provided negligible improvement in flexural strength compared to neat epoxy. the maximum improvement in flexural strength was found to be less than 10% for both clay - epoxy systems from the base flexural strength of neat epoxy. similar observation has been reported in the literature, where addition of nanoclay particles did not significantly improve the flexural strength of the system. furthermore, when 2.0 wt% of clay is added to the nanocomposite, flexural strength value dropped to a lower value than the neat epoxy. increasing the amount of nanoparticles more than a certain amount has been found to reduce the flexural strength in earlier studies. it can also be observed that moisture absorbed nanocomposites showed significant reduction in flexural strength. for 1.0 wt% of i.28e clay - epoxy nanocomposites, reduction in flexural strength of nanocomposites after moisture absorption has been previously reported in the literature [4, 43, 5456 ] and has been attributed to the degradation of interface region, which in turn reduces stress transfer between the nanofiller and the matrix. for redried samples, as most of the moisture is driven out of the system and plasticization effect was minimal, flexural strength for these samples recovered almost fully. for instance, flexural strength recovers to 95% of its original value for 1.0 wt% of nanomer i.28e clay - epoxy nanocomposite. almost similar trend was observed for somasif mae clay - epoxy nanocomposites, where addition of clay did not change the property significantly, and after 24 h of hygrothermal aging property decreased to a lower value compared to the as - prepared samples. however, the severity of degradation was much less in both clay - epoxy systems compared to neat epoxy system. well dispersed high aspect ratio clay platelets have the capability of crack deflection and crack arresting, which can lead to the observed higher flexural strength in wet clay - epoxy samples in comparison to neat epoxy samples [48, 57 ]. for redried nanocomposites, similar trend was observed for both material systems and it was found that flexural strength recovers almost fully. in this study, flexural strength has not been largely affected by the addition of clay into the epoxy. this may as well mean that flexural strength has been primarily governed by the flexural strength of the epoxy. as the strength of neat epoxy was marginally affected by the absorption - desorption cycle, so did the strength of clay - epoxy nanocomposites. the scanning electron micrographs of the fracture surface of tested neat epoxy and clay - epoxy nanocomposites are shown in figures 48. sem micrograph of the fracture surface of the as - prepared neat polymer (figure 4(a)) showed characteristic brittle failure with a smooth fracture surface. this mirror - like fracture surface is an indication of poor fracture toughness of epoxy and has been reported in previous studies conducted on epoxy polymers. for the redried neat epoxy specimen (figure 4(b)), a network of microcracks throughout the fracture surface is found. according to their study, absorbed moisture enhances craze initiation and propagation in polymer which can result in the formation of microcracks or fibrils in the polymer system. in this study, lower fracture toughness for dried neat polymer compared to unaged neat polymer can be attributed to the formation of these microcracks. the sem micrographs of clay incorporated epoxy systems showed significantly rougher fracture surface compared to the neat polymer. clay, if present in a system, physically blocks and slows down the crack propagation and the resulting fracture surface of the nanocomposite shows river - markings instead of smooth fracture surface found in the neat polymer. these river - markings provide clear indication of the enhanced toughening mechanism in polymeric materials due to clay incorporation and support the observed higher fracture toughness for clay - epoxy nanocomposites compared to the neat epoxy polymer. fracture surfaces of nanofiller reinforced polymer nanocomposites showing higher surface roughness have been reported in prior studies [4, 6, 59 ]. comparing the fracture surfaces of the as - prepared nanomer i.28e and somasif mae nanocomposite systems (figures 5(a), 6(a), 7(a), and 8(a)), it was observed that the as - prepared somasif mae clay nanocomposite has considerably less amount of river - markings, which is indicative of poor toughening in somasif mae clay nanocomposites. this observation supported the difference of critical stress intensity factor measured for these two nanocomposite systems. poor adhesion between somasif mae clay and epoxy resulted in less energy requirement during new surface formation, which can be attributed as the reason of these nanocomposites showing less fracture toughness than nanomer i.28e clay nanocomposites for the same clay loading percentage. fracture surface of moisture absorbed nanomer i.28e clay nanocomposites (figures 5(b) and 7(b)) showed less number of river - markings (i.e., lower critical stress intensity factor) than the as - prepared nanocomposites. sem micrograph of somasif clay - epoxy nanocomposites (figures 6(b) and 8(b)) showed the presence of shear leaps. as shear yielding requires less energy to form new surface, moisture absorbed specimens had lower fracture toughness than the as - prepared specimens. although shear yielding was found to be the principle mechanism of failure in these specimens, some form of crack bifurcation and crack pinning was also present in these fracture surfaces. fracture surface micrographs of redried nanomer i.28e nanocomposites (figures 5(c) and 7(c)) showed more roughness than the moisture absorbed specimens indicating higher fracture energy absorbance for these specimens. micrographs of redried somasif mae clay - epoxy samples (figures 6(c) and 8(c)) showed significantly less rough fracture surface than the redried nanomer i.28e samples. it is important to note that these redried somasif mae clay - epoxy fracture surfaces showed very little resistance against the crack propagation even after most of the water was driven out of the system. this observation led to the speculation that absorbed moisture could have weakened the interface of the somasif mae clay particles and the epoxy matrix. the replacement of oh (hydroxyl) groups from the octahedral layer of the clay by the f (fluorine) groups makes the somasif mae clay particles much more hydrophobic compared to the nanomer i.28e clay particles. the higher hydrophobicity of the somasif mae clay particles could have exerted an additional force on the moisture absorbed interface and weakened the bond between the clay particles and the epoxy chains. this might have in turn resulted in the poor adhesion / less fracture energy absorption in the redried somasif mae clay - epoxy nanocomposites. ftir spectra of the as - prepared and redried neat epoxy are shown in figure 9. from the ftir spectra, it was evident that as a result of the absorption - desorption cycle the epoxy underwent some permanent chemical changes. the drying cycle used in this study removed most of the water that was absorbed by the polymer system, and it was believed that the remaining water (only about 7% of the total moisture uptake) is chemically bound to the polymer system. ftir spectra (figure 7) showed band at 32003400 cm, which is characteristic oh stretching of the hydroxyl group. figure 10 shows the ftir spectra for nanomer i.28e and somasif mae clay powder before and after the absorption - desorption cycle. nanomer i.28e clay showed another peak at 915 cm region, which is characteristic al oh peak. in somasif mae, the oh groups present in the corner of the octahedral layer of nanomer i.28e were substituted with f, which explains the absence of the peak at 915 cm. both clays in as - is condition showed 32003400 cm band for hydroxyl groups. however, the band was weaker in case of somasif mae compared to nanomer i.28e clay because of the hydrophobic nature of the somasif mae clay. it was interesting to note that the overall changes the clay powders underwent as a result of absorption - desorption cycle are fairly small and can be considered as insignificant. property deterioration due to moisture absorption has been one of the most important areas of interest in polymer research for the last few years. to be used as structural materials, it is of utmost importance to understand the reversible and irreversible changes occurring in polymeric materials as a result of moisture absorption. this study was conducted to elucidate the effect of moisture absorption on the mechanical properties of clay reinforced epoxy polymers. fracture toughness, flexural strength, and flexural modulus were determined for two different clay - epoxy nanocomposites following the astm standards. the effects of hygrothermal aging and subsequent redrying on the mechanical properties of these polymer nanocomposites were investigated. after removing the free water by drying, the irreversible effect or the permanent damage due to hygrothermal aging on the clay - epoxy nanocomposite systems was determined. irrespective of the clay reinforcement type, all the studied properties were degraded due to hygrothermal aging. several physical and chemical changes, such as interface weakening, hydrolysis, and chain scission, are responsible for the observed effect. the permanent damage or degradation was severe in case of fracture toughness and flexural modulus. permanent damage was found to be the highest for somasif mae clay reinforced specimens between two clay - epoxy nanocomposite systems. after studying the sem micrographs of the fracture surfaces, it was speculated that moisture absorption had higher negative impact on the interface of somasif mae clay and epoxy matrix compared to the other clay - epoxy system. the hydrophobic nature of the somasif mae clay due to the presence of f (fluorine) in the structure may have created additional tension between the polymer crosslinks in presence of moisture. ftir spectra of the clay particles treated with the same absorption - desorption cycle provided proof that both nanoparticles undergo minimal chemical change and retain their respective original chemistry. although incorporation of clay in epoxy matrix did not fully stop the degradation, it had positive effects to some extent. it was observed that the studied properties in general were less severely degraded for clay - epoxy nanocomposites compared to neat epoxy samples. therefore, clay particles could be successfully used to reinforce polymer materials to reduce the severity of property deterioration caused by the moisture absorption. however, the chemistry between the clay particles and polymer matrix and more specifically the chemical structure of the clay particles should be carefully considered to attain the best possible resistance against the property deterioration. | hydrophilic nature of epoxy polymers can lead to both reversible and irreversible / permanent changes in epoxy upon moisture absorption. the permanent changes leading to the degradation of mechanical properties due to combined effect of moisture and elevated temperature on epon 862, nanomer i.28e, and somasif mae clay - epoxy nanocomposites are investigated in this study. the extent of permanent degradation on fracture and flexural properties due to the hygrothermal aging is determined by drying the epoxy and their clay - epoxy nanocomposites after moisture absorption. significant permanent damage is observed for fracture toughness and flexural modulus, while the extent of permanent damage is less significant for flexural strength. it is also observed that permanent degradation in somasif mae clay - epoxy nanocomposites is higher compared to nanomer i.28e clay - epoxy nanocomposites. fourier transform infrared (ftir) spectroscopy revealed that both clays retained their original chemical structure after the absorption - desorption cycle without undergoing significant changes. scanning electron microscopy (sem) images of the fracture surfaces provide evidence that somasif mae clay particles offered very little resistance to crack propagation in case of redried specimens when compared to nanomer i.28e counterpart. the reason for the observed higher extent of permanent degradation in somasif mae clay - epoxy system has been attributed to the weakening of the filler - matrix interface. |
some newborn and older children are seen with ambiguous genitalia and it may be very difficult to assign them to either male or female gender. in the newborn, rapid gender assignment is essential to minimize emotional trauma in the family and also to diagnose and promptly treat potential life threatening situations. female pseudohermaphroditism is ambiguous genitalia with a normal karyotype (46,xx), normal ovarian, uterine and vaginal anatomy and virilized external genitalia believed to be due to exposure to excessive androgens in the first trimester of pregnancy. most of the conditions are associated with congenital adrenal hyperplasia (cah), but a small proportion of 46,xx females have an aromatase (cyp19) deficiency. aromatase is expressed in a tissue - specific manner and a placental deficiency abolishes its function in protecting the female fetus from masculinization and the mother from virilization due to an excess of androgens. here we report a case of both the mother and the newborn having been virilized. from the clinical features and biochemical data, we endocrinologically diagnosed her as having an aromatase deficiency. the mother denied receiving any medication during the pregnancy. from 16 wk of gestation, the mother and her relatives had noticed her voice became lower, development of mild acne and coarsening of facial features, although the obstetrician had not noticed these changes. these symptoms became progressively worse until delivery. at 35 wk and 5 d of gestation, ultrasonographic examination demonstrated a normal growth rate and no abnormal findings in the fetus. at 39 wk after delivery, the maternal manifestations of virilization disappeared gradually except the lowered voice. at birth, the baby had an enlarged clitoris (1.5 cm long ; prader v), complete fusion of posterior scrotolabial folds, and a single meatus on the top of the clitoris. at a local hospital the baby was assigned as a male with a micropenis and unpalpable testes. at 7 wk of age, the baby s karyotype was examined for the ambiguous genitalia and revealed to be 46, xx and fluorescence in situ hybridization (fish) analysis revealed negative sry. at 4 mo of age, this infant was referred to our hospital for further examination. at that time, the infant s length was 59.2 cm (1.04 sd) and weight was 5.5 kg (1.3 sd). the infant had ambiguous genitalia ; there was 1 cm long 0.6 cm wide phallus - like structure, complete posterior labial fusion, and a single meatus opening at the top of a phallus - like clitoris. single meatus is opening at the top of a phallus - like clitoris.). genital appearance on admission. a small phallus - like structure and complete labioscrotal fusion were observed. serum electrolytes and lipid profile were within the normal range ; other serum chemistry and blood cell count were also normal. t was below 5 ng / dl, and the serum e2 level was 22.7 pg / ml. after a single bolus intravenous acth administration, serum 17-hydroxy progesterone (17ohp) and progesterone levels increased from 4.9 to 37 ng / ml, and from 12 to 110 ng / ml (table 1table 1 baseline steroid levels and steroid responses to a 0.25 mg / m bolus of acth). dehydroepiandrosterone (dhea), 17-hydroxy pregnenolone (17ohpreg) and deoxycorticosterone (doc) were within the normal ranges (table 1). on a human cg stimulation test (4000 iu / m intramuscularly for three consecutive days) both basal lh and fsh levels were with in the normal range and lh showed a slightly hyper - response to a lhrh loading test (table 2table 2 gnrh loading test). the patient s urinary steroids profile measured by gas chromatography / mass spectrometry (gc / ms) is shown in table 3table 3 urinary steroids profile. although pregnanetriolone (ptl) was higher than the control, it did not increase as much as in 21-oh deficiency. sixteen -hydroxy dehydroepiandrosterone (16hd), 17-hydroxy pregnenolone (17-hp), 5-tetrahydro-11deoxy cortisol (5bths) and pregnenetriol (pt5) did not increase. an ultrasonographic examination revealed enlarged ovaries (right 3.8 2.4 cm, left 2.5 1.7 cm). abdominal magnetic resonance imaging (mri) showed that she had a uterus and cystic ovaries (fig. 2fig. 2 enlarged cystic ovaries (arrow) and normal sized uterus (arrowhead) were observed in sagittal t2 image.). enlarged cystic ovaries (arrow) and normal sized uterus (arrowhead) in the present case, the clinical manifestations were characterized by female pseudohermaphro - ditism and the mother s mild virilization during pregnancy. her karyotype was 46, xx and the sry gene were negative by fish. differential diagnoses of female ambiguous genitalia were as follows : virilizing form of cah ; { 21-hydroxylase deficiency, 11-hydroxylase deficiency, 3-hydroxysteroid dehydrogenase deficiency (3-hsd) }, true hermaphroditism, aromatase deficiency, transplacental virilizing drugs (androgens and progesterone), maternal androgen production (6, 7), and others. as for the 21-hydroxylase deficiency, serum 17ohp and urinary ptl levels were relatively low for genital virilization, and urinary pt5 and 16hd did not increase. normal levels of urinary 5bths and serum doc indicated that 11-hydroxylase activity was normal. furthermore, no increase in dhea or 17-hp was observed, suggesting that 3-hsd could be excluded. usually, maternal virilization does not occur with fetal cah during pregnancy. upon human cg loading test, the lack of a rise in serum testosterone levels was incompatible with true hermaphroditism. considering both fetal and maternal virilization leading to female pseudohermaphro - ditism, we can consider an aromatase deficiency, transplacental drugs, luteoma of pregnancy, adrenal tumors, and ovarian tumors. the mother denied receiving any medication during pregnancy and her virilization disappeared after delivery. these phenotypes are known to be caused by a placental aromatase deficiency. from the clinical features and biochemical data, we endocrinologically diagnosed her as having an aromatase deficiency. the aromatase gene is now under investigation for definite diagnosis. to the best of our knowledge, 6 affected females with an aromatase deficiency have been reported (table 4table 4 review of the clinical features of aromatase deficiency in genetic females) (8,9,10,11,12,13). these cases were severely virilized (prader iv~v) and their mothers had virilization with facial acne, hirsutism, whole body pigmentation, and hoarseness. these symptoms progressed with advancement of gestation and all of them improved except for the lowered voice after delivery. three of the cases were found to have cystic ovaries. in the other 3 cases low dose estrogen replacement therapy resulted in suppression of gonadotropin levels and resolution of the cystic ovaries (13). therefore, the etiology of these ovarian changes seemed to be hypersecretion of gonadotropin due to positive feedback caused by low estrogen levels. similarly, our patient had cystic ovaries discovered by ultrasonography and mri, and hypersecretion of lh after lhrh loading. if enlargement of cystic ovaries is noted, estrogen supplement will then be taken into consideration. in past reports, the value was within the normal range except for one case (10). we suppose that obstruction of the adrenal and genital cascade in steroidogenesis may occur due to an aromatase deficiency, resulting in high 17ohp and progesterone. in summary, we report a patient with aromatase deficiency who had ambiguous genitalia and maternal virilization during pregnancy. from the clinical features and biochemical data, we endocrinologically diagnosed her as having an aromatase deficiency. we finally agreed that aromatase deficiency should be suspected when both the mother and the newborn have been virilized. | female pseudohermaphroditism is caused by several etiologies. here we report a case of aromatase deficiency who showed ambiguous genitalia and maternal virilization during pregnancy. the mother had noticed her own virilization from 16 wk of gestation without androgen exposure and had low urinary estriol levels (5~10 g / ml at 35 wk of gestation). at birth, the patient presented severe virilization (prader v), and was assigned as a male with a micropenis and unpalpable testes but the patient had a normal female karyotype and a uterus and cystic ovaries found by magnetic resonance imaging. the patient had a increase in serum 17-hydroxy progesterone levels (basal 4.9 37 ng / ml after a single 0.25 mg / m2 infusion of acth), but the increase in adrenal androgen was not sufficient to virilize the external genitalia. dehydroepiandrosterone, 17-hydroxy pregnenolone and deoxycorticosterone were within the normal ranges. these findings suggested a diagnosis of nonadrenal female pseudohermaphroditism. from the clinical features and biochemical data, we endocrinologically diagnosed her as having an aromatase deficiency. the aromatase gene is now under investigation for definite diagnosis. we finally agreed that aromatase deficiency should be suspected when both the mother and the newborn have been virilized. |
malnutrition in elderly patients has a large number of negative consequences on health : it can often influence the prognosis of different pathologies, reduce health - related quality of life, and increase morbidity / mortality and hospital admissions.1,2 malnutrition is common among residents in hospitals and nursing homes. in europe, considering the time of admission to hospital, prevalence of malnutrition ranges from 10%80%, with an average value of 35% upon hospital admission, and it tends to worsen in most cases during hospitalization, while, in long - term - care (ltc) settings and in nursing homes, the average prevalence is 30%.3 the malnutrition prevalence has been reported to be 3%5% of free - living older adults4,5 and 21.3% in home care patients.6 the etiology is multifactorial and involves physiological aging7 and socioeconomic and psychological factors,8 as well as comorbid conditions typical of the elderly.9,10 dementia is itself a risk factor for malnutrition. a recent study shows that the nutritional state of institutionalized dementia patients is worse than those not institutionalized of the same age and with a normal cognitive state or mild cognitive impairment.11 the relationship between weight loss and dementia is complex and not completely clear ; in fact, weight loss can be different according to the type of dementia, the stage of the disease, and the living situation of the patients. studies examining food intake in the dementia population report varying results regarding the extent of weight loss and the adequacy of diet and/or energy intake.12,13 grundman found a significant association between low body mass index and cerebral cortex atrophy in the areas involved in control of eating behavior among patients with alzheimer s disease (ad).14 white explored the association between ad and weight changes, studying 362 subjects affected by ad and 317 control subjects for 2 years.15 they found that almost twice as many patients with ad lost more than 5% total body weight compared to controls, and that patients with more severe forms of ad are six to seven times more likely to suffer progressive loss of weight.15 poor nutritional status further undermines the functional state of dementia promoting musculoskeletal damage (sarcopenia and osteopenia), causing immunosuppression and, ultimately, decreases in respiratory and cardiac capacity.16 malnutrition becomes more evident in later stages of the disease when dysphagia or complications, such as pressure ulcers, repeated infections, and immobility syndrome, that further worsen the nutritional state of the patient, occur.17 about 78% of residents in nursing homes and extensive rehabilitation organizations associated with the national association of third age structures (anaste) calabria (an italian association of nursing home and rehabilitation to care for third age) were found to suffer from cognitive deterioration of different etiologies and gravities, of which 52% had severe dementia. diagnostic reevaluation of these patients, according to the criteria of the national institute of neurological and communicative disorders and stroke and the alzheimer s disease and related disorders association (nincds - adrda),18 showed that 56.14% were suffering from ad : in particular, 45.45% met the criteria for possible ad ; 28.12% met the criteria for probable ad ; and 37.5% met the criteria for probable - uncertain ad.19 few studies have focused on the relationship between patients nutritional states and severity of dementia, comorbidity and functional state in institutionalized demented.16 patients suffering from malnutrition have an increased need for nursing care due to the increased incidence of complications and reduced quality of life.20 the need for assistance may be calculated by an index of case mix and load assistance, expressed in minutes of assistance / day and multidisciplinary team in ltc.21,22 the purpose of the present study was to investigate the relationship between cognitive deficit and nutritional state in a cohort of elderly residents in ltc on functional state and the load of assistance. a network of ltc facilities for the care of frail elderly, consisting of nursing homes and extensive rehabilitation organizations, operates in calabria, italy. access to these facilities is regulated according to the guidelines provided by the calabria region (dgr 685/2002, dgr 695/2003, lr 29/2008, dgr 3137/1999). the present study was a 6-month observational, descriptive study carried out on residents across ten anaste calabria nursing homes in january 2010. the customary care practices provided to all patients who belong to anaste calabria ltc facilities were conducted throughout the study. at the moment of admission to ltc, informed consent of the patients and/or their caregiver was acquired for daily care practices and use of their personal data. baseline and follow - up data comprised a battery of validated indices chosen to establish an overview of health state. a sample of 174 residents, 122 female (70.1%) and 52 male (29.8%), was subjected to multidimensional and multidisciplinary evaluation. the diagnosis of dementia was investigated in an interview covering detailed personal and family history and was subsequently confirmed by the administration of psychometric tests. all patients fulfilled the criteria for dementia as described in the diagnostic and statistical manual of mental disorders, fourth edition, text revision (dsm - iv - tr).23 cognitive evaluation was conducted by a neuropsychologist who used folstein s mini - mental state examination (mmse).24 the patients were assessed as affected by severe, moderate, or slight cognitive impairment, based on mmse scores (010, 1020, and 2023, respectively). functional state was evaluated with the use of the activity daily living (adl)25 and barthel index (bi),26 in each of which a lower score indicates a worse functional state. the affective state was scored using the geriatric depression scale (gds), in which a high score (> 6) denotes a depressive state.27 comorbidity was examined according to the indices of severity and complex comorbidity of cumulative illness rating scale (cirs), in which higher scores indicate greater comorbidity.28 health care need was calculated in terms of minutes of assistance through evaluation by resource utilization groups (rug)-iii.22 finally, the number of different medications per patient was determined. the nutritional state evaluation included several biochemical values in serum, albumin (g / dl), cholesterol (mg / dl), iron (g / dl), and hemoglobin (g / dl), quantified by the mini nutritional assessment (mna).29,30 the mna has been previously validated for screening and assessment of malnutrition in older people. harris noted that the mna test demonstrates 80% sensitivity and 90% specificity.31 patients are classified in mna as follows : normal nutritional state (score of 30) ; at risk of malnutrition (score of 1723.5) ; and malnutrition (score of 6) denotes a depressive state.27 comorbidity was examined according to the indices of severity and complex comorbidity of cumulative illness rating scale (cirs), in which higher scores indicate greater comorbidity.28 health care need was calculated in terms of minutes of assistance through evaluation by resource utilization groups (rug)-iii.22 finally, the number of different medications per patient was determined. the nutritional state evaluation included several biochemical values in serum, albumin (g / dl), cholesterol (mg / dl), iron (g / dl), and hemoglobin (g / dl), quantified by the mini nutritional assessment (mna).29,30 the mna has been previously validated for screening and assessment of malnutrition in older people. harris noted that the mna test demonstrates 80% sensitivity and 90% specificity.31 patients are classified in mna as follows : normal nutritional state (score of 30) ; at risk of malnutrition (score of 1723.5) ; and malnutrition (score of 0.05), whereas there was a significant correlation between the group with severe cognitive deficit and the group with moderate cognitive impairment (r=0.33 ; p 4% body weight is an independent factor of morbidity and mortality.43 the incidence of medical problems associated with malnutrition in elderly people in nursing homes is 27%, whereas it is about 16% in patients who are well nourished, although mortality is three times higher in the former.44 in elderly patients, the effectiveness of nutritional interventions is reduced and recovery of malnutrition is difficult to achieve.45 in patients with dementia, a state of malnutrition must be prevented or at least improved by an early and appropriate intervention strategy.46,47 the results of the present study show that it is necessary to set appropriate nutritional interventions in patients with mild - to - moderate dementia ; in fact, these patients have more opportunities for therapeutic response than patients suffering from severe dementia. in anaste calabria ltcs, the multidisciplinary team is trained to carry out the assessment of nutritional state. this team is also trained to identify as risk factors for malnutrition both the cognitive impairment and worsening of functional state. malnutrition plays an important role in the progression of cognitive decline ; early recognition and treatment of malnutrition or risk of malnutrition are important preventive measures to increase the quality of care and quality of life of patients with dementia. | objectivethe interaction between dementia and nutritional state is very complex and not yet fully understood. the aim of the present study was to assess the interaction between cognitive impairment and nutritional state in a cohort of residential elderly in relationship with functional condition of patients and their load of assistance in long - term - care facilities of the national association of third age structures (anaste) calabria.methodsone hundred seventy - four subjects (122 female and 52 male) were admitted to the long - term - care anaste calabria study. all patients underwent multidimensional geriatric assessment. nutritional state was assessed with the mini nutritional assessment (mna), whereas cognitive performance was evaluated by the mini - mental state examination (mmse). the functional state was assessed by barthel index (bi) and activity daily living (adl). the following nutritional biochemical parameters were also evaluated : albumin, cholesterol, iron, and hemoglobin. all patients were reassessed 180 days later.resultsa severe cognitive impairment in mmse performance was displayed in 49.7% patients, while 39.8% showed a moderate deficit ; 6.9% had a slight deficit ; and 3.4% evidenced no cognitive impairment. in mna, 30% of patients exhibited an impairment of nutritional state ; 56% were at risk of malnutrition ; and 14% showed no nutritional problems. malnutrition was present in 42% of patients with severe cognitive impairment, but only 4% of malnourished patients showed moderate cognitive deficit. the statistical analysis displayed a significant correlation between mna and mmse (p<0.001), as did mmse correlated with activity daily living (p<0.001) and bi (p<0.05). mna correlated with bi (p<0.001) and albumin (p<0.001). the follow - up showed a strong correlation between cognitive deterioration and worsening of nutritional state (p<0.005) as well as with the functional state (p<0.05) and mortality (p<0.01).conclusionthe present study clearly shows that malnutrition may play an important role in the progression of cognitive loss. |
s. typhimurium infection in mice is a standard laboratory model for human typhoid, and previous studies have shown that s. typhimurium mutants which are unable to survive in murine macrophages are avirulent. thus, survival of salmonella in macrophages appears to be a critical step in the induction of typhoid. the salmonella phop / phoq regulon regulates genes located on salmonella pathogenicity island 2 (spi-2) which encode proteins needed for survival of salmonella inside of macrophages and salmonellae which have mutations in their phop / phoq regulon are avirulent in mice. the affect of phop on salmonella survival is multifaceted but studies by svensson. have shown that phop mutation induces increased nitrite ion production by macrophages compared with nitrite ion production induced by the parent strain but this study did not investigate the mechanisms behind this phenomenon. studies using inos and nadph mice indicate that reactive nitrogen species (rns) are important in controlling salmonella later in the infection and this is preceded by a reactive oxygen species (ros)-dependent control phase [5, 6 ] and it is also known that nitric oxide increases the sensitivity to cellular acid by phop mutants. taken together these studies indicate that the ability of salmonella to down - regulate nitrite ion production by host macrophages may be due to the effect of spi-2 proteins under the control of phop and that this confers survival advantage to the salmonella at some point in the infection, but the underlying inductive mechanism has not been reported. for example, nuclear factor kappa b (nfb) and activator protein-1 (ap-1) are both known to transcribe the inos gene [8, 9 ] but nothing has been reported regarding the activity of these transcription factors in relation to inos and in the context of wild type or phop mutant salmonella. furthermore, there are no reported comparisons between typhoidal and nontyphoidal salmonella serovars with regard to inos suppression. the aim of this study was to investigate the effect of wild type salmonella and phop mutants on nfb and ap-1 activity and their subsequent downstream effect using inos as a biological readout. we also compared the effect of serovars which cause typhoid in mice with those which do not. bacteria were grown in luria bertani (lb) broth (life technologies ltd, paisley, uk) for 18 h at 37c under agitation. the bacteria were then subcultured in fresh lb for 4 h to late log phase (established by conventional counts of cfu / ml). prior to incubation with j774.2 cells, bacteria were adjusted to the multiplicity of infection (moi) of 10. during this study, the following strains / mutants were used : s. typhimurium 14028 (atcc strain), s. typhimurium cs022 (phop mutant of 14028, a gift from dr s. i. miller, university of washington, usa), which does not survive in macrophages. in a separate study, the effect of other murine typhoid - inducing (s. typhimurium 4/74, s. enteritidis kms1977, s. dublin 2229, and s. choleraesuis a50) and nontyphoid inducing strains (s. gallinarum 9, s. kedougou gp, and s. montevideo kms) was analysed. j774.2 cells were grown to confluence in 96 well plates (nunc, naperville, il, usa) containing rpmi 1640 media at 37c in co2 (5% v / v). the cells were then washed 3 times in phosphate - buffered saline (pbs), to remove media and nonadherent cells, and incubated in pbs at 22c for 15 min prior to infection. nitrite ion concentration in j774.2 supernatants were measured by griess reagent kit (promega, madison, wi, usa) as per manufacturer instructions. briefly, 50 l of supernatant were mixed with 50 l of sulfanilic solution and incubated at 22c in darkness for 10 min. 50 l of napthyl ethylenediamine solution was added and incubated for a further 10 min and the reaction was read on a plate reader (anthos labtech instruments, hamburg, germany) at 550 nm. the nitrite ion concentration was determined by comparison with a nitrite ion standard curve with a limit of 10005 m. the activity of inos in infected and uninfected j774.2 cells was determined by standard immunocytochemical methods. salmonella - infected cells, which had or had not been incubated with 100 u / ml ifn-, were washed free of media and permeabilised for 10 min at ambient temperature in 0.05% v / v triton x-100 after 2, 7, 12, and 24 h postinfection. the cells were then fixed for 15 min in 5% v / v paraformaldehyde at ambient temperature and washed in pbs. fixed cells were then incubated in the dark on an end - to - end shaker for 60 min at 4c with mouse anti - inos igg (autogen bioclear, uk) pbs - tween 20 (0.02% v / v tween, pbs - t) to give a final antibody concentration of 1/100. the cells were then washed three times in pbs - t and incubated in identical conditions but with secondary anti - mouse antibody conjugated to fluorescein isothiocyanate (fitc, sigma). samples were viewed on a tcs nt confocal laser scanning microscope equipped with an argon laser (leica, cambridgeshire, uk). controls included j774.2 cells which had not been infected or incubated with ifn- and also uninfected cells which were incubated with 100 u / ml ifn-. expression of inos protein in salmonella - infected j774.2 cells was assessed by western blotting. j774 cells were lysed in cold water containing protease inhibitors (pepstatin 1 g / ml ; phenylmethylsulphonyl fluoride 1 mm and leupeptin 10 m) (sigma) and protein concentration was determined using a bicinchoninic acid (bca) assay (pierce, cheshire, uk). 10 g / ml j774.2 protein was electrophoresed by standard methods on two 10% acrylamide gels ran back - to - back. to test for parity in protein loading, one gel was stained with coomassie blue whilst proteins on the second gel were transferred to hybond - c nitrocellulose paper (amersham, buckinghamshire, uk) by a trans - blot sd, semi - dry transfer cell (bio - rad, hertfordshire, uk). the nitrocellulose was blocked at room temperature for 60 min in pbs - t containing bovine serum albumin (5% w / v, sigma). after blocking, the nitrocellulose was washed 3 times for five min in pbs - t on an end - to - end shaker at room temperature. the nitrocellulose was then incubated for 60 min in pbs - t containing 1/200 dilution of anti - mouse inos (autogenbioclear, uk), washed three times as before and incubated for 60 min at room temperature in pbs - t containing rabbit anti - mouse igg conjugated to horseradish peroxidase (sigma, 1/12000 dilution). the nitrocellulose was then washed and developed using an enhanced 3, 3 diaminobenzidine (dab) kit (vector laboratories, burlingame, ca, usa). inos expression in j774.2 cells infected with salmonella was measured by reverse transcription polymerase chain reaction (rt - pcr) using a previously reported method. briefly, 6 10 j774 cells were suspended in 3 ml tri reagent (sigma) and stored at 70c until required (used within 14 days). samples were centrifuged at 12,000 g for 10 min in a bench top centrifuge at 4c. the supernatants were transferred to separate tubes, and 200 ml chloroform was added per ml tri reagent prior to incubation for 10 min at 22c. the sample was then centrifuged at 12,000 g and 4c for 15 min, the aqueous phase was removed, and an equal volume of propan-2-ol was added. the sample was centrifuged at 12,000 g for 10 min, and the rna pellet was washed in a mixture of 1 vol 75% ethanol : 1 vol sterile water. the mixture was then centrifuged for 10 min at 7,500 g, and, after removal of the supernatant, the pellet was allowed to air dry for further 10 min. rna purity was measured using an ultraspec iii spectrophotometer (pharmacia, hertfordshire, uk) and was found to have a typical 260/280 nm ratio of 1.9 to give yields of around 100 g / ml. rna concentration was adjusted to 1 g/l prior to the rt reaction. rt reactions were performed on a thermal cycler (eppendorf, hamburg, germany) using the following previously reported primer sequences : forward : 5-gta aac tgc aag aga acg gag aac-3. reverse : 3-gag ctc ctc cag agg ggt ag-5.as a loading control, the following glyceraldehyde-3-phosphate dehydrogenase (gapdh) primer sequences were used : forward : 5-gtt cag ctc ttg gat gac ctt gcc-3. reverse : 3-tcc tgc acc acc aac tgc tta gcc-5. forward : 5-gta aac tgc aag aga acg gag aac-3. reverse : 3-gag ctc ctc cag agg ggt ag-5. forward : 5-gtt cag ctc ttg gat gac ctt gcc-3. reverse : 3-tcc tgc acc acc aac tgc tta gcc-5. nuclear lysates were prepared by scraping cells into 5 ml pbs and washing once with buffer i (hepes 10 mm, kcl 10 mm, mgcl2 1.5 mm, ph 7.9). the cells were disrupted by freeze - thawing twice in 1 ml buffer i containing nonidet p-40 (np-40) (5%, sigma). the lysate was centrifuged for 5 min at 2000 rpm in a bench - top microfuge. the pellet was then washed twice with buffer i and np-40 before being centrifuged at 12,000 rpm for 5 min to obtain the nuclear pellet. nuclear proteins were extracted from the pellet for 10 min with 3040 l extraction buffer (hepes 20 mm, nacl 420 mm, mgcl2 1.5 mm, edta 0.2 mm, 25% glycerol, ph 7.9). after mixing, the supernatant was then diluted in 4060 l dilution buffer (hepes 20 mm, kcl 50 mm, edta 0.2 mm, glycerol 20%, ph 7.9). protease inhibitors (pepstatin 1 g / ml, phenylmethylsulphonyl fluoride 1 mm, leupeptin 10 m, sigma) were added to the lysate which was then used immediately or stored at 70c for up to 14 days. emsa was used to determine dna binding of nfb (p50) and activating protein (ap)-1 (c - jun) to j774.2 dna following infection by salmonella and/or incubation with ifn- (100 u / ml). emsa reactions were performed using a kit as per manufacturer instructions (promega, usa) using the following oligonucleotide sequences : ap-1 (c - jun) 5-cgc ttg atg agt cag aag gaa-3 3-gcg aac tac tca gtc ggc ctt-5 5-cgc ttg atg agt cag aag gaa-3 3-gcg aac tac tca gtc ggc ctt-5 nfb (p50) 5-agt tga ggg gac ttt ccc agg c-3 3-tca act ccc ctg aaa ggg tcc g-5kit controls included hela cell nuclear lysate with consensus oligo (positive control) and hela cell nuclear lysate without consensus oligo (negative control). as an additional negative control, 5-agt tga ggg gac ttt ccc agg c-3 3-tca act ccc ctg aaa ggg tcc g-5 digital image analysis was performed using a phoenix 1d analyser using a power scanner v.3 (phoretix, newcastle upon tyne, uk). mann - whitney analysis (minitab) was used to measure significant difference at the 95% confidence limit between different test groups and between the same test groups at different time points. nitrite ion production by macrophages was significantly (p < 0.05) lower when the cells were cultured with wild type s. typhimurium 14028 compared to nitrite ion production by macrophages cultured with 14028 phop mutant. however addition of ifn- to culture media significantly increased (p < 0.05) nitrite ion concentrations in supernatants recovered from both wild type and phop - cultured macrophages (figure 1(a)). the numbers of wild type 14028 recovered from macrophages were also between 1 - 2 logs greater after 1224 h culture compared with the numbers of 14028 phop mutants recovered from cells over the same period (figure 1(b)). when macrophages were cocultured with ifn-, the numbers of wild type 14028 recovered from cells decreased and were comparable to the numbers of 14028 phop mutants recovered over the same time period (figure 1(c)). increased inos mrna expression was detected in macrophages cultured with 14028 phop mutants when compared with 14028 wild type salmonella and in both cases the inos mrna signal was increased by coculture with ifn- (100 u / ml) (figure 2(a)). however, when adjusted for gadph expression, inos mrna expression, in j774 cells, induced by ifn- and 14028 wild type, was still slightly lower than was induced by ifn- alone. confocal microscopy data also clearly showed that a phop mutation (figure 2(d)) caused a more intense inos protein signal in the cytoplasm of j774.2 cells when compared to uninfected control macrophages (figure 2(b)) or wild type 14028-infected cells (figure 2(c)). the intensity of inos signal was increased by co - culture of macrophages with wild type 14028 and ifn- (figure 2(e)) and was even more intense in macrophages which were co - cultured with 14028 phop mutants and ifn- (figure 2(f)). the results obtained by confocal were also repeated by western blotting (figure 2(g)) and in this case j774 cells co - cultured with 14028 wild type and ifn- produced a greater amount of inos protein when compared to j774 cells cultured only with ifn-. our results show that mutation in the salmonella phop regulon increased the amount of nfb and ap-1 bound to macrophage dna after 2 h, when compared to wild type 14028 (figure 3(a)). when compared to a positive control, nfb / dna interaction following wild type 14028 infection was reduced by a mean of 68% but when infected macrophages were co - cultured with ifn- (100 u / ml), nfb / dna interaction was reduced by 42% (figure 3(a)). in comparison, when macrophages were infected with 14028 phop mutants, nfb / dna interaction was only reduced by a mean of 22% (relative to the positive control) and this remained constant following co - culture of infected cells with ifn- (figure 3(a)). after macrophages were cultured with wild type 14028 for 12 h, nfb / dna interaction was reduced by a mean of 66% but when infected macrophages were co - cultured with ifn-, nfb / dna interaction was reduced by 76% (figure 3(b)). when macrophages were infected for 12 h with 14028 phop mutants, nfb / dna interaction was only reduced by 40% (relative to the positive control) and this also remained constant following co - culture of infected cells with ifn- (figure 3(b)). in macrophages infected for 2 h with wild type 14028, results obtained for ap-1/dna binding was equivalent to those obtained for a negative control which contained no oligonucleotide (92% reduction in dna binding compared to the positive control) and this was altered very little by coculture with ifn- (88% reduction compared to the positive control) (figure 4(a)). however, when macrophages were infected with 14028 phop mutants for 2 h, ap-1/dna binding was only reduced by 39% relative to the positive control and this level was maintained following co - culture with ifn- (figure 4(a)). after macrophages were cultured with wild type 14028 for 12 h, ap-1/dna interaction was increased with a reduction relative to the positive control of 62.9% and ap-1/dna interaction was increased further following co - culture of infected macrophages with ifn- for 12 h, in which a mean reduction of 55% (relative to the positive control) was measured (figure 4(b)). however, ap-1/dna interaction in macrophages infected with 14028 phop mutants for 12 h was reduced only by 53% compared to the positive control but, following co - culture with ifn-, ap-1/dna interaction was increased with a mean reduction of only 21% relative to the positive control (figure 4(b)). when macrophages were cultured with salmonella serovars which are known to induce murine typhoid, nitrite ion concentrations were reduced in cell supernatants compared to supernatants isolated from macrophages cultured with nontyphoidal strains over the same time period (figure 5). however s. choleraesuis, which is known to induce murine typhoid, was an exception to this rule since it stimulated nitrite ion production by macrophages at similar levels to those measured in supernatants isolated from macrophages cultured with nontyphoidal serovars (figure 5). eriksson. have previously reported that hyper - survival mutants of s. typhimurium tt16729, obtained by multiple passage through j774 cells, induced lower nitrite ion concentrations than did the parent strains, although an attenuated salmonella mutant was not studied for comparison. however, svensson. reported that wild type s. typhimurium 14028 induced lower nitrite ion production in murine bone marrow derived macrophages when compared with an s. typhimurium constitutive phop mutant but that the survival of the phop was only marginally different (< 0.5 log) to the 14028 wild type. in contrast to this result, we show that there is a clear increase in survival rates of wild type 14028 compared to its phop mutant in j774.2 macrophages. however, the differences we observe may conceivably be due to differences in cell type or moi, since our moi was constant at 10 : 1 whereas the previous study used 14028 at an moi of 15 : 1 and phop at an moi of 17 : 1. in contrast to these studies, a study by das. has shown that in the murine macrophage cell line raw264.7, wild type salmonella inhibit ifn--induced no production via the virulence gene nirc and that this correlates with increased cellular survival. however our study shows that inos mrna, inos protein, and nitrite ion production are increased when wild type 14028-infected j774 cells are co - cultured with ifn- but overall wild type infected cells elicit much weaker responses than do 14028 phop - infected macrophages, and this occurs in the presence or absence of ifn-. we have also found that 14028 wild type and 14028 phop have comparable sensitivity to exogenous nitrite ions, as shown by bacterial growth curves obtained at different nitrite ion concentration (data not shown). it is possible that the inhibition of no production via inos suppression may have relevance later in the infection as shown by mastroeni. or it is also possible that the inhibition of inos per se (rather than downstream no) may have immediate impact on other factors. for example, inos - dependant induction of 8-nitroguanosine 3,5-cyclic monophosphate (8-nitro - cgmp) has been shown to induce heme oxygenase 1 (ho-1) which has both cytoprotective and antimicrobial effects in murine salmonellosis. no production may also enhance other antimicrobial pathways, for example, via interaction with reactive oxygen species. our study also attempted to relate changes in inos to the activity of two critical transcription factors (nfb and ap-1) in infected j774 macrophages, with or without co - culture with ifn-. the inos gene has previously been shown to be transcribed by nuclear factor kappa b (nfb) (p50/65) which may translocate to the cell nucleus following stimulation by interferon gamma (ifn-) [17, 18 ] and this may act synergistically with bacterial lipopolysaccharide (lps), depending on relative concentrations. as well as nfb binding sites, promoter sequences on murine inos genes also contain binding sites for ap-1 and ifn- response elements (-ire) (reviewed). however, ap-1 activation during salmonella invasion of macrophages has not been comprehensively studied but temporal changes in the heterodimeric composition of ap-1 during culture of murine macrophages with s. typhimurium lps or porins have been reported. our data was interesting for a number of reasons ; firstly we show that wild type 14028 suppresses nfb / dna interaction within the first 2 h postinfection of j774 macrophages but this is not the case when phop mutation is induced. this suggests that the ability to suppress nfb activity is, therefore, phop - dependent. however, the inherent suppression of nfb activity by wild type 14028 is overcome when the macrophages are co - cultured with ifn-, and this is consistent with one study which has shown that ifn- suppresses phop transcription in wild type salmonella. interaction of nfb with macrophage dna was also reduced further after 12 h culture with wild type 14028 and co - culture with ifn- had no effect, whereas phop mutants maintained relatively strong nfb / dna interaction (although this was also reduced by about half when compared to nfb / dna interactions measured after 2 h). these results suggest that an intact phop regulon promotes significant changes in the ability of nfb to interact with dna and this probably had a significant impact on the inos suppression we observed in wild type - infected cells. have shown that ifn- induces nuclear translocation of interferon regulatory factor 1 (irf-1) which then synergises with nfb to transcribe inos. therefore, it is possible that this is an additional mechanism by which ifn- increases inos transcription in our study, as well as the increased nfb / dna interaction we have shown. however, our study also indicates that there is temporal separation between the induction of nfb / dna interaction and ap-1/dna interaction in 14028 wild type infected macrophages but this was not observed in macrophages infected with 14028 phop mutants. after 2 h postinfection with wild type 14028, no discernable interaction between ap-1 and macrophage dna was measured but after 12 h post - infection ap-1/dna interaction was measured and this was increased when the cells were co - cultured with ifn-, although this was on average 5563% lower than that measured for the positive control. in contrast, when the macrophages were cultured with 14028 phop mutants, ap-1/dna interaction was only about 53% lower than the positive control but when the cells were co - cultured with ifn-, ap-1/dna binding was only 21% lower than the positive control. these results also indicate that an intact phop regulon prevents long - term interaction of ap-1 with dna and ifn--induced increase in ap-1/dna interaction (as was the case with nfb). we, therefore propose that the affect of the phop regulon to prevent long - term and strong interaction of macrophage dna with nfb and ap-1 and ifn--stimulated upregulation of this interaction will have a profound effect on inos expression and nitrite ion production via reduced exposure of inos promoter sequences within macrophage dna to these essential transcription factors. no previously published data exists which has compared inos activity and nitrite ion production in murine macrophages cultured with typhoidal and non - typhoid salmonella serovars. however, eisenstein. have shown that s. typhimurium and s. dublin both inhibit nitrite ion production by murine splenocytes, and although this study at least considered different typhoid inducing serovars, a comparison between typhoid - inducing and non - inducing serovars was not reported. the one surprising exception in our study was s. choleraesuis which failed to down- regulate nitrite ion production by macrophages. s. choleraesuis causes typhoid - like systemic disease in a much wider range of mammalian hosts than do s. typhimurium, s. dublin, or s. enteritidis and we can not, as yet, explain why this serovar does not down regulate inos. however, all other typhoid - inducing serovars were able to down - regulate nitrite ion production by murine macrophages whereas those which do not induce typhoid were unable to do so. this may suggest that the ability of typhoid - inducing salmonella may allow dissemination to deeper tissues, via a phop - induced suppression of nfb and ap-1 and subsequent suppression of inos. our data may be relevant in the future treatment of typhoid in humans, since it suggests a possible role for the adjunctive use of ifn- (and antibiotic) to overcome phop - dependent inos suppression. thus, increasing nuclear translocation of essential transcription factors needed for transcription of the inos gene and nitrite ion production. | background. there are no reported data to explain how salmonella suppress nitrite ion production in macrophages or whether this phenomenon is unique to typhoidal or non - typhoidal serovars. the aims of this study were, therefore, to investigate these phenomena. methods. we measured survival of s. typhimurium 14028 and its phop mutant in murine j774 macrophages, cultured with or without interferon gamma. we compared expression of inducible nitric oxide synthase (inos) mrna and protein, and nitrite ion production and also examined binding of nuclear factor b (nfb) and activator protein 1 (ap-1) to macrophage dna. results. s. typhimurium 14028 inhibited binding of nfb and ap-1 to dna in murine j774. a macrophages via an intact phop regulon. this correlated with increased survival and reduced inos expression. suppression of nfb activity was ameliorated in macrophages cultured with ifn- and this correlated with increased expression of inos mrna and nitrite ion production, although ifn- had no effect on ap-1/dna interaction. we show, that with one exception, suppression of inos is unique to typhoidal serovars. conclusion. s. typhimurium inhibit nfb and ap-1 interaction with macrophage dna via the phop regulon, this reduces nitrite ion production and is principally associated with typhoidal serovars. |
carcinomas of the vagina are rare, accounting for only about 2% of gynecologic malignancies. the most common histological type is squamous cell carcinoma (75 - 90%), followed by adenocarcinoma (5 - 10%), melanoma (3%) and sarcomas (3%). it is recommended that smooth muscle tumors > 3 cm in diameter, with five or greater mitoses per 10 high - power fields (hpfs), moderate or marked cytologic atypia and infiltrating margins be classified as leiomyosarcoma. herein, we add another case of leiomyosarcoma of the vagina to the available sparse literature. a 39-year - old woman presented with complaints of dyspareunia, vaginal discomfort and difficulty of micturition for 2 years. pelvic examination revealed a 4 cm 4 cm firm, globular mass located on the middle part of the right lateral vaginal wall. the patient then underwent vaginal myomectomy and histopathological examination revealed atypical leiomyoma with a definite risk of recurrence. the surgical margins were free of tumor. the patient was kept on close follow - up with regular pelvic examination and magnetic resonance imaging (mri) done at 6 months postvaginal myomectomy was normal. however, at 11 months follow - up, she presented with recurrence of vaginal growth. mri was suggestive of a lobulated lesion on the upper and middle part of the right lateral vaginal wall, abutting closely the right cervical lip. the patient then underwent exploratory laparotomy with total abdominal hysterectomy and bilateral salpingo - oophorectomy plus resection of recurrent tumor and partial vaginectomy. cytology of the peritoneal washings and biopsies obtained from the ovaries, omentum and pelvic lymph nodes were negative for tumor. histologically, it was composed of spindle - shaped cells arranged in the form of varying - sized nodules and interlacing fascicles with marked pleomorphism and nuclear atypia [figure 1a ]. the tumor cells comprised of spindle - shaped nuclei with blunt ends, coarse nuclear chromatin and prominent nucleoli [figure 1b ]. many bizarre cells and multinucleated cells were also seen. mitotic activity was approximately 12 per 10 hpfs [figure 1c ] and there was no evidence of coagulative necrosis. based on the histopathological results, the tumor was diagnosed as a leiomyosarcoma of high grade. postoperatively, the patient received adjuvant chemotherapy with adriamycin and ifosfamide for three cycles followed by pelvic radiotherapy of 50 gy in 25 fractions and then followed by completion of chemotherapy up to a total of six cycles. the patient continues to be on follow - up in our outpatient clinic every 3 months. contrast - enhanced computed tomography performed at last follow - up at 29 months did not show any evidence of disease. (a) the tumor is composed of fascicles of spindle cells showing marked pleomorphism (200). (b) the tumor cells comprised of spindle - shaped nuclei with blunt ends, coarse nuclear chromatin and prominent nucleoli. smooth muscle tumors, although rare, are reported to be the most common benign and malignant mesenchymal tumors in adult women, and leiomyosarcoma is the most common vaginal sarcoma in adult women. the literature on vaginal sarcoma consists principally of few case reports and small case series. the rarity of these tumors has contributed to the paucity of information regarding the clinical features, mode of management and prognosis of patients. most patients with vaginal leiomyosarcoma present with an asymptomatic vaginal mass, but may also experience vaginal, rectal or bladder pain, vaginal discharge or bleeding, difficulty in micturition or, rarely, dyspareunia. the vaginal leiomyosarcoma spreads by local invasion and hematogenous metastasis. the average age at diagnosis is about 50 years, with a range extending from 21 to 86 years. most sarcomas arise de novo, with malignant transformation of a benign mesenchymal tumor being a very rare event. however, in our case, the swelling has recurred at the same site after 11 months. miyakawa., have described that the sarcomatous degeneration of a leiomyoma of the uterus occurs in less than 1% of all cases. because of the risk of recurrence and future transformation of leiomyoma to leiomyosarcoma, as well as diagnosis as malignant after surgery, these tumors should be removed completely. the primary treatment for vaginal smooth muscle tumor is resection of disease with adequate margins and of the site of possible uterine sarcoma, but the extent of resection may not be related to clinical course. peters., found that only patients treated with pelvic exenteration had long - term survival, and suggested that exenteration be considered in patients able to tolerate the procedure. in the review of the literature by ciaravino.,66 cases of vaginal leiomyosarcoma were identified, of which 48 had follow - up data. they also supported considering pelvic exenteration as a treatment option and emphasized that tumor grade should remain a consideration in patient management. in the same study, younger age, low stage and surgical resection, compared with chemotherapy or radiotherapy, stage was shown to be an independent predictor of survival. moreover, there was no difference in survival between patients who had undergone surgery followed by adjuvant radiotherapy and/or chemotherapy and patients who had been treated with surgery alone. the role of adjuvant radiotherapy and chemotherapy is not clearly defined in vaginal sarcomas, primarily due to the limited number of case reports and series, and even fewer data are available regarding chemotherapy used as the primary treatment rather than as salvage therapy at recurrence. adjuvant radiation therapy seems to be indicated in patients with high - grade sarcomas, low - grade recurrent tumor and if the tumor extends beyond the surgical margins. postoperative radiotherapy has been used for the management of soft tissue sarcomas to reduce the incidence of local recurrences. in most series, patients treated with adjuvant radiation presumably had higher risk factors thus biasing the data against radiotherapy. however, a phase iii randomized trial in stage i and ii uterine sarcomas reported that postoperative pelvic radiotherapy did not improve the overall survival for leiomyosarcoma when compared with observation. the largest series on vaginal sarcomas reported to date included 17 cases, of which 10 were leiomyosarcoma, four were mixed mesodermal tumors and three were other sarcoma types. these results underscore the importance of local therapy because pelvis was the first site of recurrence in all 14 treatment failures and the only site of failure in 50%. hensley reinstated the need for primary surgery plus adjuvant radiation to decrease local recurrence rates for high - grade lesions and for cases in which the surgical margins were positive for tumor, adding a recommendation for chemotherapy for persistent and recurrent disease. the role of adjuvant chemotherapy is also controversial ; however, it has been utilized because of the high risk of systemic relapse. in the recent updates of meta - analysis, pervaiz. this update showed that a combination of ifosfamide and adriamycin resulted in risk reduction of death from 41% to 30%. the authors concluded that there is marginal efficacy of chemotherapy in localized resectable soft tissue sarcoma with respect to local recurrence, distant recurrence and overall survival. ngan., found that neither chemotherapy nor radiotherapy seemed to affect the outcome in late or recurrent disease. our case report describes a stage i, grade iii leiomyosarcoma of the vagina treated with combined modality treatment. the rarity of vaginal leiomyosarcoma has contributed to the paucity of information regarding the appropriate management and prognosis of these patients. the treatment options must be individualized and tailored to the needs of each individual patient. each case must be reported in the literature to add more to our understanding of this rare disease. | primary malignant lesions of the vagina are uncommon, and vaginal sarcomas are even rarer. we describe a rare case of stage i, high - grade leiomyosarcoma of the vagina treated with combined modality treatment. a 39-year - old female presented with vaginal mass and underwent resection. histopathological examination revealed atypical leiomyoma of the vagina with definite risk of recurrence. eleven months later, she presented with a recurrent vaginal mass and underwent exploratory laparotomy with total abdominal hysterectomy and bilateral salpingo - oophorectomy plus resection of recurrent tumor and partial vaginectomy. the detailed histopathological examination was suggestive of leiomyosarcoma of the vagina. the patient received adjuvant radiotherapy and chemotherapy. the patient is alive and disease - free 29 months postsurgery. experience with vaginal leiomyosarcomas is limited. the optimal treatment methods have not yet been established because of the rarity of the tumor. we add another case of leiomyosarcoma of the vagina to the limited existing literature. |
obesity as a common problem in menopausal women is a major risk factor for the development of cardiovascular diseases. this is due to the importance of estrogen in the determination of body fat distribution. previous studies showed that deficiency of this hormone leads to an accumulation of visceral fat, and consequently, a decrease in insulin sensitivity. obesity is often associated with cardiovascular disease risk factors such as hypertension, dyslipoproteinemia, diabetes, and elevated inflammation markers [79 ]. accumulating evidence suggests that altered vitamin d homeostasis may also contribute to an increased cardiovascular disease risk in obese subjects. there are positive correlations between low 25-hydroxyvitamin d [25(oh)d ;. also, vonhurst. reported that as a result of taking 4,000 iu of vitamin d every day for 26 weeks, the insulin and blood glucose levels of patients with diabetes were significantly reduced. although our previous studies and others showed adequate effectiveness by regular physical activity in improving insulin resistance, ovx + at + d showed a better response. there are some mechanisms for the effects of vitamin d : the presence of vitamin d receptors on pancreatic cells, vitamin d - activating 1hydroxylase is expressed in pancreatic cells, the vitamin d response element in the insulin gene due to the presence of vitamin d receptors in skeletal muscle and the fact that 1,25(oh)d increases the transcription of insulin receptor genes and also suppresses the renin gene, reducing hyperglycemic - induced increases in renin levels in pancreatic cells. blockade of renin - angiotensin activity has been proposed as a novel target for the treatment of diabetes. the protective effects of vitamin d on obesity may be due to well known effects of vitamin d such as its anti - inflammatory properties, its effects on calcium and phosphorus metabolism and its regulation of the insulin receptor gene. it seems that vitamin d increases the calcium content of cells, in turn leading to the increased transport of glucose into the muscle. vitamin d also regulates nuclear ppar (peroxisome proliferative activated receptor), which has an important role in insulin sensitivity. it would be useful though to undertake further studies to discover more about the mechanism and the effect of vitamin d on both alpha and islet beta - cell functions and also on the mechanisms that determine insulin resistance. aerobic training and vitamin d supplementation contribute to increases in the metabolic rate by improving cardiovascular capacity and suppressing pro - inflammatory cytokine production, in addition to increasing the release of nitric oxide and the consumption of free fatty acids, thus enhancing insulin sensitivity. in general, inadequate vitamin d may promote greater adiposity through other metabolic effects, such as the regulation of pth and the modulation of adipogenesis. moderate to severe vitamin d deficiency leads to increased pth, which may promote an increase in free intracellular calcium in adipocytes and thereby enhance lipogenesis. the question that needs to be answered is whether or not vitamin d deficiency prevents the beneficial effects of exercise especially after menopause. recently, numerous interventional and epidemiological studies have shown that calcium and vitamin d consumption effectively reduce abdominal fat and metabolic syndrome risk. it has been reported that vitamin d plays an essential role in the homeostasis control of calcium. vitamin d was closely connected to the metabolic mechanism of calcium, and increased blood calcium concentration consequently affects blood lipid concentration and insulin resistance. one limitation of the present study was the lack of a group of ovariectomized animals treated with hormone replacement therapy to confirm the positive effects of at and vit d on plasma lipid profiles and insulin resistance. further studies should be conducted to more accurately elucidate and characterize the metabolic energy imposed by this aerobic training protocol and vitamin d supplementation in rats and humans. in conclusion, aerobic training and vitamin d supplementation successfully attenuate some metabolic syndrome profiles and might prevent cardiovascular risk factors caused by ovariectomy. from the clinical point of view, this study suggests that vit d supplementation is a very useful strategy to prevent obesity and menopause - associated metabolic disturbances. | purposethe purpose of this study was to determine the interaction effects of aerobic exercise training and vitamin d supplementation on indices of obesity and plasma lipid profiles in ovariectomized (ovx) rats.methodsforty female wistar rats were divided into 5 groups : aerobic training (3 days / week for 8 weeks ; at ; n = 8), aerobic training and vitamin d supplementation (ovx + at + vit d ; n = 8), vitamin d supplementation (ovx + vit d ; n = 8), ovariectomized control (ovx + c, n = 8) and sham (n = 8). after blood sampling, visceral fat was taken from the abdominal cavity and weighed immediately. data was statistically analyzed by one - way anova and repeated measure anova tests with a 0.05 significance level.resultsbody weight, visceral fat, bmi and food intake decreased significantly in ovx + at + vit d (p < 0.001) ; whereas these variables increased significantly in ovx + c (p < 0.001) and sham (p < 0.023) groups. at the end of two - months of follow - up, we observed significant differences in tc, tg, hdl - c, ldl - c, glucose, insulin, and homa - ir in all groups.conclusionit seems that aerobic training with vitamin d, due to the involvement of muscle mass and exposure to dynamic pressure on the bones and muscles, increased energy expenditure, stimulated insulin exudation and glucose homeostasis, decreased insulin resistance and improved the lipid profile in ovariectomized rats. |
type 2 diabetes mellitus (t2 dm) is a complex disease in which both genetic and environmental factors interact in determining impaired -cell insulin secretion and peripheral insulin resistance [13 ]. t2 dm is also a metabolic disorder between pro- and anti - inflammatory characterized by chronic hyperglycemia and increased or decreased levels of circulating cytokines. the rise in the proinflammatory cytokines (e.g., interleukin- (il-) 1, il-6, tumor necrosis factor- (tnf-), c - reactive protein (crp), transforming growth factor- (tgf-), and leptin) or the fall in anti - inflammatory cytokines (e.g., interleukin-1 receptor antagonist (il-1ra), il-4, il-10, il-13, and adiponectin) is the essential step in glucotoxicity and lipotoxicity induced mitochondrial injury, oxidative stress, and beta cell apoptosis in t2 dm [24 ]. these pro- and anti - inflammatory cytokines can enhance insulin resistance directly in adipocytes, muscle, and hepatic cells, leading to systemic disruption of insulin sensitivity and impaired glucose homeostasis. many single nucleotide polymorphisms (snps) in various genes including those of pro- and anti - inflammatory cytokines have been reported as a risk for t2 dm [13 ]. but not all snps have been confirmed by unifying results in different studies and wide variations have been reported in various ethnic groups [24 ]. genetic polymorphisms of c - reactive protein (crp) and their association with prediabetes and t2 dm have been widely studied. il-6 was regarded as a kind of pro- and anti - inflammatory factor ; one of the common polymorphisms in the il-6 gene promoter (c-174 g) was considered as risk factors for t2 dm development. il-10 is an anti - inflammatory cytokine protecting against t2 dm and inflammation ; several variants in the il-10 gene promoter region have been identified and showed the association with the development of t2 dm [3, 4 ]. it is reported that tnf- is a possible mediator of insulin resistance and diabetes since it inhibits insulin signaling and impairs its secretion [24 ]. one of the snps in tnf- gene showed a twofold increase in transcriptional activity and an association of tnf- snps with t2 dm. genetic variants in some inflammatory factors associated with t2 dm may provide a rationale for further studying their roles as biomarkers for disease early risk prediction and therapeutic targets for t2 dm and related complications. although accumulating evidences support the pathological role of inflammatory cytokines in t2 dm, most studies only focused on a few specific inflammatory factors and were done in relatively small population groups in a particular ethnic group. in our previous studies, we have explored the associations between few several inflammatory cytokines (e.g., crp, immunoglobulin e (ige), chymase, and tryptase) with prediabetes and t2 dm and concluded that ige and crp are risk factors of prediabetes and t2 dm only based on a small sample of cross - sectional study design [6, 7 ]. in this study, we extend the analysis of the role of pro- and anti - inflammatory cytokines in prediabetes and newly diagnosed t2 dm based on a larger sample of cross - sectional study design, by measuring the levels of several proinflammatory cytokines (e.g., ige, hs - crp, il-6, tnf-, and tryptase) and anti - inflammatory cytokines (e.g., il-4, il-10, and forkhead / winged helix transcription factor 3 + (foxp3 +)) in prediabetes and t2 dm, and correlate them with other laboratory and clinical biochemical indicators. our findings will lend support to the hypothesis that some specific inflammatory factors may play an etiological role in the pathogenesis of t2 dm and also will offer new insights into the potential clinical value of these inflammatory factors as biomarkers for disease early risk prediction and therapeutic targets for t2 dm and related complications. the study is part of the diabetes intervention project (dip) started in 2012 from school of nursing and medicine, huzhou university, zhejiang, china. from march to december 2012, a total of 7054 rural residents aged 5075 years from eight rural communities in the city of huzhou participated in physical examination. based on the criteria of fasting plasma glucose (fpg) < 5.6 mmol / l, 898 (12.73%) were classified into prediabetes. during july to august 2013, after excluding subjects with known dm or receiving hypoglycaemic medications or with cardiovascular disease, cerebrovascular disease, malignant disease, chronic liver disease, or kidney failure, or those under medications, 825 cases of 898 prediabetes subjects and another 300 randomized sampling normal glucose subjects were invited for fpg, 2-hour oral glucose tolerance test (2 h ogtt), and haemoglobin a1c (hba1c) tests as part of the prediabetes and t2 dm screening. but only 560 subjects would participate in this study, according to the following clinical criteria based on fpg, 2 h ogtt, and hba1c test : 219 (39.11%) had normal glucose tolerance (ngt), 215 (38.39%) were diagnosed as prediabetes subjects, and 126 (22.5%) were diagnosed as t2 dm subjects. this study was approved by the huzhou city ethics committee and all subjects gave written, informed consent prior to participating in the study. trained staff interviewed participants using a self - designed questionnaire to obtain information on demographic characteristics and anthropometric and lifestyle variables. physical activity assessments were performed using self - reported total energy expenditure questionnaire (teeq), a nine - step scale where every step was assigned a fixed value in terms of multiple of metabolic energy turnover (met). anthropometric measurements (body height and weight, waist circumference (wc), hip circumference, and blood pressure) were attained at the initial screening visit. two sitting blood pressure measurements were taken for each participant using a mercury sphygmomanometer according to a standard protocol. the biochemical parameters, including fpg, 2 h ogtt, hba1c, fasting insulin, plasma total cholesterol (tc), triglyceride (tg), low - density lipoprotein cholesterol (ldl - c), high - density lipoprotein cholesterol (hdl - c), hs - crp, and ige, were measured in the clinical biochemistry unit of huzhou first hospital, a teaching hospital of huzhou university. the cytokine concentrations, including il-6, tnf-, tryptase, il-4, il-10, and foxp3 +, were measured in patients sera using commercially available elisa double antibody sandwich method assays (r&d systems), performed according to the manufacturer 's instructions. diabetes and prediabetes were grouped according to american diabetes association 2010 (ada 2010) criteria or hba1c - based diagnosed criteria [9, 10 ]. diabetes was classified with a fasting plasma glucose (fpg) 7.0 mmol / l or 2 h ogtt 11.1 mmol / l or hba1c 6.5%, whereas prediabetes was defined as fpg 5.6 and < 7.0 mmol / l or 2 h ogtt 7.8 and subjects were classified as having a normal glucose profile if fpg < 5.6 mmol / l and 2 h ogtt < 7.8 mmol / l or hba1c < 6.0%. the mean and standard deviation (mean sd) of continuous and normal distributional variables and median and quartile range of continuous but skewed distributional variables were used. body mass index (bmi) was calculated as weight in kilograms divided by the square of the height in meters (kg / m). waist - to - hip ratio (whr) was calculated as waist divided by hip circumference. homeostasis model assessment - insulin resistance (homa - ir) was calculated as value of fpg value of fasting insulin/22.5 and homeostasis model assessment- cell function (homa-) was calculated as 20 value of fasting insulin/(fpg-3.5). based on china 2006 blood pressure control criteria and china prevention and treatment classification recommendation on dyslipidemia, hypertension was defined as systolic blood pressure (sbp) and/or diastolic blood pressure (dbp) 140/90 mmhg or as receiving blood pressure lowering medications ; high tg was defined as a fasting plasma tg 1.70 mmol / l, low hdl - c as a fasting hdl - c 0.9 mmol / l, high tc as tc 5.72 mmol / l, and low ldl - c as a fasting ldl - c 3.64 mmol / l. based on china obesity task group recommendation, overweight was classified when a body mass index (bmi) 24 kg / m and obesity was classified when a body mass index (bmi) 29 kg / m. in analyzing the relationships of inflammatory cytokines to prediabetes or t2 dm, ige was classified as normal / abnormal according to upper quartiles (p75 = 34.0 iu / l) ; crp was classified as normal / abnormal according to upper quartiles (p75 = 1.0 mg / l) ; tryptase was classified as normal / abnormal according to upper quartiles (p75 = 1699.94 ng / ml) and tnf- was classified as normal / abnormal according to upper quartiles (p75 = 325.14 pg / ml) ; il-4 was classified as normal / abnormal according to upper quartiles (p75 = 323.91 ng / l) ; il-10 was classified as normal / abnormal according to upper quartiles (p75 = 256.22 ng / l) ; foxp3 + was classified as normal / abnormal according to upper quartiles (p75 = 411.17 pg / ml). one - way analysis of covariance was used to test for differences in continuous distributional variables between three groups. the nonparametric kruskal - wallis test was used to test for differences in continuous but skewed distributional variables between two groups, and test was used to test differences in proportions between three groups. spearman correlation coefficients were calculated to evaluate associations between inflammatory cytokines and the traditional cardiovascular factors. binary logistic regression model was used to estimate the odds ratios (ors) and 95% cis for prediabetes and t2 dm according to dichotomy of inflammatory biomarkers concentration, using the lower values as the referent category. considering the potential confounding factors, we applied 10 models to assess the association between biomarkers and prediabetes or t2 dm, including unadjusted mode, age and sex - adjusted model, multivariable (age, sex, bmi, whr, sbp, dbp, tc, tg, level of physical activity, dietary intake, alcohol intake, smoking status, presence or absence of family history of diabetes, hypertension, heart disease, stroke, and hypercholesterolemia) adjusted model, and multivariable combined with inflammatory biomarkers mutually adjusted model. the descriptive characteristics of 560 study participants were presented separately for participants with normal glucose tolerance, prediabetes, and t2 dm (table 1). a total of 215 were prediabetes, 126 were t2 dm, and 219 were normal glucose subjects. overall, except smoking status, alcohol use, disease family history, physical activity level, and dietary intake, all traditional vascular risk factors were worse in the pdg or the t2dmg patients than ngg subjects. iu / l) (p < 0.05) and with pdg subjects (20 iu / l) (p < 0.05). median levels of crp in pdg subjects (0.8 mg / l) or t2dmg subjects (1.5 mg / l) were significantly higher compared with ngg subjects (0.5 mg / l) (p < 0.05). mean levels of tryptase in pdg subjects (mean sd : 1545.36 291.45 ng / ml) or t2dmg subjects (mean sd : 1524.96 286.65 ng / ml) were significantly higher compared with ngg subjects (mean sd : 1481.21 271.44 ng / ml) (p < 0.05). mean levels of il-4 in pdg subjects (mean sd : 303.70 56.84 ng / l) or t2dmg subjects (mean sd : 304.45 55.87 ng / l) were significantly higher compared with ngg subjects (mean sd : 283.45 52.50 mean levels of il-10 in pdg subjects (mean sd : 244.03 53.34 ng / l) or t2dmg subjects (mean sd : 265.04 40.42 ng / l) were significantly higher compared with ngg subjects (mean sd : 229.23 46.54 but there were no significant differences in il-6, tnf-, and foxp3 + (table 1). spearman correlation analysis between inflammatory cytokines and the traditional cardiovascular factors indicated that whether, in ngg, the pdg, or the t2dmg, there were significant relationships between most of the traditional cardiovascular factors and between most of inflammatory cytokines. however, there were seldom significant relationships between the traditional cardiovascular factors and inflammatory cytokines (tables 24 ; tables s1s6) (in supplementary material available online at http://dx.doi.org/10.1155/2016/7965317). after adjusting confounding factors by multivariable combined with inflammatory biomarkers mutually adjusted model, plasma ige was associated with t2 dm compared with ngg (or (odds ratio) : 3.46 (1.926.23, 95% ci), p < 0.001) or compared with pdg (or : 2.57 (1.544.30, 95% ci), p < 0.001) ; plasma crp was associated with pdg (or : 2.30 (1.463.62, 95% ci), p < 0.001) or t2 dm (or : 16.24 (7.9933.02, 95% ci), p < 0.001) compared with ngg or t2 dm (or : 5.93 (3.2910.72, 95% ci), p < 0.001) compared with pdg ; serum il-4 was associated with pdg (or : 1.68 (1.052.69, 95% ci), p = 0.031) or t2 dm (or : 1.94 (1.103.53, 95% ci), p = 0.023) compared with ngg ; serum il-10 was associated with pdg (or : 2.13 (1.333.41, 95% ci), p = 0.002) or t2 dm (or : 8.62 (4.5016.49, 95% ci), p < 0.001) compared with ngg or t2 dm (or : 3.04 (1.795.13, 95% ci), p < 0.001) compared with pdg (table 5). serum tryptase was associated with prediabetes (or : 1.53 (1.012.32, 95% ci), p = 0.044) before adjustment and (or : 1.63 (1.022.61, 95% ci), p = 0.041) after adjustment by multivariable + il-4, compared with ngg. serum il-6, tnf-, and foxp3 + were not associated with pdg or t2 dm (table 5). our data showed increased levels of crp, il-4, il-10, and tryptase in prediabetes subjects and increased levels of crp, il-4, and il-10 in t2 dm subjects compared with normal glucose subjects. not all inflammatory cytokines in our study were in agreement with previous findings. with the associations between inflammatory cytokines (e.g., il-6, tnf-, and hs - crp) and insulin resistance, prediabetes or t2 most studies found that subjects with insulin resistance, prediabetes, or t2 dm had increased levels of il-6, tnf-, and hs - crp [1316 ]. a study reported that high levels of inflammatory cytokines appeared in early stage of t2 dm and were capable of predicting the development of type 2 diabetes through diminishing insulin sensitivity. but a recent study showed decreased levels of il-6 and tnf- in t2 dm compared to healthy controls. the author in this paper interpreted that this discrepancy could be attributed to duration of the diseases, small sample size, and the differences in age and sex of the studied groups. elevated plasma levels of hs - crp in prediabetes and t2 dm subjects have been demonstrated in this paper (tables 1 and 5), our previous studies, and other studies [6, 7, 1421 ]. our data showed that mean levels of tnf- or il-6 in pdg or t2dmg subjects were higher but insignificant compared with ngg subjects (tables 1 and 5), which could be explained by the differences of some incompletely measured residual confounding factors or inflammation - related diseases (e.g., rheumatoid arthritis (ra) or osteoarthritis (oa)) or allergies in the studied groups. it also plays a crucial role in the pathophysiology of t2 dm [6, 7 ]. this current study demonstrated that plasma ige levels strongly correlated with t2 dm (table 5), which was in agreement with our previous studies [6, 7 ]. further large population - based prospective studies are warranted to assess the role of ige in t2 dm. il-4 was major anti - inflammatory cytokine and had been proposed to play a crucial role in the pathophysiology of t2 dm, and several candidate genes have been identified. our data showed that serum il-4 was associated with pdg or t2 dm (tables 1 and 5). il-10 was anti - inflammatory cytokine and has been shown to improve impaired insulin signaling, prevent the development of il-6, inhibit nadph oxidase, prevent pancreatic beta cell destruction, and play an important role in modulation of cardiovascular insulin resistance [2326 ]. two studies indicated that low il-10 level was associated with high hba1c and serum il-10 level may be one of the predictors of glycemia [27, 28 ]. increased level of il-10 in prediabetes and t2 dm subjects in our study was in disagreement with these findings [2, 2628 ], which could be explained by high levels of inflammatory cytokines appearing in early stage of t2 dm [14, 17 ]. t regulatory (treg) cells are a unique population of t - cells which play a crucial role in the maintenance of self - tolerance and suppression of potentially inflammatory t - cells. chronic low - grade inflammation in obesity and impaired insulin sensitivity has been associated with fewer tregs in adipose tissue [2931 ]. our study did not prove the associations between foxp3 + with the pds or t2 dm (tables 1 and 5). mast cell tryptase is an important protease that has been implicated in cardiovascular diseases [3234 ]. our previous study did not demonstrate the association between plasma tryptase with prediabetes or t2 dm [6, 7 ]. but in this paper, our data indicated that tryptase was mildly associated with prediabetes (or : 1.63 (1.022.61, 95% ci), p = 0.041) after adjustment by multivariable + il-4, compared with ngg, which could be explained for the synergistic effect of il-4. our data also showed that there were significant relationships between most of the traditional cardiovascular factors and between most of inflammatory cytokines. however, there were seldom significant relationships between the traditional cardiovascular factors and inflammatory cytokines (tables 24 ; tables s1s6), which indicated that complex interactions could exist between these inflammatory cytokines or between cardiovascular factors [13, 20, 21 ]. results of our study extend other evidences linking inflammatory cytokines to insulin resistance and risk of diabetes based on a larger sample of cross - sectional study design. our findings further lend support to the hypothesis that some specific inflammatory factors may play an important role in the pathogenesis of t2 dm and also offer new insights into the potential clinical value of these inflammatory factors as biomarkers for t2 dm early prediction and treatment [1317 ]. we assessed the associations between few inflammatory cytokines and prediabetes and t2 dm by means of plasma or serum biomarker only based on cross - sectional study design and can not demonstrate that altered plasma or serum levels of inflammatory biomarkers are predictors of incident diabetes by large prospective cohort study. our comprehensive assessment of diabetes risk factors allowed statistical control for important confounding factors in the pathogenesis of diabetes, but residual confounding could remain in the analysis. in particular, we did not consider potential inflammation - related diseases, such as ra or oa, which widely exist in the senior, or allergies which are also very common among individuals, and these unmeasured residual confounding factors might result in discrepancies. in conclusion, circulating inflammatory mediators, hs - crp, ige, il-4, il-10, and tryptase, were positively associated with prediabetes or t2 dm. further large prospective studies are warranted to assess a temporal relation between baseline levels of inflammatory biomarkers and incidence of prediabetes or t2 dm and its associated chronic diseases. | this study evaluated the inflammatory markers in prediabetes and newly diagnosed type 2 diabetes mellitus (t2 dm). inflammatory markers levels were analyzed using one - way analysis of covariance and the association with prediabetes or t2 dm risks was examined by logistic regression models. our data showed increased levels of hypersensitivity c - reactive protein (hs - crp), interleukin (il-4), il-10, and tryptase in prediabetes subjects and hs - crp, immunoglobulin e (ige), il-4, and il-10 in t2 dm subjects. we concluded that hs - crp, ige, il-4, il-10, and tryptase were positively associated with prediabetes or t2 dm. further large prospective studies are warranted to assess a temporal relation between inflammatory biomarkers and incidence of prediabetes or t2 dm and its associated chronic diseases. |
dermatofibrosarcoma protuberans (dfsp) is a mesenchymal tumor of the dermis and subcutaneous tissues (1). it is a slow growing low- to intermediate - grade malignancy with a high propensity for local recurrence, especially if negative resection margins are not achieved. because occurrence in the breast is very rare, dfsp is often misdiagnosed as a benign breast tumor, leads to treatment delay. only a few single case reports with imaging findings have been published (2). we reported a patient with dfsp of breast together with characteristic imaging features of various radiologic modalities including mammography, ultrasound, mri and fludeoxyglucose - positron emission tomography computerized tomography (fdg - pet ct), which are necessary for an accurate diagnosis and providing a differential diagnosis from other breast lesions. a 41-year - old female presented with a lump in her left breast that she had noticed it about one month previously. physical examination revealed a firm, tender nodule at 12-oclock position of the left breast. mammography showed heterogeneously dense breasts that would be categorized as breast imaging reporting and data system (bi - rads) type c. it also revealed a circumscribed, oval - shaped, equal - density mass in the subcutaneous fat layer of the upper central portion of the left breast. associated abnormal calcification or architectural distortion was not observed (figure 1a and b). subsequent ultrasonography performed using an acoustic gel pad demonstrated a 2.4 1.0 cm, circumscribed, oval - shaped mass in the skin and subcutaneous fat layer at the 12-oclock position of the left breast, 4 cm from the nipple. the mass had a heterogeneous echo pattern with a peripheral hyperechoic rim (figure 2a) and increased vascularity on color doppler sonography (figure 2b). malignancy was suspected for this lesion and it was categorized as bi - rads 4. dynamic contrast - enhanced breast - magnetic resonance imaging (mri) was performed using a 1.5-t system with a dedicated breast coil, which revealed a 2.4 2.0 cm circumscribed oval mass with a wide base to the skin. the mass was observed as low signal intensity on t1-weighted images with homogeneous strong enhancement (figure 3a and b). most sections of the tumor showed rapid initial enhancement with washout kinetics (figure 3c). 18f - fdg - pet demonstrated faint fdg uptake around the mass in the left breast and standardized uptake value (suv) was measured as 1.8 (figure 4a and b). histologic examination revealed infiltration of a 2.0 1.5 2.0 cm sized tumor into the subcutaneous fat on low - power field view and storiform arrangement of short spindle cells on high - power field view. the tumor cells were diffusely positive for cd34 immunostaining (figure 5). therefore, histologic diagnosis was dfsp. the reported incidence is around 5 cases per 1 million persons annually and is most commonly found in adults aged 20 and 50 years (1). although the tumor is a low - to - intermediate grade sarcoma, it can infiltrate into the surrounding deeper tissue with a propensity for local recurrence, especially after inadequate excision. while metastasis is rare, it can occur in distant sites, especially the lung (2 - 4). furthermore, differentiation into higher - grade sarcomas with higher metastatic potential tends to occur in poorly treated recurrent tumors (1). dfsp most commonly appears on the trunk (50 - 60%), extremities (25%) and in the head and neck (10 - 15%). although breast involvement is quite unusual, it has been reported in the literature with imaging features (1). the reported mammographic findings of dfsp are nonspecific, i.e. a skin - based or intramammary - located, oval, circumscribed mass without calcification that mimics a benign lesion (1, 2, 5). these features are consistent with our case, in which a circumscribed, equal- density, oval mass without calcification was observed in superficially subcutaneous fat layer of the upper central portion of the left breast. the characteristic ultrasonographic features of dfsp of breast were a circumscribed, hypoechoic, subcutaneous mass with increased internal blood flow in color doppler image (4, 6, 7). especially, hypervascularity of the tumor helps it to be differentiated from other benign breast lesions. although a breast abscess or complicated epidermal inclusion cyst such as infection or rupture can appear as a hypoechoic mass with a thick echogenic periphery and increased vascular flow (8). however, our patient had no clinical symptoms associated with infection such as pain, redness, a sense of heating or fever. another distinct ultrasonographic feature of dfsp of the breast is a peripheral hyperechoic band like rim in the subcutaneous tissue, which represents a mixture of tumor cells and fibrous tissue infiltrating the subcutaneous fat (2). although the signal intensity of dfsp on breast mr imaging is nonspecific, mri is the most helpful imaging modality for evaluating the tumor extent and anatomical relationships with surrounding structures. compared to ultrasound, mri represents more accurately the tumor size and the boundary because of excellent contrast with surrounding tissue. mr appearance of breast dfsp is a well - circumscribed, intermediate low - intensity tumor on both t1 and t2-weighted images with washout - type kinetics and strong enhancement (4). we also observed a low signal intensity mass on t1-weighted images with initially rapid strong enhancement with washout kinetics in our case. although dfsp has a high tendency for local recurrence after insufficient surgical excision, metastasis is rare. however, distant metastasis may occur at the lung, bone or regional lymph nodes after repeated local recurrence (9, 10). fdg - pet is a useful tool for predicting tumor recurrence and distant metastasis, detecting multicentric tumor origin and providing relevant anatomical information (11). a few reports have described pet - ct findings of dfsp including mild uptake in the skin of the trunk and head or more marked uptake at metastatic sites in the lung, retroperitoneum or muscles (12, 13). (14) reported a highly significant relationship between the highest pet suv values and cellularity and proliferative indices in sarcomas. in the present case, although a correlation between fdg uptake and histologic grade in dfsp has not been reported in the literature, we assume that low suv of the dfsp of the breast was attributable to low cellularity and proliferative indices. to our knowledge, this is the first case showing 18f - fdg pet findings of breast dfsp. in conclusion, breast dfsp is a rare sarcoma that may simulate a primary breast mass both clinically and radiographically. we described a case of breast dfsp and presented radiologic features that can potentially help provide a differential diagnosis between dfsp and primary benign breast lesions. mammography, ultrasound, mri and fdg - pet findings of a subcutaneously located, highly vascular, non - inflammatory tumor with a wide base to the skin might be suggestive of dfsp. | dermatofibrosarcoma protuberans is an uncommon malignant mesenchymal tumor of the dermis, which most commonly appears on the body and extremities. a preoperative diagnosis of dermatofibrosarcoma protuberans is extremely difficult, because it is a very rare entity and its appearance is often similar to that of benign breast lesion. here, we presented a case of breast dermatofibrosarcoma protuberans with associated radiological mammography, ultrasound, magnetic resonance imaging and fludeoxyglucose - positron emission tomography computerized tomography (fdg - pet ct) features. to our knowledge, our case is the first report of pet - ct findings of breast dermatofibrosarcoma protuberans. furthermore, we reviewed characteristic radiologic features of this rare entity, which are helpful for differentiating it from other primary benign breast lesions. |
as part of the fastest growing and largest ethnic minority group, projections indicate that 128 million latinos will reside in the united states (u.s.) by 2050.1 research on racial and ethnic disparities in health - care access and utilization constantly identifies latinos as one of the most disadvantaged ethnic groups. using measures such as usual source of care, health insurance coverage, and the quality of care received, barriers for latinas are readily identified.2 however, an inclusive understanding of health and health - care disparities must take into account gender differences, given that health and illnesses are experienced differently by men and women. if demographic trends continue, it is suggested that latinas will represent 25% of the total female population in the u.s. and make up 52% of the growing latino population.3 research addressing health disparities on the basis of ethnicity and gender can influence professional and public consciousness and put forth important considerations for health and social policy.3 as a fast - growing demographic, it is important to address the challenges and barriers that may affect the quality of health and health care among latina women. the terms latino and hispanic are used interchangeably in research to describe this fast - growing demographic.4 the u.s. census bureau introduced the use of the term hispanic as a classification of the spanish - speaking population in the 1970s.5 the census identifies hispanic origin as a person cuban, mexican, puerto rican, south or central american, or other spanish culture or origin regardless of race.6 on the other hand, latino is a self - chosen pan - ethnic identity marker, regarded as an attempt to promote an alternative to the artificial bureaucratic homogenization.5 neither term is unanimously accepted by this large and highly diverse group, resulting in the 1997 revision of the standards for the classification of federal data on race and ethnicity from hispanic to hispanic or latino. the term hispanic is highly used and recognized in spite of protests among the minority group ; some reject the label, whereas others use latino as an alternative.5 for the purpose of consistency in this review, the terms latino and latina will be used. the latino population is identified as a whole ; however, each country or subgroup has its own identity.4 women of mexican (59%) and puerto rican (10%) descent make up the two largest subgroups of latinas in the united states.3 furthermore, the population of people with roots spanning 11 other latin american countries has increased to over 1 million in the u.s. in recent years.1 in examining the health disparities of latinas, it is important to identify certain barriers that may cause disparities. comparing those issues to non - latina women in the u.s. can shed light on topics such as variance in utilization of preventative health care as well as overall attitudes on disease prevention. on average, the life expectancy of latinas is 77.1 years in contrast to asian women living 86.8 years, white women living 79.6 years, and black women for 74.9 years.3,7 recent reports estimate the life expectancy of latinos at birth to be estimated at 83.8 years, with the life expectancy of whites and blacks being 2.4 and 5.4 years lower, respectively.8 the higher life expectancy of latinas compared to that of other races supports the latino paradox. the latino health paradox (or hispanic paradox) refers to the phenomenon that despite having lower incomes and less access to health - care services, latinas in the u.s. have lower mortality rates and longer life expectancy than their non - latina counterparts.9 the latino mortality estimates and facilitated comparisons with other groups do not separately specify latino mortality patterns by nativity or national origin.10 latino immigrants generally exhibit 15%20% lower mortality than u.s.- born latinos, most likely because immigrants are self - selected on good health - enhancing attributes relative to nonimmigrants.10 the latino paradox also includes the classical immigrant assimilation model, which states that immigrants gradually adopt the behaviors of the dominant cultural group over time. linear increases in assimilation generally occur progressively across successive generations.11 in the context of a first - generation, non - citizen child developing bronchitis, subsequent generations were also significantly more likely to have bronchitis.11 in addition, compared to first - generation non - citizens, third and fourth generations were also significantly more likely to have allergies.11 the top three health - related causes of death for women as a whole are heart conditions, cerebrovascular diseases, and cancer.3 cardiovascular disease mortality rates per 100,000 as of 2013 were 78.3 for latinas, 267.6 for whites, 211.9 for blacks, and 94.1 for asians, whereas cerebrovascular disease mortality rates were considerably lower : 50.6 for whites, 39.2 for blacks, 23.8 for asians, and 16.1 for latinas.8 cancer is the leading cause of death in latinas between the ages of 25 and 54 years.3 the cancer mortality rate for latinas is 298 per 100,000 compared to 180.6 for blacks and 155 for whites.3,12 death rates for lung, colon, and ovarian cancers are lower in latinas compared to white, black, and asian women.12 this may be linked to reports that latinas have lower occurrences of alcohol consumption and cigarette - smoking habits.3 breast cancer is not only the most common cancer among women, it is also the leading cause of cancer - related deaths among latinas.13 lung cancer still ranks as the leading cause of death for non - latina women. this may indicate a correlation to the higher mortality rate from breast and cervical cancers compared to their non - latina counterparts. cervical cancer is more common in ethnic groups, particularly latina women, who currently have the highest incidence rates in the u.s.14 rates of cervical cancer screenings among latinas are significantly lower compared to those of other ethnicities.15 the use of mammography screening is proven to decrease breast cancer mortality.16 mammography screening has increased over the decades throughout the u.s. ; however, the biggest disparity has been reported among women who are uninsured, are recent immigrants, or do not have a usual source of health care.16 lower screening among women with low income was reported regardless of race or ethnic identification.16 latina women are more likely to be uninsured than non - latina women ; however, the use of screening is expected to increase among uninsured latina women in the future.16 the increase in screening rates among uninsured, low - income, and minority populations may be attributable in part to programs promoting screening in underserved populations.16 programs such as the national breast and cervical cancer early detection program aim to decrease screening disparities in low - income, uninsured, and minority populations. physical activity and good nutrition seem to be readily identified as habits of good health by latinas and that one had to walk or do other types of exercise to prevent high blood pressure and strokes.17 even though physical activity is identified as a good health measure to avoid cardiovascular and cerebrovascular disease, a study conducted in texas found that only 30.9% of their latina sample met physical activity requirements compared to 45.1% of 2007 national data.18 reasons given by latina women for lowered levels of physical activity were lack of time, no childcare, tiredness, and no self - discipline.18 hands - on programs specifically targeting latina women to promote physical activity and proper nutrition would be beneficial in bridging the gap between knowledge and action in this population. an examination of cervical cancer prevention found that latinas did not have a strong understanding of what causes cervical cancer and they would seek screening only if they were experiencing symptoms.15 in addition, latinas identified barriers such as fear of results, embarrassment of being touched, access to health care, and language issues as reasons for not obtaining regular screenings.15 recommendations for reducing the identified barriers would be a greater access to health - care clinics with female providers, lower cost screenings, and more spanish - speaking facilities.19 preventative education included in the health - care plan should be increased and expanded to include males, as risk of cervical cancer is associated with the sexual behavior of males, and condom use is lower among foreign - born latino males.20 cardiovascular and cerebrovascular diseases are other main health issues facing latinas today. cardiovascular health disparities in the latina population exist due to income barriers, language barriers, and health education barriers.21 latina women were not as knowledgeable of healthy cardiovascular measures for blood pressure, blood lipids, and fasting blood glucose levels compared to other groups. however, latina women were more likely to assist their children and others in healthy lifestyle changes over white and black women.22 in examining minority communities, studies have suggested that differences do exist in certain areas where blacks and latinos report worse health conditions than whites and asians.23 exploring the relationship between health - related quality of life and physical activity / diet among black, latino, and asian communities in new york city, health - related quality of life was measured by self - reported physical health, mental health, and social functioning in order to understand the overall health status of the population.23 latino and asian groups who live in immigrant communities report to have healthier diets but had lower weekly physical activity participation than other populations in the area.23 results indicated distinct variations among each subgroup with latinos being the most likely to participate in sufficient physical activity, consume recommended fruits and vegetables, and yet have lower overall health - related quality of life.23 in addition to quality physical activity and healthy diet, there are other factors that must contribute to the overall health - related quality of life score. all three subgroups were highly insured and reported regular health - care access but failed to meet ideal physical activity and fruits and vegetable intake recommendations.23 these results highlight the importance of understanding ethnic / racial communities in order to better promote health initiatives that target each subgroup s needs. according to census reports, the median age for latinas that live in the united states is 26.6 years ; where 56% are married and 58% have children younger than 18 years.3 latinas are at their prime for childbearing. about half of the latinos at large self reported that they speak english less than very well, defining them as individuals with limited english proficiency (lep).24 children of latino parents with lep disproportionately experience poor primary care access and quality health care compared to latino children in english - speaking families.25 one study exploring the experiences and expectations of lep latina mothers with pediatric primary care addressed the challenges of health - care disparities and made suggestions for improvement.25 a major concern identified for latina mothers was the level of attentiveness that was measured by the doctor taking his or her time with each patient. mothers reported that many times they felt rushed during doctor visits and preferred longer wait times with caring, patient doctors because quality health care is worth the wait.25 because of job demands, mothers sought out health - care providers with evening and weekend hours and preferred clinics that had spanish - speaking providers, which allow for direct communication between provider and parent. clinics without spanish - speaking providers used nurses for interpretation, and clinics without spanish - speaking staff limited communication, which results in misinformation and frustration.25 these are barriers, which if corrected, can increase quality health - care benefits for all latina populations. a thorough understanding of the socioeconomic profile of latinas will raise consciousness to the social inequalities that may potentially place them at high risk for certain health conditions. these inequalities could also limit access to quality health care.3 scholastically, 43% of latinas have a 12th grade education or less and only 11% have a bachelor s degree or higher, compared to 12% and 26%, respectively, for white women.3 without a strong educational background, latinas are more likely to be employed in low - paying, part - time, or seasonal jobs and experience twice the rate of unemployment (7.7%) compared to white women (3.3%).3 recent documented weekly income for a full - time employed latina was $ 570 compared to $ 621 for blacks, $ 745 for whites, and $ 943 for asians.26 latinas are vulnerable to poverty - related health conditions and may lack health insurance or financial means to pay for quality health care due to economic disadvantages.3 latina women are a fast - growing population and are becoming a prevalent demographic. health policies and considerations ethnic and gender - specific data and research are limited, leading to many gaps and higher health - care disparities. increased awareness and further research | analyzing the latino community and focusing on the women that make up this fast - growing demographic create a better understanding of the needs and considerations for health - care professionals and social policies. it is important that national health and health - care data on the latino ethnic group be presented by gender in order to determine areas specific to women. this review focuses on the existing health and health - care data of latino women (latinas). the ability to distinguish the health - care experiences of latinas will increase the understanding of existing barriers to their health care, the initiatives needed to overcome them, and increase the overall quality of health among latina women. |
the role of ultrasonography in detection of pleural effusion was first discovered in the 1960s (1). one decade later the diagnostic value of a - mode ultrasonography in identification of pleural effusion was reported to be 95% (2). thus, the impression that ultrasonography is not a suitable modality for detection of chest lesions was rejected at that time. afterwards, various surveys were conducted evaluating the diagnostic accuracy of ultrasonography and radiography in identification of pleural effusion and most of them found a higher diagnostic value for ultrasonography compared to radiography (3 - 5). however, ultrasonography is still not considered as the first diagnostic tool for detection of pleural effusion and the majority of physicians use radiography for this propose (6). although a radiogram captured in lateral decubitus position with horizontal rays has a high sensitivity and specificity in detection of pleural effusion but it can not be obtained from all the patients especially critically ill and trauma victims (4). other views of chest radiogram have low sensitivities in this regard (7, 8). vast improvements of technology have led to development of hand - held and pocket - size ultrasound devices (9, 10). a meta - analysis was performed in 2010 on four studies that reported the sensitivity and specificity of ultrasonography in detection of pleural effusion to be 93% and 96%, respectively (11). yet, inclusion of few surveys and lack of sensitivity analysis and publication bias confirm the necessity of an update on this subject. therefore, this study aims to conduct a systematic review of the available literature determining the diagnostic value of ultrasonography and radiography for detection of pleural effusion through a meta - analytic approach. search strategy and selection criteria this study was designed according to the consensus statement of meta - analysis of observational studies in epidemiology (moose) (12). extended systematic search was carried out in databases of medline (via pubmed), embase (via ovidsp), isi web of knowledge, scopus, cochrane library, and proquest based on the keywords obtained from medical subject heading (mesh) terms and emtree. ultrasound or chest film or chest radiograph combined with pleural effusion or effusion or pleural free fluid. the directive was to find prospective and retrospective studies assessing the diagnostic value of ultrasonography or chest radiography in detection of pleural effusion. two of the authors (m.y, p.g) independently searched for sources and contacted the authors of relevant articles to obtain further articles and data. the studies that had used radiography as their reference test were excluded and only surveys in which a final diagnosis of pleural effusion had been confirmed by computed tomography scan or surgery were included. the search yielded three surveys which had compared the diagnostic value of pocket - size ultrasonography machines with high - end ones. since these high - end ultrasonography machine had been performed by cardiologists and their results had been assessed by the final diagnosis proposed by two specialists as the reference test, these three surveys were also included in the meta - analysis. flowchart of the study charecteristics of included studies 1, (+ / -) : number of patient with pleural effusion / number of patient without pleural effusion ; 2, number are presented as mean standard deviation or (range). ct : computed tomography ; cxr : chest radiography ; ep : emergency physician ; na : not applicable ; nr : not reported ; us : ultrasonography the articles were entered into endnote x7 software and after elimination of duplicates, two reviewers (m.y, p.g) independently screened the articles. quality of the surveys were evaluated according to the guidelines proposed by 14-item quality assessment of diagnostic accuracy studies (quadas2) tool (18). each article was assessed regarding presence of various biases including selection, performance, recording, and reporting and they were classified as three grades of poor, fair or good. only fair and good surveys were included in meta - analysis. data on distribution of the study population regarding age and gender, sample size, the number of patients with / without pleural effusion according to reference tests, technique of ultrasonography, the specialty of the ultrasound device s operator, sonographic definition of pleural effusion, probe s frequency, blinding status, sampling method (consecutive, convenience) and finally number of true and false, positive and negative cases were gathered. a third author (m.h) was in charge of solving disagreements. in cases where data were not presented in the article web - based programs were used to compute true positive, false positive, true negative and false negative figures according to sensitivity and specificity. when available, data for each hemi - thorax were included in the analysis separately. statistical analysis stata 11.0 statistical software was used to analyze the data via midas module. to evaluate the adequacy of ultrasonography and radiography in detection of pleural effusion, summary receiver operative curves (sroc), sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio with 95% confidence interval (95% ci) heterogeneity between the included surveys was assessed through utilization of i2 and 2 tests and a p value of less than 0.1 along with an i2 greater than 50% were considered as positive heterogeneity (19). consequently, subgroup analysis was performed on sampling method (consecutive/ convenience), operator (emergency physician/ other specialists) or interpreting physician, frequency of the probe (1 - 5 mhz/ 5 - 10 mhz), type of ultrasound device (pocket - size / other) and sample size (less than 100 patients/ more than 100 patients). subgroup analysis of diagnostic accuracy for chest radiography and ultrasonography in detection of plural effusion p value < 0.1 was considered as significant for heterogeneity ; ci : confidence interval. study characteristics search process and the number of articles obtained in each step are presented in figure 1. after detailed evaluation of these surveys 12 studies were included in meta - analysis (3 - 5, 7 - 10, 13 - 17). the mean age of the subjects was 63.3 years (ranging from 14 to 101 years old) and 58.6% were male. all the studies were single - center (3 - 5, 7 - 10, 14 - 17)except one which was performed in two center (13). a total of 646 patients with pleural effusion and 912 subjects without it were evaluated. diagnostic accuracy of both these modalities was assessed in nine surveys (3 - 5, 8 - 10, 13 - 17), radiography was evaluated individually in one study (7) and ultrasonography was assessed individually in two articles (10, 17). 11 articles were in english (3 - 5, 7 - 10, 13 - 15, 17) and one was in farsi (16). the presence of heterogeneity was confirmed between the studies (p<0.1), but no publication bias was detected (figure 2). area under the curve of srocs for ultrasonography and chest radiography in detection of pleural effusion were 0.98 (95% ci : 0.97 - 0.99) and 0.73 (95% ci : 0.69 - 0.77), respectively (figure 3). pooled sensitivity and specificity of ultrasonography in detection of pleural effusion were 0.94 (95% ci : 0.88 - 0.97 ; i2= 84.23, p<0.001) and 0.98 (95% ci : 0.92 - 1.0 ; i2= 88.65, p<0.001), respectively. also, its pooled positive and negative likelihood ratios were calculated to be 53.96 (95% ci : 11.46 - 254.05 ; i2= 88.12, p<0.001) and 0.06 (95% ci : 0.03 - 0.12 ; i2= 84.44, p<0.001), respectively (figure 4). pooled sensitivity and specificity of radiography in pleural effusion diagnosis were 0.51 (95% ci : 0.33 - 0.68 ; i2= 91.76, p<0.001) and 0.91 (95% ci : 0.68 - 0.98 ; i2= 92.86, p<0.001), respectively. in addition, its pooled positive and negative likelihood ratios were also found to be 5.60 (95% ci : 1.14 - 27.42 ; i2= 88.14, p<0.001) and 0.54 (95% ci : 0.35 - 0.84 ; i2= 84.44, p<0.001), respectively (figure 5). subgroups analyses were performed regarding study design, patients type (critically ill / other), operator of ultrasound machine, ultrasound model and sample size. the results showed that the sensitivity was higher when the procedure was performed via an intensivist or a radiologist, using 5 - 10 mhz transducers, whereas it was lower when carried out via pocket - size devices. on the other hand, the diagnostic value of radiography was affected by the sample size and type of patients. in this regard, the sensitivity and specificity of chest radiography was found to be higher in studies with sample sizes of greater than 100 patients while both of these measures were calculated to be lower in critically ill patients (table 2). deeks ess : effective sample sizes summary receiver operative curves (sroc) for ultrasound (a) and chest radiography (b) in detection of plural effusion. auc : area under the curve ; sens : sensitivity ; spec : specificity forest plot of screening performance characteristics of chest ultrasonography in detection of plural effusion. sensitivity and specificity (a) ; diagnostic likelihood ratio (dlr) (b). ci : confidence interval forest plot of screening performance characteristics of chest radiography in detection of plural effusion. sensitivity and specificity (a) ; diagnostic likelihood ratio (dlr) (b). the present meta - analysis showed that ultrasonography has high sensitivity (94%) and specificity (98%) in detection of pleural effusion. when the analysis was limited to the surveys in which the procedure was carried out by an intensivist or a radiologist, the presented sensitivity increased (98%). while, the diagnostic value of radiography was reported to be lower than ultrasonography (sensitivity 51%, specificity 91%). when we limited the analyses to studies in which the etiology of pleural effusion was trauma, surgery or congestive heart failure, sensitivity of radiography slightly increased (58%). on this basis it can be concluded that ultrasonography is a better diagnostic tool for pleural effusion compared to radiography. in comparison with the results of the present study, grimberg. they found the sensitivity and specificity of ultrasonography to be 93% and 96% respectively while these figures for radiography were calculated to be 24% and 100% (11). the higher sensitivity of radiography presented in this study might be due to inclusion of more surveys in the analyses. grimberg s survey lacked subgroup analysis which prevents us from further comparisons. in another meta - analysis chavez. reviewed 10 articles aiming to evaluate the diagnostic value of ultrasonography in detection of pneumonia and they found a sensitivity of 94% and a specificity of 96% for this modality (20). these researchers stated that ultrasonography is a suitable diagnostic tool for ruling out pneumonia in patients referring to medical centers and emergency departments. congruent with this survey we also found a considerable diagnostic value for ultrasonography in detection of pleural effusion. one of the factors that influence sensitivity of ultrasonography is the operator of ultrasound device which has been verified by various surveys in the past years (21 - 24). an ultrasonography carried out by an intensivist or a radiologist is able to detect pleural effusion more effectively and precisely. pocket - size ultrasonography is a new technology recently being used in some medical centers and reported to be an effective modality in diagnostic procedures (25 - 27). this machine transmits high resolution pictures that help physicians to make more precise decisions regarding the patients pathology (28, 29). accordingly, we found three studies which assess the diagnostic accuracy of pocket - size ultrasonography for pleural effusion (9, 10, 17), in two of which the procedure was performed by a trained nurse (9, 10) and in one it was carried out by an internal medicine specialist (17). the first two reported a sensitivity of approximately 92 - 98% and the latter survey found a sensitivity of 62% in detection of pleural effusion. nevertheless, the sample sizes of all the three surveys were small and so further investigation is required for application of this ultrasonography device in clinical settings. in this meta - analysis the extended search in databases and bibliographies yielded 12 relevant articles. although few studies were included in this study, but the large sample population of 1554 subjects assured the validity of performed analyses to a great extent. heterogeneity between the articles was another limitation of this survey which was attempted to overcome through application of mixed - effects binary regression model and subgroup analysis. simultaneous inclusion of retrospective and prospective surveys was probable limitation of this study, but the scatterplot developed to evaluate the outlier studies according to standardized predicted random effects showed that retrospective surveys were not the source of diversity between the included articles. the present meta - analysis found chest ultrasonography to have a considerably higher screening value in detection of pleural effusion compare to radiography. being performed by an intensivist or a radiologist and using probes with frequencies of 5 - 10mhz improves the sensitivity of this imaging modality. this research has been supported by tehran university of medical sciences & health services grant number : 93 - 02 - 38 - 25618. all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors. | introduction : the role of ultrasonography in detection of pleural effusion has long been a subject of interest but controversial results have been reported. accordingly, this study aims to conduct a systematic review of the available literature on diagnostic value of ultrasonography and radiography in detection of pleural effusion through a meta - analytic approach.methods:an extended search was done in databases of medline, embase, isi web of knowledge, scopus, cochrane library, and proquest. two reviewers independently extracted the data and assessed the quality of the articles. meta - analysis was performed using a mixed - effects binary regression model. finally, subgroup analysis was carried out in order to find the sources of heterogeneity between the included studies. results:12 studies were included in this meta - analysis (1554 subjects, 58.6% male). pooled sensitivity of ultrasonography in detection of pleural effusion was 0.94 (95% ci : 0.88 - 0.97 ; i2= 84.23, p<0.001) and its pooled specificity was calculated to be 0.98 (95% ci : 0.92 - 1.0 ; i2= 88.65, p<0.001), while sensitivity and specificity of chest radiography were 0.51 (95% ci : 0.33 - 0.68 ; i2= 91.76, p<0.001) and 0.91 (95% ci : 0.68 - 0.98 ; i2= 92.86, p<0.001), respectively. sensitivity of ultrasonography was found to be higher when the procedure was carried out by an intensivist or a radiologist using 5 - 10 mhz transducers. conclusion : chest ultrasonography, as a screening tool, has a higher diagnostic accuracy in identification of plural effusion compared to radiography. the sensitivity of this imaging modality was found to be higher when performed by a radiologist or an intensivist and using 5 - 10mhz probes. |
a prolonged obstruction of either the intra- or extra - hepatic biliary duct may damage hepatic tissue and lead to the development of fibrosis and cirrhosis.1 liver repair and fibrogenesis both resemble the wound healing process. cirrhosis is an advanced stage of fibrosis that is characterized by the formation of regenerative nodules in the liver parenchyma separated by fibrotic septa. the generation of cirrhosis involves three major processes : cell death, aberrant extracellular matrix deposition (fibrosis), and vascular reorganization.2 cholestasis is associated with, and probably best defined as, the impairment of bile formation. this impairment may be accompanied by or caused by the failure to excrete bile acids into the hepatocyte canaliculus, which disrupts the circulation of enterohepatic bile acid and elevates concentrations of hepatocellular and serum bile acid.3 the impairment in hepatic regeneration in cholestatic livers is a well - known phenomenon that may be caused by altered hepatic hemodynamics (decreased portal venous flow), attenuated production of mediators that are associated with liver proliferation, increased rate of apoptosis, and blocked enterohepatic circulation. these processes are all induced by biliary obstruction,4 which reduces the portal venous flow and the production of liver growth - associated factors, increases hepatocyte apoptosis, and impairs enterohepatic circulation. the intrahepatic accumulation of toxic bile salts results in hepatocyte apoptosis via a fas - dependent mechanism.5 many toxic bile acids trigger oligomerization of the fas death receptor, which, in turn, activates the classic fas death receptor cascade.6 in addition, it has been shown that an increased expression of toll - like receptor 2 and the fas ligand in intrahepatic natural killer t cells after biliary obstruction is associated with hepatocyte apoptosis.7 these results indicate that biliary obstruction activates hepatocyte apoptosis. bile acids may directly affect mitochondria, causing mitochondrial dysfunction and activating the mitochondrial pathway of cell death.8 this information suggests that bile acids induce apoptosis in vitro. hepatic stellate cells (hscs) trigger pathogenesis by a liver damage - dependent activation.9 activated hscs transdifferentiate into myofibroblasts (mfbs), which exhibit a synthesis profile that predisposes them to increased extracellular matrix (ecm) deposition. this process allows them to proliferate upon expression of mitogenic cytokines and their receptors, alters their morphology, and increases contractility by triggering - smooth muscle actin (sma) fiber formation. this then leads to the constriction of sinusoidal blood flow and increases the synthesis and release of fibrillar collagens.10 myofibroblasts do not normally exist in the liver. myofibroblasts (mfbs) derived from small portal vessels proliferate around biliary tracts in cholestasis - induced liver fibrosis to initiate collagen deposition.11 fibrosis develops with different spatial patterns according to the different prevalent mechanisms that are associated with the diverse causes of parenchymal damage. indeed, fibrosis that is caused by chronic viral infection is initially concentrated within and around the portal tract, whereas fibrosis that occurs as a secondary effect of exposure to toxic / metabolic damage (as in bile duct obstruction) is located mainly in the centrolobular areas. additionally, it is becoming increasingly evident that different cell types are involved in the deposition of the fibrillar extracellular matrix during active hepatic fibrogenesis. hepatic stellate cells are mainly involved when hepatocellular damage is limited or concentrated within the liver lobule, whereas portal mfbs and fibroblasts contribute the most when the damage is located in the proximity of the portal tracts. in the later stages of evolution (septal fibrosis), it is likely that all extracellular matrix - producing cells will contribute to fibrogenesis.12 the reduction of expression and function of transport systems is another crucial process that plays a key role in the pathogenesis of cholestasis and may cause or maintain cholestatic injury.13,14 transport defects may be caused by hereditary genetic defects or acquired as a result of cholestatic injury, such as during inflammation, drug use, or biliary obstruction. it is also important to keep in mind that not all of the encountered changes in transporter expression are procholestatic and negative. while some of these alterations contribute to cholestasis, other changes might act as compensatory anticholestatic defense mechanisms, providing alternative excretory routes. reducing basolateral bile acid uptake while simultaneously increasing basolateral bile acid excretion might be considered a major hepatic defense mechanism counteracting bile acid accumulation within hepatocytes.15 with regard to the pathogenesis of cholestatic liver injury, the oxidative stress that is associated with increased lipid peroxidation is considered to be crucial for hepatotoxicity.16,17 lipid peroxidation, protein oxidation, and the thiol redox state of the cell are important indicators of oxidative stress.18 bile duct ligation causes changes in the equilibrium between the antioxidant and prooxidant activities ; it favors the latter, as it increases production of free radicals and causes the reduction in free radical scavengers, such as catalase, superoxide dismutase, and glutathione peroxidase 19 the accumulation of hydrophobic bile acids impairs the mitochondrial electron transport chain and enhances the production of reactive oxygen species (ros), which eventually disturb cellular energy homeostasis.20,21 lipid peroxidation causes the depletion of mitochondrial respiratory chain elements, leading to a vicious cycle that results in more ros generation.22 lipids are known to be one of the major and consistent targets of oxidative stress.1 studies on animals and humans have shown that there is a close relationship between lipid peroxidation and cholestatic liver injury.21,23 the role of lipid peroxidation products in the pathogenesis of excessive fibrogenesis in cholestasis has been, so far, the most investigated aspect of oxidative stress - induced damage. one of the major and early lipid peroxidation products to be identified was oxidized low - density - lipoprotein (oxldl), which was first identified in large vessel walls when antioxidative protective pathways had failed ; it was later popularized as a proatherogenic agent. since then, oxldl has been considered to be very important either in the initiation or perpetuation of atherosclerotic processes.24 following the identification of specific membrane receptors for oxldl on kupffer cells, hscs, macrophages, and liver endothelial cells, we previously showed that oxldl accumulates in the liver of bile duct - ligated balb / c mice.25,26 here, we used a prolonged (21-day) experimental bile duct ligation to examine the probable relationship between oxldl accumulation and hepatic fibrogenesis in rat liver. sixteen male albino wistar rats, weighing 200230 g, were used in the study. they were housed in stainless steel cages, under controlled temperature (23c) and humidity with 12-hour dark / light cycles. they were maintained on a standard laboratory diet with tap water ad libitum throughout the experiment, except for an overnight fast before surgery. the experiments were carried out according to the guidelines set forth by the ethics committee of zonguldak karaelmas university, zonguldak, turkey. group i was designated as the prolonged obstructive jaundice group (oj), while group ii was established as the sham - operated group (so). all surgical procedures were performed under strict sterile conditions, using intraperitoneal ketamine - hcl (ketalar ; parke davis, morris plains, nj, usa) as the anesthetic agent (50 mg / kg bodyweight) and a standard upper midline incision. in group i, a bile duct ligation was carried out according to the method of lee.27 in group ii, the sham operation was performed by placing a silk ligature around the common bile duct and then removing it. the abdominal incision was closed in two layers with chromic 40 cat gut and 40 silk. the rats were allowed food and water ad libitum postoperatively. on the 21 day after the operation, blood was centrifuged at 1,800 g for 15 min at 4c to obtain plasma for biochemical analysis. after blood collection, the rats were killed and the livers of the animals were removed immediately for histopathological, immunofluorescent, and biochemical examination. the serum bilirubin level was determined with cobas bio (hoffman la roche, basel, switzerland) using the bilirubin test (hoffman la roche). the serum activity of aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), albumin, and gamma glutamine transpeptidase (ggt) was estimated using commercially available kits (boehringer, mannheim, germany). total protein concentration was measured according to the method described by lowry, using serum bovine albumin as a standard.28 the liver mda level was assessed according to the method described by ohkawa and ohishi.29 in brief, the liver was homogenized with cold 1.15% kcl to make a 10% homogenate. a solution of 0.2 ml of 8.1% sds and 1.5 ml of 20% acetic acid was adjusted to ph 3.5 with naoh, and 1.5 ml of 0.8% aqueous solution of thiobarbituric acid was added to 0.2 ml of 10% tissue homogenates. the volume of the mixture was adjusted to 4.0 ml with distilled water, and then the mixture was heated in an oil bath at 95c for 60 min. after cooling with tap water, 1.0 ml of distilled water and 5.0 ml of a mixture of n - butanol and pyridine (15:1, v / v) were added, and the solution was shaken vigorously. after centrifugation at 4,000 rpm for 10 min, the organic layer was removed and its absorbance was measured at 532 nm on a shimadzu uv 1601 spectrophotometer. as a standard, 1.1.3.3-tetraetoxypropane was used. the mda concentration was calculated per gram of tissue (nmol / gr tissue). hepatic sod level was determined using the method of sun.30 the assay for sod activity involves inhibiting the reduction of nitroblue tetrazolium with xanthine and xanthine oxidase, which was used as a superoxide generator. liver tissues were weighed, and 1.15% kcl was added to make a 10% homogenate. to 0.5 ml of the supernatant, 2.45 ml sod measurement reactive solution (40 ml 0.3 mmol / l xanthine, 20 ml 0.6 mmol / l edta, 20 ml 150 mol / l nitroblue tetrazolium, 12 ml 400 mmol / l na2co3, and 6 ml, xanthine oxidase reagents were added in a volume of 0.05 ml per tube at 30 sec intervals and incubated for 20 min. the xanthine oxidase reagent was freshly prepared with ice - cold 2 mol / l nh4so4 ; the final concentration of xanthine oxidase was 167 reactions were then terminated by adding 1 ml of 0.8 mmol / l cucl2 reagent into each tube at 30 sec intervals. the absorbance of each sample was measured at 560 nm on a shimadzu uv 1601 spectrophotometer. the percent inhibition was calculated according to the formula : one unit of sod is defined as the amount of protein that inhibits the rate of nbt reduction by 50%. sod activity was calculated per mg of tissue protein (u / mg protein). the presence of oxldl in the liver tissue sections of the jaundiced and the sham - operated rats was evaluated using a special method of immunofluorescence staining. the left lobe of each rat liver was obtained and stored at 85c in a deep freezer. the slides were prepared from frozen liver biopsy sections, which were cut to a thickness of 7 m. slides were further divided into two pieces : one was used for the test, and the other was used as a negative control. primary antibody in 30 l (human polyclonal anti - oxldl immunoglobulin (ig)g, immco diagnostics, new york, ny, usa) was added only to the test slides, wheras phosphate buffered solution (pbs) in the same volume was added to the control slides. after incubating for 30 min in a humid chamber at room temperature, both the control and test slides were washed with pbs, and a fluorescein isothiocyanate labeled antihuman igg (30 l) was administered as a conjugate substance. after another 30 min at room temperature, the slides were washed with the standard pbs solution. after drying, the slides were covered with a mounting medium and examined under a fluorescence microscope (leica dmrx, wetzlar, germany). for histological examination the liver samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 m, and stained with hematoxylin and eosin. a single pathologist, blind to the study, assessed all biopsies. although we acknowledge that there are differences in the pathogenesis of cholestasis cases caused by obstruction and hepatocellular lesions caused by chronic hepatitis, we have herein evaluated a scoring system following the same categorization as described by ishak. in order to provide semi - quantitative scores to assess the degree of inflammatory activity and fibrosis.31 liver damage was categorized as mild, moderate, or severe (cirrhosis). inflammatory scores between 13 and fibrosis scores between 12 were classified as mild ; inflammatory scores between 418 and fibrosis scores between 35 were classified as moderate liver damage. sixteen male albino wistar rats, weighing 200230 g, were used in the study. they were housed in stainless steel cages, under controlled temperature (23c) and humidity with 12-hour dark / light cycles. they were maintained on a standard laboratory diet with tap water ad libitum throughout the experiment, except for an overnight fast before surgery. the experiments were carried out according to the guidelines set forth by the ethics committee of zonguldak karaelmas university, zonguldak, turkey. group i was designated as the prolonged obstructive jaundice group (oj), while group ii was established as the sham - operated group (so). all surgical procedures were performed under strict sterile conditions, using intraperitoneal ketamine - hcl (ketalar ; parke davis, morris plains, nj, usa) as the anesthetic agent (50 mg / kg bodyweight) and a standard upper midline incision. in group i, a bile duct ligation was carried out according to the method of lee.27 in group ii, the sham operation was performed by placing a silk ligature around the common bile duct and then removing it. the abdominal incision was closed in two layers with chromic 40 cat gut and 40 silk. on the 21 day after the operation, blood samples of all animals were collected using the cardiac puncture method. blood was centrifuged at 1,800 g for 15 min at 4c to obtain plasma for biochemical analysis. after blood collection, the rats were killed and the livers of the animals were removed immediately for histopathological, immunofluorescent, and biochemical examination. the serum bilirubin level was determined with cobas bio (hoffman la roche, basel, switzerland) using the bilirubin test (hoffman la roche). the serum activity of aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), albumin, and gamma glutamine transpeptidase (ggt) was estimated using commercially available kits (boehringer, mannheim, germany). total protein concentration was measured according to the method described by lowry, using serum bovine albumin as a standard.28 the liver mda level was assessed according to the method described by ohkawa and ohishi.29 in brief, the liver was homogenized with cold 1.15% kcl to make a 10% homogenate. a solution of 0.2 ml of 8.1% sds and 1.5 ml of 20% acetic acid was adjusted to ph 3.5 with naoh, and 1.5 ml of 0.8% aqueous solution of thiobarbituric acid was added to 0.2 ml of 10% tissue homogenates. the volume of the mixture was adjusted to 4.0 ml with distilled water, and then the mixture was heated in an oil bath at 95c for 60 min. after cooling with tap water, 1.0 ml of distilled water and 5.0 ml of a mixture of n - butanol and pyridine (15:1, v / v) were added, and the solution was shaken vigorously. after centrifugation at 4,000 rpm for 10 min, the organic layer was removed and its absorbance was measured at 532 nm on a shimadzu uv 1601 spectrophotometer. as a standard, 1.1.3.3-tetraetoxypropane was used. the mda concentration was calculated per gram of tissue (nmol / gr tissue). hepatic sod level was determined using the method of sun.30 the assay for sod activity involves inhibiting the reduction of nitroblue tetrazolium with xanthine and xanthine oxidase, which was used as a superoxide generator. liver tissues were weighed, and 1.15% kcl was added to make a 10% homogenate. to 0.5 ml of the supernatant, 2.45 ml sod measurement reactive solution (40 ml 0.3 mmol / l xanthine, 20 ml 0.6 mmol / l edta, 20 ml 150 mol / l nitroblue tetrazolium, 12 ml 400 mmol / l na2co3, and 6 ml, xanthine oxidase reagents were added in a volume of 0.05 ml per tube at 30 sec intervals and incubated for 20 min. the xanthine oxidase reagent was freshly prepared with ice - cold 2 mol / l nh4so4 ; the final concentration of xanthine oxidase was 167 reactions were then terminated by adding 1 ml of 0.8 mmol / l cucl2 reagent into each tube at 30 sec intervals. the absorbance of each sample was measured at 560 nm on a shimadzu uv 1601 spectrophotometer. the percent inhibition was calculated according to the formula : one unit of sod is defined as the amount of protein that inhibits the rate of nbt reduction by 50%. sod activity was calculated per mg of tissue protein (u / mg protein). the presence of oxldl in the liver tissue sections of the jaundiced and the sham - operated rats was evaluated using a special method of immunofluorescence staining. the left lobe of each rat liver was obtained and stored at 85c in a deep freezer. the slides were prepared from frozen liver biopsy sections, which were cut to a thickness of 7 m. slides were further divided into two pieces : one was used for the test, and the other was used as a negative control. primary antibody in 30 l (human polyclonal anti - oxldl immunoglobulin (ig)g, immco diagnostics, new york, ny, usa) was added only to the test slides, wheras phosphate buffered solution (pbs) in the same volume was added to the control slides. after incubating for 30 min in a humid chamber at room temperature, both the control and test slides were washed with pbs, and a fluorescein isothiocyanate labeled antihuman igg (30 l) was administered as a conjugate substance. after another 30 min at room temperature, the slides were washed with the standard pbs solution. after drying, the slides were covered with a mounting medium and examined under a fluorescence microscope (leica dmrx, wetzlar, germany). the liver samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 m, and stained with hematoxylin and eosin. a single pathologist, blind to the study, assessed all biopsies. although we acknowledge that there are differences in the pathogenesis of cholestasis cases caused by obstruction and hepatocellular lesions caused by chronic hepatitis, we have herein evaluated a scoring system following the same categorization as described by ishak. in order to provide semi - quantitative scores to assess the degree of inflammatory activity and fibrosis.31 liver damage was categorized as mild, moderate, or severe (cirrhosis). inflammatory scores between 13 and fibrosis scores between 12 were classified as mild ; inflammatory scores between 418 and fibrosis scores between 35 were classified as moderate liver damage. data are expressed as mean sd. comparisons among the groups were performed using the mann - whitney u test. no deaths were observed in groups i or ii. all animals with bile duct ligation were obviously jaundiced by 56 days after the operation. the jaundice was confirmed by assessing serum bilirubin concentration on the 21 day after bile duct ligation. the mean values of the serum ast, alt, alp, ggt, total protein, bilirubin, and albumin concentrations measured in the blood samples are presented in table 1. prolonged obstructive jaundice significantly increased the serum ast, alt, alp, ggt, and bilirubin concentrations as expected. while there were no statistically significant differences between groups in serum total protein concentration, serum albumin concentrations were significantly decreased in prolonged obstructive jaundiced animals. the mean mda and sod activities of the livers in each group are shown in table 2. while the mean liver content of sod in group i was significantly lower than that of group ii, prolonged bile - duct ligation resulted in a significant increase in the liver mda content as compared to sham - operated rats (table 2). a significant positive immunofluorescence staining for oxldl was found in the liver tissue sections of the prolonged jaundiced group (group i ; figure 1), and we did not observe any positive immunofluorescence staining in sham - operated rats (group ii ; figure 2). neither fibrosis nor any of the other criteria for inflammation and hepatocellular injury were observed in the sham - operated group, whereas features of severe hepatic injury, namely fibrosis, were determined in the liver tissues of prolonged obstructive jaundiced rats (figures 34). irrespective of the etiology, liver fibrosis is a common consequence of chronic liver injury.32 liver fibrosis is defined as the abnormal accumulation of extracellular matrix in the liver. cholestasis may result either from a functional defect in bile formation at the level of the hepatocyte or from an impairment in the secretion of bile and/or obstruction of flow at the bile duct level.33 hepatic uptake and biliary excretion of organic anions, such as bile acids and bilirubin, is mediated by hepatobiliary transport systems. the recruitment of alternative export transporters in coordination with phase i and ii detoxifying pathways provides alternative elimination pathways to counteract the accumulation of potentially toxic biliary constituents in cholestasis.32 the genes encoding for organic anion uptake (ntcp / slc10a1, oatps), canalicular export (bsep, mrp2), and alternative basolateral export (mrp3, mrp4) transporters in the liver are regulated by a complex interacting network of hepatocyte nuclear factors (hnf1, 3 and 4) and nuclear receptors (e.g., fxr, pxr, car, rar, lrh-1, shp, and gr).34 moreover, adaptive transporter changes, such as the induction of basolateral efflux pumps, may help to limit hepatic accumulation of potentially toxic biliary constituents in cholestasis by providing alternative escape routes.33 bile acids, proinflammatory cytokines, hormones, and drugs mediate these causative and adaptive transporter changes at a transcriptional level by interacting with these nuclear factors and receptors. increased matrix formation (i.e., fibrogenesis) occurs in parallel to decreased matrix degradation (i.e., fibrolysis). a landmark finding was the identification of activated hscs and mfbs as the major collagen - producing cells, which, upon activation by profibrogenic cytokines, acquire a profibrogenic phenotype and start to synthesize extracellular matrix that replaces parenchymal tissue.35 the progression of fibrosis results in the development of cirrhosis, chronic liver failure, portal hypertension, and hepatocellular carcinoma.31,36 as fibrosis develops in response to liver injury, hsc activation leads to the accumulation of a scar matrix. this process, in turn, contributes to the loss of hepatocyte microvilli and endothelial fenestrae, leading to the deterioration of hepatic function. kupffer cell (macrophage) activation contributes to the activation of hscs.37 in the cholestatic rodent, kupffer cell activation and the infiltration of neutrophils contribute to tissue injury.38 similarly, in our experimental data, neutrophil infiltration was evident in bile duct - ligated rats but not in sham - operated rats. activated hscs, portal fibroblasts, and mfbs of bone marrow origin have been identified as the major collagen - producing cells in the injured liver. these cells are activated by fibrogenic cytokines, such as angiotensin ii, tgf-1, and leptin. 39 recent reports have highlighted the important role of bile in maintaining intestinal integrity and normal intestinal immunity.40 the impaired function of the intestinal barrier in patients with biliary obstruction is restored by bile replacement.41 bile administration maintains the integrity and permeability of the intestinal mucosa and prevents villous atrophy, villous edema, and lacteal canal dilation. these results were obtained using rats with biliary obstruction.43 the lack of enterohepatic circulation during biliary obstruction also affects hepatic regeneration. the excretion rates of biliary lipids are disturbed with external biliary drainage compared to rates with internal biliary drainage, and liver regeneration rate is significantly correlated with bile flow and the excretion rates of biliary lipids.44 these results clearly indicate that the lack of enterohepatic circulation in the cholestatic liver is one of the major contributors to impaired hepatic regeneration.4 one of the most interesting targets of experimental investigation that has been undertaken so far has been the mechanism underlying cholestatic liver damage. liver fibrosis is a complex phenomenon, in which nonparenchymal cells, such as kupffer cells and hscs, are reported to play a major role.45 kupffer cells stimulate the progression of the fibrogenic process and hscs synthesize most of the matrix proteins. hepatic fibrosis develops 23 weeks after bile duct ligation in rats ; this was the length of our experimental period. mitochondrial oxidative stress and lipid peroxidation are considered to be the significant molecular players in cholestatic liver injury.46 a increase in oxidative over antioxidative biochemical activities, (i.e., during oxidative stress), has been reported to occur in liver fibrosis.1 since lipids are a major target of oxidative stress, products of lipid peroxidation seem to play a role in cholestatic liver injury. the extent of oxidative stress is thought to be a crucial factor determining various pathophysiological conditions, with the spectrum of conditions ranging from cell modulation to fibrosis, to inflammation, irreversible cell damage, and apoptosis.45 it has been observed that in chronic cholestasis, an increased concentration of biliary acids in hepatocytes induces mitochondrial toxicity and results in the overproduction of ros.47 furthermore, increased lipid peroxidation has been reported to occur in the model of bile duct ligation.48 a statistically significant correlation between total bilirubin levels and lipid peroxidation has also been described in hepatic toxicity and diseases of the biliary tract.49 the observation of increased lipid peroxide levels in the blood of children with chronic cholestasis supports the oxidative hypothesis and explains, at least in part, the cholestatic - induced hepatocellular injury.50 sokol. has also shown that exposure to hydrophobic bile acids causes a significant peroxidation of membrane lipids in isolated hepatocytes and liver mitochondria.51 in obstructive jaundice, increased hepatic lipid peroxidation, hepatocellular mitochondrial dysfunction, and decreased glutathione levels occur.52 previous reports have shown that bile duct ligation in the rat for four weeks produces cirrhosis and a sixfold increase in liver collagen content.53 if the large biliary duct is obstructed for several weeks to months, as in our experiment, cholestasis extends throughout the lobule and hepatocyte necrosis leads to lobular bile infarcts. the ductular reaction develops at the periphery of the portal tracts and extends toward the neighboring portal tracts and deep into the parenchyma. this ductular reaction is accompanied by the proliferation of periductular (myo)fibroblasts that are derived from the portal fibroblasts around the interlobular bile ducts. extensive fibrosis surrounding the ductular reaction enlarges the portal tracts and leads to the formation of porto - portal septa.2 in the septa development process of portal fibrogenesis, researchers maintain that viral hepatitis leads to the formation of porto - central septa, featuring activation of hsc, and that cholestatic liver diseases lead to portal portal septa laid down by portal - tract - derived mesenchymal cells.54 during cirrhosis, the normal architecture of the liver is completely modified by the development of fibrotic septa. the cell populations involved differ according to their pattern of fibrosis. in portal diseases, such as chronic viral hepatitis and chronic bile duct obstruction, portal fibroblasts are found in the septa and hscs are found at the interface between the septa and parenchyma.2 in the model of cholestatic fibrosis induced by common bile duct ligation, the withdrawal of intraductal pressure is sufficient to induce apoptosis in bile duct epithelial and portal fibroblastic cells, and to elicit extracellular matrix remodeling.55 these data provide insight into the mechanisms involved in the regression of cholestatic diseases and highlight the role of mechanical stimuli in cell growth and death. the experimental bile duct ligation in rats, as in our experiment, is still one of the main models that support a link between oxidative damage and fibrogenesis during chronic cholestasis, which mimics clinical and morphological aspects of fibrosis and cirrhosis secondary to extrahepatic biliary obstruction in humans. we used 21 days of obstructive jaundice, as the collagen deposition was reported to become significant only after 15 days of bile duct ligation.23 the histopathological evaluation of liver tissue from the jaundiced group exhibited marked features of hepatocellular injury and fibrosis. in bile duct - ligated rats, the presence of marked oxidative stress and oxidative damage to lipids is traditionally demonstrated with thiobarbituric acid (tba) reactive substance assays, which detect mda, a peroxidation product of polyunsaturated fatty acids.56 mda is considered to be the major product of lipid peroxidation.57 an mda assay is generally used to detect the existence of lipid peroxidation as a result of oxidative stress in any tissue. in our study, we found that 21 days of obstructive jaundice resulted in a significant elevation of the concentration of liver mda, compared with the sham - operated group (35.07 3.73 vs. 20.86 4.69 nmol / gr liver, p < 0.01 ; table 2). this result is also in agreement with other studies on obstructive jaundice conducted in animals and humans.20 moreover, the level of the antioxidant enzyme sod, which is the scavenger of ros in oxidative stress, was found to be significantly reduced in the jaundiced group (1.62 1.36 vs. 7.86 1.42 nmol / gr liver, p < 0.01 ; table 2). as the unsaturated fatty acids oxidize, they undergo molecular rearrangements and generate peroxy - fatty acids. these readily decompose, particularly in the presence of redox metals, such as iron and copper, to generate aldehydes, such as mda, 4-hydroxynonenal, and others.58 during oxidative stress, plasma ldl is oxidatively modified mainly in the subendothelial space, attracting blood monocytes to the subendothelial spaces. oxldl is taken up by macrophages via scavenger receptors.59 these scavenger receptors have been shown to exist in many cells, including kupffer cells, hscs, macrophages, and liver endothelial cells.1,25,60 these phagocytic cells are reportedly responsible for a marked increase in the steady - state concentration of fibrogenic cytokines that intensify the cross - talk with extracellular matrix - producing cells.1 the liver is the primary organ for the clearance of the oxidatively modified cytotoxic and atherogenic ldl molecules. the effective filtration of oxldl has been confirmed by the complete removal of these molecules from circulation within a few minutes when administered exogenously.60 fluorescently - labeled oxldl was essentially found to concentrate in kupffer cells and, to a lesser extent, in endothelial cells in humans and in the rat liver. following the rapid hydrolysis of cholesteryl esters from oxldl by kupffer cells, the molecule is transported to liver parenchymal cells and bile.61 therefore, it is expected that oxldl would accumulate in the liver after bile duct ligation. previously, we have shown, using fluorescent immunostaining, that oxldl accumulates in the liver of balb / c mice after 10 days of experimental cholestasis.26 in this study, we used a prolonged period of obstructive jaundice, 21 days, as it has been demonstrated that, at high degrees of oxidation of ldl, the affinity for the oxldl specific binding site is strongly enhanced and the phagocytic capacity of kupffer cells increases within 23 weeks of biliary obstruction.62 ongoing oxidative stress in prolonged cholestasis induces more ros production than usual, which may enhance the oxidative modification of ldl within the liver. free radicals not only induce oxidative stress in the liver but also initiate and maintain the oxidation of ldl.63 it has been demonstrated that there is a close relationship between the severity of fibrosis and the amount of lipid peroxidation products, which reflects the level of oxidative stress.64 moreover, there is a close link between lipid peroxidation and the activation of inflammatory cells, and a positive linear correlation exists between collagen deposition and the production of mda.23 our results related to the mda concentration are in agreement with the literature.23,64 independently from the nature of the injury, the fibrogenic pathway is similar in various tissues. kupffer cells and hscs are the key players in liver fibrosis. the phenotypic transformation of hscs into mfb - like cells is considered to be the central event of hepatic fibrogenesis. the activation of hscs results in the production of a large quantity of extracellular matrix components. in an in vitro study, it was shown that there were specific receptors for oxldl in hscs that stimulate the synthesis of the extracellular matrix, a result that is consistent with the hypothesis that oxldl might at least participate in the events leading to liver inflammation and fibrosis in cholestasis.25 in conclusion, we have demonstrated that oxldl accumulates in liver tissue sections of prolonged obstructive jaundiced rats. the accumulation of oxldl may be the consequence of either a failure to eliminate it via bile flow or a cholestasis - induced enhancement of ros production and lipid peroxidation, which may predict ongoing oxidative stress. oxidative damage, possibly in association with more than one mechanism, appears to be crucial for the progression of fibrosis through cirrhosis in cholestatic liver injury. we believe that oxldl might at least play a contributory role in the various pathophysiological mechanisms underlying the process of liver fibrosis and that the association of oxldl accumulation and hepatic fibrosis deserves to be considered as something more than coincidence in cholestasis. further studies are needed to evaluate the potential effects of oxldl in the progression to secondary biliary cirrhosis. | objectivethe aim of the present study was to examine the probable relationship between the accumulation of oxldl and hepatic fibrogenesis in cholestatic rats.introductionthere is growing evidence to support the current theories on how oxidative stress that results in lipid peroxidation is involved in the pathogenesis of cholestatic liver injury and fibrogenesis. one of the major and early lipid peroxidation products, oxldl, is thought to play complex roles in various immuno - inflammatory mechanisms.methodsa prolonged (21-day) experimental bile duct ligation was performed on wistar - albino rats. biochemical analysis of blood, histopathologic evaluation of liver, measurement of the concentration of malondialdehyde (mda) and superoxide - dismutase (sod) in liver tissue homogenates, and immunofluorescent staining for oxldl in liver tissue was conducted in bile - duct ligated (n = 8) and sham - operated rats (n = 8).resultssignificantly higher levels of mda and lower concentrations of sod were detected in jaundiced rats than in the sham - operated rats. positive oxldl staining was also observed in liver tissue sections of jaundiced rats. histopathological examination demonstrated that neither fibrosis nor other indications of hepatocellular injury were found in the sham - operated group, while features of severe hepatocellular injury, particularly fibrosis, were found in jaundiced rats.conclusionour results support the finding that either oxldls are produced as an intermediate agent during exacerbated oxidative stress or they otherwise contribute to the various pathomechanisms underlying the process of liver fibrosis. whatever the mechanism, it is clear that an association exists between elevated oxldl levels and hepatocellular injury, particularly with fibrosis. further studies are needed to evaluate the potential effects of oxldls on the progression of secondary biliary cirrhosis. |
in recent years, hcfa has revised its strategic plan, reorganized its operations, and initiated several programs aimed at ensuring that medicare beneficiaries receive the highest possible quality of health care. the m+c program, created by the 1997 bba, expands medicare 's health insurance options to include a wider range of health plan options. the bba, the government performance and results act, and other government - wide initiatives have all emphasized the themes of accountability and a renewed focus on medicare beneficiaries as customers. the m+c program has developed programs to monitor and improve the quality of ambulatory care. in an effort to better understand the experiences and needs of medicare beneficiaries, it now surveys annually a probability sample of all medicare beneficiaries and asks about their health care experiences using the cahps survey. to ensure that beneficiaries have access to the best possible health care, hcfa has initiated several quality monitoring systems, including the cahps surveys, the health outcomes survey, and the collection of measures in the health plan employer data information set (hedis). these measurement activities allow hcfa to identify quality improvement opportunities and to disseminate information to beneficiaries to help them make better health care choices. the advent of m+c presented hcfa with the formidable task of educating the nation 's 40 million medicare beneficiaries about changes in the medicare program. hcfa established the national medicare education program to inform beneficiaries, caregivers, and partners, about features of the medicare program and, in particular, about the expanded options with m+c. as part of the national medicare education program, hcfa provides comparative information on health plans, including information about enrollee experiences through the medicare & you handbook, mailed annually to medicare beneficiaries before the open enrollment period, through a telephone hotline (1 - 800-medicar[e ]), and through hcfa 's web site http://www.medicare.gov. since 1998, hcfa has conducted an annual nationwide survey of medicare beneficiaries enrolled in managed care organizations to collect information about their experiences with their health plans. cahps is the first comprehensive effort to assess consumer experiences with their health plans and services. the medicare cahps effort consists of : the managed care survey, a disenrollment survey, and the medicare (fee - for - service) survey. this article focuses exclusively on the survey of beneficiaries enrolled in the mmc plans, presents background information on the cahps survey, provides an overview of the first 3 years of data collection and analysis, and a discussion of the long - run potential for using the managed care data for performance improvement. this survey is part of a group of consumer surveys developed by a consortium of researchers from the research triangle institute, the rand corporation, and the harvard medical school through cooperative agreements with the agency for healthcare research and quality. the goal of the cahps initiative, which began in 1995, was to develop and test standardized questionnaires and report formats that could be used to collect and report meaningful and reliable information about health plans. the surveys contain core items that can be used with any population and additional items targeted to particular groups. thus, they can be used with all types of health insurance consumers, including medicaid recipients, medicare beneficiaries, those who are commercially insured, and across the full range of health care delivery systems. the core cahps survey asks respondents for four overall ratings of their doctor, specialist (if used), overall health care, and health care plan. information from most of the specific questions is combined to create composite scores on getting needed care, getting care quickly, doctor communication, courtesy and helpfulness of office staff, and paperwork, information, and customer service. in 1996 hcfa funded the cahps consortium to develop a special version of cahps for beneficiaries enrolled in the mmc plans. the initial mmc cahps survey, which was only used in the first administration of the survey, included all items in the cahps 1.0 adult core instrument and 28 additional items specific to the mmc version. items unique to the mmc cahps version ask about ease of obtaining needed medical equipment, physical, occupational or speech therapy, home health services, and prescription drugs. other mmc items concern advice to quit smoking, receipt of flu shots, pneumonia vaccinations, personal doctor 's knowledge about important medical decisions, and the health plan 's handling of complaints. most of the medicare - specific items were added to the core to capture the experience of beneficiaries with services that are used more often by patients with chronic conditions or limitations in activities of daily living. the questions related to smoking counseling, flu shots, and pneumonia vaccinations were added to obtain information from managed care enrollees to fulfill hedis reporting requirements. we present data on the ratings and composite scores here because those are the data that currently are publicly available. factor analyses of data from the first year survey confirm that these composites generally correspond to the dimensions of performance along which plans vary (zaslavsky., 2000a). because cahps 1.0 was used the first year and cahps 2.0 in subsequent years of the mmc cahps surveys, some of the composite scores are not directly comparable across years. its subcontractors westat, data recognition corporation, picker institute, and the harvard medical school to implement the mmc cahps survey in fall 1997. the first survey was fielded in february 1998 and subsequent surveys have been fielded every fall, beginning in 1998. for the most recently completed survey (fall 1999), eligible plans included all m+c organizations and continuing cost contracts with contracts in effect as of july 1, 1998. in some cases, a single contract that covers a large geographic area was divided into several geographically defined parts, which were then treated separately in the survey and analysis. hcfa drew a random sample of up to 600 medicare beneficiaries who had been enrolled in an eligible plan for at least 6 months and who were non - institutionalized. if the organization had less than 600 medicare beneficiaries, everyone in the organization was included in the sample. numerous attempts are made to reach respondents in english and spanish by mail, telephone, federal express, or priority mail. a pre - survey notification letter is sent to all sample members followed by the first survey mailing. non - respondents receive a second survey mailing and telephone interviews are conducted with sample members who have not responded to the first or second mailing. non - respondents for whom no telephone numbers are available receive a third survey by federal express or priority mail. for february of 1998, there were 237 organizations sampled and 89,804 beneficiaries responded for a response rate of 75 percent. for september of 1998, there were 311 organizations sampled and 138,354 beneficiaries responded for a response rate of 81 percent. for september of 1999, there were 367 organizations sampled and 166,072 beneficiaries responded for a response rate of 82 percent. mean scores on each of the global ratings and composites are presented for each of the 10 hcfa regions for the third year of the mmc cahps survey in table 1. for example, in year 3, the regional average doctor ratings ranged from 8.7 to 9.1 (table 2). the highest doctor scores each year tend to be in the northeast and mid - atlantic and the lowest doctor scores tend to be in the pacific and northwest regions. plan ratings tended to have less interregional variation than ratings of doctor, specialist, and health care. the absolute differences in cahps scores among regions may appear modest, but they are substantial compared with typical differences among plans and represent average differences over thousands of beneficiaries. analyses of data from the first year of the survey showed large variations among market areas within regions and large interplan variations within market areas (zaslavsky., 2000b). that is, the data confirm that the cahps survey has adequate precision to detect differences in consumer experiences across plans and geographic areas. we do not know what accounts for the variations observed, but several aspects of the results are consistent with other information we have about health care quality. the finding that scores are highest in the northeast, and lowest in the pacific and mountain regions, is consistent with patterns in other data, such as, other hedis indicators. also, the differential patterns in the measures suggest that they reflect different aspects of plan performance. for example, the relatively large differences in doctor scores may reflect well - known regional differences in practice styles (table 2). 2000b) show complementary patterns. for example, perhaps because many plans in the same market share common physicians, there is relatively little interplan variability in doctor scores. on the other hand, there are significant differences in plan scores, probably because they have distinct policies and administrative procedures. comparative information can be useful to plans as they try to identify areas in which to focus quality improvement efforts (figure 1). although a lack of differences among plans does not mean there is not a need for improvement, areas in which there are large interplan differences on a rating or composite score provide tangible evidence of the possibility of better performance and can motivate plans to improve those areas of performance. data from the mmc cahps survey provide useful information on variation among plans overall and by dimensions of plan performance. while the initial purpose of the mmc cahps was to obtain information that could be used by medicare beneficiaries to compare plan options, the information also can be a valuable tool for monitoring health plan performance and managed care quality improvement efforts. the documented variations in health plan performance, previously described, provide evidence that some plans are more effective than others in meeting the needs of their medicare enrollees. the ratings of individual plans encompass overall performance, provider performance, processes of and access to care. these data offer the opportunity to identify specific dimensions of health plan performance that are of concern and to develop mechanisms to encourage and assist these plans to improve. as an initial step in that direction, hcfa is currently providing detailed information on individual health plan ratings to medicare health plans, peer review organizations, (pros) and to hcfa staff with responsibilities for monitoring and oversight of medicare health plans. health plans receive a yearly detailed report of their mmc cahps scores on overall ratings and specific measures. in addition, to assist the health plans on how to evaluate their scores, state means of the mmc cahps scores are provided. this information can be used by each plan to assess areas of good performance and areas where members are indicating that they are less successful. ideally, health plans would use these data to identify problems and areas for improvement and would develop strategies that would result in improvements in areas where they are less successful in meeting the needs of medicare beneficiaries. since the mmc cahps is only in its third year (and medicare plans have received just two reports), there is little evidence to date about how much and in what ways the cahps information is encouraging health plans to change. pros receive an annual mmc cahps report that provides detailed information on the cahps ratings of each medicare health plan in its region. pros can use these data to identify problems in service to medicare beneficiaries enrolled in health plans, both in individual health plans and overall plans. identification of these problems could then provide a basis for developing targeted approaches for encouraging improvements in health plan performance. hcfa central and regional offices individual health plan ratings and summary ratings, by region, are provided to central and regional office staff who have responsibility for oversight and monitoring of the m+c program. the mmc cahps information, in combination with the knowledge that hcfa staff have about specific medicare health plans, offers the opportunity to assess and compare health plan performance and to identify potential problem areas in specific health plans. this information, in turn, could be used by hcfa staff to focus on particular organizational components and their operations during periodic site visits to medicare health plans, in order to evaluate the extent to which they are in compliance with hcfa requirements. additionally, hcfa staff could encourage these health plans to make changes that would improve performance, overall and by component. in addition to providing information of medicare health plan ratings to plans, pros, and internal staff, hcfa also is using mmc cahps data to better understand the relationships between medicare health plan cahps ratings and factors that affect these ratings (landon., 2001). results of these analyses may be used, in the longer term, to assist health plans, pros, and hcfa to develop strategies for better meeting the needs of all medicare beneficiaries enrolled in m+c plans. finally, hcfa is also supporting extensive research on differences in the health plan experiences of subgroups of the medicare population, including those with exceptional needs for health care (e.g., the medicare disabled under age 65, those in fair or poor health and/or with limited independence), racial and ethnic minorities (e.g., black persons, hispanics, and latinos), and medicare - medicaid dually eligible beneficiaries (cox and langwell, 2000). the purpose of these analyses is to gain insight into the performance of medicare health plans in meeting the needs of those beneficiaries who may have atypical health care needs or who may face cultural, linguistic, or other barriers to care. results may be used by hcfa to identify problems faced by beneficiaries who may require additional efforts by health plans to facilitate their effective use of services. health plans could then be encouraged to make changes that would improve access, service, and outcomes for all medicare enrollees. thus, its potential for use as a tool to assist health plans, hcfa, and pros to improve performance is still evolving. it is clear, however, that the mmc cahps could be an extremely useful means for identifying areas for medicare health plan improvement. evidence to date, suggests that hcfa and pros will use this information to design strategies for encouraging health plans to make changes in their organization and operations that will offer increasing value and service to their medicare enrollees. hcfa is moving toward value - based purchasing, which is a way to encourage and financially reward managed care organizations that provide better than average quality to m+c beneficiaries. mmc cahps, in combination with other indicators compiled by hcfa, will be used to more fully manage the m+c program and to reward plans that demonstrate high performance in serving and meeting the needs of the medicare population. to date, value - based purchasing has not been implemented by hcfa, but several possibilities are being explored. for example, hcfa could design an incentive rewards system that would offer m+c plans higher payments, reduced regulatory burden, or other rewards for maintaining high performance and for demonstrating significant improvements in key areas of quality and service. the mmc cahps offers the opportunity to design incentive rewards to improve health plan performance based on consistent, reliable, and mean scores on each of the global ratings and composites are presented for each of the 10 hcfa regions for the third year of the mmc cahps survey in table 1. for example, in year 3, the regional average doctor ratings ranged from 8.7 to 9.1 (table 2). the highest doctor scores each year tend to be in the northeast and mid - atlantic and the lowest doctor scores tend to be in the pacific and northwest regions. plan ratings tended to have less interregional variation than ratings of doctor, specialist, and health care. the absolute differences in cahps scores among regions may appear modest, but they are substantial compared with typical differences among plans and represent average differences over thousands of beneficiaries. analyses of data from the first year of the survey showed large variations among market areas within regions and large interplan variations within market areas (zaslavsky., 2000b). that is, the data confirm that the cahps survey has adequate precision to detect differences in consumer experiences across plans and geographic areas. we do not know what accounts for the variations observed, but several aspects of the results are consistent with other information we have about health care quality. the finding that scores are highest in the northeast, and lowest in the pacific and mountain regions, is consistent with patterns in other data, such as, other hedis indicators. also, the differential patterns in the measures suggest that they reflect different aspects of plan performance. for example, the relatively large differences in doctor scores may reflect well - known regional differences in practice styles (table 2). 2000b) show complementary patterns. for example, perhaps because many plans in the same market share common physicians, there is relatively little interplan variability in doctor scores. on the other hand, there are significant differences in plan scores, probably because they have distinct policies and administrative procedures. comparative information can be useful to plans as they try to identify areas in which to focus quality improvement efforts (figure 1). although a lack of differences among plans does not mean there is not a need for improvement, areas in which there are large interplan differences on a rating or composite score provide tangible evidence of the possibility of better performance and can motivate plans to improve those areas of performance. data from the mmc cahps survey provide useful information on variation among plans overall and by dimensions of plan performance. while the initial purpose of the mmc cahps was to obtain information that could be used by medicare beneficiaries to compare plan options, the information also can be a valuable tool for monitoring health plan performance and managed care quality improvement efforts. the documented variations in health plan performance, previously described, provide evidence that some plans are more effective than others in meeting the needs of their medicare enrollees. the ratings of individual plans encompass overall performance, provider performance, processes of and access to care. these data offer the opportunity to identify specific dimensions of health plan performance that are of concern and to develop mechanisms to encourage and assist these plans to improve. as an initial step in that direction, hcfa is currently providing detailed information on individual health plan ratings to medicare health plans, peer review organizations, (pros) and to hcfa staff with responsibilities for monitoring and oversight of medicare health plans. health plans receive a yearly detailed report of their mmc cahps scores on overall ratings and specific measures. in addition, to assist the health plans on how to evaluate their scores, state means of the mmc cahps scores are provided. this information can be used by each plan to assess areas of good performance and areas where members are indicating that they are less successful. ideally, health plans would use these data to identify problems and areas for improvement and would develop strategies that would result in improvements in areas where they are less successful in meeting the needs of medicare beneficiaries. since the mmc cahps is only in its third year (and medicare plans have received just two reports), there is little evidence to date about how much and in what ways the cahps information is encouraging health plans to change. pros receive an annual mmc cahps report that provides detailed information on the cahps ratings of each medicare health plan in its region. pros can use these data to identify problems in service to medicare beneficiaries enrolled in health plans, both in individual health plans and overall plans. identification of these problems could then provide a basis for developing targeted approaches for encouraging improvements in health plan performance. hcfa central and regional offices individual health plan ratings and summary ratings, by region, are provided to central and regional office staff who have responsibility for oversight and monitoring of the m+c program. the mmc cahps information, in combination with the knowledge that hcfa staff have about specific medicare health plans, offers the opportunity to assess and compare health plan performance and to identify potential problem areas in specific health plans. this information, in turn, could be used by hcfa staff to focus on particular organizational components and their operations during periodic site visits to medicare health plans, in order to evaluate the extent to which they are in compliance with hcfa requirements. additionally, hcfa staff could encourage these health plans to make changes that would improve performance, overall and by component. in addition to providing information of medicare health plan ratings to plans, pros, and internal staff, hcfa also is using mmc cahps data to better understand the relationships between medicare health plan cahps ratings and factors that affect these ratings (landon., results of these analyses may be used, in the longer term, to assist health plans, pros, and hcfa to develop strategies for better meeting the needs of all medicare beneficiaries enrolled in m+c plans. finally, hcfa is also supporting extensive research on differences in the health plan experiences of subgroups of the medicare population, including those with exceptional needs for health care (e.g., the medicare disabled under age 65, those in fair or poor health and/or with limited independence), racial and ethnic minorities (e.g., black persons, hispanics, and latinos), and medicare - medicaid dually eligible beneficiaries (cox and langwell, 2000). the purpose of these analyses is to gain insight into the performance of medicare health plans in meeting the needs of those beneficiaries who may have atypical health care needs or who may face cultural, linguistic, or other barriers to care. results may be used by hcfa to identify problems faced by beneficiaries who may require additional efforts by health plans to facilitate their effective use of services. health plans could then be encouraged to make changes that would improve access, service, and outcomes for all medicare enrollees. thus, its potential for use as a tool to assist health plans, hcfa, and pros to improve performance is still evolving. it is clear, however, that the mmc cahps could be an extremely useful means for identifying areas for medicare health plan improvement. evidence to date, suggests that hcfa and pros will use this information to design strategies for encouraging health plans to make changes in their organization and operations that will offer increasing value and service to their medicare enrollees. hcfa is moving toward value - based purchasing, which is a way to encourage and financially reward managed care organizations that provide better than average quality to m+c beneficiaries. mmc cahps, in combination with other indicators compiled by hcfa, will be used to more fully manage the m+c program and to reward plans that demonstrate high performance in serving and meeting the needs of the medicare population. to date, value - based purchasing has not been implemented by hcfa, but several possibilities are being explored. for example, hcfa could design an incentive rewards system that would offer m+c plans higher payments, reduced regulatory burden, or other rewards for maintaining high performance and for demonstrating significant improvements in key areas of quality and service. the mmc cahps offers the opportunity to design incentive rewards to improve health plan performance based on consistent, reliable, and continuously collected data. | the medicare+choice (m+c) program, created by the 1997 balanced budget act (bba), expands medicare 's health insurance options to include a wider range of health plan options. in this article, we describe the consumer assessment of health plans study (cahps) survey and its use with beneficiaries receiving care through medicare managed care (mmc) plans. we also discuss the implications of these efforts for future quality improvement efforts. |
a 24-year - old woman presented to our hospital with complaint of intermittent left hip joint catching pain and snapping sensation for 3 years. three years ago, she was admitted at our hospital with history of pedestrian traffic accident followed by fracture - dislocation of the left hip joint. after closed reduction, ct scans showed the fracture in anterior wall of the acetabulum. she underwent internal fixation for acetabular fracture using the fully threaded cannulated screw and was lost to follow - up after surgery. the plain anterior - posterior radiography showed asymmetry of the joint space and ct scans showed an intra - articular bony fragment on the left hip joints. acetabular osseo - labral fragment was widely detached from the superior to posterior acetabular rim and incarcerated in the joint space. we made standard three portals (anterior, anterolateral, posterolateral) and additional distal anterolateral portal (1 - 2 cm distal to anterolateral portal). we performed reduction of inverted labral tear and refixation using two 2.7 mm absorbable suture anchor (bioraptor ; smith & nephew, andover, ma, usa), and the bony fragment was removed. in addition, there were evidence of chronic cam type femoroacetabular impingement and osteochondral defect on femoral head. we performed femoral osteoplasty across the head - neck junction to protect secondary injury and micro - fracture in femoral head. after operation, concentric reduction and complete removal of bony fragment were confirmed using ct scans (fig. the visual analogue score (vas) for pain improved from 7 preoperatively to 3 postoperatively and 1 at 3 months follow - up. at 2 year follow - up, modified harris hip score (mhhs) was improved from 52 preoperatively to 92 postoperatively and hip outcome score of activity of daily living (hos adl) and sport related activity (hos sport) were improved from 58 and 56 preoperatively to 93 and 91 postoperatively, respectively. a 17-year - old man was referred to our hospital with a one year history of left hip joint pain, sometimes severe catching pain, and worsened with activity. physical examination revealed a hip pain when moving leg and slightly limited range of motion was observed due to pain. a year ago, the patient felt groin pain following hip fracture - dislocation cause to passenger traffic accident. in medical history, he was treated only the closed reduction of hip joint and rest in a private clinic. the plain anterior - posterior radiography showed asymmetry of the medial joint space and ct scans showed an intra - articular bony fragment on the hip joint. the patient underwent hip arthroscopy for bony fragment removal. in arthroscopic findings, acetabular osseo - labral fragment was widely detached from the superior to posterior acetabular rim and incarcerated in the joint space. we performed reposition of inverted labral tear and refixation using two 2.7 mm absorbable suture anchor (bioraptor) using standard 3 portals and additional distal anterolateral portal, and the bony fragment was removed. after operation, concentric reduction and complete removal of bony fragment were confirmed using ct scans (fig. mhhs was improved from 55 preoperatively to 93 postoperatively and hos adl and hos sport were improved from 59 and 62 preoperatively to 92 and 93 postoperatively, respectively. a 54-year - old woman presented to our hospital with complaints of right hip pain and limited range of motion. she was complaint that the pain type was different from before : she felt catching pain when moving the leg. two months ago, she was involved in traffic accident and sustained posterior dislocation of the right hip associated with a fracture of the inferior femoral - head. the hip was reduced spontaneously while the patient was being positioned for plain radiographs in local hospital. post - reduction plain radiographs showed that the femoral - head fragment was aligned satisfactorily however bony fragment and asymmetry joint space were found. in medical history, she was recommended bed rest in a local hospital because of femoral - head fracture. the patient underwent hip arthroscopy for bony fragments removal. in arthroscopic findings, acetabular osseo - labral fragment was widely detached from the superior to posterior acetabular rim and incarcerated in the joint space. we performed labral debridement and excision of bony fragments because of the labral tear was irreducible and bony fragments were small (bony deficit across the posterior acetabulum was less than 10%) (fig. 3). at 2 year follow up, the symptoms of the patient were improved but mild pain (vas 1) still remained. her mhhs was improved from 47 preoperatively to 85 postoperatively, and hos adl and hos sport were improved from 51 and 49 preoperatively to 85 and 87 postoperatively. a 24-year - old woman presented to our hospital with complaint of intermittent left hip joint catching pain and snapping sensation for 3 years. three years ago, she was admitted at our hospital with history of pedestrian traffic accident followed by fracture - dislocation of the left hip joint. after closed reduction, ct scans showed the fracture in anterior wall of the acetabulum. she underwent internal fixation for acetabular fracture using the fully threaded cannulated screw and was lost to follow - up after surgery. the plain anterior - posterior radiography showed asymmetry of the joint space and ct scans showed an intra - articular bony fragment on the left hip joints. acetabular osseo - labral fragment was widely detached from the superior to posterior acetabular rim and incarcerated in the joint space. we made standard three portals (anterior, anterolateral, posterolateral) and additional distal anterolateral portal (1 - 2 cm distal to anterolateral portal). we performed reduction of inverted labral tear and refixation using two 2.7 mm absorbable suture anchor (bioraptor ; smith & nephew, andover, ma, usa), and the bony fragment was removed. in addition, there were evidence of chronic cam type femoroacetabular impingement and osteochondral defect on femoral head. we performed femoral osteoplasty across the head - neck junction to protect secondary injury and micro - fracture in femoral head. after operation, concentric reduction and complete removal of bony fragment were confirmed using ct scans (fig. the visual analogue score (vas) for pain improved from 7 preoperatively to 3 postoperatively and 1 at 3 months follow - up. at 2 year follow - up, modified harris hip score (mhhs) was improved from 52 preoperatively to 92 postoperatively and hip outcome score of activity of daily living (hos adl) and sport related activity (hos sport) were improved from 58 and 56 preoperatively to 93 and 91 postoperatively, respectively. a 17-year - old man was referred to our hospital with a one year history of left hip joint pain, sometimes severe catching pain, and worsened with activity. physical examination revealed a hip pain when moving leg and slightly limited range of motion was observed due to pain. a year ago, the patient felt groin pain following hip fracture - dislocation cause to passenger traffic accident. in medical history, he was treated only the closed reduction of hip joint and rest in a private clinic. the plain anterior - posterior radiography showed asymmetry of the medial joint space and ct scans showed an intra - articular bony fragment on the hip joint., acetabular osseo - labral fragment was widely detached from the superior to posterior acetabular rim and incarcerated in the joint space. we performed reposition of inverted labral tear and refixation using two 2.7 mm absorbable suture anchor (bioraptor) using standard 3 portals and additional distal anterolateral portal, and the bony fragment was removed. after operation, concentric reduction and complete removal of bony fragment were confirmed using ct scans (fig. mhhs was improved from 55 preoperatively to 93 postoperatively and hos adl and hos sport were improved from 59 and 62 preoperatively to 92 and 93 postoperatively, respectively. a 54-year - old woman presented to our hospital with complaints of right hip pain and limited range of motion. she was complaint that the pain type was different from before : she felt catching pain when moving the leg. two months ago, she was involved in traffic accident and sustained posterior dislocation of the right hip associated with a fracture of the inferior femoral - head. the hip was reduced spontaneously while the patient was being positioned for plain radiographs in local hospital. post - reduction plain radiographs showed that the femoral - head fragment was aligned satisfactorily however bony fragment and asymmetry joint space were found. in medical history, she was recommended bed rest in a local hospital because of femoral - head fracture. in arthroscopic findings, acetabular osseo - labral fragment was widely detached from the superior to posterior acetabular rim and incarcerated in the joint space. we performed labral debridement and excision of bony fragments because of the labral tear was irreducible and bony fragments were small (bony deficit across the posterior acetabulum was less than 10%) (fig. 3). at 2 year follow up, the symptoms of the patient were improved but mild pain (vas 1) still remained. her mhhs was improved from 47 preoperatively to 85 postoperatively, and hos adl and hos sport were improved from 51 and 49 preoperatively to 85 and 87 postoperatively. a torn osseo - labral fragment rarely prevents consentric reduction of the hip in a traumatic dislocation and difficult to detect. kim.6) reported the mechanism of incarcerated acetabular labrum following reduction of traumatic hip dislocation : an acetabular rim fracture or detachment of the labrum from the acetabular rim results from the posteriorly dislocating femoral head ; when the hip is manually reduced, the torn labrum with or without the fractured fragment of the acetabular rim is pushed into the acetabulum, and it is trapped between the joints. and mullis and dahners5) found that 7 of 9 hip dislocation patients had loose bodies although no loose bodies were detected preoperatively and the joints were congruent on imaging. this illustrates that conventional radiographs and ct scans do not depict the extent of chondral or labral pathology associated with traumatic hip dislocation. it is important to obtain complete early reduction to prevent the rapid development of degenerative arthritis. even though the joint appears normal, we should thoroughly examine the inside of the joint for possible soft tissues incarceration. in our cases, we also missed the diagnosis radiologically at first time. however, the persistent patient 's unusual hip catching pain, despite the reduction, caused concern about the possible soft tissues incarceration. it is thus important to recognize that a difference in the width of the joint space in postreduction plain radiographs suggests soft tissues incarceration. conventional treatment of hip fractures involves opening the joint to perform reduction and fixation and excision of fracture fragments. recently, hip arthroscopy is used increasingly as a less invasive, safe and quickly able to regain mobility. nonconcentric hip reduction or intra - articular loose bodies are ideal indications for hip arthroscopy 457). hence, we also performed arthroscopic incarcerated fragment reduction and fixation and extraction in three cases. this three case reports emphasize the nonconcentric hip reduction due to osseo - labral incarceration that are not shown clearly on plain radiograph and importance of unusual pain types ; catching pain. we must consider that the hip arthroscopy is limited treatment but safe and effective as a subsequent procedure to treat incarcerated acetabular labrum following reduction of traumatic hip dislocation. it also constitutes for diagnosis of intra - articular pathologic lesions not evident on preoperative imaging studies. | traumatic hip fracture - dislocations are associated with chondral and labral pathology as well as loose bodies that can be incarcerated in the hip joint. incarceration, such as interposed labrum between acetabulum and femoral head that is not readily visualized preoperatively, is a rare but important cause of pain and can potentially be a source for early degeneration and progression to osteoarthritis. we present three cases, arthroscopic surgery of incarcerated acetabular osseo - labral fragment following reduction of traumatic hip fracture - dislocation. |
primary aldosteronism (pa) is characterized by the overproduction of the mineralocorticoid hormone aldosterone by the adrenal gland, a condition that is relatively autonomous of the renin - angiotensin system (ras), and nonsuppressible by sodium loading. the elevated circulating aldosterone levels lead to potassium loss, hypernatremia, metabolic alkalosis, and hypertension. the syndrome can be the result of bilateral hyperplasia of the adrenal glands, unilateral adrenal hyperplasia, aldosterone producing adrenal adenoma, or in rare cases, by glucocorticoid - remediable aldosteronism (gra). early epidemiologic studies have determined the prevalence of pa to be 180/100 mmhg), the prevalence of pa was 13.2%, as opposed to 1.99% in patients with stage i hypertension (systolic bp [sbp ] 140159 mmhg, diastolic bp [dbp ] 9099 mmhg). further, patients with pa had a higher bp (164/102 12/10 mmhg) than patients without pa (156/96 16/9 mmhg, p 166/101 mmhg), the prevalence of pa was 19%. in this study, screening for pa was undertaken using the arr and confirmed by non- or mild suppression of plasma aldosterone levels after saline infusion, and the elevated bp levels in the clinic were confirmed by 24-hour abpm. pa is particularly common in patients with resistant hypertension (rhtn), defined as bp that remains above goal in spite of use of at least 3 antihypertensive medications, ideally prescribed at optimal doses and one of which is a diuretic. in a large retrospective study involving 1,616 patients with rhtn who were referred to a specialty clinic in greece, the prevalence of pa (screened for using the aldosterone - to - renin ratio [arr ] and confirmed by intravenous saline loading or fludrocortisone suppression and further confirmed by assessing response to spironolactone monotherapy) was determined to be 11.3%. prospective studies have found an even higher prevalence of pa in patients with rhtn. among 88 patients with rhtn who were consecutively referred to the hypertension clinic of the university of alabama at birmingham (uab), 18 patients (20%) were confirmed to have pa, based on a high 24-hour urinary aldosterone excretion (> 12 g/24 hr) paired with a suppressed plasma renin activity level (200 meq/24 hr). this high prevalence of pa in patients with moderate to severe hypertension has been confirmed in other prospective studies done elsewhere. investigators from oslo, norway found a pa prevalence of 21% of patients with rhtn. in a university - based hypertension clinic in seattle, washington, the pa prevalence was 17% in patients with severe and/or poorly controlled hypertension (bp > 140/90 mmhg). lastly, in a study done on 100 diabetic patients with rhtn, pa was confirmed in 14% of the study patients, using oral or intravenous saline loading. overall, these studies show that pa prevalence is higher (1421%) in patients with moderate to severe hypertension on multiple antihypertensive medications, compared to the general hypertensive population. high aldosterone levels lead to increased sodium and water reabsorption (and consequently potassium loss) in the distal nephron, acting via mineralocorticoid receptors (mrs) that regulate gene transcription. aldosterone also exerts rapid, nongenomic cellular effects on mrs found in nonepithelial tissue, thereby influencing cell volume, oxidation - reduction state, and vascular function. these effects are linked to the development of vascular stiffness and fibrosis, particularly in large arteries, the heart, and the kidney. taken together, these effects act in concert to raise bp and contribute to target organ damage in hypertensive individuals with aldosterone excess and may partly explain treatment resistance in those with more severe hypertension. aldosterone excess has been linked to the development and progression of several cardiovascular diseases, notably hypertension, congestive heart failure, coronary artery disease, chronic kidney disease, and stroke. in the framingham offspring study, higher baseline serum aldosterone levels were associated with an increased risk of bp elevation or development of hypertension after four years in 1688 normotensive individuals (mean age 55 years, 58% women). using multivariate analysis, there was a 16% increased risk of an elevation in bp (p =.002) and a 17% increased risk of development of hypertension per quartile increment in the serum aldosterone level, and the highest quartile of serum aldosterone, compared to the lowest quartile, was associated with a 1.61-fold risk of development of hypertension (95% ci 1.05 to 2.46). beyond its effects on bp, the presence of pa is associated with increased carotid intima thickness, higher pulse wave velocity (a marker of increased arterial stiffness), and impaired flow - mediated brachial artery dilation (which is an indicator of endothelial dysfunction) compared to hypertensive patients without pa [19, 20 ]. left ventricular wall thickness is higher, and there is greater incidence of diastolic dysfunction as measured by tissue doppler imaging in patients with pa compared to matched hypertensive controls. in the kidney, apart from its effects on renal sodium handling, excess aldosterone induces early kidney damage. patients with pa have a higher urinary albumin excretion rate compared to matched hypertensive controls, with a preserved glomerular filtration rate (which is an early manifestation of kidney disease). comparing hypertensive patients with pa to those without biochemical evidence of aldosterone excess, the former had a greater incidence of stroke (both hemorrhagic and ischemic, odds ratio [or ] 4.2, 95% confidence interval [ci ] 2.0 to 8.6, p 10 events / hour) after overnight polysomnography (psg), and osa was more prevalent (96 versus 65%, p =.014) and more severe (mean ahi 32 versus 14, p = 0004) in men than in women. these results were confirmed in our study involving 71 patients with rhtn, where the prevalence of osa was determined to be 85% and is also more common and more severe in men than in women. increasing severity of osa also is associated with difficulty to control hypertension. in 257 patients with osa who are adherent to a stable antihypertensive regimen for 6 months, those who were ineffectively treated (bp > 140/90 mmhg) had a higher ahi (44 events / hr) compared to those with controlled bp (33 events / hr, p 5.5 meq / l). in patients given eplerenone, the serum potassium increased by a mean of 0.30 0.45 meq / l after initiation of eplerenone treatment, and mild hyperkalemia (serum potassium 5.5 to 6 meq / l) was seen in only two patients (out of 52, or 3.8%), despite being on an ace inhibitor or an arb concomitantly. beyond bp lowering, treatment with mr antagonists also reverses or attenuates cardiovascular injury mediated by excess aldosterone, particularly the nongenomic effects, which lead to tissue fibrosis, arterial stiffness, and increased oxidative stress. in patients with rhtn (with or without pa), spironolactone (at an initial dose of 25 mg / day then forcetitrated to 50 mg / day after 4 weeks) reduced left ventricular mass index at the 3rd and 6th month followup. the degree of lv regression achieved with spironolactone treatment was greater for patients with pa compared to those without pa (22 versus 12%, resp. further, spironolactone given to patients with both rhtn and pa for 6 months significantly decreased brain natriuretic peptide values from baseline, an effect that was not seen in those with normal or low aldosterone levels, indicating a prominent diuretic effect even when administered on top of chronic thiazide diuretic treatment. in another study involving patients with rhtn and pa, treatment with low - dose spironolactone (12.5 to 25 mg / day) for 3 months significantly increased flow - mediated dilation of the brachial artery, indicating improvement of endothelial function, and this effect was independent of the change in bp. lastly, spironolactone reduced the severity of osa in patients with rhtn. in a prospective evaluation involving 12 patients with rhtn in whom spironolactone (2550 mg / day) was added to a stable antihypertensive regimen, there were significant reductions in the ahi (39.8 19.5 versus 22 6.8 events / hour, p <.05), hypoxic index (13.6 10.8 versus 6.7 6.6 events / hour, p <.05), and clinic and 24-hour ambulatory bp on the 8th week followup, and plasma renin activity was increased (figure 2). although the study was small and did not have an active control group, this study lends support to the concept that aldosterone excess contributes to the severity of osa in patients with rhtn. aldosterone excess is believed to contribute significantly to uncontrolled bp in patients with rhtn, as shown by a higher prevalence of pa in patients with rhtn and a significant bp - lowering effect produced by mr antagonists, particularly spironolactone, when added to a multidrug antihypertensive regimen that typically already includes a diuretic and an ras blocker. this effect was seen in both patients with and without pa, showing that even in those patients without elevated aldosterone levels (by current laboratory standards), there is some element of relative aldosterone excess that contributes to the raised bp. further, treatment with mr antagonists produces cardiovascular benefits beyond bp lowering (including reduction of the severity of osa), showing that aldosterone also plays a direct role in the development of target organ damage, arterial stiffness, and endothelial dysfunction, mostly mediated via nongenomic mechanisms. | resistant hypertension (rhtn) is defined as blood pressure (bp) that remains uncontrolled in spite of intake of 3 antihypertensive medications, ideally prescribed at optimal doses and one of which is a diuretic. the incidence of primary aldosteronism (pa) in patients with rhtn is estimated in prospective studies to be 14 to 23%, which is higher than in the general hypertensive population. patients with pa are at an increased cardiovascular risk, as shown by higher rates of stroke, myocardial infarction, and arrhythmias compared to hypertensive individuals without pa. likewise, rhtn is associated with adverse cardiovascular outcomes, and the contribution of pa to this increased risk is undetermined. similar to pa, obstructive sleep apnea (osa) is closely associated with rhtn, and a causal link between pa, osa, and rhtn remains to be elucidated. the addition of mr antagonists to the antihypertensive regimen in patients with rhtn produces a profound bp - lowering effect, and this effect is seen in patients with or without biochemical evidence of pa, highlighting the role of relative aldosterone excess in driving treatment resistance in this group of patients. |
hypocalcemia, a common complication after thyroidectomy, results in prolongation of hospitalization and patient discomfort. it occurs transiently in up to 30%, and permanently in up to 4% of patients after this procedure.1 traditionally, predicting this common complication has been accomplished by following calcium trends every 612 hours for 2448 hours, after which levels generally plateau.2,3 since 2002, numerous studies have shown that the parathyroid hormone (pth) assay, checked minutes to hours after completing surgery, is excellent in predicting those patients who will become hypocalcemic.418 multiple algorithms detailing clinically - practical and cost - effective means to use pth assay after thyroid surgery have been documented. a recent meta - analysis of studies published from 2002 through 2007 addressed different methods.2 the authors concluded that obtaining a pth level 1 to 6 hours following total or completion thyroidectomy has excellent accuracy in determining who will become hypocalcemic. while there have been many positive studies finding utility in post - operative pth assays, a few reports have found pth level is not able to accurately predict post - thyroidectomy hypocalcemia.19,20 anecdotally, there appears to be considerable variation in practice regarding routine pth assays among surgeons performing this procedure. in light of these practice variations, this survey study was undertaken to document current us and european practice patterns regarding the use of this assay, particularly in terms of how surgeon demographics are associated with use of the assay and whether use of the assay is related to reported length of hospitalization. approval for this study was obtained from the institutional review board at walter reed army medical center. the survey outlined in table 1 was sent to 10,618 members of the american academy of otolaryngology - head and neck surgery (aao - hns) and the american association of endocrine surgeons (aaes). these surveys were distributed via email and paper copy to aaes and aao - hns surgeons twice from march 2009 thru november 2010. to ascertain the practice patterns of european physicians, the same survey (in english) was sent via online newsletter format to the 600 members of the european, italian, french, spanish and british societies of endocrine surgery in 2010 and 2011. settings were established electronically to exclude storage of ip addresses and any potential personal identifying information. survey responses were organized in database format and analyzed using sas 9.1, sas institute, cary, nc. individuals age 2030, rate of permanent hypocalcemia > 6%, and rate of autotransplantation > 75% all had n = 4 respondents and were collapsed into the closest category. for basic analyses, chi - square tests were used unless predicted values dictated use of fisher s exact test. for the adjusted analyses, logistic regression was used with pth assay usage, and rate of temporary / permanent hypocalcemia (ordered multinomial response model). discharge time was also modeled using ordered logistic regression. however, without a priori assumptions of expected contributors, automated backward selection was used with a staying criterion of an alpha of 0.10 to determine factors predictive of inpatient duration. of the 600 european physicians surveyed, 61 returned the survey, corresponding to a response rate of 10.1%. among the american physicians, most respondents had completed otolaryngology residency training programs without head and neck fellowship and were employed by either a group practice or academic institution. european physicians in the survey predominately represented general surgeons with a mixture of training levels and were based at academic institutions. american respondents reported a wide range of experience in thyroid surgery with 22% reporting 100/yr. in contrast, 70% of european physicians reported > 100 thyroidectomies per year, with the other frequencies each ~10%. routine pth assay use, whether alone or in conjunction with routine calcium monitoring, was reported by 105 (29.8%) american and 25 (41%) of european respondents. in the unadjusted analyses, assay use was correlated with sex (higher in men), years in practice (lower use in older physicians), fellowship training (higher use in fellowship - trained) and an academic practice location (higher use in academic settings). the same relationships held true in the adjusted analysis, except for academic location, which was no longer significant after controlling for the other factors. the most common usage was 1 hour following total thyroidectomy in the post - anesthesia care unit (35.5%), followed by the next day (27.5%). there was no consensus among survey respondents regarding reasons for not using the pth assay. respondents cited lack of change in management (33.8%) and unavailability of assay (24.6%) as the most common reasons for not utilizing the assay. the survey also queried self - reported estimates of the rates of temporary and permanent hypocalcemia. in the unadjusted analysis, lower self - reported temporary hypocalcemia rate was associated with a higher number of thyroidectomies performed, general surgery residency, academic practice and european physicians, but not sex, number of years in practice, or fellowship training., the available information did not significantly predict the rate of reported temporary hypocalcemia (overall p > 0.05), and therefore no individual characteristics were reported. in both the adjusted and unadjusted analyses, a lower rate of self - reported permanent hypocalcemia rate was associated with number of thyroidectomies performed annually, with an increase in rate as the number of surgeries per year increases (odds ratio = 1.256, p = 0.0451 adjusted), and with academic practice location (odds ratio = 2.589, p = 0.0010 adjusted). nationality, residency type and fellowship training were significant in the unadjusted analysis only, and years in independent practice became significant only after adjustment. routine prophylactic use of calcium supplementation regardless of calcium status for post - thyroidectomy patients was split, with 42.5% of respondents using prophylactic calcium and 57.5% of responders not using prophylactic calcium. finally, the survey explored self - reported postoperative hospitalizations. the most common time of discharge was the morning after surgery (see table 6). ordered logistic regression with backwards selection was used to evaluate for factors correlated with length of stay. shorter average post - operative hospital stay for thyroidectomy patients was associated with american physicians (or = 0.333, p = 0.0001) and the opinion that holding patients until calcium levels plateau is not necessary (or = 0.218, p 0.10). it is well established that one of the most common complications of thyroid surgery is the development of hypocalcemia. the risk of hypoparathyroidism increases in reoperative thyroid surgery and when central neck dissection is associated,21,22 decreases with the use of harmonic scalpel.23 traditionally, monitoring and managing this complication is based on patient symptoms and serum calcium levels. parathyroid hormone is widely used in clinical practice in diagnosing primary and secondary hyperparathyroidism.2426 within the last decade, the use of parathyroid hormone assay has gained increasing use as a reliable tool to predict postoperative hypocalcemia within hours of total or complete thyroidectomy.114 it was determined through a polling of the audience during an instructional course given at the 2008 american academy of otolaryngology head and neck surgery annual meeting that only about 4.5% of the surgeons in the audience used the pth assay postoperatively. thus, it was surmised that the popularity or knowledge of the pth assay might be less than expected. a formal survey was created to elaborate on the questions posed to the audience at the annual meeting with the objective of exploring demographic factors related to assay use as well as determining any trends in post - thyroidectomy hospitalization. based on the results of our combined survey, we determined that a more accurate value of pth assay utilization (alone or in combination with calcium trends) approximates 30% in the us and 40% in europe. furthermore, the provider responses indicate a significant relationship between type of surgical training and likelihood of utilizing the pth assay. it was evident that individuals with fellowship training in head and neck surgery or endocrine surgery were more likely to use the pth assay as part of their routine post - thyroidectomy monitoring. the reason for this increased pth assay utilization by fellowship - trained individuals was not specifically explored with this survey. however, multiple potential explanations for this finding may be advanced. first, fellowship trained surgeons are more likely complete their training at larger tertiary care facilities where the assay is more readily available, thus they become accustomed to using the assay and ultimately make it part of their routine plan of care. alternatively, these individuals may be more apt to practice at a tertiary care facility once completing their fellowship training, thus maintaining the availability of the assay for their use. arguing against this second hypothesis is the finding that after adjusting for other factors (including fellowship training), current practice location was not a significant predictor of pth assay use. aside from these demographic trends and assay utilization, the questions posed in this survey were aimed at determining if any factors could be identified associated with post - thyroidectomy hospitalization protocols. it was determined that practice location, use of the pth assay, nationality, and routine calcium supplementation are significantly associated with self - reported post - operative hospitalization. the relationship between the longer self - reported hospitalizations associated with pth assay use is an unexpected finding. previous studies have explored the association of shorter hospitalization and cost savings with the use of the pth assay. in particular, payne described a protocol in which 1 and 6- hour post - thyroidectomy monitoring of pth and corrected calcium have led to fewer blood tests, a 10-hour reduction in length of hospital stay and ultimately a cost savings of $ 766 per patient.27 a similar study published by cote explored using the pth assay alone as a means for cost savings at their institution.17 they determined that a 1-hour pth cutoff of < 15 ng / l should serve as an indication for prophylactic calcium supplementation. per their report, this resulted in an approximate 20% reduction in hospitalization duration that resulted in cost savings for their institution. given this background, we speculate that our findings of longer hospitalization are more appropriately interpreted in the reverse physicians with longer length of stay (los) are starting to turn to the assay in an attempt to better identify patients who can be discharged safely at an earlier time point. other factors not explored in this survey, such as complexity of surgery performed, particularly in benign vs. neoplastic disease, may also account for some of this difference. when interpreting the results of this survey, we acknowledge the inherent weaknesses of this investigation. the study design itself opens itself to bias, as all data is self - reported. we did not prevent repeated completion of the survey by each respondent ; however we doubt that many surgeons would be likely to complete the survey more than once. the response rate of 3.35% among the american physicians was low, and the european physicians were polled within a limited group, which restricts our ability to generalize our findings. these low response rates are probably results of time constraints experienced by most surgeons as well as the large number of survey studies that are received by most physicians. additionally, our attempts to measure some potential confounding factors probably omitted some contributing factors. unmeasured confounders may account for some of the relationships or lack thereof seen in this data. despite the low response rate, the results of this surgeon survey provides valuable information suggesting interesting relationships between training type and propensity for pth assay utilization, as well as post - operative hospitalization patterns related not only to training type but to pth assay utilization and post - operative calcium supplementation. these results set the stage for considering further studies aimed at considering if and how demographic and training factors, specifically training type as identified in this survey study, impact the use of the assay and ultimately change standards of care. this survey also illuminates the large degree of practice variation in this area, suggesting either that best practices have not yet been determined, or inefficiency in their adoption among surgeons performing this procedure. approximately one - third of thyroid surgeons surveyed routinely obtain pth assays for patients undergoing thyroidectomy, and the use of the assay is associated with sex, fellowship completion, and years in practice. | introductionhypocalcemia after thyroidectomy results in prolongation of hospitalization and patient discomfort but can be predicted by pth assays. however, there is considerable variation in their use.methodsthis study was undertaken to document current us and european practice patterns regarding the use of this assay. anonymous surveys were collected in 20092011 from members of the american academies of otolaryngology - head and neck surgery and endocrine surgery and the european, italian, french, spanish and british societies of endocrine surgery.resultsthere were 356 american (3% response) and 61 european (10% response) respondents. 105 (29.8%) american and 25 (41%) european respondents reported routine pth assay use. fellowship trained surgeons reported increased use of the pth assay (p = 0.004). shorter reported average post - operative hospital stay was associated with american physicians (p = 0.0001), community practice location (p = 0.0002) and routine calcium supplementation (p = 0.0015).conclusionssurgical training was associated with routine use of the pth assay. average reported hospital stay was lower for american and community practice physicians and correlated with post - operative oral calcium use. |
the risk of congenital anomalies is increased in infants of diabetic mothers (idm), and is estimated to be between 2.5 to 12%, with an over - representation of congenital heart defects.12 the incidence of malformations is the highest in the group where mothers were on insulin at the time of conception.1 idms often develop respiratory problems which need to be differentiated from the cardiovascular problems they are prone to have (structural congenital heart defect and hypertrophic cardiomyopathy) and from cardiovascular maladaptation to extra - uterine life which they may also suffer.34 congenital heart disease occurs in 5% of idm. the highest relative risk for major cardiovascular defects occurs if the mother has gestational diabetes and develops insulin resistance in the 3 trimester. it is thought that maternal diabetes, via its effects on maternal metabolism, is responsible for the increase of malformations in the offspring.5 however, using hemoglobin a1c values as an indicator of maternal diabetic control, other studies have shown that congenital heart disease in the fetus is not significantly related to maternal diabetic control.6 the most frequent cardiac anomalies in idms include ventricular septal defect, transposition of great arteries and aortic stenosis. defects involving the great arteries, including truncus arteriosus and double outlet right ventricle, are also more prevalent in idms.78 the clinical presentation, diagnostic approach and therapy depend on the type of structural heart disease present, and are no different from when they occur in neonates born to non - diabetic mothers. in idm with respiratory distress, the right ventricular pre - ejection period to ventricular ejection time ratio is elevated, suggesting an abnormality of the transitional pulmonary circulation.3 the closure of the ductus arteriosus and postnatal decrease in pulmonary artery pressure are also delayed in these neonates.4 this may partially explain the frequent occurrence of respiratory problems and the slowness of the recovery in such infants. primary pulmonary hypertension may be associated with and aggravated by the polycythemia which is also frequently present in these neonates. 3.1 while symptomatic hypertrophic cardiomyopathy (hc) occurs in 12.1% of idm, when routinely searched for with an echocardiographic scan it is found in 30%.9 the left ventricular mass and contractility are increased and there is left ventricular outflow tract (lvot) obstruction with apposition of the anterior leaflet of the mitral valve to the interventricular septum during systole. cardiac output is significantly reduced, secondary to reduced stroke volume and is directly related to the degree of septal hypertrophy.10 this asymmetric septal enlargement, with a disproportionally hypertrophic septum, is an anabolic result of fetal hyperinsulinemia triggered by maternal hyperglycemia during the third trimester. cardiac septum hypertrophy correlates with maternal glycosylated haemoglobin levels and high levels of fetal insulin better than with macrosomia (fig. 1). large hypotonic infant of diabetic mother, lying in a frog - like position, with some bruising of the left arm due to shoulder dystocia 3.2 the severity of idm cardiomyopathy can vary from an incidental finding on echocardiography (30% of cases) to an infant with severe symptoms of congestive heart failure (12% of cases).11 hc is usually benign and consists of systolic murmur and transitory cardiomegaly. sometimes, overt congestive heart failure develops, with tachypnea, tachycardia, gallop rhythm and hepatomegaly. 3.3 cardiomegaly is invariably present radiologically when hc is symptomatic, with pulmonary congestion (fig. chest x - ray showing significant cardiomegaly and pulmonary venous congestion as a result of hypertrophic cardiomyopathy in an infant of diabetic mother echocardiography is essential for the diagnosis of hc, it will also assess the left ventricular function, the degree of lvot obstruction and it will also rule out structural cardiac malformations. the left ventricular mass is significantly greater, with the interventricular septum being significantly thicker (mean (sd) 4.77 (1.4) mm) when compared with those born to non - diabetic mothers (2.5 (0.7) mm).14 two - dimensional echocardiogram showing septal hypertrophy which partially obstructs the left ventricular outflow tract in hypertrophic cardiomyopathy 3.4 the natural history of hc is that of spontaneous regression of symptoms and septal hypertrophy.15 most of the infants need only supportive care, with fluid restriction, diuretics and oxygen. if pharmacologic intervention is deemed necessary, beta adrenergic blocking agents such as propranolol, are the drugs of choice, as they reduces heart rate, left ventricular contractility and wall stress, with total relief of symptoms obtained with standard doses of propranolol in 30% of affected infants.1516 3.5 the natural history of hc is benign, with resolution of symptoms within two to four weeks and resolution of septal hypertrophy during the first 2 to 12 months of life, irrespective of therapy.1415 early or progressive hypertrophy of the lv posterior wall is a predictor of poor outcome, regardless of associated disease.17 the highest relative risk for major cardiovascular defects occurs if the mother has gestational diabetes and develops insulin resistance in the 3 trimester. it is thought that maternal diabetes, via its effects on maternal metabolism, is responsible for the increase of malformations in the offspring.5 however, using hemoglobin a1c values as an indicator of maternal diabetic control, other studies have shown that congenital heart disease in the fetus is not significantly related to maternal diabetic control.6 the most frequent cardiac anomalies in idms include ventricular septal defect, transposition of great arteries and aortic stenosis. defects involving the great arteries, including truncus arteriosus and double outlet right ventricle, are also more prevalent in idms.78 the clinical presentation, diagnostic approach and therapy depend on the type of structural heart disease present, and are no different from when they occur in neonates born to non - diabetic mothers. in idm with respiratory distress, the right ventricular pre - ejection period to ventricular ejection time ratio is elevated, suggesting an abnormality of the transitional pulmonary circulation.3 the closure of the ductus arteriosus and postnatal decrease in pulmonary artery pressure are also delayed in these neonates.4 this may partially explain the frequent occurrence of respiratory problems and the slowness of the recovery in such infants. primary pulmonary hypertension may be associated with and aggravated by the polycythemia which is also frequently present in these neonates. 3.1 while symptomatic hypertrophic cardiomyopathy (hc) occurs in 12.1% of idm, when routinely searched for with an echocardiographic scan it is found in 30%.9 the left ventricular mass and contractility are increased and there is left ventricular outflow tract (lvot) obstruction with apposition of the anterior leaflet of the mitral valve to the interventricular septum during systole. cardiac output is significantly reduced, secondary to reduced stroke volume and is directly related to the degree of septal hypertrophy.10 this asymmetric septal enlargement, with a disproportionally hypertrophic septum, is an anabolic result of fetal hyperinsulinemia triggered by maternal hyperglycemia during the third trimester. cardiac septum hypertrophy correlates with maternal glycosylated haemoglobin levels and high levels of fetal insulin better than with macrosomia (fig. 1). large hypotonic infant of diabetic mother, lying in a frog - like position, with some bruising of the left arm due to shoulder dystocia 3.2 the severity of idm cardiomyopathy can vary from an incidental finding on echocardiography (30% of cases) to an infant with severe symptoms of congestive heart failure (12% of cases).11 hc is usually benign and consists of systolic murmur and transitory cardiomegaly. all symptoms usually spontaneously regress within a few weeks. sometimes, overt congestive heart failure develops, with tachypnea, tachycardia, gallop rhythm and hepatomegaly. 3.3 cardiomegaly is invariably present radiologically when hc is symptomatic, with pulmonary congestion (fig. chest x - ray showing significant cardiomegaly and pulmonary venous congestion as a result of hypertrophic cardiomyopathy in an infant of diabetic mother echocardiography is essential for the diagnosis of hc, it will also assess the left ventricular function, the degree of lvot obstruction and it will also rule out structural cardiac malformations. the left ventricular mass is significantly greater, with the interventricular septum being significantly thicker (mean (sd) 4.77 (1.4) mm) when compared with those born to non - diabetic mothers (2.5 (0.7) mm).14 two - dimensional echocardiogram showing septal hypertrophy which partially obstructs the left ventricular outflow tract in hypertrophic cardiomyopathy 3.4 the natural history of hc is that of spontaneous regression of symptoms and septal hypertrophy.15 most of the infants need only supportive care, with fluid restriction, diuretics and oxygen. if pharmacologic intervention is deemed necessary, beta adrenergic blocking agents such as propranolol, are the drugs of choice, as they reduces heart rate, left ventricular contractility and wall stress, with total relief of symptoms obtained with standard doses of propranolol in 30% of affected infants.1516 3.5 the natural history of hc is benign, with resolution of symptoms within two to four weeks and resolution of septal hypertrophy during the first 2 to 12 months of life, irrespective of therapy.1415 early or progressive hypertrophy of the lv posterior wall is a predictor of poor outcome, regardless of associated disease.17 the highest relative risk for major cardiovascular defects occurs if the mother has gestational diabetes and develops insulin resistance in the 3 trimester. it is thought that maternal diabetes, via its effects on maternal metabolism, is responsible for the increase of malformations in the offspring.5 however, using hemoglobin a1c values as an indicator of maternal diabetic control, other studies have shown that congenital heart disease in the fetus is not significantly related to maternal diabetic control.6 the most frequent cardiac anomalies in idms include ventricular septal defect, transposition of great arteries and aortic stenosis. defects involving the great arteries, including truncus arteriosus and double outlet right ventricle, are also more prevalent in idms.78 the clinical presentation, diagnostic approach and therapy depend on the type of structural heart disease present, and are no different from when they occur in neonates born to non - diabetic mothers. in idm with respiratory distress, the right ventricular pre - ejection period to ventricular ejection time ratio is elevated, suggesting an abnormality of the transitional pulmonary circulation.3 the closure of the ductus arteriosus and postnatal decrease in pulmonary artery pressure are also delayed in these neonates.4 this may partially explain the frequent occurrence of respiratory problems and the slowness of the recovery in such infants. primary pulmonary hypertension may be associated with and aggravated by the polycythemia which is also frequently present in these neonates. 3.1 while symptomatic hypertrophic cardiomyopathy (hc) occurs in 12.1% of idm, when routinely searched for with an echocardiographic scan it is found in 30%.9 the left ventricular mass and contractility are increased and there is left ventricular outflow tract (lvot) obstruction with apposition of the anterior leaflet of the mitral valve to the interventricular septum during systole. cardiac output is significantly reduced, secondary to reduced stroke volume and is directly related to the degree of septal hypertrophy.10 this asymmetric septal enlargement, with a disproportionally hypertrophic septum, is an anabolic result of fetal hyperinsulinemia triggered by maternal hyperglycemia during the third trimester. cardiac septum hypertrophy correlates with maternal glycosylated haemoglobin levels and high levels of fetal insulin better than with macrosomia (fig. 1). large hypotonic infant of diabetic mother, lying in a frog - like position, with some bruising of the left arm due to shoulder dystocia 3.2 the severity of idm cardiomyopathy can vary from an incidental finding on echocardiography (30% of cases) to an infant with severe symptoms of congestive heart failure (12% of cases).11 hc is usually benign and consists of systolic murmur and transitory cardiomegaly. all symptoms usually spontaneously regress within a few weeks. sometimes, overt congestive heart failure develops, with tachypnea, tachycardia, gallop rhythm and hepatomegaly. 3.3 cardiomegaly is invariably present radiologically when hc is symptomatic, with pulmonary congestion (fig. chest x - ray showing significant cardiomegaly and pulmonary venous congestion as a result of hypertrophic cardiomyopathy in an infant of diabetic mother echocardiography is essential for the diagnosis of hc, it will also assess the left ventricular function, the degree of lvot obstruction and it will also rule out structural cardiac malformations. the left ventricular mass is significantly greater, with the interventricular septum being significantly thicker (mean (sd) 4.77 (1.4) mm) when compared with those born to non - diabetic mothers (2.5 (0.7) mm).14 two - dimensional echocardiogram showing septal hypertrophy which partially obstructs the left ventricular outflow tract in hypertrophic cardiomyopathy 3.4 the natural history of hc is that of spontaneous regression of symptoms and septal hypertrophy.15 most of the infants need only supportive care, with fluid restriction, diuretics and oxygen. if pharmacologic intervention is deemed necessary, beta adrenergic blocking agents such as propranolol, are the drugs of choice, as they reduces heart rate, left ventricular contractility and wall stress, with total relief of symptoms obtained with standard doses of propranolol in 30% of affected infants.1516 3.5 the natural history of hc is benign, with resolution of symptoms within two to four weeks and resolution of septal hypertrophy during the first 2 to 12 months of life, irrespective of therapy.1415 early or progressive hypertrophy of the lv posterior wall is a predictor of poor outcome, regardless of associated disease.17 with congenital heart disease occuring in up to 5% of fetuses of diabetic mothers, and with 90% of the cardiac lesions identifiable prenatally, it has been suggested that detailed fetal echocardiography is offered to all diabetic women during pregnancy.1822 however, when studied in relation to maternal initial hba1c, the overall sensitivity for identifying congenital heart disease was 50% and specificity 54% and no critical level of hba1c that provided optimal predictive power for congenital heart disease screening was identified.23 it has also been suggested that the fetal cardiac septum thickness should be measured in utero by sonocardiography in all diabetic pregnancies.24 careful management of diabetes in pregnancy may reduce the severity of hypertrophic cardiomyopathy.1125 however, other studies found no relationships between the echocardiographic results and the metabolic control of pregnancy or fetal characteristics, suggesting that strict maternal diabetes control may not prevent accelerated fetal cardiac growth and abnormal development of cardiac function.2627 | congenital anomalies occur more commonly in infants born to diabetic mothers, and cardiac defects predominate. although respiratory problems are also frequently found in those infants, they need to be differentiated from cardiovascular problems that such patients may also have, which include cardiovascular maladaptation to extra - uterine life, congenital heart defects and hypertrophic septal cardiomyopathy. a high index of suspicion is required as the specific management may vary and digoxin, or inotropic agents which may be used in heart failure associated with structural heart defects are contraindicated if hypertrophic cardiomyopathy is present. this article reviews the epidemiology, pathophysiology, clinical presentation, prognosis and available diagnostic and therapeutic modalities. the need for antenatal fetal echocardiography in pregnant diabetic mothers is also reviewed, as well as the controversial role of maternal glycemic control in the prevention of these anomalies. |
municipal solid waste workers (mswws) or refuse collectors, universally expose to many work related health hazards and safety risks, notably allergic and other diseases of the respiratory system. health impacts could also entail musculoskeletal, gastro - intestinal and infectious diseases as well as injuries caused by work - related accidents. communal wastes comprise organic dust and bio - aerosol stuffed with micro - organisms (bacteria, viruses and fungi), endotoxins, and various toxic organic and inorganic chemicals that are aggravated in leachate resulting from a sanitary landfill. however, control of these work conditions and enforcement of appropriate hygienic measures are difficult due to the lack of hygiene standards for biohazardous materials present in the air of the workplace. workers can be protected by employing safety procedures in the workplace and ensuring the use of adequate personal protective equipment. health impact and morbidity data about occupational exposure to solid waste among mswws is scarce. no studies have been conducted thus far in egypt regarding exposure and health effects among workers engaged in solid waste collection, processing and disposal. according to the information available, this work provides a description of the health impact of communal solid waste management among hundreds of employees in the main municipality company in alexandria - egypt in order to estimate the potential excess in risks to which mswws can be exposed and adverse health outcomes and to consequently call for corrective measures, occupational safety and health hazard evaluation and monitoring program using health risk reduction behaviors models. a cross - sectional study was conducted between january and april 2013, among mswws in the main municipality company in alexandria (egypt). this company was established in 2001 to deal with all types of solid waste [figure 1 ] generated from different sources ; residential, shops, industrial, agricultural, and hospitals medical waste in all municipal districts of alexandria. its total area is about 14 square kilometers comprising three waste storage station, three sorting and recycling plants, one factory for fertilizer production and one landfill site. the daily solid waste weight disposed at each discharge site is from 3 to 3.5 thousand tons. the company comprises a large laboring force of about 6000 permanent workers serving in several sectors concerned with waste collection, transportation, sorting, recycling, treatment, incineration, landfill and fertilizer production. health hazards encountered by mswws in the collection and sorting of waste and recyclables the municipality was contacted and asked to participate in the study by a document describing the purpose and concerns of the study. the study comprised all current workers who were employed for 1 year or more by the municipality. we categorized the participants into two occupational groups (exposed ; n = 186 and non - exposed ; n = 160) on the basis of the direct exposure to solid waste. the exposure group included those working in the collection of mixed domestic and communal waste, street sweepers, workers engaged in loading, transfer and evacuation, sorting, incineration, dumping, landfilling, bin / scoop washing, and fertilizer production. group 2 included transportation workers (vehicle / truck drivers), technical workers (mechanics, car and equipment maintenance, heavy equipment operators), welders, inspectors, timekeepers and other office workers who are supposed to be indirectly exposed. participants were interviewed (face to face) using a standardized pre - designed questionnaire form. the questionnaire was tested on a subset of 5 workers prior to starting to obtain information that might improve the work plan and facilitate the execution of the study. this pilot also enabled the adaptation of the questionnaire, the estimation of the time needed for interviewing the participants (20 min) and performing the physical examination. after necessary questionnaire modifications, the final data collection sheet was completed for each participant and covered general information about (age, gender, residence, education, marital status, socioeconomic level, work activity, duration of work, complete medical history and work related complaint, occurrence of risky occupational exposures and wearing of personal protective clothes : disposable thick gloves, head covers, boots, masks goggle, or overalls). data were analysed using spss (statistical package for the social sciences) software version16.0 (spss inc., chicago, il, usa). differences in proportions were evaluated by pearson 's chi - square test ; p < 0.05 was considered to be statistically significant. or was used as a measure of the strength of association. a cross - sectional study was conducted between january and april 2013, among mswws in the main municipality company in alexandria (egypt). this company was established in 2001 to deal with all types of solid waste [figure 1 ] generated from different sources ; residential, shops, industrial, agricultural, and hospitals medical waste in all municipal districts of alexandria. its total area is about 14 square kilometers comprising three waste storage station, three sorting and recycling plants, one factory for fertilizer production and one landfill site. the daily solid waste weight disposed at each discharge site is from 3 to 3.5 thousand tons. the company comprises a large laboring force of about 6000 permanent workers serving in several sectors concerned with waste collection, transportation, sorting, recycling, treatment, incineration, landfill and fertilizer production. the municipality was contacted and asked to participate in the study by a document describing the purpose and concerns of the study. the study comprised all current workers who were employed for 1 year or more by the municipality. we categorized the participants into two occupational groups (exposed ; n = 186 and non - exposed ; n = 160) on the basis of the direct exposure to solid waste. the exposure group included those working in the collection of mixed domestic and communal waste, street sweepers, workers engaged in loading, transfer and evacuation, sorting, incineration, dumping, landfilling, bin / scoop washing, and fertilizer production. group 2 included transportation workers (vehicle / truck drivers), technical workers (mechanics, car and equipment maintenance, heavy equipment operators), welders, inspectors, timekeepers and other office workers who are supposed to be indirectly exposed. participants were interviewed (face to face) using a standardized pre - designed questionnaire form. the questionnaire was tested on a subset of 5 workers prior to starting to obtain information that might improve the work plan and facilitate the execution of the study. this pilot also enabled the adaptation of the questionnaire, the estimation of the time needed for interviewing the participants (20 min) and performing the physical examination. after necessary questionnaire modifications, the final data collection sheet was completed for each participant and covered general information about (age, gender, residence, education, marital status, socioeconomic level, work activity, duration of work, complete medical history and work related complaint, occurrence of risky occupational exposures and wearing of personal protective clothes : disposable thick gloves, head covers, boots, masks goggle, or overalls). data were analysed using spss (statistical package for the social sciences) software version16.0 (spss inc., chicago, il, usa). differences in proportions were evaluated by pearson 's chi - square test ; p < 0.05 was considered to be statistically significant. or was used as a measure of the strength of association. the study was approved by the institutional review board of the high institute of public health - alexandria university. permission to conduct the study all the medical waste handlers were given verbal information about the study and were assured about the confidentiality, protection and anonymity of their data. individuals found with physical ill - health were referred to hospital 's physicians for management. household and health care waste collection in alexandria is performed in a traditional way, with most domestic, waste products generated at homes being mixed together without being sorted (as followed in most of modern countries where three separate bins are used to separate paper, plastic and cardboard from broken glass or food waste). the mixed domestic waste is stored in plastic bags, to be stored in special plastic bins or metal containers in the street and collected every day by waste collectors. waste collection vehicle is a compactor truck with a closed container or scoop for storage of the waste. loading system comprises automatic fork that align carefully with sleeves of waste containers through a set of levers. the containers is lifted till it get to top, the contents are slipped upside down and is emptied into vehicle hopper. bulky garbage is compacted hydraulically forward by moving walls that oscillate to push waste to the rear of the vehicle. the collection vehicle transport waste to be evacuated at the discharge site that receives wastes from all municipal districts, where it is reloaded into loaders, weighed and transported to plants for sorting and recycling. part of un - reusable waste is used for fertilizer production and the remaining is transported to the dumping site for final disposal in pre - designed landfill cells. a network of pipes and wells are present to collect methane gas to be then burnt. most of them 344 (99.4%) were males and only 2 (0.6%) females. the majority were married 316 (91.1%) and almost above 30 (82.4%). about one - fourth of the participants were illiterate, one half (55.4%) were beyond primary education, while only 25 (7.2%) had a university education. according to the modified social scoring of fahmy and el - sherbini, comparable percentages had low (51.4%) and very low (41%) socio - economic level. the socio - demographic characteristics of the group directly exposed to solid waste differed significantly from that of the indirectly exposed group [table 1 ]. younger ages, rural residence, lower educational level, low socio - economic standard and being unmarried (single or divorced) were more encountered among the exposed group. sociodemographic characteristics of enrolled mswws about two - thirds of the workers (60.7%) were using tools during work. the waste collectors and street cleaners used to use dust - pans (32.4%), sweepers (30.6%) and sometimes tweezers or tongs (8.4%). the use of hand brush and rigid receptacles for picking up sharp objects was infrequent and reported by only 16 (4.6%). about 1.7% were using floor cloth and a wiper for cleaning building floors and bathrooms. five street sweepers (1.4%) claimed that when the dust - pans are not provided they make use of two woody plates that they adapt for collecting swept garbage. workers in sorting and recycling plants were using a hock (15.3%) at one hand to catch and open plastic bags containing waste as they run along a conveyer belt where manual sorting starts. nevertheless, a considerable number of the mswws were handling waste by their hands (with or without gloves) [figure 2 ]. tools used by municipal solid waste workers during work about 75 (21.7%) workers were not wearing the recommended personal protective clothing [figure 3 ]. this should include 36 (10.4%) personnel (inspectors and office workers) not exposing to waste. safety boot and long sleeve gown (overall) were the most frequent protective clothing often used by almost two thirds of workers (61.6% and 66.2% respectively). thick (heavy duty) gloves were used by only 29.8% of the workers ; mainly sorters (38.4%), collectors (14.1%), welders (14.1%), sweepers (8.1%) and those handling medical waste. head cover in the form of protective helmet or the ordinary cap was used by 30.3% of the workers. eye protection was assured by safety goggles for 9.5% of mswws engaged in welding (50%), technical work (18.7%) or dumping (9.4%). likewise, face mask was provided for 12.4% and was mainly used by sorters (39.5%), collectors (14%), technical workers (18.7%), those involved in medical waste autoclaving and incineration tasks (7%), landfilling (4.7%) and by inspectors (4.7%). available personal protective equipment used by municipal solid waste workers basic personal hygiene is essential for reducing the risks of ill health from handling waste. the majority of workers used to often wash their hands before eating (85.8%), if their hands become contaminated (77.5%) and before taking medications (56.1%). hand washing was also often emphasized by mswws before using the toilets (43.4%), drinking (35.3%), wearing gloves (16.8% ; 47.2% of those using gloves), using the cell phone (5.2%) and smoking (3.5% ; 8.1% of smokers) [figure 4 ]. household and health care waste collection in alexandria is performed in a traditional way, with most domestic, waste products generated at homes being mixed together without being sorted (as followed in most of modern countries where three separate bins are used to separate paper, plastic and cardboard from broken glass or food waste). the mixed domestic waste is stored in plastic bags, to be stored in special plastic bins or metal containers in the street and collected every day by waste collectors. waste collection vehicle is a compactor truck with a closed container or scoop for storage of the waste. loading system comprises automatic fork that align carefully with sleeves of waste containers through a set of levers. the containers is lifted till it get to top, the contents are slipped upside down and is emptied into vehicle hopper. bulky garbage is compacted hydraulically forward by moving walls that oscillate to push waste to the rear of the vehicle. the collection vehicle transport waste to be evacuated at the discharge site that receives wastes from all municipal districts, where it is reloaded into loaders, weighed and transported to plants for sorting and recycling. part of un - reusable waste is used for fertilizer production and the remaining is transported to the dumping site for final disposal in pre - designed landfill cells. a network of pipes and wells are present to collect methane gas to be then burnt. most of them 344 (99.4%) were males and only 2 (0.6%) females. the majority were married 316 (91.1%) and almost above 30 (82.4%). about one - fourth of the participants were illiterate, one half (55.4%) were beyond primary education, while only 25 (7.2%) had a university education. according to the modified social scoring of fahmy and el - sherbini, comparable percentages had low (51.4%) and very low (41%) socio - economic level. the socio - demographic characteristics of the group directly exposed to solid waste differed significantly from that of the indirectly exposed group [table 1 ]. younger ages, rural residence, lower educational level, low socio - economic standard and being unmarried (single or divorced) were more encountered among the exposed group. about two - thirds of the workers (60.7%) were using tools during work. the waste collectors and street cleaners used to use dust - pans (32.4%), sweepers (30.6%) and sometimes tweezers or tongs (8.4%). the use of hand brush and rigid receptacles for picking up sharp objects was infrequent and reported by only 16 (4.6%). about 1.7% were using floor cloth and a wiper for cleaning building floors and bathrooms. five street sweepers (1.4%) claimed that when the dust - pans are not provided they make use of two woody plates that they adapt for collecting swept garbage. workers in sorting and recycling plants were using a hock (15.3%) at one hand to catch and open plastic bags containing waste as they run along a conveyer belt where manual sorting starts. nevertheless, a considerable number of the mswws were handling waste by their hands (with or without gloves) [figure 2 ]. tools used by municipal solid waste workers during work about 75 (21.7%) workers were not wearing the recommended personal protective clothing [figure 3 ]. this should include 36 (10.4%) personnel (inspectors and office workers) not exposing to waste. safety boot and long sleeve gown (overall) were the most frequent protective clothing often used by almost two thirds of workers (61.6% and 66.2% respectively). thick (heavy duty) gloves were used by only 29.8% of the workers ; mainly sorters (38.4%), collectors (14.1%), welders (14.1%), sweepers (8.1%) and those handling medical waste. head cover in the form of protective helmet or the ordinary cap was used by 30.3% of the workers. eye protection was assured by safety goggles for 9.5% of mswws engaged in welding (50%), technical work (18.7%) or dumping (9.4%). likewise, face mask was provided for 12.4% and was mainly used by sorters (39.5%), collectors (14%), technical workers (18.7%), those involved in medical waste autoclaving and incineration tasks (7%), landfilling (4.7%) and by inspectors (4.7%). available personal protective equipment used by municipal solid waste workers basic personal hygiene is essential for reducing the risks of ill health from handling waste. the majority of workers used to often wash their hands before eating (85.8%), if their hands become contaminated (77.5%) and before taking medications (56.1%). hand washing was also often emphasized by mswws before using the toilets (43.4%), drinking (35.3%), wearing gloves (16.8% ; 47.2% of those using gloves), using the cell phone (5.2%) and smoking (3.5% ; 8.1% of smokers) [figure 4 ]. analysis of health hazards that may be encountered in waste management revealed that mswws are exposed daily to domestic waste including papers, cardboard, plastics, canes, rage, food waste (60.9%), feces in nappies or incontinence pads and blood in sanitary pads used by women during menses (59.8%), animal and domestic pets excreta, waste produced from pens or hutches, poultry and fish shops (52.5%), dead animals and rodent carcasses (51.9%, 6 workers stated that they sometimes find dead human bodies, particularly newborns in the waste stream), pins and used syringe / needles (53.9% ; 32.7% reported needle stick injury), broken glass and other sharp items (59.2%), laboratory waste (39.4%), hospital waste including papers, cardboard, plastics, canes, rage, food waste (41.4%), dust (41.3%) and repugnant odor (63.8%), sludge, debris, decayed matters remaining in vehicle hopper and containers (14%), leachates and sludge that sometimes contains worms (15%) and tan (13.8%). we compared prevalence of health disorders between mswws in two groups according to the direct exposure to solid waste ; exposed and non - exposed. the most frequent complains encountered among mswws were cough (29.1%), fatigue (27.5%), headache (21.1%) and a number of constitutional symptoms notably fever, malaise, night sweating, anorexia and dizziness that were experienced by (9.5%, 8.9%, 3.5% and 6.9% and 0.6% respectively). these probably constitute symptoms of organic dust toxic syndrome. prevalence and or for self - reported health outcomes among enrolled mswws gastrointestinal (git) complaints frequent attacks of abdominal colics, diarrhea, dyspepsia, vomiting, or dysentery were reported by (25.5%, 24.5%, 24.3%, 10.9% and 10.9% respectively). low back pain and sciatic pain constituted 17.3% (one case had definitive lumbar disc prolapse), whereas generalized bone aches and arthralgia affecting mainly knee, hip, shoulder and neck joints were reported by 3.2%. other complains included upper respiratory infection (sore throat, sinusitis) (2.3%), dysuria, pyuria, renal colic (4.2%), dyspnea (2.6%), hemoptysis (0.9%) and weight loss (3.5%). tinnitus was a complaint of 3 workers (0.9%) in sorting and recycling plant who expose to excessive noise. two office employees complained of angina and chest tightness and cardiac catheterization revealed coronary obstruction. hypotension was more prevalent among the exposed group (or = 2.3, 95% ci = 0.9 - 5.5) who are supposed to exert more physical activity and lose water and salt in sweating, unlike the non - exposed group performing sedentary work more that showed a higher risk of hypertension. likewise, weight loss (or = 2.7, 95% ci = 0.73 - 10.2) and low bmi (or = 8.1, 95% ci = 0.4 - 151.7) were evident among the exposed group whereas over weight and obesity were more prevalent among the other group. head and neck examination revealed that 1.2% of mswws have acne and scalp furunculosis, 0.9% have eye infection ; most probably conjunctivitis and 0.6% have mouth ulcers. abdominal distention (1.4%), hepatomegaly (2.9%), splenomegaly (1.7%) and abdominal tenderness (5.5%) were evident in abdominal examination, whereas chest auscultation revealed rhonchi and wheezes in 1.2% of the evaluated participants. on skin examination, fungal infections were frequent (tinea versi color [7.2% ], tinea corporis [2.6% ], tinea cruris and [1.7% ] and onchomycosis [0.6% ]). scabes, maculopapular rash, eczema / dermatitis, furunculosis (boils), infected wound, foot bullae / cracks and vitiligo, were seen in (4.2%, 6.3%, 5.4%, 0.9%, 1.7%, 1.2% and 0.9% respectively). about 3% of the mswws were complaining of pruritus due to skin allergy. history of work related accident or trauma namely contusions, lacerations, sprains, fracture, or bites was reported by 46.5% of the workers whereas needle stick injury constituted 32.7%. distribution of health disorder by category of work service is detailed in [table 1 ]. municipal waste management is a livelihood to poor people of low educational levels, inadequate housing conditions in rural residence, large family size with insufficient family income. in egypt, this job is mainly and formally performed by male employees and the same applies in other countries. workers involved in waste collection, sorting, treatment and landfill and thus directly exposed to solid waste had lower socio - economic standard, lower educational level and resided rural areas where life conditions lags behind urban communities. being single or divorced also reflects poor life conditions and inability to afford marriage expenses. the workers indirectly exposed to solid waste were involved more in technical work that necessitates certain degree of specialized knowledge and thus higher levels of education and socioeconomic life. mswws are exposed to more occupational health and safety risks than workers in many other industries. in this study, we show that mswws performing different tasks in waste management differ in risk of acquiring adverse health effects. participants from 15 work services were evaluated and statistically categorized into two groups (directly and indirectly exposed groups or potential high and low exposure groups). those not directly engaged in the waste handling such as truck drivers claimed that they used to help collectors and reloading workers during truck loading and evacuation. moreover, mechanics, welders and technicians stated that they can repair broken vehicle or rolling conveyer while being loaded with waste. these workers do not often put their personal protective clothes and hence tend to handle waste with bared hands and without making use of any tool. although strongly recommended, personal protective equipment was not regularly used by a considerable number of the participants. some reasoned that for the shortage in its supply that they can receive pair of thick heavy duty gloves every 3 month, if they are torn during work, not replaced. others (especially technical worker and truck drivers) claimed that they prefer working without wearing gloves as they limit their free movement during work or cause skin irritation and dryness. most of the participants were aware of the importance and timing of hand washing and hygienic practices. nevertheless, no convenient washing facilities (with warm water and soap) were available near the collection points, work station in landfill site, or for those working in the street. moreover, lay people of low socio - economic standard do not always practice hand or mouth washing. this is the first comprehensive study in egypt that reviewed the health problems encountered among the mswws that entailed git, respiratory, muscloskeletal, cutaneous and constitutional symptoms as well as allergies and accidents. git symptoms such as diarrhea, dysentery, vomiting and nausea are well - known problems among sewage, waste water treatment and mswws workers exposed to high concentrations of airborne gram negative bacteria since waste handling may cause dust full of microorganisms and bacterial endotoxins to become aerosolized. moreover, direct skin contact of these waste matter stuffed with fungal spores, bacteria, viruses and parasitic ova that can cause diarrhea via feco - oral route. in agreement with reports from poulsen., and 1995, hours., 2003, the exposed group were at higher risk of diarrheal disease compared with the non - exposed group. did not find any excess of git symptoms among the household waste collectors possibly due to lower concentration of bacteria they estimated in the waste. helicobacter pylori was incriminated as a causative agent of dyspepsia and epigastric pain among waste water treatment and sewage workers and hence we can hypothesis that the high prevalence of dyspepsia among mswws especially sorting workers and collators in the present study is due to h. pylori infection. some of the participants indicated factors in their work environment, generally the repugnant odor of rotten waste that they inhale are the causes of hurt burn and git perturbations. palpable liver and spleen found on examination in 2.9% and 1.7% respectively of participants and significantly higher in the non - exposed group are not related waste exposure rather than consequences of bilharzias. this disease is endemic in egypt and was proved to infect 13.3% of studied participants where schistosoma mansoni egg was detected in their stool (eassa. several studies have pointed to the possible effects of waste on respiratory function and correlated that to the high dust levels, micro - organisms, fungal spores and endotoxins or the presence of higher levels of total suspended particulate matter than the local standards that can impair lung function on chronic exposure. higher prevalence of respiratory complains particularly cough (dry or productive) were found amongst collectors, sorting workers, street cleaners, truck drivers, landfill workers, welders, and vehicle technicians who are subjected to variable direct exposure to both chemical and biological sensitizers in the workplace. more specifically, collectors who ride on footplates on the backs of the trucks and truck drivers are exposed to exhaust fumes, cleaners are exposed to street dust, sorters are exposed to molds and bioaerosole, welders are exposed to reactive oxygen species, carbon mono - oxide and welding fumes containing heavy metals, mechanics are exposed to benzene and paints, and workers in dumping sites are exposed to bioaerosole, dust and harmful methane gas. some workers experienced breathlessness probably due to air way obstruction and inflammation as stated earlier. no significant difference was observed between both evaluated groups albeit with higher rate among the exposed group. this was seldom described in the literature where observed skin diseases pointed to callused palms, bruises, dermatitis, folliculitis, abrasions, and xerosis. previous studies has assessed fungal air contamination in waste stations and confirmed the presence of three fungal species : aspergillus flavus, aspergillus fumigatus, and stachybotrys chartarum which cause pulmonary disease. since tineas are known to be caused by dermatophytes, further studies are needed to investigate their contamination to work environment. given their exposure to other air - borne contaminants pruritus due to skin allergies were more prevalent among the non - exposed groups especially the technical workers that included mechanics, vehicle and equipment maintenance workers, heavy equipment operators who directly expose to skin irritants and used to work without the use of protective gloves. this category experienced 8.4 increased risk of work related accidents as compared to the exposed group (p = 0.004) due to manipulation of heavy and moving objects, repeated pressure, pounding, friction and vibration. dermal injuries comprised needle stick injuries caused by disposable needles originating from residential and health care sources and accounted for 32.7% of the claims. cut wounds that were also found infected in six workers were reported to be caused by sharp objects bumped into skin, disposable razors, broken glass, pins, sharp can lids, thorns, or broken tree limbs. workers described loose needles and such materials as commonly protruding from uncontained disposal or ruptured garbage bags in the waste stream and puncture skin, when handled by collectors or during manual sorting in recycling plants. waste collection is also a task which requires repeated heavy physical activity such as heavy lifting, carrying, pulling / pushing of bins and containers that involve static muscle contraction and hence increases the risk of musculoskeletal problems.. showed in a recent study conducted in egypt higher percentage of musculoskeletal complaints (60.8%) among mswws particularly waste collectors where low back pain was the most frequently reported (22.5%). we report high prevalence (17.3%) of low back pain and sometimes sciatica among mswws that was significantly higher among the exposed group (or = 3.5, 95% ci = 1.8 - 7). this could be attributed to the large volume of refuse they have to pack manually and hold above shoulder level or due to un - acquaintance with the proper safety maneuver that should be adopted while being in duty. moreover, in egypt workers tend to use old and traditional equipment and depend mainly on the physical power. the existence of low back pain, bone aches and arthralgia did not significantly correlate with the duration of work (r = 0.041, p = 0.44) suggesting that musculoskeletal disorders are related to exerting improper physical maneuver rather than the frequency of sustained muscle action. moreover, workers get their positions upgraded (crew chef, inspectors) after spending certain number of years in employment. the present work is limited by the use of cross - sectional design, the lack of specific exposure assessment and a standard threshold level for exposure and the healthy worker effect. the high prevalence of accidents and skin injuries revealed in the present study calls for new standardized guidelines and measures to improve work safety in hazardous waste collection, transport and handling. most participants especially those directly involved in waste handling reported receiving job safety and hygiene training. however, this compulsory pre - employment training should be expanded to cover measures to be taken if exposed to biohazards and unexpected medical waste. although medical waste is collected and disposed in different settings, observation of medical waste (disposable needles, syringes, blades, iv lines, bandages and blood bags) in the general waste stream was common. this indicates the need for proper handling, containment or destruction of these dangerous materials by users prior to disposal. campaign must be organized to educate citizens about the importance of sorting waste before disposal. action must focus on improving and intensifying preventive measures to minimize bioaerosol levels at work stations by installing vacuum cleaning system and enclosing conveyers. wearing of internationally recommended personal protective dressing should be enforced, making the protection of skin, eyes, eyes and respiratory airway more effective. special filters should be provided for welding, cleaners and landfill personnel as respiratory protective devices that will significantly reduce the risk of ill health. the transfer of two - wheeled container to be hydraulically lifted and emptied by the truck should be carried out by two to three persons instead of one. the hopper should not be over loaded by waste to ovoid over spill that is then gathered by bared hands of drivers and transfer workers. effective personal hygiene must be maintained by provision of adequate including mild soap, towels or even cleansing wipes on collection vehicles and effective skincare regime using mild cleansers and pre - work and after - work barrier skin creams to keep the skin moisturized. physician should intensify medical supervision and organize health education sessions for diffusing clear job guidelines concerning number of working hours and maximum weight limits for packing, carrying or pulling to avoid muscle and joint strains. they should also consult mswws about how to avoid musculoskeletal disorders and educate the correct physical movement while pulling, carrying or pushing heavy objects, also the early signs of joint affection. future research should elaborate more on these emerging issues and seek the construction of a health risk reduction behaviors models to help protect the health of mswws and drive authorities to adopt safer management techniques. | background : solid waste management has emerged as an important human and environmental health issue. municipal solid waste workers (mswws) are potentially exposed to a variety of occupational biohazards and safety risks. the aim of this study was to describe health practices and safety measures adopted by workers in the main municipal company in alexandria (egypt) as well as the pattern of the encountered work related ill health.methods:a cross - sectional study was conducted between january and april 2013. we interviewed and evaluated 346 workers serving in about 15 different solid waste management activities regarding personal hygiene, the practice of security and health care measures and the impact of solid waste management.results:poor personal hygiene and self - care, inadequate protective and safety measures for potentially hazardous exposure were described. impact of solid waste management on health of mswws entailed high prevalence of gastrointestinal, respiratory, skin and musculoskeletal morbidities. occurrence of accidents and needle stick injuries amounted to 46.5% and 32.7% respectively. the risk of work related health disorders was notably higher among workers directly exposed to solid waste when compared by a group of low exposure potential particularly for diarrhea (odds ratio [or ] = 2.2, 95% confidence interval [ci ] = 1.2 - 3.8), vomiting (or = 2.7, 95% ci = 1.1 - 6.6), abdominal colic (or = 1.9, 95% ci = 1.1 - 3.2), dysentery (or = 3.6, 95% ci = 1.3 - 10), dyspepsia (or = 1.8, 95% ci = 1.1 - 3), low back / sciatic pain (or = 3.5, 95% ci = 1.8 - 7), tinnitus (or = 6.2, 95% ci = 0.3 - 122) and needle stick injury (or = 3.4, 95% ci = 2.1 - 5.5).conclusions : workers exposed to solid waste exhibit significant increase in risk of ill health. physician role and health education could be the key to assure the mswws health safety. |
dystocia or abnormal progress of labor is the most common maternal problem and cause of burden in low - income countries. it is estimated that 600,000 maternal deaths occur due to pregnancy and delivery disorders each year in the world, of which 95% is reported from developing countries, and in 30% of the cases, the problem is cephalopelvic disproportion. this is a case control prospective double - blind study conducted on 447 nulliparous women referring to maternity ward of omolbanin hospital in mashhad with term gestational age (38 - 42 weeks), single tone pregnancy, and vertex presentation during nov 2008-july 2009. the research project was approved by the ethical consideration committee of mashhad university of medical sciences and an informed consent was taken from all the subjects. sample size, after a pilot study with confidence level of 99% and optimum error of 1%, was determined by ratio estimation formula. the women with history of pelvis fracture, asymmetric pelvis, foot lameness, clearly pelvic contraction in clinical examination, bmi > 30 kg / m, 18 50%) as the cut - off points. so, sensitivity and specificity for percentiles and quarters of various anthropometric measurements in the study population were calculated, and in the second percentile, transverse diagonal of michaelis sacral rhomboid area 9.6 cm was considered as its cut - off point. in the third percentile, maternal height 155 cm was considered as its cut - off point. in the fourth percentile, intertrochanteric diameter 31 cm ; in the fourth percentile, ratio of height to fundal height 4.7 ; in the third percentile, lower limbs length 78 cm ; in the sixth percentile, ratio of head circumference to height 35.05 ; in the first quarter, foot length 23 cm ; in the second quarter, fetal estimated weight by johnson 's formula 3255 g ; in the second quarter, fetal weight estimated by multiplication of uterine height by abdominal circumference 3255 g ; in the sixth percentile, fundal height > 33 cm ; in the second quarter, mother 's bmi > 22 kg / m ; in the second quarter, vertebral length 58.5 cm ; in the sixth percentile, abdominal circumference > 98.6 cm ; in the second quarter, head circumference > 55 cm ; in the second quarter, vertical diagonal of michaelis sacral rhomboid area 9.5 cm ; in the sixth percentile, lower limb length to height ratio 50.6 ; and in the s econd quarter, vertebral length to height ratio 36.8 were considered as the anthropometric measurement cut - off points. comparison of mean anthropometric measurements in the two groups of normal delivery and dystocia was conducted by mann various percentiles and quarters of height and pelvic diameters were calculated, and the sensitivity and specificity, positive and negative indicative values, and their accuracy were manually calculated. in this research, 527 nulliparous women entered the study of whom 80 were excluded due to cs from any other cause except dystocia, such as meconium fetal amniotic fluid (n = 25), fetal distress (n = 16), fetal macrosomia (n = 4), abruption placenta (n = 2), severe vaginal bleeding (n = 3), brow presentation (n = 1), birth weight 33 cm with an accuracy of 73.7%, combination of mothers height with estimation of fetal weight by johnson 's method with an accuracy of 73.3%, combination of mothers height with lower limb length with an accuracy of 71.5%, combination of mothers height with abdominal circumference with an accuracy of 71.5%, and combination of mothers height with foot length with an accuracy of 69.2%, in comparison with mothers height alone, led to a better predictor for dystocia. combination of height with other anthropometric measurements in comparison with mothers height alone did not result in a better predictor [table 3 ]. combination of estimated fetal weight by johnson 's method with pair combination of height and other anthropometric measurements led to a better predictor for dystocia, and the highest diagnostic value obtained in the present study was for combination of the third percentile of mothers height with the second percentile of transverse sacral michaelis diameter and the second quarter of fetal weight estimation through johnson 's formula with a sensitivity of 70%, specificity of 93.7%, and accuracy of 90.5% [table 4 ]. diagnostic values of combining of maternal height with other maternal anthropometric measurements diagnostic values of combining different deciles and percentiles of maternal height with pelvic diameters and pelvic diameters with each other by the highest validity in the present study, with the goal of achieving better predictors for dystocia, in addition of height, we calculated the other maternal anthropometric measurements : mothers head circumference, head circumference to height ratio, lower limb length, lower limb to height ratio, vertebral length, vertebral length to height ratio, transverse and vertical diagonals of michaelis sacral rhomboid area, intertrochanteric diameter, height to symphysio - fundal height ratio, and abdominal girth. the accuracy obtained for mothers height 155 cm was 68.2%, with a sensitivity of 50% and specificity of 70.8%. among the various maternal anthropometric measurements, the transverse diagonal of the michaelis sacral rhomboid area 9.6 cm with an accuracy of 81.2%, sensitivity of 60.7%, and specificity of 84.1% was the best predictor for dystocia, and had a high accuracy, sensitivity, and specificity compared to mothers height. height to symphysio - fundal height ratio 4.7, lower limb length 78 cm, maternal head circumference to height 35, fetal weight estimation 3255 g (johnson 's method), fetal weight estimation 3255 g by multiplication of symphysio - fundal height with abdominal girth, symphysio - fundal height > 33 cm, mothers bmi > 22 kg / m, vertebral length 58.5 cm, abdominal girth > 98.6 cm, intertrochanteric diameter 31 cm, and vertical diagonal of michaelis sacral rhomboid area 9.5 cm had a higher sensitivity in the prediction of dystocia compared to mothers height, but their obtained specificity and accuracy were lower than those of mothers height. mothers foot length 23 cm and head circumference > 55 cm had lower sensitivity, specificity, and accuracy compared to mothers head. few studies have compared the diagnostic value of maternal anthropometric measurements with height. in the study of liselele., (2000) the cut - off points of pelvic diameters were selected based on percentile 10 of their society, and their obtained sensitivity, specificity, and positive likelihood ratio for mothers height were 21.%, 93.8%, and 3.5, respectively. transverse diagonal of michaelis sacral rhomboid area with a sensitivity of 42.9%, specificity of 91.1%, and positive likelihood ratio of 4.8, and intertrochanteric diameter with a sensitivity of 38.1%, specificity of 89.4%, and positive likelihood ratio of 3.6 were better predictors for dystocia compared to mothers height. in rozenholc., (2007) report, mothers height with a sensitivity of 28.6%, specificity of 98.4%, and positivity likelihood ratio of 18.4 was the best predictor for dystocia and other maternal anthropometric measurements had lower diagnostic value. for transverse diagonal of michaelis sacral rhomboid area, they found a sensitivity of 45.9%, specificity of 92.7%, and positivity likelihood ratio of 6.3. although it had a higher sensitivity compared to mothers height, its specificity and positive likelihood ratio were lower, and the intertrochanteric diameter with a sensitivity of 26.5%, specificity of 88.9%, and positive likelihood ratio of 2.4 had lower diagnostic value compared to mothers height. the calculated sensitivity for mothers height and transverse diagonal of michaelis sacral rhomboid area in our study was more than the sensitivity obtained in the aforementioned studies and is not consistent with them, possibly due to different determined cut - off points in our study, which have been obtained based on the best sensitivity, specificity, and accuracy calculated by various percentiles and quarters. in our study, the diagnostic value of transverse diagonal of michaelis sacral rhomboid area was higher than the diagnostic value of mothers height, which is consistent with the results of liselele. benjamin., (2011) determined the cut - off point of maternal anthropometric measurements based on rock 's curve, and calculated sensitivity, specificity, and positive predictive value for mothers height 155.5 cm as 70.4%, 52.1%, and 15.4%, respectively. they reported that mothers foot length 23 cm with a sensitivity of 77.8, specificity of 58.6%, and positive predictive value of 18.6% had a better prediction value compared to mothers height and transverse diagonal of michaelis sacral rhomboid area 10.4 was not a better predictor for dystocia compared to mothers height, which is not consistent with the present study. in the study of rozenholc., (2007) and benjamin., (2011) symphysio - fundal height, mothers foot length, vertical diagonal of michaelis sacral rhomboid area 10.1, fetal weight estimation by johnson 's formula and abdominal girth had lower specificity compared to mothers height. in the study of van bogaert. (1999), the mean lengths of lower limb in the groups of natural delivery and dystocia were 91.3 and 89.3 cm, respectively (p = 0.014), the mean lengths of vertebra in normal delivery and dystocia groups were 75.2 and 73.8 cm, respectively (p = 0.0003), and the mean mothers heights in normal delivery and dystocia groups were 157.6 and 154.1 cm, respectively (p = 0.0001). in the study of barnhard., (1997) the mean ratios of height to symphysio - fundal height in normal delivery and dystocia groups were 7 and 3.7, respectively (p = 0.02). the mean mothers head circumference values in normal delivery and dystocia groups were not significantly different, but the mean ratios of head circumference to height in normal delivery and dystocia groups were 34 and 35.1, respectively (p = 0.001). some researchers have argued that an increase in the ratio of head circumference to height in animals is a risk factor for dystocia. they reasoned that the women with high ratio of head circumference to height possibly have faced a growth disorder in their fetal period, leading to an imbalance in their ratio of head circumference to height, and consequently, this growth disorder may have affected their pelvis size. the ratio of lower limb to height is an important predictor for nutrition and health status of individuals, so women with malnutrition face shortness of vertebra and acute shortness in their lower limb as well as a reduction in the ratio of lower limb to height. malnutrition in childhood is an important risk factor for bones growth and shortness, which can be associated with growth disorder of pelvic bones. adolf michael suggested the importance of michaelis sacral rhomboid area in the evaluation of pelvic capacity for the first time in 1851. abnormal size of michaelis sacral rhomboid area is a predictor for mothers shape and abnormal pelvic size, and in pelvises with stenosis, its transverse diameter is shorter than its vertical diameter. the distance between femoral great trochanters is associated with transverse pelvic diameter and in a number of studies has been reported to have a better diagnostic value compared to mothers height. fetal size is estimated through different measurement methods such as measurement of symphysio - fundal height, abdominal girth, calculation of fetal weight by johnson 's formula, and multiplication of symphysio - fundal height with abdominal girth. fetal size alone is not counted as an appropriate criterion for an unsuccessful delivery, as in most of the cases, cephalopelvic disproportion is observed among the fetuses with their weight in a normal range. therefore, evaluation of the imbalance of fetal size with mothers pelvis can be a better criterion to predict dystocia compared to fetal weight alone. these results are in accordance with those of the present study which showed the ratio of height to symphysio - fundal height was a better predictor for dystocia compared to symphysio - fundal height alone. in the present study, combination of mothers height with different anthropometric measurements led to better predictors for dystocia compared to mothers height alone. combination of mothers height 155 cm with transverse diagonal of michaelis sacral rhomboid area 9.6 cm with an accuracy of 86.2%, sensitivity of 58.3%, and specificity of 89.9% yielded the best predictor. after this, combination of cut - off points of mothers height with symphysio - fundal height > 33 cm, mothers height with fetal weight 3255 g by johnson 's formula, mothers height with lower limb length 78 cm, and mothers height with abdominal girth > 98.6 cm were better predictors concerning sensitivity, specificity, and accuracy, respectively. in rozenholc., (2007) and liselele., (2003) study, combination of mothers height with transverse diagonal of michaelis sacral diameter, intertrochanteric diameter, mothers foot length, and symphysio - fundal height resulted in better predictors concerning sensitivity and specificity. benjamin., (2011) suggested combination of mothers height with fetal weight estimation by johnson 's formula and measurement of mothers foot length as better predictors compared to mothers height alone. in the present study, maternal anthropometric measurements in addition to height and fetal weight estimation by johnson 's formula were combined, and among the triple combinations, the highest accuracy (90.5%) was for combination of mothers height, transverse diagonal of michaelis sacral rhomboid area, and estimated fetal weight by johnson 's formula, with a sensitivity of 70% and specificity of 93.7%, which was better than the combination of height and transverse diagonal of michaelis sacral rhomboid area concerning specificity and accuracy. triple combination of fetal estimated weight with height and lower limb length with a sensitivity of 64%, specificity of 78.7%, and accuracy of 76.3%, triple combination of fetal estimated weight with height and vertebral length with a sensitivity of 73.6%, specificity of 71.6%, and accuracy of 72%, and triple combination of fetal estimated weight with height and intertrochanteric diameter with a sensitivity of 75%, specificity of 70%, and accuracy of 70.6% resulted in better predictors concerning sensitivity, specificity, and accuracy, compared to each of the paired combinations. (2011). in the investigation of diagnostic value of each maternal anthropometric measurement, the accuracy obtained for mothers height was 68.3. except for transverse diagonal of michaelis sacral rhomboid area, combination of mothers height with the other anthropometric measurements led to better predictors compared to mothers height alone. the best predictor was pair combination of mothers transverse diagonal of michaelis sacral rhomboid area and height with a sensitivity of 58.3%, specificity of 89.9%, and accuracy of 86.2. combinations of mothers height with symphysio - fundal height (accuracy = 73.4%), lower limb length (accuracy = 71.5%), and abdominal girth (accuracy = 71.5%) were the best predictors compared to mothers height alone. in the present study, the best predictors concerning sensitivity, specificity, and accuracy were obtained by triple combination of maternal anthropometric measurements and height with fetal weight estimation by johnson 's method, and the best predictor was related to combination of third percentile of mothers height (155 cm), the second percentile of transverse diagonal of michaelis sacral rhomboid area (9.6 cm), and fetal estimated weight (3255 g) by johnson 's method, which had accuracy of 90.5%, sensitivity of 70%, and specificity of 93.7%. based on the results of the present study, mothers height alone is not an appropriate predictor for dystocia, and its combination with other maternal anthropometric measurements and estimation of fetal weight yields better predictors to predict dystocia. | background : dystocia is one of the important causes of maternal morbidity and mortality in low - income countries. this study was aimed to determine the diagnostic accuracy of maternal anthropometric measurements as predictors for dystocia in nulliparous women.materials and methods : this prospective cohort study was conducted on 447 nulliparous women who referred to omolbanin hospital. several maternal anthropometric measurements such as height, transverse and vertical diameters of michaelis sacral rhomboid area, foot length, head circumference, vertebral and lower limb length, symphysio - fundal height, and abdominal girth were taken in cervical dilatation 5 cm. labor progression was controlled by a researcher blind to these measurements. after delivery, the accuracy of individual and combined measurements in prediction of dystocia was analyzed. dystocia was defined as cesarean section and vacuum or forceps delivery for abnormal progress of labor (cervical dilatation less than 1 cm / h in the active phase for 2 h, and during the second stage, beyond 2 h or fetal head descend less than 1 cm / h).results : among the different anthropometric measurements, transverse diameter of the michaelis sacral rhomboid area 9.6 cm, maternal height 155 cm, height to symphysio - fundal height ratio 4.7, lower limb length 78 cm, and head circumference to height ratio 35.05 with accuracy of 81.2%, 68.2%, 65.5%, 63.3%, and 61.5%, respectively, were better predictors. the best predictor was obtained by combination of maternal height 155 cm or the transverse diameter of the michaelis sacral rhomboid area 9.6 cm and johnson 's formula estimated fetal weight 3255 g, with an accuracy of 90.5%, sensitivity of 70%, and specificity of 93.7%.conclusions : combination of other anthropometric measurements and estimated fetal weight with maternal height in comparison to maternal height alone leads to a better predictor for dystocia. |
giant intraabdominal cysts in adolescents are very rare entities and arise most commonly from the small bowel mesentery, the omentum, or the ovary. although they are almost always benign, full midline celiotomy has been the conventional surgical approach for resection. a 16-year - old african american female presented to us with progressive abdominal distension over 1 year., she appeared cachectic with a markedly distended, very tense, nontender abdomen along with dilated collateral veins on the chest wall and upper thighs (figure 1). ct scan of the abdomen and pelvis showed a giant multiseptated cystic mass in the abdomen measuring 22.5x30x40.5 cm (figure 2). this cyst had a significant mass effect on the adjacent organs, causing mild intrahepatic ductal dilatation and severe right - sided hydronephrosis. a laboratory workup overall was normal, including b - hcg, cea, afp, and ca 19 9, except for an elevated ca 125 (152 u / ml). a bowel preparation was performed, and the patient was taken to the operating room for laparoscopic management. computed tomographic scan of the abdomen showing the giant cystic mass along with the mass effect and right hydronephrosis. a pursestring suture was placed in the cyst wall, then the cyst entered at the center of the pursestring and decompressed with careful attention to avoid spillage ; 15 liters of murky brown fluid were aspirated. the cyst wall opening was closed, the midline fascia was partially closed, a hasson cannula was introduced, and the abdomen was insufflated. the cyst was noted to be arising from the left ovary ; the right ovary was grossly normal. the left round ligament was divided with electrocautery, and the leaves of the broad ligament were separated, opening the pelvic sidewall. the ovarian artery was divided with a harmonic scalpel ; then the dilated ovarian vein was clipped and divided. the cyst was still attached to the omentum superiorly, so the patient was placed in a reverse trendelenburg position, and the omentum was dissected free by using a harmonic scalpel. a remaining loculation required laparoscopic drainage, and the cystic mass was removed through the umbilical incision. the postoperative course was uneventful, and the patient was discharged home on postoperative day 1 with minimal pain and tolerating a regular diet. pathology revealed a mature cystic teratoma (2851 grams) containing intestinal mucosa, smooth muscle, and mucinous cystadenoma. giant intraabdominal cysts in children and adolescents may arise from different sources, including most commonly ovarian, gastrointestinal, urological, and lymphatic processes. ovarian cysts are traditionally labeled as large when they are over 5 cm and giant or voluminous when they are over 15 cm ; a better designation for giant cysts may however relate the size of the cyst to the size of the peritoneal cavity in these growing young patients. ninety percent of large ovarian cysts in children (simple or complex) will either decrease in size or resolve and may be safely followed with serial pelvic ultrasonography, although some recommend cystectomy when the size is over 5 cm. giant ovarian cysts however always require resection, usually oophorectomy, because of the associated symptoms, the complications due to mass effect, the doubt about ovarian origin despite imaging studies, or for all of these reasons. minimally invasive surgical techniques have been applied to the management of these giant cysts, but only a few cases have been reported ; all reported techniques include decompression of the cyst to allow for room to work, facilitate manipulation of the cyst and ovary, and prevent inadvertent perforation and spillage. salem reported an experience with 15 cases of large ovarian cysts reaching above the level of the umbilicus. all cysts were benign, and aspiration of the cyst fluid was done after puncture of its wall, then the cyst was removed as usual. two additional cases of giant ovarian cysts, 21 cm and 22 cm, managed laparoscopically have been reported ; one patient underwent prelaparoscopy cyst drainage with a suprapubic catheter followed by laparoscopic cystectomy, the other had paracentesis performed during diagnostic laparoscopy, followed by laparoscopic removal of the cyst and left adnexa. to our knowledge, the 40-cm ovarian cyst reported on herein is the largest intraabdominal cyst managed laparoscopically reported to date. we suggest drainage of these cysts via a minilaparotomy, which allows for a more controlled approach to minimize spillage as compared with percutaneous techniques ; in addition, prelaparoscopy decompression is necessary to allow establishment of pneumoperitoneum when these cysts become so voluminous. although slow - growing cysts that reach this giant size are almost always benign, suspicion of malignancy based on preoperative imaging and tumor markers should preclude this laparoscopic approach. as was the case in our patient, decompression of the cyst followed by diagnostic laparoscopy will easily determine the source of the cyst, and laparoscopic techniques can still be used to manage the pathology at hand ; laparoscopic management of giant mesenteric and omental cyst has been reported. this approach should provide all the benefits of laparoscopic techniques including less pain, shorter hospital stay, in addition to a significantly better cosmetic result in these adolescent patients. giant ovarian cysts can be managed laparoscopically regardless of the size of the cyst when a normal tumor marker profile and benign imaging appearance excludes the possibility of malignancy. | introduction : giant intraabdominal cysts are very rare, and conventional treatment is full midline laparotomy. we present a case of complete laparoscopic extirpation of a giant ovarian cyst.case report : a 16-year - old female presented with progressive abdominal distension for 1-year along with early satiety, constipation, and significant weight loss. a ct scan showed a giant multiseptated cystic mass in the abdomen measuring 22.5x30x40.5 cm with significant mass effect causing intrahepatic ductal dilatation and right hydronephrosis. the mass was decompressed via a minilaparotomy in a controlled fashion, removing 15 liters of fluid. a laparoscopic left oophorectomy was then performed. the postoperative course was uneventful, and the patient was discharged home on postoperative day 1 with minimal pain and tolerating a regular diet. pathology examination revealed a mature cystic teratoma.conclusion:giant ovarian cysts can be managed laparoscopically when a normal tumor marker profile and benign imaging appearance exclude the possibility of malignancy. |
it is used in the form of almond - violet oil in traditional persian medicine (tpm) since ancient times. almond - violet oil was used for the treatment of insomnia, headache, cough, and fever based on tpm textbooks. the first is macerating voila flowers in oil for several days under the sunlight. in this study, after mixing the violet flowers with in 1:2 proportions, almond - violet oil was obtained under pressure. fatty acid ingredients of almond - violet oil were analyzed by gas chromatography (gc) technique. analysis of almond - violet oil by gc method showed some major components such as oleic acid (70.54%), linoleic acid (omega-6 fatty acids) (18.22%), palmitic acid (8.51%), stearic acid (1.58%), and palmitoleic acid (0.69%). monounsaturated fat consumption has been considered to decrease low - density lipoprotein (ldl) cholesterol. linoleic acid lipid radicals can also be used to act as an antioxidant agent in natural phenols. on the other hand, oleic acid may be responsible for the hypotensive (blood pressure reducing) effects. palmitoleic acid is a beneficial fatty acid not only to increase insulin sensitivity by suppressing inflammation, but also to inhibit the destruction of insulin - secreting pancreatic beta cells. in some aspects, the result of the present study does not fully match with the standards of the europe pharmacopoeia. this could be due to differences associated with the environment and cultivation of the plants. | background : viola odorata l. belongs to violaceae family and is native to iran. it is used in the form of almond - violet oil in traditional persian medicine (tpm) since ancient times. almond - violet oil was used for the treatment of insomnia, headache, cough, and fever based on tpm textbooks. there are two methods for the preparation of almond - violet oil. the first is macerating voila flowers in oil for several days under the sunlight. the second method is cold pressing of violet flowers and.methods:in this study, after mixing the violet flowers with in 1:2 proportions, almond - violet oil was obtained under pressure. fatty acid ingredients of almond - violet oil were analyzed by gas chromatography (gc) technique.results:analysis of almond - violet oil by gc method showed some major components such as oleic acid (70.54%), linoleic acid (omega-6 fatty acids) (18.22%), palmitic acid (8.51%), stearic acid (1.58%), and palmitoleic acid (0.69%). monounsaturated fat consumption has been considered to decrease low - density lipoprotein (ldl) cholesterol. linoleic acid lipid radicals can also be used to act as an antioxidant agent in natural phenols. on the other hand, oleic acid may be responsible for the hypotensive (blood pressure reducing) effects. palmitoleic acid is a beneficial fatty acid not only to increase insulin sensitivity by suppressing inflammation, but also to inhibit the destruction of insulin - secreting pancreatic beta cells.conclusion:in some aspects, the result of the present study does not fully match with the standards of the europe pharmacopoeia. this could be due to differences associated with the environment and cultivation of the plants. such differences should be considered whilst studying native plants. |
statin therapy is a well established treatment for hyperlipidemia. in the uk, the joint british society (jbs) has developed national guidelines on prevention of cardiovascular disease (cvd) in clinical practice. in these guidelines, the jbs has taken account of scientific evidence from both epidemiologic and randomized controlled studies, and preference has been given to evidence that is based on systematic reviews or meta - analyses rather than single observational studies or trials. the guidelines help to risk - stratify patients who will benefit most from drug therapy.1 total cvd risk for an asymptomatic individual is estimated from several risk factors, ie, age, gender, smoking habit, systolic blood pressure, and ratio of total cholesterol (tc) to high - density lipoprotein (hdl) cholesterol. this is expressed as a probability (percentage chance) of developing cvd over a defined period of time, which in the jbs guideline is calculated over 10 years. the logic for estimating total cvd risk based on major risk factors is that cvd is multifactorial in origin, risk factors tend to cluster, and coexistent risk factors tend to have a multiplicative effect on cvd risk.2 according to the jbs, statin therapy in the cvd risk range of 10%20% is appropriate on the basis of scientific evidence, although absolute benefits will be small. when the cvd risk is 9%), elevated blood pressure requiring antihypertensive therapy, raised tc (6.0 mmol / l), features of metabolic syndrome (central obesity and fasting triglycerides > 1.7 mmol / l [non - fasting > 2.0 mmol / l ], and/or hdl cholesterol 0.05, figure 3). tc levels were also similar for the different dosages of statins (p > 0.05). on further analysis of the data, we noticed that patients whose tc levels were 6 mmol (n = 34) received simvastatin (n = 21, 61%), atorvastatin (n = 9, 26%), and pravastatin (n = 4, 11%). patients whose tc levels were > 6 mmol (n = 17) received atorvastatin (n = 13, 76%) and simvastatin (n = 4, 24%). this study shows that statin prescriptions for primary prevention in secondary care vary with dose and choice of statin. a preference for atorvastatin and simvastatin in patients whose tc levels were > 6 mmol was identified. this prescription tendency may be influenced by local guidelines for secondary prevention (cheshire and merseyside cardiac network), which actually suggest a starting dose of simvastatin at 40 mg (baseline cholesterol 6 mmol) and of atorvastatin at 40 mg (baseline cholesterol 6 mmol). these variations in prescribing may occur because there is no set guidance for starting or maintenance doses of statins for primary prevention. the jbs cvd risk prediction charts or similar risk scoring tools should be used before initiating statins for primary prevention. this approach would not only identify the group of patients who would benefit most from statins but also limit side effects due to inappropriate use, and also have huge financial implications within the national health service. future clinical trials to compare various statins and their optimum doses (also in various ethnic groups) for primary prevention are needed to improve the effectiveness of statin prescriptions for primary prevention. statin therapy for primary prevention should be offered to patients who are at increased risk of cardiovascular disease the jbs cvd risk scoring system helps to risk - stratify asymptomatic patients who would benefit the most from treatment clinicians are keen to treat cholesterol to target levels ; however, patients are not risk - stratified appropriately and may receive unnecessary treatment regional guidelines for secondary prevention appear to influence statin prescriptions for primary prevention clinicians appear to favor a higher starting dose of statin for primary prevention. only 14% of patients received statin therapy appropriately for primary prevention, and the rest of the prescriptions were issued to patients with a low to medium cardiovascular composite risk score. we noticed that the choice of statin and dosage were not related to tc levels or jbs cvs risk score. however, we identified a preference for atorvastatin and simvastatin in patients whose tc levels were > 6 mmol. we noticed an influence of secondary prevention guidelines in primary prevention prescriptions, which could potentially lead to more side effects and noncompliance with statins, especially in a group of patients who are otherwise healthy.10 | introduction : statin therapy is a well established treatment for hyperlipidemia. however, little is known about prescribing of statins for primary prevention in the real world, and even less about what happens to patients requiring primary prevention who are seen in a secondary care setting. the purpose of this research was to investigate the appropriateness of statin prescriptions by using the joint british society cardiovascular disease (jbs cvd) risk score for primary prevention in a large secondary care center.methods:we retrospectively analyzed 500 consecutive patients in whom a statin prescription was initiated over a four - month period. we excluded patients who met secondary prevention criteria. we used the jbs cvd risk prediction chart to calculate 10-year composite risk. we also studied which statins were prescribed and their starting doses.results:of 500 patients consecutively started on statins in secondary care, 51 patients (10.2%) were treated for primary prevention. of these, seven (14%) patients had a 10-year composite cardiovascular event risk of more than 20% (high - risk category), and were hence receiving appropriate therapy. three main statins were prescribed for primary prevention, ie, atorvastatin (22 patients, 43%), simvastatin (25 patients, 49%), and pravastatin (four patients, 8%). the statins prescribed were initiated mainly at the 40 mg dose.conclusions:statin prescribing in secondary care for primary prevention is limited to about 10% of initiations. there is some overprescribing, because 86% of these patients did not require statins when risk - stratified appropriately. the majority of the prescriptions were for simvastatin 40 mg and atorvastatin 40 mg. |
the effects of several pesticides, mammary carcinogens, and antiestrogens on 17beta - estradiol (e2), 16alpha- and 2-hydroxylase activities, and 16alpha-/2-hydroxyestrone (ohe1) ratios were investigated in mcf-7 cells using a radiometric assay. the mammary carcinogens 7,12-dimethylbenz[a]anthracene (dmba) and benzo[a]pyrene (bap), respectively, increased and decreased 16alpha-/2-ohe1 ratios at some concentrations. the 16alpha-/2-ohe1 metabolite ratios for 10(-5) m kepone, atrazine, p, p'-dde, o, p'-dde, o, p'-ddt, and beta - hexachlorocyclohexane were 1.82 + /- 0.060, 0.71 + /- 0.027, 0.66 + /- 0.030, 1.56 + /- 0.089, 1.14 + /- 0.059, and 0.69 + /- 0.052 (mean + /- standard error), respectively, and did not show any specific trend. the effects of a series of direct and indirect acting antiestrogens on 16alpha-/2-ohe1 metabolite ratios were also investigated, and the results were compound specific. indole-3-carbinol, tamoxifen, 4'-hydroxytamoxifen, and 9-cis, retinoic acid decreased the ratio ; the effects of all trans - retinoic acid and 2,3,7,8-tetrachlorodibenzo - p - dioxin were concentration dependent ; the antiestrogen ici 182,780 increased the 16alpha-/2-ohe1 metabolite ratio. the results indicate that in mcf-7 cells treated with pesticides, mammary carcinogens, and antiestrogens, there were both increased and decreased 16alpha-/2-ohe1 metabolite ratios for each class of chemicals and the assay did not predict mammary carcinogens.imagesfigure 1figure 2figure 3figure 4 |
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cd117 is a kit membrane tyrosine kinase receptor codified by the gene c - kit and expressed on the cell plasma membrane. in the adult, the so - called interstitial cells of cajal (icc) and the mast cells are among the cell types that are c - kit (or cd117)-positive. the icc form networks within the gastrointestinal (gi) muscle coat and there is a general agreement [18 ] that they (i) are the cells described and called interstitial cells or interstitial neurons by cajal, (ii) are c - kit - positive, (iii) have a characteristic ultrastruc - ture and (iv) are directly involved in the gi motor activities. cd34 is a sialylated transmembrane glycoprotein and its expression is a hallmark of haematopoietic stem cells [1012 ]. cd34 expression decreases as these cells differentiate and it is no more expressed by the mature blood cells. interestingly, cd34 is also expressed by cells outside the haematopoietic system. among them, the endothelial cells, cells identified as fibroblasts, the so - called iclc (interstitial cajal - like cells) described in several organs [1525 ], and some cells observed in the gi wall [26, 27 ]. most of these cells, in particular the iclc and the gi cells located in the connective tissue among the muscle bundles and around the myenteric plexus ganglia and nerve bundles, have an elongated and ramified body resembling the icc ; however, a sure identification of these cells is still lacking. it has been suggested that both gists and egists (gastrointestinal and extra - gastrointestinal stromal tumours) originate from the icc ; this hypothesis was built on the observation that almost all these tumours were made by cd117-positive cells [2833 ]. moreover, cd34-positive cells might be present in these tumours, and, therefore, the question rose on whether in the gists and egists also these cells are to be considered as icc. up - to - now, this question remains disputed because only according to reports obtained in the gists [see 34 and 35 for review of literature ] the icc are positive for both kit and cd34 whereas, according to the findings obtained in controls the icc never express the cd34 [26, 27 ]. all the reports on the cd34-positive cells are based on immunohistochemical identification and, curiously, nobody has considered the possibility that the cells described in the gut by cajal might be comprehensive of two cell types : the c - kit- and the cd34-positive cells. the present study was aimed to build a map of the cd34-positive cells within the human gi tract, and, in particular, to morphologically characterize those present in the muscle coat where icc are undoubtedly present and well recognizable for their location, c - kit - positivity and ultrastructure. at this aim, we made immunohistochemistry for both cd34 and cd117 receptors, verifying their expression under light and fluorescence microscope, and performed a routine and a cd34-immuno - electron microscopy. the results obtained would allow establishing whether the cd34-positive cells can be considered a subpopulation of the icc or, instead, a novel independent cell type. furthermore, a definitive morphological characterization of this cell type could be useful to define its nature, to propose a role and to better understand which are the cell types present in the gists. samples of gastric (fundus, corpus and antrum) and intestinal (ileum, ascending and descending colon) wall were obtained from patients operated for cancer. patients were 6 (3 females and 3 males, mean age 68 years) considering this study did not require a high number of cases. care was taken in taking specimens far from the tumour and in choosing the areas devoid of inflammation. specimens were processed for light, fluorescence and electron microscope immunohistochemistry and for routine electron microscopy. four - micron - thick sections were cut from formalin - fixed and paraffin - embedded tissue blocks. they were mounted on gelatinized slides, de - waxed in xylene and re - hydrated through a graded series of ethanol. for heat - induced antigen retrieval, the samples were treated for 20 min. at 9092c in buffer tris 10 mm / l and edta 1 mm / l ph 9.0. for light microscope detection, endogenous peroxidase and then the sections were pre - incubated in 1% bovine serum (bsa) in pbs for 30 min. cd117, c - kit rabbit poly - clonal antibody (dako, mi, italy), was used at a final dilution of 1 : 400 ; cd34 mouse monoclonal antibody (dako, mi, italy) was used at a final dilution of 1 : 25. all the antibodies were diluted in bsa 0.1% in pbs and applied for 30 min. at room temperature. after washing, the sections were incubated with a polyclonal biotinylated secondary antibody (vector laboratories, burlingame, ca, usa) diluted 1 : 1000, or with a monoclonal biotinylated secondary antibody (vector laboratories, burlingame, ca, usa) diluted 1 : 300 in 0.1% bsa in pbs, for 90 min. at room temperature. subsequently, the sections were washed and abc reagent (vectastain abc / elite kit, vector laboratories, burlingame, ca, usa) was added for 20 min. and developed with diaminobenzidine (dab) (sigma, st. sections not exposed to the primary antibody were included as negative controls for antibody specificity. all the sections were counterstained with haematoxylin for nuclei labelling and observed under the zeiss axioscop light microscope. for fluorescence microscope detection, some sections were pre - incubated in 1% bsa in pbs for 30 min. the c - kit rabbit polyclonal antibody (dako, mi, italy) was used at a final dilution of 1 : 100 and the cd34 mouse monoclonal antibody (dako, mi, italy) was used at a final dilution of 1 : 25. all the antibodies were diluted in bsa 0.1% in pbs and applied overnight at 4c. at the end of the incubation, the sections were washed in pbs and incubated with fluorescein cy2-conjugated affinipure f(ab1)2 fragment goat anti - rabbit igg (h+l ; jackson immunoresearch, west grove, pa, usa) secondary antibody, diluted 1 : 50 or with texas red conjugated affinity purified igg (h+l) anti - mouse secondary antibody raised in horse (vector laboratories, burlingame, ca, usa) diluted 1 : 100. all the secondary antibodies were diluted in bsa 0.1% in pbs and applied for 2 hrs, in the dark, at room temperature. for cd117/cd34 double labelling, the sections were incubated as above mentioned putting together the primary antibodies. all the sections were then mounted in an aqueous medium (gel mount, biomeda corp., foster city, ca, usa) and observed under an epifluorescence zeiss axioskop microscope and photographed. for conventional electron microscopy, full - thickness strips of the muscle coat of the stomach and intestine were immersed in a fixative solution of 2% cacodylate - buffered glutaraldehyde (ph 7.4) and kept in this solution for 6 hrs. then, they were rinsed in a cacodylate - buffered solution supplemented with sucrose, post - fixed with 1% phosphate - buffered oso4 (ph 7.4) for 2 hrs, dehydrated with graded alcohol, clarified in propylene oxide and embedded in epon using flat moulds. for pre - embedding immuno - electron microscopy, full - thickness thin sections of the muscle coat were fixed for 20 min. at room temperature with the plp fixative that was prepared by mixing immediately before use equal volumes of paraformaldehyde 8% in distilled water and 0.2 m pbs containing 0.055 g / l naio4 and after washing in 0.1 m pbs, the specimens were pre - incubated in 0.1 m pbs (ph 7.4), added with 1% bsa, for 15 min. at room temperature and, then, incubated in the presence of the cd34 primary antibody. after washing, the samples were incubated in the presence of horseradish peroxidase (hrp) biotinylated anti - rabbit secondary antibody (vectastain abc kit ; vector laboratories, burlingame, ca, usa), 1 drop/5 ml 0.1 m pbs, for 30 min. at room temperature and the specimens were then washed and treated with avidin - biotin complex system (vectastain abc kit ; vector laboratories) for 10 min. and louis, mo, usa) 0.015 g in 10 ml of 0.5 m tris buffer (ph 7.4) and two drops of h2o2 3%. samples were observed under a light microscope to verify the presence of the immunoreaction and the selected areas were cut and post - fixed in 1% phosphate - buffered oso4 (ph 7.4) for 2 hrs at room temperature, dehydrated and embedded as described above for conventional electron microscopy. the semi - thin sections of both types of samples were obtained with a lkb nova ultra - microtome, either unstained or stained with a solution of toluidine blue in 0.1 m borate buffer, and then observed under a light microscope. ultra - thin sections of the selected areas were obtained with the same ultra - microtome using a diamond knife and for conventional electron microscopy stained with an alcoholic solution of uranyl acetate, followed by a solution of concentrated bismuth subnitrate ; for the immunostained specimens, some sections were observed without staining them and some others were stained with a solution of lead citrate. all the ultra - thin sections were examined under a jeol 1010 electron microscope and photographed. everywhere in the gi wall, the endothelial cells are cd34-positive and c - kit - negative (fig. 1a and c), and mast cells are cd34-nega - tive and c - kit - positive (fig.. moreover, other types of cd34-positive cells are present in the submucosa (fig. 1e and f) and either cd34- or c - kit - positive cells are located in the muscularis propria. a, c, e and f : cd34-immunoreactivity ; b and d : c - kit - immunoreactivity. (a) (b) intestinal villous, mast cells only are c - kit- positive (arrows). (c - f) submucosa. most of the cd34-positive cells are sparsely distributed (c, stomach, e, large intestine, f, small intestine) and some others form a thin and continuous layer that encircles large vessels (c, stomach, f, small intestine) and submucous plexus ganglia (e, large intestine). bar : a - d = 35 m ; e, f = 20 m. examined, the cd34-positive cells are located around and within the muscle bundles (fig. these cells are polymorphic, but have in common an elongated shape, a small body and thin processes (fig. the body contains the nucleus and a variable amount of cytoplasm and its shape might be oval or triangular. processes are thin and long and their number is highly variable, as well as that of their ramifications (fig. the cd34-positive cells make an almost continuous sheath around myenteric ganglia (fig. 2c and g) and a discontinuous layer at the submucosal border of the circular muscle layer (fig. although often close to each other, these cells do not clearly form networks, either within the muscle coat (fig. 2a and b) or at the myenteric plexus level (fig. 2 g). on the contrary, the c - kit - positive icc, although sharing similar morphologies with the cd34-positive cells being elongated and provided of processes, form networks either intramuscularly or at the myenteric plexus level (fig. 2a) are only occasionally encountered at the level of the deep muscular plexus (dmp), where c - kit - positive cells identifiable as icc - dmp are numerous and form a network. by double labelling, cd34- and c - kit - positivity c), although the cd34- and the c - kit - positive cells are often so close to each other to suggest that a single cell has a c - kit - positive body and cd34-positive processes or a cd34-positive body and c - kit - positive processes. moreover, it can be undoubtedly appreciated when the bodies and the processes of the cd34-positive and the c - kit - positive cells are strictly intermingled (fig. 3b) as well as when the cd34-positive cells run in rows intercalated with the c - kit - positive cells (fig. a - g : cd34-immunoreactivity ; h : c - kit - immunoreactivity a : small intestine. the cd34-positive cells form a discontinuous layer at the submucosal border of the circular muscle layer and some of them are intramuscularly located (arrows). cd34-positive cells (arrowheads) located in the connective septa form a discontinuous layer close to muscle bundles ; some other cells (arrows) are within the muscle bundles. c, d and g : cd34-pos - itive cells make an almost continuous sheath around myenteric ganglia (c, small intestine and g, stomach) and nerve strands (d, large intestine). at the level of nerve strands and in between the ganglia these cells form a discontinuous three - dimensional network (d and g). e and f : everywhere in the gi tract, the cd34-positive cells have an elongated shape, a small body and a variable number of thin and long processes. h : small intestine ; a high number of c - kit - positive cells at the myenteric plexus level where form networks. mp : ganglia at the myenteric plexus ; n : nerve strands at the myenteric plexus. bar : a = 35 m ; b - g = 20 m ; h = 80 m. (b) the cd34-positive and the c - kit - positive cells are often very close to each other. arrows indicate the processes of a cd34-positive cell passing over the body of a c - kit - positive cell. c : a row formed by a c - kit - positive cell intercalated between two cd34-positive cells. : cd34-positive cells ; : c - kit - positive cells. none of the other connective tissue cells is cd34-positive in the mucosa, while most of them are cd34-positive in the submucosa (fig. moreover, some of the cd34-positive cells characteristically form a thin and almost continuous layer that encircles large vessels (fig. at all level examined, there are cells identifiable as icc for their ultrastructural characteristics [3639 ]. moreover, other cells frequently run close to the icc (figs. 4 and 5), thus sharing a similar distribution. 4b) and entering nerve strands where form an apparently discontinuous three - dimensional network (fig. 4a). some of these cells form a discontinuous layer at both the submucosal and mesothelial borders of the circular (fig. 5a and b) and longitudinal muscle layers. in the stomach (antrum), cells with these features surround groups made by nerve bundles and icc (fig. 5c and d) and some of their processes apparently contribute to form the aforementioned submucosal sheath (fig. 4c) are located in the intramuscular connective septa, where they form a discontinuous sheath around muscle bundles in continuity with the submucosal one. finally, some other cells are sparsely distributed among the smooth muscle cells (fig. all these cells, at variance with the icc, have a very small oval or triangular body containing the nucleus (figs. 4a d, 5c, d) and extremely long and thin processes (fig. 5). the cytoplasm is scarce and contains cisternae of the rough endo - plasmic reticulum (figs. 4a, d, 5c), especially numerous in the nucleated portion, a small golgi apparatus and mitochondria sparsely distributed (figs. 4 and 5). triangularly shaped enlargements (knobs) occur all along the processes, where both mitochondria and rough endoplasmic reticulum cisternae are contained (figs. 4a and 5d). 4a) and between these processes and the icc body can be seen ; on the contrary, none of these cells establishes specialized contacts with smooth muscle cells and is in contact with nerve endings. as already described by one of us, the intramuscular icc are often intercalated with these cells. a : cells (iclc) with a small oval or triangular body, long and thin processes and fibroblastic features cover nerve strands (a) and myenteric ganglia (b). along the processes arrowheads indicate contact areas between the cell processes, one of which is enlarged in the inset. c : one fibroblast - like cell (iclc) located in a connective septum, in the vicinity with a nerve bundle (n) and an icc (icc). d : two interconnected fibroblast - like cells (iclc) located among the smooth muscle cells and in the vicinity of a nerve bundle (n). bar : a - c = 1.3 m ; d = 0.6 m ; inset = 0.3 m. a and b : thin processes of the iclc cover the submucosal border of the circular muscle layer. c and d : cells with fibroblastic features (iclc) surround nerve bundles and groups of icc (icc) located at the submucosal border of the circular muscle layer of the antrum. in d, some of the iclc processes run towards the circular muscle (cm) to cover its submucosal surface. bar : a and b = 0.9 m ; c = 1.3 m ; d = 2 m. the labelling is exclusively present on the plasma membrane and distributed all along of the entire cell contour, caveolae included. the plasma membrane appears as a dark and thick layer from which regularly distanced spherules protrude within the glycocalyx (fig. cd34-immunoelectro - labelling is present on cells with iclc features (a - f) and on the endothelium (c). the labelling appears as an electron - dense material distributed all along the plasma membrane, from which spherules protrude outside. bar : a and b = 1 m ; c = 2 m ; d, e = 0.5 m ; f = 0.4 m. everywhere in the gi wall, the endothelial cells are cd34-positive and c - kit - negative (fig. 1a and c), and mast cells are cd34-nega - tive and c - kit - positive (fig.. moreover, other types of cd34-positive cells are present in the submucosa (fig. 1e and f) and either cd34- or c - kit - positive cells are located in the muscularis propria. a, c, e and f : cd34-immunoreactivity ; b and d : c - kit - immunoreactivity. (a) (b) intestinal villous, mast cells only are c - kit- positive (arrows). (c - f) submucosa. most of the cd34-positive cells are sparsely distributed (c, stomach, e, large intestine, f, small intestine) and some others form a thin and continuous layer that encircles large vessels (c, stomach, f, small intestine) and submucous plexus ganglia (e, large intestine). bar : a - d = 35 m ; e, f = 20 m. examined, the cd34-positive cells are located around and within the muscle bundles (fig. these cells are polymorphic, but have in common an elongated shape, a small body and thin processes (fig. the body contains the nucleus and a variable amount of cytoplasm and its shape might be oval or triangular. processes are thin and long and their number is highly variable, as well as that of their ramifications (fig. the cd34-positive cells make an almost continuous sheath around myenteric ganglia (fig. 2c and g) and a discontinuous layer at the submucosal border of the circular muscle layer (fig. although often close to each other, these cells do not clearly form networks, either within the muscle coat (fig. 2a and b) or at the myenteric plexus level (fig. 2 g). on the contrary, the c - kit - positive icc, although sharing similar morphologies with the cd34-positive cells being elongated and provided of processes, form networks either intramuscularly or at the myenteric plexus level (fig. 2a) are only occasionally encountered at the level of the deep muscular plexus (dmp), where c - kit - positive cells identifiable as icc - dmp are numerous and form a network. by double labelling, cd34- and c - kit - positivity c), although the cd34- and the c - kit - positive cells are often so close to each other to suggest that a single cell has a c - kit - positive body and cd34-positive processes or a cd34-positive body and c - kit - positive processes. moreover, it can be undoubtedly appreciated when the bodies and the processes of the cd34-positive and the c - kit - positive cells are strictly intermingled (fig. 3b) as well as when the cd34-positive cells run in rows intercalated with the c - kit - positive cells (fig. a - g : cd34-immunoreactivity ; h : c - kit - immunoreactivity a : small intestine. the cd34-positive cells form a discontinuous layer at the submucosal border of the circular muscle layer and some of them are intramuscularly located (arrows). cd34-positive cells (arrowheads) located in the connective septa form a discontinuous layer close to muscle bundles ; some other cells (arrows) are within the muscle bundles. c, d and g : cd34-pos - itive cells make an almost continuous sheath around myenteric ganglia (c, small intestine and g, stomach) and nerve strands (d, large intestine). at the level of nerve strands and in between the ganglia these cells form a discontinuous three - dimensional network (d and g). e and f : everywhere in the gi tract, the cd34-positive cells have an elongated shape, a small body and a variable number of thin and long processes. h : small intestine ; a high number of c - kit - positive cells at the myenteric plexus level where form networks. mp : ganglia at the myenteric plexus ; n : nerve strands at the myenteric plexus. bar : a = 35 m ; b - g = 20 m ; h = 80 m. (b) the cd34-positive and the c - kit - positive cells are often very close to each other. arrows indicate the processes of a cd34-positive cell passing over the body of a c - kit - positive cell. c : a row formed by a c - kit - positive cell intercalated between two cd34-positive cells. : cd34-positive cells ; : c - kit - positive cells. none of the other connective tissue cells is cd34-positive in the mucosa, while most of them are cd34-positive in the submucosa (fig. moreover, some of the cd34-positive cells characteristically form a thin and almost continuous layer that encircles large vessels (fig. at all level examined, there are cells identifiable as icc for their ultrastructural characteristics [3639 ]. moreover, other cells frequently run close to the icc (figs. 4 and 5), thus sharing a similar distribution. 4b) and entering nerve strands where form an apparently discontinuous three - dimensional network (fig. 4a). some of these cells form a discontinuous layer at both the submucosal and mesothelial borders of the circular (fig. 5a and b) and longitudinal muscle layers. in the stomach (antrum), cells with these features surround groups made by nerve bundles and icc (fig. 5c and d) and some of their processes apparently contribute to form the aforementioned submucosal sheath (fig.. 4c) are located in the intramuscular connective septa, where they form a discontinuous sheath around muscle bundles in continuity with the submucosal one. finally, some other cells are sparsely distributed among the smooth muscle cells (fig. all these cells, at variance with the icc, have a very small oval or triangular body containing the nucleus (figs. 4a d, 5c, d) and extremely long and thin processes (fig. 5). the cytoplasm is scarce and contains cisternae of the rough endo - plasmic reticulum (figs. 4a, d, 5c), especially numerous in the nucleated portion, a small golgi apparatus and mitochondria sparsely distributed (figs. 4 and 5). triangularly shaped enlargements (knobs) occur all along the processes, where both mitochondria and rough endoplasmic reticulum cisternae are contained (figs. 4a and 5d). 4a) and between these processes and the icc body can be seen ; on the contrary, none of these cells establishes specialized contacts with smooth muscle cells and is in contact with nerve endings. as already described by one of us, the intramuscular icc are often intercalated with these cells. a : cells (iclc) with a small oval or triangular body, long and thin processes and fibroblastic features cover nerve strands (a) and myenteric ganglia (b). along the processes arrowheads indicate contact areas between the cell processes, one of which is enlarged in the inset. c : one fibroblast - like cell (iclc) located in a connective septum, in the vicinity with a nerve bundle (n) and an icc (icc). d : two interconnected fibroblast - like cells (iclc) located among the smooth muscle cells and in the vicinity of a nerve bundle (n). bar : a - c = 1.3 m ; d = 0.6 m ; inset = 0.3 m. b : small intestine. a and b : thin processes of the iclc cover the submucosal border of the circular muscle layer. c and d : cells with fibroblastic features (iclc) surround nerve bundles and groups of icc (icc) located at the submucosal border of the circular muscle layer of the antrum. in d, some of the iclc processes run towards the circular muscle (cm) to cover its submucosal surface. bar : a and b = 0.9 m ; c = 1.3 m ; d = 2 m. the labelling is exclusively present on the plasma membrane and distributed all along of the entire cell contour, caveolae included. the plasma membrane appears as a dark and thick layer from which regularly distanced spherules protrude within the glycocalyx (fig. cd34-immunoelectro - labelling is present on cells with iclc features (a - f) and on the endothelium (c). the labelling appears as an electron - dense material distributed all along the plasma membrane, from which spherules protrude outside. bar : a and b = 1 m ; c = 2 m ; d, e = 0.5 m ; f = 0.4 m. in the present study, the distribution of the cd34-positive cells in the wall of the entire human gi tract has been identified by immunohistochemistry. some differences, however, were seen between the cells located in the muscularis propria and those located in the submucosa. indeed, in the muscularis propria, the cd34-positive cells shared some morphological characteristics similar to those of another cell type present at the same location, the c - kit - positive cell or icc, while in the submucosa, where the icc are absent, the cd34-positive cells resembled fibrocytes/ fibroblasts. moreover, routine electron microscope examination allowed providing an unequivocal identification and an ultrastructural characterization of the cd34-positive cells. under the electron microscope, we presently observed cells not yet identified and having specific characteristics. their ultra - structure is simple and mainly similar to that of both the fibrocytes (quiescent cells) and the fibroblasts (activated cells) and different from that of the typical human icc [3639 ]. these cells, therefore, might correspond to the cells recently described in the gi muscle coat as fibroblast - like cells. importantly, under transmission electron microscope, only the cells with these features were cd34-positive and the ultrastructural distribution of the cd34-immunoreactivity is in agreement with the fact that cd34 is a sialylated transmembrane glycoprotein. indeed, labelling was specifically located within the thickness of the plasma membrane and on spherules protruding outside at the level of the glycocalyx. recently, popescu group [1524 ] described in several organs of humans a cell type, named iclc (interstitial cajal - like cells), having ultrastructural features practically identical to those presently observed for the cd34-positive cells located within the muscle coat of the human gi tract. briefly, we can reasonably conclude that the cd34-positive fibroblast - like cells we identified in the gi muscle coat of man might be identified as iclc. in the table 1, the ultrastructural differences and similarities among the human icc, the iclc, the fibroblasts and the fibrocytes are summarized. interestingly, due to the fact that the iclc have been described in a great variety of organs, these cells can be considered as a ubiquitous cell type. popescu group has also studied these cells by immunohistochemistry and found a larger numbers of cells stained for cd34 than for c - kit, suggesting not all of the iclc co - express the two molecules. moreover, in the iclc isolated from the fallopian tube, these authors could see that in the double - labelled cells the c - kit antibody mainly stained the cell body, while cd34 antibody preferentially stained the cell processes. ultrastructural characteristics of the icc, iclc, fibrocytes and fibroblasts of the human gi tract the question on whether the cd34-positive cells are or not to be considered as icc is solved by the present data. in agreement with vanderwinden.[26, 27 ], the c - kit- and the cd34-immunoreactivities were never seen to co - localize at any level of the human gi tract, although some cells seemed to possess the two immunoreactivities due to the vicinity of the cell bodies and the intermingling cell processes. the absence of a co - localization is in contrast with other reports according to which icc or at least a large subset of them are cd34-positive in the human gut. these studies, however, are mainly conducted in gists and egists [see 3336 for review of literature ] and the hypothesis of a co - localization is based on the fact that the c - kit- and the cd34-positive cells share a similar localization rather than on a double labelling. moreover, it has also to be considered that most of the reports are in favour for a co - expression in these tumours and not in controls. in this regard, it has never been considered that the cd34-positive cells present in the tumours might be the endothelial cells. finally, the presence in the gists of cd34- and c - kit - positive cells might also be explained not by a cd34/c - kit co - localization, rather by the presence of both cell types which, as presently shown, are normally resident and intermingled in the gi muscle coat. cajal described in the gut the cells, which are now known as icc and similar cells in other organs. cajal called all of them interstitial cells (neurons) and used methods, such as methylene blue vital staining and silver impregnations, which are highly capricious. as an example, these methods do not label, at least in the gut, all the icc populations and work differently in the various animal species. as already stressed, we must accept that the cells corresponding to the gi interstitial cells of cajal are the cells that in the gut are c - kit - positive, have a specific ultrastructure and a well ascertained role in gi motility. nobody has up to now considered the possibility that the interstitial cells cajal described in the gut might be a mixture of c - kit- and cd34-positive cells. the novelty of the present study is that in the muscle coat of the human gi tract, together with the typical icc, there is another type of cell we consider to correspond to the iclc described by popescu group and, even though also this cell might belong to the interstitial cells ' described by cajal, it has not to be confused with the icc. we have presently provided a detailed description of the ultra - structural features of this new cell type but it still remains to understand its role. the iclc located in the rat mesentery have been proposed to form a three - dimensional network at the same time resistant and deformable following stretches consequent to intestine movements, mainly avoiding blood vessels closure. yamazaki and eyden suggested for the network of cd34-positive interstitial cells located in human fallopian tube a putative immune surveillance role. both roles might be proposed also for the iclc we found in the gi muscle coat, in particular the presence at the myenteric plexus level of a three - dimensional network resistant to and deformable following intestine movements is easily conceivable and in this case these cells might correspond to the covering cells '. conversely, it is hard to identify the role of the iclc accompanying nerve bundles and icc at the submucosal border of the circular muscle layer of the gastric antrum. these iclc seem to continue with those covering the entire submucosal border of the circular muscle layer and with the septal ones. at present finally, since some of the intramuscular icc and iclc seem to be part of a unique network in which icc only are innervated, we would suggest these intramuscular iclc play a role in neurotransmission, possibly contributing to spread the slow waves generated by the icc. taking into account the formers, another more intriguing role might be hypothesized for the gastrointestinal iclc. it has been reported, both in humans and laboratory mammals, that the immature icc are fibroblast - like during the pre- and post - natal life [43, 44 ] ; similarly, after bac treatment, the newly differentiating icc have fibroblast - like features. furthermore, although icc undergo to apoptosis, their number does not change significantly with aging ; nevertheless, dividing icc have never been observed either in the foetal stages or in the adults whereas mitotic figures were found in both the smooth muscle and fibroblast - like cells (faussone - pellegrini, personal observations). keeping in mind all these data, especially considering that the iclc share fibroblast - like features with the immature icc and are often located in proximity or even intercalated with the icc, we hypothesize that the iclc might be identified with the mesenchymal stromal cells derived from the mesenchymal cells and which are present in the adults and reasonably comprehensive of a pool of icc precursors. in agreement with this hypothesis, it has been recently reported that murine immature gastric cells expressing in vitro cd34 at high level and c - kit at very low level might differentiate into icc by stopping to express cd34 and increasing c - kit labelling, and it has also been proposed that the starting cell is the mesenchymal stem cell and the intermediate cell the fibroblast - like cell. in conclusion, cells we identified as iclc are present within the human gut. in the muscle coat, these cells are very often close to the icc and also intercalated with them ; however, their ultrastructure is different from that of the human icc. these cells are sometimes in contact with the smooth muscle cells but never with nerve endings. several roles can be proposed : a mechanical supporting role, to be involved in neurotransmission, to be important for icc renewal and gists genesis possibly representing a pool of icc precursors. | cd117 (or c - kit) is expressed by the interstitial cells of cajal (icc), which are located within the gastrointestinal (gi) muscle coat and directly involved in its motility. cd34 is expressed by several cell types some of which have features and location resembling the icc ; however, a sure identification of these cells is still lacking. in order to establish whether the cd34-positive cells of the human gi tract are to be considered as icc subpopulation or a novel independent cell type, and to hypothesize their nature and role, we verified cd34 and cd117 receptor expression under light and fluorescence microscope and performed a routine and a cd34-immuno - electron microscopy. cd34-positive cells were seen in the entire human gi tract. in the muscularis propria, shared morphologies similar to the c - kit - positive cells, in the submucosa, resembled fibroblasts. their ultrastructure resembled that of the fibrocytes / fibroblasts and of the interstitial cajal - like cells (iclc). double labelling and immunoelectro - microscopy demonstrated that they are unequivocally different to the icc and, due to the similarities with the iclc, we identified them as iclc. the novelty of these results is that two types of interstitial cells are present in the gi muscle coat of humans : the icc and the iclc. we hypothesize a mechanical role for the septal iclc, those at the myenteric plexus level and those bordering the muscle layers ; a helping role in neurotransmission is proposed for the iclc intercalated with the intramuscular icc, possibly in spreading the slow waves generated by the icc. furthermore, the possibility that the iclc represent the adult mesenchymal stromal cells able to guarantee the icc renewal deserves to be considered. |
fixed partial denture generally consists of two parts, the enduring substructure or coping and the esthetic veneer overlaid on the coping to match the neighboring tooth color. different types of dental alloys have been used as substructures for metal - ceramic restorations. because of their high fracture resistance and high reliability, metal - ceramic restorations have shown exceptionally high success rates even for a long - term treatment.1 - 3 however, it is more difficult to create a metal - based restoration with a natural tooth color and translucency when compared to an all - ceramic prosthesis because a metal substructure does not allow light transmission through a restoration.4,5 in contrast, various core ceramics have been shown to have varied degree of translucency, ranging from a very translucent material to an opaque core ceramic.4 - 6 the varied translucency of these core ceramics has made it uncomplicated to fabricate a restoration that can match its neighboring teeth in terms of color and translucency. the translucency of ceramic materials depends on several factors such as relative refractive index, wavelengths of the light sources, numbers and sizes of porosity and inclusion etc.7 contrast ratio is an optical parameter that is used to represent the degree of translucency of a material.7,8 several studies have used this parameter to compare the light transmission capability or the masking ability of tooth - colored dental restorative materials.4 - 6,9,10 for determining the contrast ratio, the ratio of light reflectance of a material over black and white backgrounds (yb / yw) are measured using a spectrophotometer.7,8 while the contrast ratio for a completely opaque material is 1, a lower contrast ratio represents a more translucent substance. for tooth enamel and dentin, the contrast ratio was approximately 0.55 at 1 mm thickness.11 as previously mentioned, color match between a dental restoration and the adjacent natural teeth depends partly on the degree of translucency of dental restorative material. however, the ability to conceal the discolored abutment tooth or a metal post and core is also a subject of interest for all - ceramic restorations. the masking or covering ability of materials can be defined as a measure of the capability of a coating to hide a colored background and the contrast ratio of 0.98 of a covering layer is proposed for the perfect masking ability in industrial production.8 if the color of an underlying structure can be observed, it would result in the color difference between the covering material and the target color. the color difference in cielab units is given by the following equation;12 (1)eab = [(l) + (a) + (b) ] when l, a and b are the differences in lightness, chroma in red - green axis (a), and chroma in blue - yellow axis (b) of the measured colors of two objects. in dentistry, the color difference or eab is used to evaluate the color match between a dental restoration and the adjacent natural teeth. the perceptible thresholds (eab 2.6 - 3.7) were set as guidelines for color matching determination according to the results from few in vivo studies.13,14 the perceptible thresholds obtained from in vitro studies were lower (eab 0.4 - 1) because of their better viewing and measuring conditions.15,16 however, the color differences were reported even for the matched natural teeth.17 for a perfect match between natural upper central incisors, the reported eab values ranged from 0.1 to 1.6. for a perfect match between an upper natural central incisor and a contralateral all - ceramic crown, eab values varied from 0.2 to 2.9 with an average of 1.6. zirconium dioxide or zirconia has presently received considerable attention from dental practitioners because of its high fracture resistance and excellent biocompatibility. most zirconia - based core materials obtained from different manufacturers are yttria - stabilized tetragonal zirconia polycrystals or y - tzp. even though they are all polycrystalline materials with comparable compositions, they could have slightly different microstructures.18 as a result, y - tzps could have different degree of opacity because of an increase or decrease in light scattering caused from the microstructural variations.19 for example, an increase in the light scattering inside the bulk material results from an increase in porosities or inclusions or the discontinuity of refractive indices at the grain boundary.19 in an esthetic viewpoint, a ceramic material with limited light transmission is not desirable because it does not imitate the optical characteristics of a natural tooth. on the contrary, a high opacity ceramic material is required when a restoration is made on an abutment such as a discolored tooth or a metal post and core. for zirconia - based core materials, they appear to be opaque materials and they could be used to mask the dark colors of an underlying substructure.3,20 however, there is limited information about the translucency of zirconia core materials. the objective of this study was to determine the effect of color of an underlying substructure on the overall color of zirconia all - ceramic crowns. the protocol for this study was approved by the mahidol university institutional review board (mu - irb 2008/031.0506). twenty adult subjects, of good to excellent dental health, were recruited from the pool of subjects on the waiting list of the faculty of dentistry at mahidol university in bangkok, thailand. all selected subjects had healthy periodontal tissues, were free of active periodontal disease and caries, and showed no evidence of bruxing. eligible subjects had at least one posterior endodontically treated tooth opposed by natural dentition in the maxillary or mandibular arch. this endodontically treated tooth was used as an abutment for an all - ceramic crown. after obtaining an informed consent to participate in the study, color shade of a contralateral or an adjacent tooth was determined for use as an all - ceramic crown shade. all abutment teeth were prepared either for a metal cast post and core or a prefabricated fiber post with a composite core build - up as foundations for the all - ceramic crowns. the following minimal guidelines for the final tooth preparation were followed ; axial tooth reduction of 1 mm, shoulder or deep chamfer design, occlusal reduction of 1.5 - 2 mm, and rounded inner angles / edges / transitions. the ceramic material used in this study was a zirconia - based core ceramic and a compatible glass - based veneering ceramic (zeno, wieland dental and technik gmbh & co, germany, and ips e. max ceram, ivoclar vivadent ag, liechtenstein). twenty one crowns were made using a layering technique according to the manufacturer 's instructions. these crowns were divided into three groups according to the remaining abutment tooth structure and the appropriate reconstruction of the core foundation as shown in table 1. after try - in and adjustment to produce proper anatomical contours and proper occlusion, each crown was glazed before insertion. the all - ceramic crowns were cemented using resin cement which had simply one shade (multilink, ivoclar vivadent ag, liechtenstein). only one shade resin cement was used in order to limit the influence of cement shade on the color of all - ceramic crowns. examinations of the overall prosthesis, the marginal area, the adjacent gingival tissues, and the occlusion were evaluated before cementation. the thicknesses of the core and veneering materials were measured before cementation at the middle 1/3 of the buccal, lingual and occlusal surfaces of each ceramic crown. color measurements of all crowns were made using a shade measuring device (shadeeye ncc, shofu inc., l represents the lightness of a material, + a represents color in the red axis and -a indicates color in the green axis. this shadeeye ncc intraoral shade measuring device has been used in some previous studies with an acceptable performance.21,22 the color of all - ceramic crowns was measured using a pulsed xenon lamp as an optical light source and a vertical light receiving system. the instrument was calibrated against a standard calibration according to a manufacturer 's recommendation before each color measurement. the contact plastic tip, having a diameter of 3 mm, was positioned at the middle 1/3 of the buccal surface of each crown, the tip made an intimate contact with the crown surface during the measurement. after each measurement, l, a and b value was obtained and used for calculation of color differences between before try - in, before and after cementation of all - ceramic crowns. a repeated measure anova was used for a statistical analysis of a color change between before try - in, before and after cementation of all - ceramic crowns at =.05. in order to obtain the optical properties of a zirconia - based material used in this study, 24 zirconia core ceramic (zeno, wieland dental and technik gmbh & co, germany) were prepared in a laboratory. these rectangular core specimens (15 mm 15 mm) with four different thicknesses (0.4, 0.6, 0.8 and 1.0 mm) were prepared considering a shrinkage during the sintering process. after sintering, zirliner and dentine shade a3 (ips e. max ceram, ivoclarvivadent ag, liechtenstein) was applied onto the specimens and fired in a furnace (programat p100, ivoclarvivadent ag, liechtenstein) according to the manufacturer 's firing instructions. a lithia - disilicate - based core ceramic (15 mm 15 mm 0.8 mm, empress 2, ivoclar vivadent ag, liechtenstein) and a base metal alloy (wiron 99, bego, germany) were also prepared as controls. for a metal - ceramic system, six rectangular specimens, with a dimension of 15 mm 15 mm 0.3 mm lithia - disilicate - based ceramic and metal samples were veneered with dentin porcelains (ips eris, ivoclar vivadent ag, liechtenstein and vita vmk95, vita zahnfabrik, germany). after veneering, all specimens were ground to a final thickness of 1.5 0.1 mm using a milling machine (schick dentalgerate s master 3, vacalon, usa) and a diamond grinding disc with a grit size of 80 m (s327010, bredent gmbh & co. kg, senden, germany). all specimens were glazed by applying a thin layer of the glaze paste onto the grinding surface and fired according to the recommended schedule. after firing, the thickness of each specimen was measured four times and the mean thickness was calculated prior to color measurement. the contrast ratios of all specimens were measured before and after veneering, respectively, using a spectrocolorimeter (colorflex, model 45/0, hunter associates laboratory, inc., all specimens were measured using the 45/0 geometry with cie illuminant d65 and 2 degree observer function. calibration of the machine was made using a black glass and a white tile as recommended by the manufacturer. each specimen was placed at the specimen port with the measuring window of 13 mm in diameter. the spectral reflectance data was obtained in the range of 400 - 700 nm at 10 nm intervals. the mean contrast ratios before and after veneering of each group were calculated and statistical analysis of the data was performed using a mixed / split - plot design anova (spanova) test at =.05. the tukey 's multiple comparison test was used to determine the rank of each group. for all - ceramic crowns, the mean thicknesses of core ceramic at the buccal surface were 0.6, and 0.7 0.1 mm for premolar and molar crowns, respectively. the mean thicknesses at the occlusal surface of the core ceramic were 0.7 0.1 for premolar, and 0.8 0.2 mm for molar crowns. the total thicknesses of all - ceramic crowns after veneering were 1.8 0.3 mm for premolars, and 2.0 0.3 mm for molars at the buccal surface. l, a and b values of all - ceramic crowns cemented on a metal cast post and core are shown in fig. 1, fig. 2 and fig. l, a, and b values did not show a significant change when the values obtained before try - in, before and after cementation were compared. however, l value of all - ceramic crowns with a metal cast post and core appeared to be decreased (-l1) and a tended to be increased (+ a1) when compared between before try - in and before cementation. in contrast, b was not affected much by the color of a background substructure. for the metal cores in these groups, the height of the metal part was approximately 2 - 3 mm covered the remaining tooth structure. l and b values of molar crowns with prefabricated post and composite core build - up also did not show a significant change when compared the values obtained before try - in, before and after cementation. but a was significantly increased when compared between before try - in and before cementation (+ a1). the color differences or eab between the colors of an all - ceramic crown obtained before try - in and before cementation (e1), and between before try - in and after cementation (e2) were determined using equation 1. for in vitro ceramic specimens, the mean contrast ratios before and after veneering of all - ceramic and metal - ceramic materials are summarized in table 3. the contrast ratio of zirconia core specimen was significantly increased from 0.71 to 0.86 as their thickness was increased from 0.4 to 0.8 mm, and no significant difference was found at the thickness of 0.8 and 1.0 mm. after veneering to a final thickness of 1.5 mm, their contrast ratios was increased to 0.92 - 0.95. compared with a translucent lithia disilicate - based all - ceramic system, zirconia core with a thickness of 0.4 mm had a level of contrast ratio similar to this system after veneering. the metal - ceramic system was used as a control group for a completely opaque system, and its contrast ratio was 1.00. even the zirconia - based restorations have significantly higher fracture resistance than other all - ceramic systems, but their limited light transmission as reported from few previous studies is their critical disadvantage.6,20 the contrast ratio of 1.00 was reported for one commercially available zirconia - based core ceramic, whereas the results from another study have shown that a zirconia - based restoration might not be as opaque as expected. an excellent match between a veneered zirconia crown and the adjacent natural anterior tooth has been reported.17 however, the information about the opacity of the zirconia core materials is still limited even though there are many zirconia core systems that are currently available in the market. different levels of light transmission were allowed for dissimilar polycrystalline zirconia - based specimens because of their microstructural dissimilarities.20 these microstructural dissimilarities such as grain sizes, the amount of porosity or inclusions, and the extent and orientation of the grain boundary, are the key factors which responded for the changes in light reflected and transmitted through zirconia materials.19,20 the variation in grain sizes and porosity of y - tzp used in dentistry has been reported in few previous studies.18,23 for posterior zirconia - based all - ceramic crowns used in this study, the changes of l, a and b values were detected after try - in and cemented on the abutment teeth with either metal post and core or prefabricated post and composite core build - up, even though these changes were not statistically significant. the color differences (e1 and e2) values were determined from the changes of l, a and b values. regarding to e1, these values indicated that the color of a background substructure could affect the overall color of posterior zirconia - based all - ceramic crowns. the cement layer would have minor influence on the overall color because e2 was comparable to e1. however, the mean e1 and e2 values (1.2 - 3.1) did not exceed the clinically acceptable limit (eab < 3.7). this result implied that the color modification observed on all - ceramic crowns in this study was instrumentally detectable, but it would still be clinically acceptable. in a previous study conducted in both in vivo and in vitro environments, the contrast ratio could be related to the masking ability of ceramic veneers placed on the discolored abutment.10 even the results from that study showed that the masking ability of 1 mm thick veneer was insufficient to conceal the discolored teeth but the threshold contrast ratio was determined to indicate the value above which the restoration could mask the discolored background. in order to produce eab value of less than 3.7, the contrast ratio of a material should be at least 0.93 - 0.94 as indicated by the results from that previous study.10 in this study, the contrast ratio of zirconia all - ceramic crowns should be at least 0.945 at the core thickness of 0.6 - 0.8 mm according to the results from the in vitro investigation (table 3). therefore, the masking ability of the zirconia crowns was acceptable at these clinically relevant thicknesses. for a thin zirconia coping (0.4 mm) that had comparable opacity as that of a lithia - disilicate - based ceramic, it would be advisable to use a tooth - colored core materials to prevent the traceable dark shadow of a restoration. as mentioned earlier that the changes of l, a and b values were detected after try - in and cemented on the abutment teeth with either metal post and core or prefabricated post and composite core build - up, but the optical properties of metal and nonmetallic material are different because of the differences in their atomic structures.24 for metallic materials, the incident light is absorbed superficially and then reflected from the surface. therefore, metals are opaque and highly reflective and the metallic color could be effectively reflected through overlying translucent materials. for nonmetallic substance, the occurrence of refraction, and transmission of light at the interface and inside the bulk material is unavoidable. as a result of refraction phenomenon, the amount of light scattering plays an important role on the optical properties of nonmetallic materials. for non - zirconia - based all - ceramic crowns, the effects of the core and cement shades were investigated in several in vitro studies.25 - 33 the results from few studies indicated that the core shade and color of the luting agents had minor influence on the overall color of all - ceramic restorations, especially when the ceramic thickness was more than 1.5 mm.25 - 27 on the contrary, the effect of the core shades and cement layer on the overall color of all - ceramic materials was significant in other studies as represented by the high eab values that exceeded the acceptable limits.28 - 33 however, a similar suggestion has been drawn from these studies that the thickness of non - zirconia - based all - ceramic materials is a vital factor for this effect because they are translucent materials. with the thickness less than 1.5 mm, the background shade could be partly detectable through the all - ceramic materials. when the thickness of a ceramic is more than 1.5 mm, the final color of an all - ceramic crown would not be significantly affected by the color of a background substructure or cement. the effects of the core and cement shades were also investigated in anterior zirconia - based all - ceramic crowns.33 the perceptible color difference caused from the substrate and cement shades was observed in that study. because the limited numbers of zirconia crown were observed in this study, the results obtained from this study were the preliminary information for only one type of a zirconia restorative material. another limitation of this study would be a geometric limitation of color measurement. to compensate the discrepancy between the measuring window and specimen size, the covering opaque backgrounds were used during the contrast ratio measurement and it could minimize the edge - loss effect. future researches in this topic are required to obtain more information that can be used in choosing and designing of materials for all - ceramic fixed partial dentures. no significant differences were observed between the l, a and b values obtained before try - in, before and after cementation of posterior zirconia crowns cemented either on a metal cast post and core or a prefabricated post and composite core. however, the color of a background substructure could affect the overall color of premolar and molar zirconia restorations with clinically recommended core thickness based on the changes of eab in this study. | purposethe objective of this study was to determine the effect of the color of a background substructure on the overall color of a zirconia - based all - ceramic crown.materials and methodstwenty one posterior zirconia crowns were made for twenty subjects. seven premolar crowns and six molar crowns were cemented onto abutments with metal post and core in the first and second group. in the third group, eight molar crowns were cemented onto abutments with a prefabricated post and composite core build - up. the color measurements of all - ceramic crowns were made before try - in, before and after cementation. a repeated measure anova was used for a statistical analysis of a color change of all - ceramic crowns at =.05. twenty four zirconia specimens, with different core thicknesses (0.4 - 1 mm) were also prepared to obtain the contrast ratio of zirconia materials after veneering.resultsl, a, and b values of all - ceramic crowns cemented either on a metal cast post and core or on a prefabricated post did not show significant changes (p>.05). however, the slight color changes of zirconia crowns were detected and represented by eab values, ranging from 1.2 to 3.1. the contrast ratios of zirconia specimens were 0.92 - 0.95 after veneering.conclusionno significant differences were observed between the l, a, and b values of zirconia crowns cemented either on a metal cast post and core or a prefabricated post and composite core. however, the color of a background substructure could affect the overall color of posterior zirconia restorations with clinically recommended core thickness according to eab values. |
desmopressin acetate, also known as ddavp (1-deamino-8-d - arginine vasopressin), a selective agonist of type 2 vasopressin receptors, is sometimes used prior to percutaneous renal biopsy to reduce the risk of bleeding complications [14 ]. ddavp was first used in the 1970s in patients with haemophilia a and von willebrand disease prior to surgical interventions to minimize the need for blood products. studies have demonstrated that infusion of ddavp elicits a rapid, transient increase in the levels of von willebrand factor (vwf) and plasma factor viii (fviii), reaching a maximum between 90 min and 2 h post - administration [3, 5 ]. ddavp has been shown to enhance platelet adhesion to vessel walls and transiently releases tissue plasminogen activator into plasma. uraemia has been associated with a prolonged bleeding time, and the use of intravenous ddavp has been shown to normalize bleeding time for up to 8 h despite these patients having normal factor viii and vwf. a recent randomized controlled trial suggested that ddavp administration decreases the risk of bleeding and haematoma size in patients undergoing percutaneous kidney biopsy. potential side effects of ddavp include headache, facial flushing, hypotension and tachycardia. as a synthetic antidiuretic hormone, ddavp increases free water reabsorption in renal collecting ducts and this may lead to water intoxication or dilutional hyponatraemia [5, 8 ]. therefore, the use of ddavp for anti - haemophilic purposes always requires a tight control of fluid balance. we present two cases of ddavp use prior to percutaneous renal transplant biopsy, which were associated with severe hyponatraemia and neurological sequelae. a 67-year - old female, with end - stage renal failure secondary to iga nephropathy, who was 30 months out from receiving a deceased - donor renal transplant, was scheduled to have a transplant biopsy due to deteriorating graft function. mol / l over 3 months, with a corresponding reduction in estimated glomerular filtration rate (egfr) from 60 to 34 ml / min/1.73 m according to the modification of diet in renal disease (mdrd), and this was associated with proteinuria (330 mg in 24 h) although no donor - specific antibody was detected. she had been compliant with her immunosuppression regimen of azathioprine 25 mg once daily and ciclosporin 75 mg twice daily. she had recently been commenced on nortriptyline 10 mg once daily for persistent headaches but was not taking diuretic therapy. other medications included 1-alfacalcidol 0.25 g once daily, aspirin 75 mg once daily, doxazosin 8 mg twice daily, epoetin alfa 2000iu twice weekly, ferrous sulphate 200 mg three times a day, irbesartan 150 mg once daily, simvastatin 20 mg once daily, beclometasone inhaler two puffs twice daily and salbutamol inhaler as required. of note, she had been drinking 45 l of fluid per day in an attempt to improve her graft function and her initial sodium concentration was 127 she was given an infusion of 12 g of intravenous ddavp over 20 min in preparation for biopsy and she was observed for 6 h post - biopsy, remaining haemodynamically stable with no macroscopic haematuria. during her journey home clonic seizure at home and was admitted to the intensive therapy unit (itu) of her local hospital, with a glasgow coma scale (gcs) of 7/15 and a sodium concentration of 107 mmol / l on admission. after eliminating other possible causes of hyponatraemia with biochemical testing, ct and mri scans of the brain and investigation for potential para - neoplastic syndromes, the hyponatraemia was presumed to be related to a combination of excess fluid intake and administration of ddavp. fortunately, the patient recovered following a short itu stay with fluid restriction leading to a gradual correction of hyponatraemia, without any permanent neurological sequelae. a 69-year - old female with end - stage renal failure secondary to hypertension was scheduled for a renal transplant biopsy to investigate deteriorating graft function 13 months after receiving a deceased - donor renal transplant. her creatinine rose to 205 mol / l from a baseline creatinine of 78 mol / l over 8 months (mdrd egfr 67 ml / min/1.73 m to 22 ml / min/1.73 m) in the presence of a donor - specific antibody but no significant proteinuria. she had been compliant with her immunosuppression regimen of prednisolone 5 mg once daily and ciclosporin 35 mg twice daily. she was not receiving diuretic therapy and other medications included alfacalcidol 0.25 g, aspirin 75 mg, atorvastatin 20 mg, bisoprolol 1.25 mg, co - trimoxazole 480 mg and ranitidine 150 mg twice daily. of note, she had been advised to increase her daily oral fluid intake in an attempt to improve graft function. she presented for a day - case renal transplant biopsy and was given an infusion of 12 g intravenous ddavp over 20 min prior to the procedure. the following day, she was found collapsed at home and had a witnessed self - terminating generalized tonic clonic seizure. mmol / l on admission to her local hospital (normal range, 135145 mmol / l). she was treated with fluid restriction and despite initial correction of the hyponatraemia, 2 months later, her sodium concentration remains 132 mmol / l. in both of these cases, patients had been encouraged to increase oral fluid intake in light of the declining graft function and in preparation for biopsy. sodium levels were < 130 mmol / l on pre - admission blood tests prior to ddavp administration, although they had been previously within the normal range. they had not received clear enough instructions to alter their water intake following the use of ddavp and both patients continued to consume their standard post - transplant fluid intake. although these cases are relatively uncommon in our experience of elective renal transplant biopsies, the severity of the clinical presentations render them clinically significant. since ddavp is being used in several renal centres in the uk prior to percutaneous renal biopsy, these cases highlight the need for increased awareness regarding side effects, in particular hyponatraemia. it is common practice to encourage oral fluid intake in the presence of declining graft function and in preparation for biopsy. as a synthetic antidiuretic hormone, ddavp increases free - water reabsorption in renal collecting ducts and this may lead to dilutional hyponatraemia, if appropriate fluid restriction is not instigated. certainly, there have been reports, predominantly in the paediatric literature, of hyponatraemia - related seizures following treatment with ddavp. ddavp itself does not cause hyponatraemia, and it is only with subsequent excessive water intake that this complication can develop. however, dilutional hyponatraemia is largely preventable with appropriate fluid intake restriction. according to the u.s. food and drug administration agency, the half - life of ddavp in patients with severe renal impairment is 9 h. therefore, it follows that patients should be advised to restrict fluid intake from 1 h before to 9 h after administration of ddavp. the use of ddavp as a haemostatic agent to reduce bleeding time prior to percutaneous kidney biopsy continues to be a common practice with potential to decrease the risk of bleeding and haematoma size post - biopsy [13, 7 ] thiazide diuretics, selective serotonin reuptake inhibitors, tricyclic antidepressants, antipsychotics, non - steroidal anti - inflammatory, carbamazepine, sulphonylureas, dopamine agonists, opiates, oxytocin, theophylline, clofibrate, angiotensin - converting enzyme inhibitors, angiotensin - ii receptor antagonists, melphalan, proton pump inhibitors and amiodaroneendocrine disorders hypothyroidism, addison 's diseasesyndrome of inappropriate antidiuretic hormone secretionheart failurechronic liver diseasethe use of ddavp in these patients requires careful consideration of sodium concentrations and current fluid balance as well as the likely fluid intake over the next 24 h and, given the usual practice of liberal fluid intake in renal transplant recipients, ddavp should be used with extreme caution and close monitoring in any such individual, particularly those with low or borderline - low serum sodium levels. in retrospect, the use of ddavp in case 1 who had a pre - biopsy serum sodium of 127 mmol / l should have been contraindicated. thiazide diuretics, selective serotonin reuptake inhibitors, tricyclic antidepressants, antipsychotics, non - steroidal anti - inflammatory, carbamazepine, sulphonylureas, dopamine agonists, opiates, oxytocin, theophylline, clofibrate, angiotensin - converting enzyme inhibitors, angiotensin - ii receptor antagonists, melphalan, proton pump inhibitors and amiodarone endocrine disorders hypothyroidism, addison 's disease syndrome of inappropriate antidiuretic hormone secretion chronic liver disease all patients receiving ddavp therapy should be observed for symptoms associated with hyponatraemia, such as headache, nausea / vomiting, restlessness, fatigue, lethargy, disorientation, depressed reflexes, loss of appetite, irritability, muscle weakness, muscle spasms or cramps and abnormal mental state such as agitation, hallucinations, decreased consciousness and confusion. severe symptoms may include one or a combination of the following : seizure, coma and/or respiratory arrest. patients should be advised to contact a doctor immediately if they feel unwell or experience unusual symptoms. in summary, these cases highlight the importance of appropriate fluid restriction and monitoring following administration of ddavp prior to renal biopsy. to prevent recurrence of similar cases, we have altered our hospital policy to include the points listed below and have included information on the side effects of ddavp in our renal biopsy patient information leaflet. review sodium concentrations prior to administration of ddavp and avoid ddavp use if serum sodium is < 130 mmol / lcaution in patients predisposed to polydipsiafluid restrict patients 1 h prior to and 9 h after administration of ddavpadvise patients to be vigilant of any non - specific symptoms in the first 24 h following ddavp use, with advice to contact a doctor to have repeat serum sodium measurements. review sodium concentrations prior to administration of ddavp and avoid ddavp use if serum sodium is < 130 mmol / l caution in patients predisposed to polydipsia fluid restrict patients 1 h prior to and 9 h after administration of ddavp advise patients to be vigilant of any non - specific symptoms in the first 24 h following ddavp use, with advice to contact a doctor to have repeat serum sodium measurements. this work received no grant from any funding agency in the public, commercial or not - for - profit sectors. the authors of this manuscript have no conflicts of interest and no involvements that might raise the question of bias in the work reported or in the conclusions, implications or opinions stated. the case reports presented in this paper have not been published previously. | desmopressin acetate (ddavp), a selective agonist of type 2 vasopressin receptors, is sometimes used prior to percutaneous renal biopsy to reduce the risk of bleeding complications. ddavp increases free water reabsorption in renal collecting ducts, potentially leading to water intoxication or dilutional hyponatraemia. we present two cases, where ddavp was used prior to percutaneous renal transplant biopsy and was associated with severe hyponatraemia and neurological sequelae. with ddavp being advocated in many centres prior to percutaneous renal biopsy, these cases highlight the need for increased awareness regarding side effects. in this report, we provide suggestions on strategies to minimize hyponatraemia in this context. |
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